International Microbiology by Institut d'Estudis Catalans

CONTENTS IÄã ÙÄ ã®ÊÄ ½ M® ÙÊ ®Ê½Ê¦ù (2014) 17:185-260 ISSN (print): 1139-6709. e-ISSN: 1618-1095 www.im.microbios.org

Volume 17, Number 4, December 2014

RESEARCH ARTICLES

Rizzo A, Carratelli CR, De Filippis A, Bevilacqua N, Tufano MA, Buommino E Transforming activities of Chlamydia pneumoniae in human mesothelial cells Ramió-Pujol S, Ganigué R, Bañeras L, Colprim J Impact of formate on the growth and productivity of Clostridium ljungdahlii PETC and Clostridium carboxidivorans P7 grown on syngas Berlanga M, Domènech Ò, Guerrero R Biofilm formation on polystyrene in detached vs. planktonic cells of polyhydroxyalkanoate-accumulating Halomonas venusta Pérez-Través L, Lopes CA, Barrio E, Querol A Stabilization process in Saccharomyces intra- and interspecific hybrids in fermentative conditions González-Toril E, Santofimia E, López-Pamo E, García-Moyano A, Aguilera A, Amils R Comparative microbial ecology of the water column of an extreme acidic pit lake, Nuestra Señora del Carmen, and the Río Tinto basin (Iberian Pyrite Belt) Cámara B, Suzuki S, Nealson KH, Wierzchos J, Ascaso C, Artieda O, de los Ríos A Ignimbrite textural properties as determinants of endolithic colonization patterns from hyper-arid Atacama Desert

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PERSPECTIVES

ALLEA statement on enhancement of Open Access to scientific publications in Europe

ANNUAL INDEXES and REVIEWERS

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Journal Citations Reports The 2012 Impact Factor of INTERNATIONAL MICROBIOLOGY is 2,556. The journal is covered in several leading abstracting and indexing databases, including the following ones: Agricultural & Environmental Biotechnology Abstracts; ASFA/Aquatic Sciences & Fisheries Abstracts; BIOSIS; CAB Abstracts; Chemical Abstracts; SCOPUS; Current Contents/Agriculture, Biology & Environmental Sciences; EBSCO; EMBASE/Elsevier Bibliographic Databases; Food Science & Technology Abstracts; ICYT/CINDOC; IBECS/ BNCS; ISI Alerting Services; MEDLINE/Index Medicus; Latindex; MedBioWorld; PubMed; SciELO-Spain; Science Citation Index Expanded; SciSearch.

Front cover legends UPPER LEFT. Analysis of a transfer mechanism exploited by the human immunodeficiency virus type 1 (HIV-1) to infect new target immune cells. Electron microscopy micrograph showing the intimate contact between a mature dendritic cell exposed to HIV-1 (top) and a CD4+ T cell (bottom). HIV-1 particles captured by the mature dendritic cell are polarized to the cell-to-cell contact area, favoring CD4+ T cell infection throughout the formation of an infectious synapse. Micrograph by M. Teresa Fernández-Figueras and Núria Izquierdo-Useros, Pathology Dept. at HUGTIP and AIDS Research Institute IrsiCaixa, Barcelona, Spain. (Magnification, ca. 20,000×)

CENTER. The Atacama Desert is located in south Perú and north Chile, covering a 1,000-km strip of land on the Pacific coast, west of the Andes mountains. Most of the desert is composed of stony terrain, salt lakes (salares), sand, and felsic lava. The Salar de Atacama is the largest salt flat in Chile. It is located at 55 km south of San Pedro de Atacama. The salt flat encompasses 3000 km2 and is about 100 km long and 80 km wide, which makes it the third largest in the world, after Salar de Uyuni in Bolivia (10,582 km2) and Salinas Grandes in Argentina (6000 km2). Its average elevation is about 2300 m above sea level. Some areas of the salt flat, form part of Los Flamencos National Reserve. [See article by Camara et al., pp. 235-247, this issue]

ders of the phylum Axostylata, specifically Trichomonadida, Hypermastigida, and Oxymonadida. Photograph (dark-field microscopy) by Rubén Duro (Center for Microbiological Research, CIM, Barcelona). See covers of Int. Microbiol. vol. 14 (2011) and R. Guerrero, L. Margulis, M. Berlanga, Int. Microbiol. 15(2013):133-143. (Magnification, ca. 1500×) LOWER RIGHT. Scanning electron micrograph of a 24-h mixed biofilm containing Candida albicans hyphae and blastoconidia of Candida glabrata. Photo by Cristina Marcos Arias. Faculty of Medicine, University of the Basque Country, UPV/EHU, Bizkaia Campus, Bilbao. (Magnification, ca. 5000×)

UPPER RIGHT. Transmission electron micrograph of Escherichia coli with amyloid inclusions of the prionoid REPA-WH1. Gold particles for immunodetection map the distribution of molecules of chaperone DnaK (Hsp70), involved in the conformational dynamics of the protein, generating, from globular amyloid aggregates, a variant amyloid that is less cytotoxic. Micrograph by Rafael Giraldo, Department of Cellular and Molecular Biology, CIB–CSIC, Madrid. (Magnification, ca. 32,000×) LOWER LEFT. Micrograph of Trychonympha sp., a protist from the intestine of the lower termite Reticulitermes grassei. Lower termites have a symbiotic protist–bacteria community in their hindgut, that allows them to digest cellulose. The protists belong to basal eukaryotic taxa, i.e., flagellate or-

Back cover: Pioneers in Microbiology Federico Lleras Acosta (1877–1938), Colombia Portrait of Federico Lleras Acosta (1877– 1932), considered the father of Colombian microbiology and pioneer of public health in his country, who contributed significantly to the development of modern medicine in Colombia at a time when clinical physicians still lacked confidence in the laboratory. He was born in Bogotá on April 27, 1877, from Federico Lleras Triana and Amalia Restrepo in a family that has given to Colombia scientists and other intellectuals as well as politicians (Lleras Acosta’s son, Carlos Alberto [1908–1994] was the president of Colombia in 1966–1977). He studied at the School of Veterinary Medicine, which depended on the School of Medicine and Natural Sciences of the University of the United States of Colombia and had been founded in 1884 by Claude Véricel, a French veterinary doctor. Along with the first microscope, Véricel had taken to Colombia laboratory reagents and media for bacteriological cultures. Graduates from that school were indeed the Colombian first microbiologists and worked mainly in fields related to microbiology, including food control and hygiene, the production of sera and vaccines, infectious and parasitic disease diagnostics, and public health. Lleras Acosta, who defended his master thesis on “La inspección sanitaria de

las carnes” (“Sanitary inspection of meat”) in 1902, specialized in serology and bacteriology, invested his savings in buying a modern microscope and founded the first clinical laboratory in Bogotá. He worked in various fields related to medical microbiology and public health; studied carbuncle, which affected the cattle, and the presence of Koch’s rod in urine, analized the quality of water in Bogotá, contributed to the diagnostic of plague, had to face an epidemics of enterocolitis which affected children in Bogotá, and spent time and efforts in the search for a method to culture Mycobacterium leprae, the leprosy rod. In 1936, he reported the cultivation of an acid-fast bacillus from the blood of patients suffering from cutaneous leprosy, but there was a high controversy about the cultivation of the true etiological agent of leprosy, whose culture in the laboratory has not yet been achieved. He also worked to set up a serological reaction for leprosy diagnostic. In 1934, Colombia’s President Alfonso Pérez Pumarejo created the Laboratorio Central de Investigaciones de la Lepra (Central Laboratory for Research on Leprosy) and Lleras Acosta was appointed its director. His pioneer work in microbiology was recognized by the University of Antioquia—the oldest university in Colombia, founded in 1803—which awarded him an honorary doctorate. Lleras Acosta died in Marseille, France, on March 18, 1938, on his way to Cairo to participate in the IV International Conference on Leprosy. Several hospitals and research centers have been named after him, the first—Federico Lleras Acosta Institute for Medical Research—having been inaugurated the very year of his death.

Front cover and back cover design by MBerlanga & RGuerrero

RESEARCH ARTICLE IÄã ÙÄ ã®ÊÄ ½ M® ÙÊ ®Ê½Ê¦ù (2014) 17:185-193 doi:10.2436/20.1501.01.221. ISSN (print): 1139-6709. e-ISSN: 1618-1095

www.im.microbios.org

Transforming activities of Chlamydia pneumoniae in human mesothelial cells Antonietta Rizzo1*, Caterina Romano Carratelli1, Anna De Filippis1, Nazario Bevilacqua2, Maria Antonietta Tufano1, Elisabetta Buommino1 Department of Experimental Medicine, Second University of Naples, Naples, Italy. 2 Department of Clinical Pathology, A. Cardarelli Hospital, Naples, Italy

Received 17 June 2014 · 29 October 2014

Summary. Knowledge in viral oncology has made considerable progress in the field of cancer fight. However, the role of bacteria as mediators of oncogenesis has not yet been elucidated. As cancer still is the leading cause of death in developed countries, understanding the long-term effects of bacteria has become of great importance as a possible means of cancer prevention. This study reports that Chlamydia pneumoniae infection induce transformation of human mesothelial cells. Mes1 cells infected with C. pneumoniae at a multiplicity of infection of 4 inclusion-forming units/cell showed many intracellular inclusion bodies. After a 7-day infection an increased proliferative activity was also observed. Real-time PCR analysis revealed a strong induction of calretinin, Wilms’ tumour gene 1, osteopontin, matrix metalloproteinases-2, and membrane-type 1 metalloproteinases gene expression in Mes1 cell, infected for a longer period (14 days). The results were confirmed by western blot analysis. Zymography analysis showed that C. pneumoniae modulated the in-vitro secretion of MMP-2 in Mes1 cells both at 7 and 14 days. Cell invasion, as measured by matrigel-coated filter, increased after 7 and 14 days infection with C. pneumoniae, compared with uninfected Mes1 cells. The results of this study suggest that C. pneumoniae infection might support cellular transformation, thus increasing lung cancer risk. [Int Microbiol 2014; 17(4):185-193] Keywords: Chlamydia pneumoniae · cytotoxicity · human mesothelial cells · cellular transformation · tumoral markers

Introduction Cancer is commonly defined as the uncontrolled growth of abnormal cells that have accumulated enough DNA damage to be freed from the normal restraints of the cell cycle. Although viral infections have been strongly associated with cancer [34,35], bacterial associations are also significant. Im-

Corresponding author: A. Rizzo Department of Experimental Medicine Second University of Naples Via Santa Maria di Costantinopoli, 16 80138 Napoli, Italy Tel. +39-815665656. Fax +39-815665662 E-mail: antonietta.rizzo@unina2.it *

portant mechanisms by which bacterial agents may induce carcinogenesis include chronic infection, immune evasion, and immune suppression. Several pathogenic bacteria, particularly those that can establish a persistent infection, can promote or initiate abnormal cell growth by evading the immune system or suppressing apoptosis [22]. In particular, some species or their toxins can alter host cell cycles or stimulate the production of inflammatory substances linked to DNA damage [7,37]. A separate discussion applies to intracellular pathogens that survive by evading the ability of the host to identify them as foreign. Chlamydia pneumoniae is a Gram-negative bacillus and a compulsory intracellular parasite. Chlamydia pneumoniae infection is acquired during childhood, and the prevalence gradually increases to reach a maximum in middle age; it causes

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respiratory infection in more than 50% of adults, leading to a higher incidence of pneumonia, as well as bronchitis, sinusitis, rhinitis, and exacerbation of chronic obstructive pulmonary disease [12]. The route of transmission is usually by aerosol and, in most cases, these infections are mild. The resulting clinical course is acute symptomatic illness followed by chronic respiratory symptoms. After acute infection, C. pneumoniae intracellular life cycle is characterized by the development of metabolically inert (antibiotic-resistant) atypical “persistent” inclusions. Persistent infection is a permanent source of bacterial antigen, promoting chronic inflammation; C. pneumoniae infections are thought to induce a state of persistent inflammation [38]. It has been suggested that persistent C. pneumoniae inflammation would correlate with increased risk of lung cancer, by inducing chronic pulmonary inflammation [20,25]. In the complex framework of interactions between the infective agent and immune response, superoxide oxygen radicals, TNF-a, IL-1 and IL-8 play an essential role, contributing to lung tissue damage and DNA damage that eventually result in carcinogenesis [36]. The time between acquiring the infection and cancer development is most often years or even decades as seen in cancers associated with Helicobacter pylori, Salmonella typhi, and Streptococcus bovis infections. The association of C. pneumoniae infection with lung cancer risk has been variable [21]; this could reflect the retrospective nature of some studies, small sample sizes, or inadequate adjustment for confounding due to smoking [27]. In addition, modest reliability of serologic assays and the lack of a validated marker for chronic infection have precluded an exact estimation of the etiologic role of C. pneumoniae [27]. Further information on the role of C. pneumoniae in lung cancer could be provided by studies using additional markers of infection and inflammation. The implementation of molecular biomarkers in the early diagnosis of lung cancer has been a long standing goal. Particular focus was given in identifying such biomarkers in bronchial washings in individuals with a high risk of developing lung cancer [33]. Calretinin (CR) is a vitamin D-dependent calcium-binding protein involved in the physiological buffering of excess cytosolic calcium ions, calcium transport, and protection against calcium ion overload [42]. CR is expressed in a subpopulation of neurons in the central and peripheral nervous system and is consistently up-regulated in reactive mesothelial cells and in epithelioid malignant mesothelioma (MM). Until now CR has been mostly considered as a highly useful marker for the identification of MM and based on the fact that CR is not expressed, or it is undetectable, in normal mesothelial cells in vivo.

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The Wilms’ tumor gene 1 (WT1) is a major regulator of cell growth and development in the embryo kidney, adult urogenital system and central nervous system [41]. The transcription factor WT1 has been found activated also in some human neoplasias, including Wilms tumor, gastrointestinal and pancreatobiliary tumors, urinary and male genital tumors, breast and female genital tumors, brain tumors, soft tissue sarcoma, osteosarcoma, malignant melanoma [32], and in mesothelioma cells [2]. WT1 expression has been reported to be markedly low in cells of normal healthy individuals, with the exception of the CD34+ hematopoietic progenitors [18]. WT1 protein expression has been observed in endothelial cells during angiogenesis, thus it can be a useful marker to distinguish between vascular proliferations and vascular malformations. Osteopontin (OPN) is a secreted phosphoglycoprotein that binds the integrin and CD44 families of receptors and plays a major role in tumorigenesis, tumor invasion, and metastasis [44]. Increasing data have shown that high expression levels of OPN are associated with invasion, progression, or metastasis in malignant tumors of the pancreatic cancer, gastric cancer, liver cancer, and lung cancer [43]. Mesothelioma progression depends on an interaction with mesothelial cells that provide membrane-type 1 metalloproteinases (MT1-MMP) necessary to activate pro-matrix metalloproteinases 2 (pro-MMP-2) to facilitate migration through an extracellular matrix layer. In particular, MT1-MMP predominantly converts pro-MMP-2 to the intermediate forms but not to mature MMP-1 form. MMP-2 has been reported as a characteristic for pleural malignant mesothelioma, and has been suggested as a predictive marker for poor prognosis [14]. On the basis of the results reported in the literature, we were prompted to investigate if chlamydial infection could contribute to in vitro cellular transformation by up-regulating the gene expression of three known biomarkers of the on-going neoplastic transformation, that is CR, WT1 and OPN. Here we report experimental evidence that sustained C. pneumoniae infection may cause cellular transformation as evaluated through the induced expression of CR, WT1 and OPN.

Materials and methods Cell culture and treatments. Primary cultures of mesothelial cells (Mes1) were isolated and developed from pleural biopsy of a patient who was cytologically, histologically and immunohistochemically confirmed as having non-malignant pleural mesotheliomas [4]. Tissue specimens were minced and incubated in growth medium 1:1 composition of DMEM and Ham’s F12 medium (Invitrogen) supplemented with 20% fetal calf serum (GIBCO BRL.

C. PNEUMONIAE IN HUMAN MESOTHELIAL CELLS

Grand Island, NY), penicillin (0.1 mg/ml), streptomycin (0.1 mg/ml), epidermal growth factor (10 μg/ml), insulin (5 mg/ml) and hydrocortisone (0.2 mg/ ml). Cell cultures were incubated at 37°C in a humidified atmosphere of 5% CO2 for 14 days to achieve 75% confluence. Mes1 cells displayed a highly flattened cellular morphology composed of tightly packed non-overlapping cells, which covered the entire surface of the culture dish following confluence. Mes1 was analyzed by RT-PCR for the expression of carcinoembryonic antigen (CEA, negative marker), WT1, mesothelin and calretinin. Propagation of Chlamydia pneumoniae. Chlamydia pneumoniae (AR39) was propagated in HEp-2 cell monolayers as described by Roblin et al [39]. In brief, C. pneumoniae was inoculated onto a pre-formed monolayer of HEp-2 cells in 35-mm diameter wells, centrifuged at 1000 ×g at 25°C for 60 min and incubated at 37°C with 5% CO2 for 1h. Supernatants were replaced with growth medium consisting of RPMI-1640 containing 1 g/ml cycloheximide. Infected cultures were incubated at 37°C in 5% CO2 for 3 days. Chlamydia pneumoniae was harvested by disrupting HEp-2 cells with glass beads followed by sonication and centrifugation at 250g to remove cellular debris. Supernatants containing C. pneumoniae were further centrifuged at 20,000g for 20 min to pellet elementary bodies (EB). The EB pellet was then suspended in sucrose–phosphate–glutamate buffer, aliquoted and stored at -70°C. Infectivity titers of chlamydial stocks were evaluated by the titration of the inclusion-forming units (IFU) per millilitre in HEp-2 cells. These titers were used to determine the infectious doses for the cell line studied. Cell cultures and chlamydial stocks were confirmed to be free of Mycoplasma infections using 4,6-diamidino-2-phenylindole fluorescent staining (SigmaAldrich S.r.l., Milan, Italy). In addition, contamination with Mycoplasma was excluded regularly by Mycoplasma-PCR using specific primers (MWG Biotech, Martinsried, Germany). In vitro infection. Mes1 cells were seeded onto coverslips in 24-well plates at a density of 5 ×104 cells/well in the growth medium. The cells were then infected with C. pneumoniae by centrifugation at 1000g for 60 min at a multiplicity of infection (MOI) of 4 IFU/cell (a preliminary study showed this MOI to be the optimum rate) and incubated for 3 days. For some experiments, determinations were performed at 3 days post infection because of the complicated, biphasic developmental cycle lasting up to 3 days. The count of IFU chlamydial was evaluated as described by Salin et al. [40]. In brief, at indicated times, the medium was removed from the wells and the coverslips were washed twice with PBS and fixed in methanol for 10 min. The coverslips were allowed to dry and the chlamydial inclusions were stained with fluorescein isothiocyanate (FITC)-conjugated anti-MOMP monoclonal antibody (Dako Cytomation, Milan, Italy), according to manufacturer’s instructions. The stained inclusions were examined under a fluorescence microscope (Axioskop 2, Carl Zeiss, Milan, Italy) at 400×. The number of the formed inclusions was counted from four eye fields of each coverslips and calculated using the following formula: [(inclusions in control – inclusions in treated sample) / inclusions in control] ×100. Cell proliferation and cell viability. Cell viability was evaluated with methyltetrazolium (MTT), which determines the activity of cellular (mitochondrial) respiration, and can be considered as a metabolic rate of cells. Mes1 cultures were incubated with DMEM alone (negative control) or with C. pneumoniae (MOI = 4) at 37°C in 5% CO2 . The number of living cells was determined by colorimetric MTT assay (3-[4.5-dimethyl-2.5 thiazolyl]-2.5 diphenyltetrazolium bromide; Sigma–Aldrich S.r.l.) according to the procedure of Boonyanugomol, et al. [3] after 3, 7 and 14 days. The absorbance of a formazan product in the tissue culture media was measured at 650 nm using a microplate reader, and the results were expressed as the mean percentage of the control cells. MTT assay data were confirmed by counting infected and uninfected cells in a Bürker chamber. For viability evaluated by microscopy examination, the cells were observed at a magnification of 200×

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(CK 40 Olympus Microscope). The viability of the cells infected with C. pneumoniae and those of the controls were confirmed by the activity of the lactate dehydrogenase (LDH) released in the supernatants [10]. Briefly, 50 l of the aliquots of cell supernatants were mixed with 25 l of LDH reagent (Sigma-Aldrich S.r.l.) and incubated at room temperature for 30 min. The LDH activity was calculated by measuring the increase in absorbance at 490 nm and was expressed as a percentage of the control values. Morphological analysis. To monitor whether Mes1 cells were capable of supporting the growth of C. pneumoniae in vitro, at 3 days post infection, the infected cells were fixed with 100% methanol and stained for the inclusion bodies using a fluorescein–isothiocyanate (FITC)-conjugated antiMOMP monoclonal antibody (Dako Cytomation, Milan, Italy). Morphological features of Mes1 cells infected with C. pneumoniae were examined at a magnification of 400× by confocal fluorescence microscopy (Axioskop 2). Determinations were performed at MOI = 4 and after 3 days post infection because this multiplicity and time of infection had been found to be the best in preliminary experiments. Real-time PCR analysis. Semi-confluent Mes1 cells (106 /well) were infected with C. pneumoniae for 7 and 14 days. Total RNA was isolated with the High Pure RNA Isolation Kit (Roche Diagnostics, Milan, Italy) from Mes1 cells, infected and non-infected with C. pneumoniae. Three hundred nanograms of total cellular RNA were reverse-transcribed (Expand Reverse Transcriptase, Roche Diagnostics) into complementary DNA (cDNA) using random hexamer primers (Random hexamers, Roche Diagnostics), at 42°C for 45 min according to the manufacturer’s instructions. Real-time PCR was carried out with the LC Fast Start DNA Master SYBR Green kit (Roche Diagnostics; LightCycler 2.0 Instrument) using 2 l of cDNA, corresponding to 10 ng of total RNA in a 20 l final volume, 3 mM MgCl2 and 0.5 M each primer (final concentration). Primer sequences and annealing temperatures are shown in Table 1. A melting curve was made at the end of each amplification to ensure the absence of non-specific reaction products. The accuracy of mRNA quantification depends on the linearity and efficiency of the PCR amplification. Both parameters were assessed using standard curves generated by increasing amounts of cDNA. Quantification was based on the threshold cycle values, measured in the early stage of the exponential phase of the reaction. All quantifications were normalized to the housekeeping gene -actin. The percentage of gene expression increase was calculated using the following formula: [(gene expression in unstimulated conditions – target gene expression) / gene expression in unstimulated conditions] ×100. Protein extraction and western blot analysis. Semi-confluent Mes1 cells (106 /well) were infected with C. pneumoniae for 7 and 14 days. Cells were scraped with 1 ml PBS, and the cell pellet was homogenized with 300 l ice-cold buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1% glycerol, 1% Triton, 1.5 mM MgCl2, 5 mM EGTA) supplemented with 20 mM sodium pyrophosphate, 40 g/ml aprotinin, 4 mM PMSF, 10 mM sodium orthovanadate, 25 mM NaF. Total extracts were cleared by centrifugation at 10,000 rpm for 30 min at 4°C and assayed for the protein content by Bradford’s method. Fifty g of protein from each cell lysate were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Filters were then stained with 10% Ponceau S solution for 2 min to verify equal loading and transfer efficiency. In addition, protein normalization was verified by densitometric analysis of bands. Blots were blocked overnight with 5% non-fat dry milk, then incubated with anti-MMP-2 (H-76) rabbit polyclonal antibody, OPN (sc-21742) mouse monoclonal, WT1 (sc-192) rabbit polyclonal, calretinin (sc-365956) mouse monoclonal and anti-tubulin mouse monoclonal antibody (Santa Cruz Biotechnology) 1:200 in TBS (150 mM NaCl, 20 mM Tris-HCl, pH 8) for 2 h at room temperature. After washing with 0.1% Tween-20 PBS, the filters were incubated with 1:2500 peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulins for 1 h at

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Table 1. Human sense and antisense primer sequences. Sequences and conditions of the oligonucleotide primers used in real time-PCR analysis Sense and antisense sequences

Conditions

5′-CATACTACGGATGTTTGACTT-3′ 5′-TCACGCTCTCTGAGTCTGG-3′

40 cycles at 95°C for 5s 56°C for 8s, 72°C for 17s

5′-CTCTTGTACGGTCGGCATCT-3′ 5′-CAGCTGGAGTTTGGTCATG-3′

40 cycles at 95°C for 5s 56°C for 8s, 72°C for 17s

5′-CACCTGTGCCATACCAGTTAAAC-3′ 5′-GGTGATGTCCTCGTCTGTAGCATC-3′

40 cycles at 94°C for 5s 53°C for 11s, 72°C for 21s

5′-TGACGGTAAGGACGGACTC-3′ 5′-TGGAAGCGGATTGGAAAC T-3′

40 cycles at 94°C for 5s 57°C for 7s, 72°C for 14s

5′-CTGGGCCATGCCCTGGGGCTC-3′ 5′-CAGGAACAGAAGGCCGGGAGG-3′

40 cycles at 94°C for 5s 64°C for 4s , 72°C for 8s

5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′ 5′-CTAGAAGCATTGCGGTGGACGATGGAGGG-3′

40 cycles at 95°C for 5s 64°C for 8s , 72°C for 20s

22°C. They were extensively washed and finally analyzed using the ECL system (Amersham). Gelatin zymography. Semi-confluent Mes1 cells were plated in 6-well plates (35 mm diameter) and infected with C. pneumoniae for 7 and 14 days. Gelatinolytic activity of MMP-2 was determinated using the method of Heussen and Dowdle [13] adapted for minigel format. Conditioned media of each sample were centrifuged at 6000 rpm for 20 min and the protein content of the supernatant was estimated by Bradford’s method. Twenty g of sample were mixed with an equal volume of 2×non-reducing sample buffer and each sample was separated by 10% (w/v) polyacrylamide gel containing 2 mg/ml of gelatine (Sigma). After electrophoresis, the gel was incubated in 2.5% Triton X-100 for 1hour to remove SDS and then overnight at 37°C in the developing buffer (50mM Tris-HCl, pH 7.6, containing 0.2 M NaCl, 5mM CaCl2 and 0.02% (w/v) Brij-35). The gel was stained for 45 min in 40% methanol/10% glacial acetic acid containing 0.1% (w/v) Coomassie Brilliant Blue R 250 and de-stained in the same solution without Coomassie Brilliant Blue.

Results

Cell invasion assay. Cell invasion assays were carried out in Boyden chambers under serum-free conditions as previously described [1]. The 10m pore-size-polycarbonate filters were coated with 5 g/ml fibronectin and then with Matrigel (BD Bioscences) 25 g/ml. After 14 days C. pneumoniae infection, Mes1 cells were trypsinized and placed in the upper compartment of the Boyden chamber in serum-free medium, and FBS 10% was placed in the lower compartment as the chemoattractant. Cells were allowed to attach and spread at 37°C in 5% CO2 for 24 h. The cells on the upper surface of the filter were completely removed by wiping with a cotton swab, while those that had traversed the Matrigel and attached to the lower surface of the filter were fixed in ethanol, stained with hematoxylin and counted in 10 random fields/filter at 200×. In parallel, the control cells were assessed for viability and counted with trypan blue. The number of cells that had invaded was normalized to analyze the effects on cell viability.

Mes1 cells proliferation and viability Chlamydia pneumoniae–infected. Confocal fluorescence microscopy using an anti-Chlamydia monoclonal antibody revealed that Mes1 cells had many intracellular inclusion bodies after 3 days of infection (Fig. 1B) compared with control cells (Fig. 1A), whereas at 1 and 2 days post infection the cells showed only few intracellular inclusion bodies (data not shown). We examined the effect of C. pneumoniae infection on the proliferative activity of Mes1 cells, by both the colorimetric MTT and LDH assay. After 3 days exposure to C. pneumoniae, the cell number was slightly modified compared to test cultures and controls (Fig. 1C–E). After 7 days of incubation with C. pneumoniae, an increased proliferative activity was observed. In particular, during this period, the proliferative response of Mes1 cells incubated with C. pneumoniae, determined by colorimetric assay and cell counting, showed an increase of 34.1% and 29.4%, respectively, compared to the cells alone (Fig. 1C,D), while the viability, determined by LDH activity, increased by 35.2% compared to the control cells (Fig. 1E). After 14 days of incubation, cell proliferation and viability still increased reaching values of 40.9%, 37.8% and 40.4%, respectively.

Statistical analysis. Each experiment was performed at least three times. The results are expressed as means ± standard deviations (SD). Student’s t test was used to determine statistical differences between the means, and P < 0.01 was considered a significant difference.

Expression of CR, WT1 and OPN in infected Chlamydia pneumoniae Mes1 cells. To investigate whether chlamydial infection might promote cellular transfor-

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mation we analysed CR and WT1 gene expression, two known biomarkers of the on-going neoplastic transformation. As shown in Fig. 2A and C, Mes1 cell infected for 14 days showed a strong induction of CR and WT1 gene expression. Shorter period of infection (7 days) induced only a slight modification of the biomarkers. On contrary, prolonged period of infection (21 days) were not investigated since Mes1 cells morphology was modified, showing the typical feature of cell suffering (data not show). To reinforce the result obtained we went to analyse osteopontin (OPN) gene expression, a marker reported to play an important role in tumorigenesis. Our results demonstrated that OPN was also up-regulated in infected Mes-1 cells (Fig. 2E), resulting in a major increase after 14 days C. pneumoniae infection. Finally, the results obtained on CR, WT1 and OPN gene expression were all confirmed by western blot analysis (Fig. 2B, 2D and 2F). CR and WT1 overexpression increases the in vitro invasive potential of Mes1 cells. In order to evaluate whether CR and WT1 overexpression might favour cell invasion we examined gelatinase MMP-2 gene expression and its activator membrane type1-MMP (MT1-MMP). As shown in Fig. 3A and 3B, MT1-MMP and MMP-2 gene expression were both induced after 7 and 14 days of Mes1 cells infection. However, the expression of these genes was stronger after 14 days infection, as it happened for the other markers analyzed. Also western blot analysis (Fig. 3C) showed that MMP-2 pro-enzyme and the mature 62k-Da enzyme of MMP-2 were more strongly increased after 14 days in Mes1 infected cells, compared with the point at 7 days and the control, suggesting the activation of the gelatinase. To confirm the result obtained we analyzed by zymography whether C. pneumoniae affects the in-vitro secretion of MMP2 by Mes1 cells. As shown in Fig. 3D, MMP-2 secretion increased after 7 days infection, with a stronger increase after 14 days, compared with uninfected cells. To strengthen the results we had obtained, we investigated the ability of C. pneumoniae to increase Mes1 cell invasion. Cell invasion, as measured using a modified Boyden chamber with a Matrigel-coated filter, was increased after 7 and 14

Fig. 1. Representative experiments of Mes1 cells infected with Chlamydia pneumoniae. (A) untreated Mes1 cells. (B) three days after incubation the infected cells were fixed with methanol and stained for the inclusion bodies using an anti-Chlamydia monoclonal antibody. Intracellular inclusion bodies (arrow). Images were collected using confocal fluorescence microscopy at ×400 magnification. (C-E) effect of Mes1 cells infected or uninfected (Ctrl) with C. pneumoniae (MOI = 4) on proliferation (C); cell counts (D); and cell viability (E). (C) Proliferation was determined by a colorimetric MTT assay (OD at 650 nm) after 3, 7 and 14 days of culture of infected and uninfected cells. Results are expressed as the mean percentage of control cells. Data are means ± SD of three different experiments. (E) Viability was determined by LDH activity and is expressed as percentage of control values after 3, 7 and 14 days culture of infected cells. Data are means ± SD of three independent experiments. The asterisk indicates a statistically significant difference between the experimental test and the control test. * P < 0.05 ** P < 0.01 versus Mes1 cells alone.

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Fig. 2. Real time PCR analysis using specific primers for CR (A), WT1 (C) and OPN (E). Mes1, cells infected or not (Ctrl) with Chlamydia pneumoniae for 7, 14 days; Ctrl, untreated Mes1 cells. The columns are the mean values from three independent experiments with three duplicates. Significant differences compared to untreated cells are indicated as follows: ** P < 0.01. (B, D and F) western blot analysis of CR, WT1 and OPN in Mes1 cells after exposure to C. pneumoniae. Line 1, untreated cells; lines 2-3, Mes1 cells infected with C. pneumoniae for 7 and 14 days respectively.

days infection with C. pneumonia (26% and 60% respectively), compared with uninfected Mes1 cells (Fig. 4).

Discussion Chlamydia pneumoniae infection has been suggested to be strongly associated with lung carcinoma. However, only seroepidemiological studies have indicated such a potential re-

lation [20]. In fact, high C. pneumoniae antibody titers have been observed in lung cancer. Specifically, high IgA against C. pneumoniae were reported to be correlated with squamous cell carcinomas and to a lesser extent with small cell carcinomas and adenocarcinomas of the lung [28]. To our knowledge, up to now there is not any report about C. pneumoniae infection inducing transformation of human mesothelial cells. In our study, Mes1 cells infected with C. pneumoniae showed many intracellular inclusion bodies, confirming that

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Fig. 3. Real time PCR analysis using specific primers for MT1-MMP (A) and MMP-2 (B). Mes1, cells infected or not (Ctrl) with Chlamydia pneumoniae for 7 and 14 days; Ctrl; untreated Mes1 cells. the columns are the mean values from three independent experiments with three duplicates. Significant differences compared to untreated cells are indicated as follows: * P < 0.05 and ** P < 0.01. (C): western blot analysis of MMP-2 in Mes1 cells after exposure to C. pneumoniae. Line 1, untreated cells; lines 2-3, Mes1 cells infected with C. pneumoniae for 7 and 14 days respectively. (D) MMP-2 secretion in Mes1 cells after exposure to C. pneumoniae. Line 1, untreated cells; lines 2-3, Mes1 cells infected with C. pneumoniae for 7 and 14 days respectively.

the microorganism was able to invade and replicate in this cellular type. In addition, an increased proliferative activity was demonstrated in C. pneumoniae-infected Mes1 cells. It is known that C. pneumoniae infection causes irregular apoptosis in tissues by unknown mechanisms [5]. Apoptosis and cellular proliferation have a pivotal role in carcinogenesis. Hyperproliferation simultaneously reduces the time available to repair mutations in DNA and also increases the risk of spontaneous mutation due to errors in DNA replication [29]. It has been reported that C. pneumonia infection of endothelial cells triggers both vascular smooth muscle cells proliferation and the mitogenic activity of platelet-derived growth factor. There is also evidence that C. pneumoniae infection in endothelial cells induces the production of different mediators of inflammation, among which MMP, which contributes to plaque destabilization [8]. Using molecular biomarkers for the early diagnosis of lung cancer has been a long standing objective. Particular focal point was given in identifying such biomarkers in bronchial washings in individuals with a high risk of

developing lung cancer. The WT1 gene was originally identified as a tumor suppressor gene, recently proposed to act as a chameleon gene in malignancies, i.e. functioning also as an oncogene [17]. It is expressed in a small number of human tissues [31] and in various cancer cells [32]. The marker has been usually considered to be positive in mesothelioma. Calretinin is one of the first markers that have proven to be useful in the diagnosis of malignant mesothelioma [9], it being positive in 80â&#x20AC;&#x201C;100% of malignant mesotheliomas [23,30]. Mesothelin and calretinin are proteins strongly expressed in mesothelial cells and mesotheliomas, whereas WT1 is mainly expressed in mesothelioma cells [11]. The expression of mesothelin and calretinin in our primary cell culture confirmed the mesothelial differentiation (data not shown). Our results showed a strong induction of CR and WT1 gene expression in Mes1-infected cells , thus confirming the ability of C. pneumoniae to induce cellular transformation. To further support our results, we analysed another marker of tumor progression, OPN, and found a significant up-regulation of OPN gene ex-

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Fig. 4. Inhibitory effect of Chlamydia pneumoniae infection on Mes1 invasion. Control and C. pneumoniae-infected Mes1 cells were plated onto a Matrigel modified Boyden chamber. Cells were allowed to attach and spread for 24 h. Only cells that had passed through the Matrigel were stained and counted. The average number of cells per field is expressed as a percentage of the control after normalizing for cell number. The results are the mean values of three different experiments. Significant differences compared to untreated cells are indicated as follows: * P < 0.05 and ** P < 0.01.

pression in infected Mes1 cells. This result is of interest since OPN has been shown to bind and/or activate pro-matrix metalloproteinase-3 (pro-MMP-3) and pro-MMP-9, and to activate phosphatidylinositol 3-kinase (PI3K)/protein kinase B pathway, promoting cell migration and cell survival [16]. Thus, OPN might contribute to sustained cell proliferation by contrasting apoptotic cell death and the elimination of acquired mutations. MMP-2 and its activator membrane type1MMP (MT1-MMP) are molecules known to be linked to aggressive tumor progression, poor survival, and high risk for metastasis [14,46]. Interestingly, our results demonstrated that MMP-2 and MT1-MMP gene expression were both induced after 14 days of Mes1 cells infection. The results were confirmed measuring the enzymatic activity by zymography. Finally, we found out that C. pneumoniae influenced the invasive behavior of Mes1 cells. The results here reported indicate that C. pneumoniae infection might support cell transformation. Epidemiological data in the literature support the idea that C. pneumoniae infection might trigger lung carcinoma. Laurila, et al. [24] reported that C. pneumoniae infection was present principally in patients with small-cell and squamous cell carcinomas, among 230 smokers with lung carcinoma. According to some studies, smoking assists C. pneumoniae to invade the lung [20]. Chlamydia infection is believed to increase lung cancer risk by inducing chronic pulmonary inflammation. Inflamma-

tory mediators, while offering protection by destroying invading pathogens, can inhibit apoptosis and enhance cell proliferation, both of which can promote mutation and carcinogenesis [19,26]. Another study suggests that chronic inflammation could be responsible for the observed link between Helicobacter pylori and carcinogenesis [15]. Similarly, C. pneumoniae infection might represent a risk factor aggravating the condition, in particular, of some classes of workers exposed to asbestos fibres at high risk of developing mesothelioma. Note that many of the bacterial infections that support oncogenesis are often asymptomatic. When the pathways toward malignancy start and when they become irreversible, though, are aspects not yet fully understood [6]. Two other intracellular bacteria, Mycoplasma fermentans and M. penetrans, phylogenetically close relatives of Chlamydiae, have been reported to transform C3H mouse embryo cells in vitro by a multistage progression characterized by C-myc mRNA over-expression [45]. Because lung carcinoma and mesothelioma usually carry a dismal prognosis, there is urgent need to develop early diagnostic markers and effective therapies against chronic C. pneumoniae infections. To our knowledge, this is the firs report of C. pneumoniae infection inducing trasformation of human mesothelial cells, which supports the idea that C. pneumoniae infection might increase the risk of lung carcinoma. Further information on the role of C. pneumoniae in lung cancer could be provided by studies using additional markers of infection and inflammation. Competing interests. None declared.

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27. Littman AJ, Jackson LA, Vaughan TL (2005) Chlamydia pneumoniae and lung cancer: epidemiologic evidence. Cancer Epidemiol Biomarkers Prev 14:773-778 28. Littman AJ, Thornquist MD, White E, Jackson LA, Goodman GE, Vaughan TL (2004) Prior lung disease and risk of lung cancer in a large prospective study. Cancer Causes Control 15:819-827 29. Lowe SW, Lin AW (2000) Apoptosis in cancer. Carcinogenesis 21:485-495 30. Lugli A, Forster Y, Haas P et al (2003) Calretinin expression in human normal and neoplastic tissues: a tissue microarray analysis on 5233 tissue samples. Hum Pathol 34:994-1000 31. Menke AL and Schedl A (2003) WT1 and glomerular function. Semin. Cell Dev Biol 14:233-240 32. Nakatsuka S, Oji Y, Horiuchi T et al (2006) Immunohistochemical detection of WT1 protein in a variety of cancer cells. Mod Pathol 19:804-814 33. Nikolaidis G, Raji OY, Markopoulou S et al (2012) DNA methylation biomarkers offer improved diagnostic efficiency in lung cancer. Cancer Res 72:5692-5701 34. Ord RA, Blanchaert RH Jr (2001) Current management of oral cancer. A multidisciplinary approach. J Am Dent Assoc 132:19S-23S 35. Pujol FH, Devesa M (2005) Genotypic variability of hepatitis viruses associated with chronic infection and the development of hepatocellular carcinoma. J Clin Gastroenterol 39:611-618 36. Redecke V, Dalhoff K, Bohnet S, Braun J, Maass M (1998) Interaction of Chlamydia pneumoniae and human alveolar macrophages: infection and inflammatory response. Am J Respir Cell Mol Biol 19:721-727 37. Rizzo A, Di Domenico M, Carratelli CR, Mazzola N, Paolillo R (2011) Induction of proinflammatory cytokines in human osteoblastic cells by Chlamydia pneumoniae. Cytokine 56:450-457 38. Rizzo A, Domenico MD, Carratelli CR, Paolillo R (2012) The role of Chlamydia and Chlamydophila infections in reactive arthritis. Intern Med 51:113-117 39. Roblin PM, Dumornay W, Hammerschlag MR (1992) Use of HEp-2 cells for improved isolation and passage of Chlamydia pneumoniae. J Clin Microbiol 30:1968-1971 40. Salin O, Alakurtti S, Pohjala L et al (2010) Inhibitory effect of the natural product betulin and its derivatives against the intracellular bacterium Chlamydia pneumoniae. Biochem Pharmacol 80:1141-1151 41. Scharnhorst V, van der Eb AJ, Jochemsen AG (2001) WT1 proteins: functions in growth and differentiation. Gene 273:141-161 42. Schwaller B (2007) Emerging functions of the “Ca2+ buffers” parvalbumin, calbindin D-28k and calretinin in the brain. In: Lajtha A, Banik N (eds) Neurochemistry and molecular neurobiology. Neural protein metabolism and function, pp 197-222 43. Takenaka M, Hanagiri T, Shinohara S et al (2013) Serum level of osteopontin as a prognostic factor in patients who underwent surgical resection for non-small-cell lung cancer. Clin Lung Cancer 14:288-294 44. Weber GF, Lett GS, Haubein NC (2010) Osteopontin is a marker for cancer aggressiveness and patient survival. Br J Cancer 103:861-869 45. Zhang B, Shih JW, Wear DJ, Tsai S, Lo SC (1997) High-level expression of H-ras and c-myc oncogenes in mycoplasma-mediated malignant cell transformation. Proc Soc Exp Biol Med 214:359-366 46. Zhong J, Gencay MM, Bubendorf L et al (2006) ERK1/2 and p38 MAP kinase control MMP-2, MT1-MMP, and TIMP action and affect cell migration: a comparison between mesothelioma and mesothelial cells. J Cell Physiol 207:540-552

RESEARCH ARTICLE IÄã ÙÄ ã®ÊÄ ½ M® ÙÊ ®Ê½Ê¦ù (2014) 17:195-204 doi:10.2436/20.1501.01.222. ISSN (print): 1139-6709. e-ISSN: 1618-1095

www.im.microbios.org

Impact of formate on the growth and productivity of Clostridium ljungdahlii PETC and Clostridium carboxidivorans P7 grown on syngas Sara Ramió-Pujol1,2, Ramon Ganigué1*, Lluís Bañeras2, Jesús Colprim1 LEQUIA, Institute of the Environment, University of Girona, Girona, Spain. 2Group of Molecular Microbial Ecology, Institute of Aquatic Ecology, University of Girona, Girona, Spain

Received 14 September 2014 · 15 November 2014

Summary. The current energy model based on fossil fuels is coming to an end due to the increase in global energy demand. Biofuels such as ethanol and butanol can be produced through the syngas fermentation by acetogenic bacteria. The present work hypothesizes that formate addition would positively impact kinetic parameters for growth and alcohol production in Clostridium ljungdahlii PETC and Clostridium carboxidivorans P7 by diminishing the need for reducing equivalents. Fermentation experiments were conducted using completely anaerobic batch cultures at different pH values and formate concentrations. PETC cultures were more tolerant to formate concentrations than P7, specially at pH 5.0 and 6.0. Complete growth inhibition of PETC occurred at sodium formate concentrations of 30.0 mM; however, no differences in growth rates were observed at pH 7.0 for the two strains. Incubation at formate concentrations lower than 2.0 mM resulted in increased growth rates for both strains. The most recognizable effects of formate addition on the fermentation products were the increase in the total carbon fixed into acids and alcohols at pH 5.0 and pH 6.0, as well as, a higher ethanol to total products ratio at pH 7.0. Taken all together, these results show the ability of acetogens to use formate diminishing the energy demand for growth, and enhancing strain productivity. [Int Microbiol 2014; 17(4):195-204] Keywords: Clostridium carboxidivorans · Clostridium ljungdhalii · syngas fermentation · biofuels · formate

Introduction The current energy model based on fossil fuels is coming to an end due to the increase in global energy demand, the depletion of primary oil reserves and its large price fluctuation [8]. Another important issue of fossil fuels as energy source is the emission of greenhouse gases and their negative impact on

Corresponding author: R Ganigué, University of Girona Campus Montilivi E-17071 Girona, Spain Tel. +34-972419549 Email: ramon.ganigue@lequia.udg.cat

global warming [1]. In recent decades, this growing concern has led to the development of alternative fuel sources. Currently, ethanol and butanol are considered two of the most promising alternative biofuels. Biofuels can be obtained from renewable raw materials, such as molasses, starch, cellulose and lignin through hydrolysis and subsequent fermentation. However, the low efficiency of the cellulose and lignin conversion processes, as well as the high feedstock cost of molasses and starch and the ethical issues arising from their use for fuel production, call the viability of these technologies into question [23]. An alternative to these processes is the stepwise process of gasification and microbial fermentation. In gasification, organic matter from a

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variety of sources, i.e. residual agricultural biomass, municipal solid waste or tires, is gasified to synthesis gas (or syngas: a mixture of primarily CO, CO2, and H2) [22]. Some studies have highlighted the ability of some acetogenic bacteria to produce volatile fatty acids and alcohols using solely syngas in a fermentation metabolism [5,17]. Acetogens are anaerobic bacteria that use the autotrophic Wood-Ljungdahl pathway (WL) for the production of acetylCoA. Depending on the metabolic demand of the cell, acetylCoA can be used as a precursor of cellular biomass or further converted into organic acids, such as acetate and butyrate, and alcohols, such as ethanol and butanol, through a pathway similar to the classical acetone-butanol-ethanol (ABE) fermentation pathway [13,28]. The WL pathway consists of two separate branches, the methyl and the carbonyl branch. One molecule of CO2 is reduced by several steps to a methyl group in the methyl branch, while the carbonyl branch involves the incorporation of a carbon monoxide molecule. The bound methyl group and the carbonyl are condensed with coenzyme A (CoA) to make acetyl-CoA. Among acetogens, Clostridium carboxidivorans P7 and Butyribacterium methylotrophicum produce a mixture of acetate, butyrate, ethanol and butanol from syngas; however, the production of alcohols by B. methylotrophicum is scarce [5,10,18]. Clostridium ljungdahlii strains PETC and ERI2, as well as Alkalibaculum bacchi strains CP11, CP13 and CP15 are also acetogens that transform syngas into acetate and ethanol, but none of these strains have been shown to produce butyrate or butanol [19,27,34]. The first steps in the methyl and carbonyl branches of the WL pathway, formate synthesis and the carbon monoxide formation, are recognized as reducing equivalent sinks that may diminish the growth capacity of cells and the conversion of inorganic carbon into valuable chemicals [7]. The use of hydrogen and carbon monoxide as sources of reducing equivalents is maximized during autotrophic growth. In this condition, the assimilation of formate as a partially reduced carbon source has the potential to reduce the hydrogen/CO demand for formate dehydrogenase activity in the WL pathway [4,31]. We hypothesize that the excess hydrogen can further be diverted to acetate reduction, increasing biofuel production. The synthesis of formic acid from CO2 has been accomplished in a bioelectrochemical system (BES) using a purified formate dehydrogenase enzyme [33]. Electrosynthesis cou-

RAMIĂ&#x201C;-PUJOL ET AL.

pled to fermentation by carboxydotrophic bacteria, has been proven but not studied in detail [25]. However, the concept of enzymatic electrocatalysis involving energy applications is gaining in prominence, especially in the direction of enzymatic electrosynthesis of desired chemicals and fuels under nonlimiting reducing power supply. Formate has been reported as an inducer of acetate production in Clostridium acetobutylicum. Maximum effects of formate on acetate production in C. acetobutylicum were obtained under acidic conditions (at pH = 4.8) [2]. Despite this example, growth on weak organic acids is rather difficult for most microorganisms and inhibition occurs at very low concentrations. Inhibition effects are higher at low pH values where higher concentrations of the undissociated acid forms exist, which can freely diffuse to the cytoplasm of the cell eventually causing the dissipation of energy gradients built across the cell membrane [13]. Additionally, formate can cause sub-lethal damage in some bacteria and has been used as an antibacterial agent [35]. PETC has been grown chemoorganotrophically in a medium containing 5 g/l of formate and 1 g/l of yeast extract [34]. However, similar experiments have never been done autotrophically with this strain. Moreover, B. methylotrophicum can also use formate as substrate for growth, but it is unclear whether other acetogenic bacteria, including P7, can use formate when growing either organo- or autotrophically [15,18]. In this light, the present work hypothesizes that the addition of formate, as a partially reduced C1 compound, would positively impact kinetic parameters for growth and alcohol production in C. ljungdahlii PETC and C. carboxidivorans P7 by diminishing the need for external reducing equivalents. The aim of this work was to provide experimental evidence to evaluate formate addition as a potential enhancer of alcohol production in C. ljungdahlii PETC and C. carboxidivorans P7.

Materials and methods Bacterial strains. Clostridium ljungdahlii PETC (DSM13528T) and C. carboxidivorans P7 (DSM15243T) strains were obtained from DSMZ [www. dsmz.de]. Media and culture conditions. Bacteria were cultured in an anaerobic mineral medium similar to ATCC1754 [34]. The used medium differed from ATCC1754 in: (i) all soluble carbon sources, i.e., yeast extract, fructose,

CLOSTRIDIUM GROWN ON SYNGAS

and NaHCO3, were excluded from the original formulation; and (ii) 2-(Nmorpholino)ethanesulfonic acid (100 mM, final concentration) was used as pH buffer. Resazurin (1 mg/l) was used as an indicator of anaerobic conditions, and the pH of the medium was initially adjusted to 5.0, 6.0 or 7.0 with 1 M NaOH or HCl. Liquid medium was prepared and distributed anaerobically in Hungate tubes. Formate was added to the medium as sodium formate (Merck, Darmstadt, Germany) at different concentrations as indicated below. In all cases, tubes head-space were flushed with synthetic syngas consisting of a mixture of 32% CO, 32% H2, 28% N2, and 8% CO2 of high purity (Praxair Technology Ltd, Spain). All culture manipulations and inoculation of freshly prepared media were done inside an anaerobic chamber (Coy Lab Products, Michigan, USA). PETC and P7 cultures were incubated in 125 ml serum bottles containing 25 ml of modified ATCC1754 medium and syngas in the head-space at an overpressure of 100 kPa. Cultures were maintained active by a 4% weekly transfer into new serum bottles. Fermentation experiments. Exponentially growing C. ljungdahlii PETC and C. carboxidivorans P7 cultures were used as inocula for batch experiments to test for formate effects on growth and alcohol production. Fermentation experiments were conducted in 25 ml anaerobic tubes containing 6 ml of organic-carbon-free ATCC1754 medium. A 10% inoculum of either PETC or P7 strains was used in all experiments. Culture tubes were inoculated in anaerobic conditions and thoroughly flushed with syngas mixture reaching a final headspace overpressure of 100 kPa. Syngas was injected only at the beginning of the experiment. Sodium formate solutions adjusted at the desired pH were aseptically added to the medium at final concentrations of: 0.1, 1.0, 2.2, 5.5, 7.6, 10.9, 15.0, 20.0, 27.2, 54.5, and 109.0 mM. In all batch tests, tubes containing no sodium formate were included as controls for growth kinetics under fully autotrophic conditions. Experiments were carried out for the two bacterial species at three pH values, 5.0, 6.0 and 7.0. The cultures were incubated at 35ºC under mild agitation on a rotary shaker Stuart incubator SI500 at 100 rpm (Bibby Scientific Ltd., OSA, UK). Tubes were placed horizontally to enhance gas-liquid mass transfer. All experimental conditions were assayed in triplicate using three independent inoculated cultures. Growth was monitored on a daily basis by measuring the absorbance at 600 nm using a CE1021 spectrophotometer (CECIL, Cambridge, UK). Growth experiments finished once cultures reached the stationary growth phase, which was considered to occur 48 to 72 h after growth cessation. Samples for the determination of organic acids (formate, acetate and butyrate) and alcohols (ethanol and butanol) concentrations were obtained at the beginning and at the end of the incubation experiments, filtered using nylon filters (0.2μm diameter, Millipore, Germany) and stored at 4ºC until analyzed. Finally, the pH of the medium was measured using a BASIC 20 pHmeter (Crison, Spain). Additionally, an independent experiment was conducted at five sodium formate concentrations: 13.7, 17.2, 21.9, 30.0 and 97.5 mM at pH 6.0 using PETC strain to test the consumption of formate throughout growth. The samples for the determination of formate concentration were obtained every 48– 72 h, as well as, at the beginning and at the end of the incubation experiment. Determination of growth variables. Linear regression of transformed absorbance readings (lnAt) at time intervals of 72 h (t) were used to estimate the changes of growth rate, according to equation (1): lnAt = lnAinit + μ · t (1) Growth rates (μ, h–1) were calculated for each incubation experiment as a measure of the growth capacity of the bacterial cultures at the conditions set in the experiment. Duration of the lag phase (days) was estimated as the time interval between the inoculations of tubes and the time at which the calculated maximum growth rate was observed.

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Analytical methods. The total amount of formic acid/formate (sum of formic acid and formate) was measured by using a spectrophotometric method [32]. Formate concentrations were measured at the beginning and the end of incubation experiments. Undissociated formic acid (HCOOH) concentration at initial conditions was calculated based on the measured pH and the total formic acid/formate measurements according to the equilibrium equation (2). HCOOH = Ft – (10(pH-pKa) ·Ft) / (10(pH-pKa) + 1) (2) Where Ft is the concentration of sodium formate added in each experiment and pKa is the equilibrium constant. In this study, a value of 3.76 was used, corresponding to the equilibrium constant at 35ºC [16]. The fermentation products (acetate, ethanol, butyrate, and butanol) were analyzed quantitatively using a gas chromatograph (Agilent 7890A GC system, Agilent Technologies, Spain) equipped with a fused-silica capillary column (DB-FFAP, 30 m × 0.32 mm × 0.5 μm) and a flame ionization detector (FID) using helium as carrier gas. The injector and detector temperatures were set at 250ºC and 275ºC, respectively. The oven temperature was initially kept at 40ºC for 1 min, and subsequently increased following a ramp of 5ºC min-1 until temperature reached 70ºC, at 10ºC min–1 from 70ºC to 180ºC, and at 35ºC min–1 from 180ºC to 250ºC. Finally, the temperature was maintained at 250ºC for 5 min. Statistical analyses. All statistical analyses were conducted using SPSS 15.0 statistical package for Windows (LEAD Technologies Inc., EEUU). Significance levels were established for P ≤ 0.05. ANOVA tests were used to analyze differences of maximum growth rate in relation to the initially added formic acid concentration, as well as, the alcohol to total product ratio and the products concentration in relation to the initially sodium formate concentration. Multiple comparisons between initial formic acid and sodium formate concentrations were further analyzed using a T3 of Dunnet post-hoc test assuming not equal variance or Bonferroni post-hoc test assuming equal variance between treatments. Pearson correlation tests were used to analyze the correlation of the acids and solvents production, as well as, alcohols to total products ratio with the initial sodium formate concentration.

Results and Discussion Growth in the presence of formate. Clostridium ljungdahlii PETC was able to grow under all experimental conditions tested except at sodium formate concentrations of 54.5 and 109.0 mM at pH 6.0 (Table 1). On the contrary, C. carboxidivorans P7 showed a more restricted range of growth conditions and no increase in absorbance was observed in many of the experimental conditions, especially at low pH values. Growth of P7 was restricted to formate concentrations lower than 10.9 mM at pH 6.0, and no growth was observed at any of the formate concentrations tested at pH 5.0. Main differences observed in growth curves for the two strains were the decrease in the optical density at the stationary phase and the duration of the lag phase depending on the formate concentration (Fig. 1). The rapid decrease in absorbance values during the stationary phase was observed for both Clostridium species when incubated at pH 7.0. Reasons for this decrease were not inves-

0.6

0.8

7.6

10.9

1.7 ± 1.2

2.3 ± 1.2

1.0 ± 0.0

2.0 ± 0.0

1.0 ± 0.0

9.0 ± 1.4

9.5 ± 0.7

7.0 ± 4.4

6.0 ± 0.0

1.7 ± 0.6

1.8 ± 0.8

0.027 ± 0.008

0.029 ± 0.007

0.036 ± 0.003

0.025 ± 0.008

0.035 ± 0.002

0.045 ± 0.007

0.037 ± 0.002

0.037 ± 0.003

0.028 ± 0.006

0.063 ± 0.020

0.030 ± 0.018

0.035 ± 0.003

ng ng ng ng

1.64 ± 0.13 5.26 ± 0.42 5.95 ± 0.30 8.83 ± 0.11

ng ng

16.75 ± 0.05 21.29 ± 0.00

1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0

0.95 ± 0.35 2.11 ± 0.08 5.23 ± 0.19 6.87 ± 0.27 7.59 ± 0.12

ng 0.23 ± 0.14

13.21 ± 0.08

4.0 ± 1.4

7.71 ± 0.32 nm

5.8 ± 2.9

1.0 ± 0.0

0.70 ± 0.20 nm

1.7 ± 1.2

0.09 ± 0.24

1.0 ± 0.0

ngc

1.19 ± 0.21

1.10 ± 0.30

7.0 ± 3.0

Lag phase (days)

0.76 ± 0.42

∆ formate concentration (mM)

Values calculated according to equation 2; b n = 6; c na-not applicable, nm-not measured, ng-no growth.

0.4

120

109.0

5.5

54.5

0.17

27.2

2.2

20.0

0.081

15.0

1.0

10.9b

6.5

7.6

0.0

5.7

5.5b

0.049 ± 0.002

0.058 ± 0.004

0.050 ± 0.003

0.049 ± 0.008

0.036 ± 0.003

0.032 ± 0.006

0.033 ± 0.005

0.042 ± 0.002

0.060 ± 0.010

0.027 ± 0.005

Maximum growth rate (h1)

C. ljungdahlii PETC

0.055 ± 0.002

0.063 ± 0.010

0. 053 ± 0.005

0.049 ± 0.010

0.050 ± 0.006

0.053 ± 0.005

0.034 ± 0.008

0.043 ± 0.012

0.053 ± 0.004

0.062 ± 0.008

0.055 ± 0.008

0.051 ± 0.009

nac

0.023 ± 0.005

Maximum growth rate (h1)

7.01 ± 1.21

6.67 ± 0.34

4.92 ± 0.39

2.31 ± 0.04

0.87 ± 0.07

0.36 ± 0.49

7.34 ± 0.52

1.73 ± 0.15

0.41 ± 0.22

0.18 ± 0.08

1.26 ± 0.30

nmc

∆ formate concentration (mM)

C. carboxidivorans P7

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7.0

1.6 ± 0.6

2.2

1.0 ± 0.0

1.1

3.0 ± 0.0

2.2 ± 0.4

6.5 ± 0.7

1.0

10.9

3.0 ± 0.0

0.1

7.6

2.3 ± 1.2

0.1

5.5

1.0 ± 0.0

2.2

1.0 ± 0.0

1.7 ± 0.6

Lag phase (days)

0.0b

1.0

6.0

0.0

5.0

Formic acid (mM)a (×102)

Sodium formate (mM)

Initial pH

Table 1. Concentration of formate and formic acid at the beginning of the experiments; mean values ± SD (n = 3) of lag phase, maximum growth rate attained and formate concentration consumed during the experiment of Clostridium ljungdahlii PETC and Clostridium carboxidivorans P7

198 RAMIÓ-PUJOL ET AL.

Fig. 1. Selected growth curves (mean values and SD, n ≥ 3) of Clostridium ljungdahlii PETC (top) and Clostridium carboxidivorans P7 (bottom) at different pH and formate concentrations.

tigated in detail but a thorough inspection of those cultures under phase-contrast microscopy revealed the presence of cell clumps and lysed cells (results not shown), both contributing to the decrease in the absorbance. The duration of the lag phase varied from 1 to 12 days for both P7 and PETC, and was directly correlated with the increase in the sodium formate concentration of the culture, especially at low pH values (Table 1). The increase on the lag phase of bacteria is generally recognized as an adaptation phase, during which bacteria stimulate transcription of new genes to resume growth under the new environmental conditions [30] . For instance, it has been reported that 10 mM of formic acid at pH 5.0 caused bacteriostasis in Escherichia coli, and growth resumed only after a 2 h incubation period proving its adaptation to formic acid [6]. However, adaptation to increasing formate concentrations may be complex since organic acids can serve as both additional carbon substrates and inhibitory compounds, depending on the concentration, pH of the media and/or the cell resistance to the acid, which

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CLOSTRIDIUM GROWN ON SYNGAS

could explain such long lag phases. Moreover, the addition of sodium formate, particularly at high concentrations, could have caused a significant increase in the ionic strength of the culture medium thus causing an additional stress for cell growth. The effect of sodium chloride concentration on the growth and alcohol production of Clostridium autoethanogenum in a completely autotrophic medium has been previously tested using a Plakett-Burmann experimental design; even if that work reports a positive effect of NaCl in ethanol production, it is not significant in the range of 0.4 to 1.0 g/l [9]. The maximum estimated growth rates for PETC and P7, were 0.063 ± 0.020 h–1 and 0.063 ± 0.010 h–1, respectively (Table 1). The calculated maximum growth rates agree with the values obtained in previous works using the same strains [15,17,27]. In both strains at low pH values, low concentrations of sodium formate (<2.2 mM) resulted in a slight increase in the growth rate compared to the formate free media, although the observed differences were only significant for PETC cultures at pH 5.0 (P < 0.05, Bonferroni test, n ≥ 3).

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According to the observed growing capacity, PETC was more tolerant to formate concentration than P7, and complete growth inhibition was only observed at 54.5 mM at pH 6.0. As stated above, a potential effect of added salt concentration could also contribute to growth inhibition. However, this is not expected to occur at sodium formate concentrations lower than 10.9 mM, according to the results obtained at pH 7.0, at which no significant growth inhibition occurred in any of both strains. This observation suggests that low sodium formate concentrations at pH 6.0 and 5.0 might enhance the growth of PETC. Extra-cellular formic acid diffuses across the lipid bilayers and dissociates inside the cell based on the intra-cellular pH [36]. The distribution of dissociated and undissociated forms on the two sides of the cell membrane is proportional to the pH [11]. The most common metabolic processes to circumvent organic acid diffusion into cells includes the use of specific transporters functioning as efflux pumps [14,24]. However, and at least for enterobacteriaceae, several other strategies exist including aminoacid decarboxylases and other protective mechanisms [3]. An inspection of public genome sequences of PETC and P7 have confirmed the presence of putative formate transporters, although with differences in the two bacterial species [17,26]. One single gene encoding for a hypothetical formate/nitrite transporter (WP_013240353) was identified in the PETC genome (NC_014328), whereas, the P7 draft genome (PRJNA48985; PRJNA29495; PRJNA55755; PRJNA33115) contains at least three genes coding

Fig. 2. Formate consumption during growth of Clostridium ljungdahlii PETC. Optical density (black dots) and formate concentration (white dots) are shown as mean values of two replicates. Error bars indicate SD.

for formate transporters, two formate/nitrite transporters (WP_007062507 and WP_007063385) and one oxalate/formate antiport (WP_007061997). The alignment of the four retrieved amino acid sequences revealed that the unique nitrite/formate transporter found in PETC had a highly similar homolog (>80%, Blosum62 matrix) in C. carboxidivorans P7 (results not shown). Note that PETC showed a much faster adaptation and higher tolerance to formate, which might be explained to some extent by differences in the other two transport proteins detected. Formate consumption. Formate concentrations were measured once growth stopped and were compared to the initial concentration to assess its net consumption or production (Table 1). A net production was detected for both strains when incubated under completely autotrophic conditions or at low formate concentration (<1.0 mM). This production was probably due to the activity of formate dehydrogenase (FDH), which converts CO2 into formate in the first step of the WL pathway. Net production ranged from 1.1 to 0.09 mM in PETC and from 1.26 to 0.18 mM in P7. On the contrary, formic acid consumption was observed in most of the treatments where formate had been added and growth resumed after the lag phase. This net formate consumption was significant for both strains, accounting for more than 80% of the added sodium formate. Time course experiments were carried out to elucidate whether formate consumption occurred during the lag or the exponential phase. No net formate consumption was observed during the

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201

additional reducing power source, which would diminish the total energy requirements to incorporate new carbon molecules [28]. The most favorable reaction to obtain reducing power in acetogenic bacteria is the oxidation of CO to CO2 by the carbon monoxide dehydrogenase (CODH) [12], but no analyses of the composition of the gas phase were done to confirm this hypothesis in this experiment. Cultivation of PETC and P7 in the same media composition as the used here, but no formate added, resulted in a complete depletion of CO, which was

Int Microbiol

lag phase, it being mostly consumed during the exponential growth phase at any of the concentrations tested (Fig. 2). The observed formate consumption could be related to its use as an alternative carbon or energy substrate in addition to H2, CO2 and CO, as has been proven for some acetogenic bacteria. Theoretically, formate uptake would partially circumvent the use of hydrogen/CO and make the first step in the WL pathway unnecessary. Moreover, formate oxidation to CO2 via formate dehydrogenase (FDH) would provide an

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Fig. 3. Concentrations of organic acids (acetate and butyrate) and alcohols (ethanol and butanol) produced by Clostridium ljungdahlii PETC (left) and Clostridium carboxidivorans P7 (right) according to initial formate concentration. Incubations at different pH values are shown. Different letters above bars indicate significant differences of acetate production between experiments within each bacterial species according to Bonferroni or T3 of Dunnet post-hoc test assuming equal or not equal variance respectively. Bars show mean value of 3 replicates. Error bars indicate SD.

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mostly converted into CO2 and used as the main source of reducing power instead of H2 (results not shown). In this respect, the incorporation of formate would considerably lower the CO oxidation as a reducing power source and increase the carbon available for fixation into cellular biomass. This probably occurred at low formate concentrations, at which an increase in the growth rate and the growth yield of C. ljungdahlii was observed. Most presumably, the positive effect of formate addition on growth was masked by the activation of resistance mechanisms to circumvent potential inhibition effects of either increased formic acid concentration or ionic strength. Production of acids and alcohols. The concentration of acids (acetate and butyrate) and alcohols (ethanol and butanol) produced by C. ljungdahlii PETC and C. carboxidivorans P7 measured at the end of the incubation experiments is shown in Fig. 3. Acetate production by PETC increased with increasing concentrations of formate at pH 5.0 and 6.0. Maximum acetate production (337.9 mg C/l and 584.7 mg C/l, respectively) was observed at 2.18 mM of formate, representing an increase of 104.5% and 52.4% compared to the control. On the contrary, acetate production was negatively affected by increasing concentration of sodium formate at pH 7.0 (P < 0.05, Pearson correlation test, n = 18). Acetate production of PETC decreased from 546.1 mg C/l (control test) to 116.3 mg C/l (10.9 mM formate). Regarding ethanol, the highest production occurred at pH 5.0, with maximum concentrations slightly over 230.0 mg C/l. Nevertheless, even if differences in alcohol production were observed at different pH values, changes were not related to the initial formate concentration (P > 0.05, ANOVA test, n ≥ 3). Formate concentrations higher than 2.18 mM can positively affect the ethanol production due to the changes in salt concentrations [9]. Acetate and ethanol production in C. carboxidivorans P7 was lower than in PETC, but different formate dependence trends were observed depending on pH. At pH 6.0, acetate production remained almost invariable between 0 and 10.9 mM. However, acetate concentration showed a negative correlation (P < 0.05, Pearson correlation test, n = 18) with added formate at pH 7.0. Maximum butyrate was 14.9 mg C/l, and was obtained at a sodium formate concentration of 2.18 mM and pH 6.0. Neither pH nor initial formate concentration did play a major role in butyrate production, except at pH 7.0 where production decreased more than 50% in the presence of formate. Alcohols production of PETC at pH 6.0 showed some significant differences, although no correlation to the sodium formate addition was observed (P > 0.05, ANOVA test, n ≥ 3) (Fig. 3).

RAMIÓ-PUJOL ET AL.

Overall, the addition of formate increased the acid production of C. ljungdahlii PETC at pH 5.0 and 6.0, although solvent production remained unaffected. This could be explained by the “acid crash” effect, during which the fast accumulation of acids results in a failure of the switch from acidogenic phase to solventogenic phase to occur, and no solvent are produced by clostridia. Formic acid has been reported to play a major role in triggering the acid crash of ABE fermentations [20,37]. This phenomenon was not observed for C. carboxidivorans, as the concentrations of acids produced in the different experiments were never higher than that of the control. The addition of formate at pH 7.0 caused the opposite effect in both strains, and accumulation of acids decreased with increasing formate concentrations. These observations seem to be in disagreement with the measure OD, lag phases and growth rates (Table 1), which shows that, at pH 7.0, undissociated formic acid concentration remained low and inhibitory effects were clearly diminished in both PETC and P7 strains. Ideally, the energy saved by the use of formate as a substrate could be utilized to increase cellular ATP production. It has been long recognized that autotrophic growth by the WL pathway must be linked to an energygenerating anaerobic respiratory process, since during autotrophic growth there is no net ATP synthesis by substrate-level phosphorylation [28]. Thus, the use of partially reduced compounds could have also allowed higher available reducing power and/or ATP, so reducing the need of acetate production. However, this was not clearly confirmed and further work would be needed to test this hypothesis. The highest alcohol to total product ratios were obtained at pH 5.0 for both strains, and they decreased significantly at higher pH values. The statistical tests (P > 0.05, Dunnet T3 test, n ≥ 3) proved that such differences were not linked to the presence of sodium formate at P7 strain, but to incubation pH. The highest alcohol to total product ratio through all the experiment was 0.53, corresponding to the PETC control experiment at pH 5.0. This ratio was negatively influenced by the addition of formate because the productivity enhancement led to the production of mainly acetate. Finally, alcohols/products ratio at pH 7.0 significantly increased from 0.09 to 0.36 (P < 0.05, Dunnet T3 test, n ≥ 3) in PETC. However, the reason for such an increase was related to the reduction of acetate production rather than to an increase in the net alcohol production. Although the total amount of carbon fixed into synthesized products was lower, such operational conditions could be beneficial when aiming at alcohol production. Downstream separation processes account for a large part of operational costs, therefore the decrease in acetate production in the fermentation medium could ease alcohol separation [29].

CLOSTRIDIUM GROWN ON SYNGAS

Implications and future prospects. The present work assessed the impact of the addition of formate on C. ljungdahlii PETC and C. carboxidivorans P7. Results showed the higher tolerance of PETC to formate, in addition to the enhancement of its growth rate and productivity at low formate concentrations at pH 5.0 and 6.0. This is of interest from the biotechnological point of view as the ability of the PETC strain to use formate as a feedstock opens up potential to upgrade carboxydotrophic fermentation process with external formate supply. Of special interest could be the combination of enzymatic electrocatalysis to produce formic acid [33] and microbial electrosynthesis [21]. In fact this could be a major breakthrough in the production of added-value compounds from carbon dioxide via bio-electrochemical fermentation. In this light, PETC could be cultivated at moderately acid pH in a BES with low formic acid production to accelerate its metabolism and enhance carbon fixation into products. However, further studies are required to elucidate the metabolic fate of formate, and to understand the impact of formate assimilation on the cell energy and reducing power balances. Acknowledgements. The authors thank the Autonomous Government of Catalonia (Generalitat de Catalunya) (2013 FI-DGR) and the Spanish Ministry of Science and Innovation (Best-Energy, CTQ2011-23632, CTM201343454-R) for their financial support in this study. LEQUIA and IEA have been recognized as consolidated research groups by the Catalan Government (2014-SGR-1168 and 2014-SGR-2016). RG gratefully acknowledges support from Beatriu de Pinós fellowship (BP-2011-B) and FP7 Marie Curie Career Integration Grants (PCIG13-GA-2013-618593).

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7. 8. 9.

10.

11. 12.

13. 14. 15.

16.

17.

18.

19.

Competing interests. None declared. 20.

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27. Perez JM, Richter H, Loftus SE, Angenent LT (2013) Biocatalytic reduction of short-chain carboxylic acids into their corresponding alcohols with syngas fermentation. Biotechnol Bioeng 110:1066-1077 28. Ragsdale SW, Pierce E (2008) Acetogenesis and the Wood-Ljungdahl pathway of CO(2) fixation. Biochim Biophys Acta 1784:1873-1898 29. Ramachandriya KD, Kundiyana DK, Wilkins MR, Terrill JB, Atiyeh HK, Huhnke RL (2013) Carbon dioxide conversion to fuels and chemicals using a hybrid green process. Appl Energy 112:289-299 30. Rolfe MD, Rice CJ, Lucchini S, Pin C, Thompson A, Cameron ADS, Alston M, Stringer MF, Betts RP, Baranyi J, Peck MW, Hinton JCD (2012) Lag phase is a distinct growth phase that prepares bacteria for exponential growth and involves transient metal accumulation. J Bacteriol 194:686-701 31. Schuchmann K, Müller V (2013) Direct and reversible hydrogenation of CO2 to formate by a bacterial carbon dioxide reductase. Science 342:1382-1385 32. Sleat R, Mah RA (1984) Quantitative method for colorimetric determination of formate in fermentation media. Appl Environ Microbiol 47:884-885

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33. Srikanth S, Maesen M, Dominguez-Benetton X, Vanbroekhoven K, Pant D (2014) Enzymatic electrosynthesis of formate through CO2 sequestration/reduction in a bioelectrochemical system (BES). Bioresour Technol. doi: 10.1016/j.biortech.2014.01.129 34. Tanner RS, Miller LM, Yang D (1993) Clostridium ljungdahlii sp. nov., an acetogenic species in clostridial rRNA homology group I. Int J Syst Bacteriol 43:232-236 35. Thompson JL, Hinton M (1997) Antibacterial activity of formic and propionic acids in the diet of hens on salmonellas in the crop. Br Poult Sci 38:59-65 36. Walter A, Gutknecht J (1984) Monocarboxylic acid permeation through lipid bilayer membranes. J Membr Biol 77:255-264 37. Wang S, Zhang Y, Dong H, Mao S, Zhu Y, Wang R, Luan G, Li Y (2011) Formic acid triggers the “Acid Crash” of acetone-butanol-ethanol fermentation by Clostridium acetobutylicum. Appl Environ Microbiol 77:1674-1680

RESEARCH ARTICLE IÄã ÙÄ ã®ÊÄ ½ M® ÙÊ ®Ê½Ê¦ù (2014) 17:205-212 doi:10.2436/20.1501.01.223. ISSN (print): 1139-6709. e-ISSN: 1618-1095

www.im.microbios.org

Biofilm formation on polystyrene in detached vs. planktonic cells of polyhydroxyalkanoateaccumulating Halomonas venusta Mercedes Berlanga1*, Òscar Domènech2, Ricardo Guerrero3,4 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Barcelona, Spain. 2Physical Chemistry Laboratory V, Faculty of Pharmacy, University of Barcelona, Spain. 3Laboratory of Molecular Microbiology and Antimicrobials, Department of Pathology and Experimental Therapeutics, Faculty of Medicine, University of BarcelonaIDIBELL, Barcelona, Spain. 4Barcelona Knowledge Hub, Academia Europaea, Barcelona, Spain

Received 15 September 2014 · Accepted 20 October 2014

Summary. Biofilm development is characterized by distinct stages of initial attachment, microcolony formation and maturation (sessile cells), and final detachment (dispersal of new, planktonic cells). In this work we examined the influence of polyhydroxyalkanoate (PHA) accumulation on bacterial surface properties and biofilm formation on polystyrene in detached vs. planktonic cells of an environmental strain isolated from microbial mats, Halomonas venusta MAT28. This strain was cultured either in an artificial biofilm in which the cells were immobilized on alginate beads (sessile) or as free-swimming (planktonic) cells. For the two modes of growth, conditions allowing or preventing PHA accumulation were established. Cells detached from alginate beads and their planktonic counterparts were used to study cell surface properties and cellular adhesion on polystyrene. Detached cells showed a slightly higher affinity than planktonic cells for chloroform (Lewis-acid) and a greater hydrophobicity (affinity for hexadecane and hexane). Those surface characteristics of the detached cells may explain their better adhesion on polystyrene compared to planktonic cells. Adhesion to polystyrene was not significantly different between H. venusta cells that had accumulated PHA vs. those that did not. These observations suggest that the surface properties of detached cells clearly differ from those of planktonic cells and that for at least the first 48 h after detachment from alginate beads H. venusta retained the capacity of sessile cells to adhere to polystyrene and to form a biofilm. [Int Microbiol 2014; 17(4):205-212] Keywords: Halomonas venusta MAT-28 · PHA · alginate beads · cell surface physicochemical characteristics · adhesion on polystyrene

Introduction Our perception of bacteria as planktonic life forms is deeply rooted in the axenic (“pure”) culture paradigm. Growth in liqCorresponding author: M. Berlanga Department of Microbiology and Parasitology Faculty of Pharmacy, University of Barcelona Av. Joan XXIII, s/n 08028 Barcelona, Spain Tel. +34-934024497. Fax +34-934024498 E-mail: mberlanga@ub.edu *

uid culture has been exploited to study many bacterial activities. However, in nature, bacteria rarely grow as axenic planktonic cultures; rather, the normal mode of bacterial growth involves attachment to a surface, followed by the development of a microbial community and biofilm formation [1,21,22,34]. Microorganisms in biofilms are coordinated functional communities that are much more efficient than mixed populations of floating planktonic organisms. In fact, biofilms resemble the tissues formed by eukaryotic cells with respect to their physiological cooperation and the extent to which they are protected from variations in bulk-phase condi-

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tions by a primitive homeostasis afforded by the biofilm matrix of exopolysaccharides [13]. Biofilm development is characterized by distinct stages of attachment, growth, and detachment (the dispersal of planktonic cells) [2,28,30]. Naturally occurring immobilized microbial cells, such as those in biofilms, differ physiologically from planktonic (free swimming) cells in a variety of ways. These modifications occur at the beginning of biofilm formation, following cell adhesion, when the cells develop surfacesensing responses [18]. The immobilization of cells in alginate beads, which serve as artificial biofilms, bypasses the adhesion step. However, the similar physiological responses of artificially and naturally immobilized microorganisms, as evaluated based on protein expression patterns, support the existence of a specific metabolic behavior by “sessile” cells [18,20,39]. The functional strategies and physiological versatility of bacterial populations growing in biofilms allow the cells to resist changing conditions within their environment [16]. In fact, prokaryotes have evolved numerous mechanisms of resistance to stress conditions. For example, many microorganisms have an inherent ability to form resting stages (e.g., cysts and spores) [25]; others, such as the spirochete Spirosymplokos deltaiberi, become swollen and form refractile resistant bodies on exposure to air [26]. The accumulation of intracellular storage polymers such polyhydroxyalkanoates (PHA) increases cell survival in changing environments [2,5,19] by enhancing bacterial environmental fitness, especially under environmental stress conditions such as UV irradiation and osmotic, thermal, and oxidative stress [33,35,38]. PHAs are energy- and carbon-rich storage compounds that accumulate as intracellular granules, but they can be mobilized and used under unfavorable conditions. Under stress conditions, bacterial cells with a higher PHA content survive longer. than those with a lower PHA content. Among the many bacterial species that are able to accumulate PHAs are members of the genus Halomonas, which belongs to the family Halomonadaceae, a Gammaproteobacteria. Members of the Halomonadaceae are gram-negative, chemoorganotrophic, aerobic or facultative anaerobic, and moderately halophilic, haloalkaliphilic, halotolerant, or nonhalophilic. In this work, we used the PHA-accumulating strain H. venusta MAT-28, isolated from a microbial mat (a complex biofilm) from the Ebro Delta [4]. Halomonas venusta MAT-28 was cultured in an artificial biofilm in which the cells were immobilized on alginate beads and as free-swimming (planktonic) cells. Cells detached from

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alginate beads and their planktonic counterparts were used to: (a) investigate adhesion to polystyrene and (b) determine whether cells that accumulate PHA are better able to adhere to a new surface than those that do not.

Material and methods Cell immobilization by alginate beads. The moderately halophilic bacterium used in this study was Halomonas venusta MAT28, which was isolated from microbial mats in the Ebro Delta, Spain. Sodium salt alginic acid from Macrocystis pyrifera (61% mannuronic acid and 39% guluronic acid; Sigma-Aldrich, St. Louis, MO, USA) was prepared as previously described [6]. The conditions used for bead preparation favored the leakage of entrapped bacteria while maintaining the integrity of the beads (Fig. 1A, B). Growth mode: alginate beads and planktonic cells. Three different growth media were used in the assays: tryptic soy broth (TSB; Oxoid, Barcelona, Spain) + 3% NaCl, TSB-30 + 3% NaCl, and minimal medium + glucose + 3% NaCl. Only the last one allows PHA accumulation; the two TSB-based media do not [6]. The three culture media are referred to in the following as TSB, TSB-30, and MM, respectively. Two modes of bacterial growth were investigated. Thus, the cells were immobilized on alginate-beads as an artificial biofilm and cultured as free-swimming (planktonic) cells. Two Erlenmeyer flasks each containing 50 ml of one of the three culture media were prepared: one for cells immobilized on alginate beads and the other for planktonic cells. The flasks were incubated at 30ºC for 24 h (for MM, the incubation time was 48 h to allow PHA accumulation). Alginate bead cultures contained approximately 8 beads/ml. Planktonic cultures were prepared from a 1:1000 ml dilution of an overnight culture. After 24 h (or 48 h for PHA accumulation) of incubation, the affinity of detached and planktonic cells for different solvents and their adhesion properties were assayed. Microbial affinity for solvents (MATS). The method employed was described by Giaouris et al. [15]. Detached and planktonic cells were harvested by centrifugation (7500 rpm, 10 min), washed twice with phosphate-buffered saline (PBS), pH 7.0, and resuspended in the same solution at a final optical density (OD600) of 0.8. Each bacterial suspension (2.4 ml) was mixed for 60 s at maximum intensity on a vortex-type agitator with 0.4 ml each of chloroform, hexadecane, diethyl ether, and hexane (Panreac, Barcelona, Spain). The samples were allowed to stand for 60 min to ensure complete separation of the two phases. One ml was carefully removed from the aqueous phase of each sample and the optical density was measured at 600 nm. The microbial affinity for each solvent was calculated using the formula: % affinity = (OD0 − ODf / OD0) × 100 where OD0 is the optical density of the bacterial suspension before mixing with the solvent and ODf is the absorbance after mixing and phase separation. Each measurement was performed in duplicate and the experiment was repeated three times with independent bacterial cultures. The following pairs of solvents were assayed: (a) chloroform (an acidic solvent) with hexadecane (an apolar solvent); (b) diethyl ether (a strong basic solvent) with hexane (an apolar solvent). The monopolar solvents used had similar Lifshitz–van der Waals surface tension components (Table 1) [3].

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Fig. 1. Micrograph of immobilized cells of Halomonas in alginate beads and cultured in TSB-30 at 30ºC for 24 h. The SEM micrograph shows microcolonies formed at the surface of a bead that are about to detach.

Biofilm formation. Aliquots (150 ml) of the bacterial suspensions (1:100 dilutions of the detached and planktonic cells) were transferred to each well of a microtiter plate (plastic). The bacterial suspensions were prepared in fresh TSB, TSB-30, or MM, depending on the culture medium used previously. After a 24-h static incubation at 30ºC, the biofilm index was determined as described by O’Toole and Kolter (1998) [27]. Atomic force microscopy (AFM). AFM images were recorded using a Multimode microscope controlled with Nanoscope V electronics (Bruker AXS, Santa Barbara, CA) equipped with a 10-μm piezoelectric scanner. The bacteria were immobilized by adsorption onto a clean mica surface. A 50-μl aliquot of a bacterial suspension was placed on 1 × 1 cm of cleaved mica and incubated for 5 min at room temperature. Non-adsorbed bacteria were eliminated by gentle rinsing of the surface with PBS. The sample was dried under a nitrogen stream. All images were taken in air in contact mode with a silicon cantilever with a nominal spring constant of 40 N·m−1. The ap-

plied force was kept as low as possible to minimize damage of the sample. The images were processed using commercial Nanoscope software.

Results Influence of the growth mode on the physicochemical properties of the bacterial surface. The cell surface of Halomonas venusta growing in TSB had a higher Lewis-acid character, with a high affinity for chloroform and the lowest affinity for diethyl ether. Cells growing in TSB-30 and MM also exhibited Lewis-acid characteristics, both detached and planktonic cells, but their affinity for chlo-

Table 1. Properties of microbial affinity for solvents (MATS)-type solvents [3] MATS solvent

Formula

Lifshitz-van del Waals (mJ/m2)

Electron donor (γ–) (mJ/m2)

Electron acceptor (γ+) (mJ/m2)

Chloroform

CHCl3

27.2

3.8

Diethyl ether

C4H8O2

16.7

16.4

Hexane

C6H14

18.4

Hexadecane

C16H34

27.7

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Table 2. Lewis acid-base and hydrophobicity surface characteristics MATS-type solvent Strain (growth mode)

Chloroform

Hexadecane

Diethyl ether

Hexane

62.4 ± 5.0

24.3 ± 4.4

41.8 ± 2.9

28.9 ± 6,8

64.7 ± 5.6

25.9 ± 2.6

42.7 ± 6.2

30.2 ± 3.5

54.97 ± 4.68

17.2 ± 3.06

47.71 ± 5.63

18.41 ± 3.34

56.90 ± 3.97

26.2 ± 4.66

49.75 ± 3.75

26.31 ± 1.35

Halomonas-P (PHA)

52.3 ± 4.4

12.6 ± 2.4

44.3 ± 3.6

17.1 ± 4.6

Halomonas-DC (PHA)

55.5 ± 3.5

24.1 ± 4.4

48.8 ± 6.7

25.3 ± 3.5

Halomonas-P Halomonas-DC Halomonas-P Halomonas-DC

P, planktonic cells; DC, detached-cells. PHA, polyhydroxyalkanoate accumulation. TSB growth medium. b TSB-30 growth medium. c Minimal medium allowing PHA accumulation. a

roform was lower in either of these media than in TSB medium. In general, H. venusta was hydrophilic, as indicated by its low affinity for apolar solvents such as hexadecane and hexane. However, detached cells displayed a slowly increasing hydrophobicity compared to planktonic cells (Table 2). Similar results have been previously reported [30].

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Influence of growth mode on surface adhesion. Static biofilms from H .venusta were allowed to develop on polystyrene microplates. Detached cells were better able to adhere to plastic than cells growing planktonically (Fig. 2).

Detached cells of PHA-accumulating H. venusta-PHA were less adherent to plastic than detached H. venusta cells that did not accumulate PHA, perhaps because of the slight differences in surface hydrophobicity (Table 2). Similar results were obtained in Pseudomonas [14]. Cells that accumulated PHA were more hydrophilic (lower affinity for apolar solvents) than those that did not. This result can be explained by a redirection of the carbon flux to fatty acid biosynthesis in cells that did not accumulate PHA. According to Chang et al. [10], a higher fatty acid accumulation is related to enhanced cell-surface hydrophobicity. In TSB medium, detached and

Fig. 2. Biofilm index. Adhesion to polystyrene by Halomonas venusta MAT28 growing planktonically (P) or on alginate beads (detached cells, DC). The height of each column is the mean of the results of three independent experiments. The standard deviations are indicated by the bars.

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Fig. 3. Atomic force microscopy images. (A) Halomonas planktonic cells growing under conditions that did not support polyhydroyalkanoate (PHA) accumulation. (B) A detail of a cell from A. (C) A surface detail from the image in panel B. (D) Halomonas detached cells growing in a culture medium that facilitates PHA accumulation. (E) Detail of a cell from D. (F) Surface detail from panel E.

planktonic cells did not differ in their adhesion to polystyrene. AFM produces a three-dimensional topographic image of the specimen surface with nanometer resolution. Figure 3A shows a three-dimensional topographic image of the surface of H. venusta grown in TSB-30 deposited on a mica surface. The bacteria are seen as ellipsoid structures with a mean length of 3.3 ± 0.3 μm, a mean width of 1.7 ± 0.2 μm, and a mean height of 290 ± 30 nm. These values, especially bacterial height, may be underestimations because of probable deformation of the cells after they attach to a flat surface. A thin film with a step height of 12 nm from mica surface was also observed. A representative zoom image of a cell is shown in Fig. 3B. The cell surface is flat and smooth, with a nanostructure of small protrusions of 6 ± 2 nm (Fig. 3C). Figure 3D shows cells detached from alginate-beads that had been suspended in MM medium, which facilitates PHA accumulation, and deposited on a mica surface. The rounded structures (white arrows) inside the bacteria are probably PHA granules. A representative image of bacteria with the above-described protrusions is shown in Fig. 3E (white arrows). Material of an undetermined nature is seen outside the cell adsorbed on mica (black arrows). Closer inspection of the bacterial surface (Fig. 3F)

showed the nanostructures to be small round particles with a mean diameter of 58 ± 9 nm and a mean height of 3.8 ± 0.5 nm. Those nanoscale surface differences between H. venusta without PHA and with PHA may have resulted in a modification of the cells’ surface properties that accounted for the difference in their adhesion capacity on polystyrene (see Table 2, Fig. 2). To determine whether the growth mode (alginate beads or planktonic) of H. venusta resulted in differences in polystyrene adhesion, a principal components analysis (PCA), as a multivariate data analysis tool, was applied. The results obtained with the adhesion datasets are shown in Fig. 4 as a two-dimensional plot. A cloud of points is observed, with each point representing H. venusta growing under different conditions with respect to culture medium and growth mode (alginate beads and planktonic). Despite the large dispersion of the dataset (Fig. 4), a general grouping depending on the growth mode can be seen. The exception was detached and planktonic cells growing in TSB, as there were no difference is adhesion under these conditions (see Fig. 2). Otherwise, the characteristics of the detached cells were clearly different from those of planktonic cells.

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Fig. 4. Analysis of the main components of Halomonas detached (DC) and planktonic (P) cells, based on the physicochemical characteristics of the cell surface and cellular adhesion capability. Halomonas were grown in TSBa, TSB-30b, or minimal medium (MM)c. Only MM allowed PHA accumulation.

Discussion Bacterial adhesion is a complex process that is affected by the characteristics of the bacteria (hydrophobicity, surface charge, fimbriae, production of exopolysaccharides, etc.), the surfaces properties of the material (surface charge, hydrophobicity, roughness, etc.), and environmental factors (temperature, pH, time of exposure, bacterial concentration, ionic strength, etc.) [8,11,12,24,31]. The initial adhesion of microbial cells is the product of non-covalent Lifshitz-van der Waals, electrostatic, Lewis acid-base, and hydrophobic interactions [12,24,31]. Halomonas venusta growing on TSB had the highest biofilm index on polystyrene microplates, without significant difference in adhesion between detached and planktonic cells (Fig. 2). Similar results have been reported for Listeria [29]. TSB is a complex mixture of casein and soy peptide hydrolyzates, along with other macromolecules that adsorb to polystyrene. The heterogeneity of the absorbed peptides in particular likely creates a variety of charged areas on the substratum that can interact with bacterial surface proteins through electrostatic as well as hydrophobic interactions. The ionic strength of the solution influences the extent of bacterial adhesion to a surface. Increasing the ionic strength decreases the thickness of the electrostatic layer surrounding a bacterium

and a surface [7]. As a result, the bacterial cell can come close enough to the surface such that the strength of the van der Waals attraction may overcome the repulsive energy barrier between two negatively charged surfaces and result in bacterial adhesion [11]. The detached cells were slightly better electron acceptors (affinity for chloroform) than their planktonic counterparts, which implies an increased number of protonated groups such as NH3 and of OH groups exposed on the bacterial surface [17]. The surfaces of the detached cells were also more hydrophobic (increase affinity for apolar solvents such as hexadecane and hexane). These differences could facilitate adhesion and biofilm formation on polystyrene, which has a negative surface charge, is hydrophobic, and is an electron donor [32]. In previous work, we showed that PHA accumulation in H. venusta MAT-28 reached steady-state concentrations after 48 h of incubation in MM [4] and that PHA accumulation was higher in detached (from alginate beads) than in planktonic cells [6]. From an ecological point of view, increased PHA accumulation is a natural strategy to increase survival in stressed environments [33,35]. Although detached-PHA cells were more adherent than planktonic-PHA cells, there was no difference in the ability of PHA-accumulating vs. non-accumulating cells in their adhesion to polystyrene (Fig. 2). PCA combines two or more correlated factors into a new

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variable, the principal component. Thus in PCA, the dimensionality of the dataset is reduced by replacing the original variables with a smaller number of newly formed variables that are linear combinations of the original ones and that explain the majority of the information (variability) from the experiment. PCA is an instrument of observation of multidimensional space, rather than a statistical technique [36]. As seen in Fig. 4, detached cells and planktonic cells clearly differed in their adhesion onto polystyrene, even differences in their Lewis acid-base characteristics and hydrophobicity were not substantial. The increased biofilm index of the detached cells may reflect differences in cell-surface properties and ionic strength at the cell-substratum interface [9,12,37]. However, for bacterial cells the most important factor resulting in greater adhesion is to have derived from a biofilm, including an artificial biofilm such as alginate beads. Detached cells from a biofilm represent a transitional phenotype between the sessile and planktonic state. They maintain their biofilm-forming capacities and constitute a general response adopted by different species of bacteria [30,40]. In this study, detached Halomonas cells retained the ability of sessile cells to adhere to surfaces and to form biofilms for at least 48 h after detachment. Compared to permanently planktonic cells, this ability facilitates the colonization of new surfaces.

Acknowledgements. This work was supported by grant CGL200908922 (Spanish Ministry of Science and Technology) to RG.

17.

Competing interests. None declared.

18.

10.

11.

12.

13. 14.

15.

16.

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RESEARCH ARTICLE IÄã ÙÄ ã®ÊÄ ½ M® ÙÊ ®Ê½Ê¦ù (2014) 17:213-224 doi:10.2436/20.1501.01.224. ISSN (print): 1139-6709. e-ISSN: 1618-1095

www.im.microbios.org

Stabilization process in Saccharomyces intraand interspecific hybrids in fermentative conditions Laura Pérez-Través1,2, Christian A. Lopes1,3, Eladio Barrio1,2, Amparo Querol1* 1 Institute of Agrochemistry and Food Technology (IATA-CSIC), Food Biotechnology Department, Paterna, Valencia, Spain. 2Genetics, University of Valencia, Valencia, Spain. 3Institute for Research and Development in Process Engineering, Biotechnology and Alternative Energy, CONICET-UNCo, Faculty of Agricultural Sciences and Faculty of Engineering, National University of Comahue, Neuquen, Argentina

Received 8 September 2014 · Accepted 18 November 2014

Summary. We evaluated the genetic stabilization of artificial intra- (Saccharomyces cerevisiae) and interspecific (S. cerevisiae  S. kudriavzevii) hybrids under wine fermentative conditions. Large-scale transitions in genome size and genome reorganizations were observed during this process. Interspecific hybrids seem to need fewer generations to reach genetic stability than intraspecific hybrids. The largest number of molecular patterns recovered among the derived clones was observed for intraspecific hybrids, particularly for those obtained by rare-mating. Molecular marker analyses revealed that unstable clones could change during the industrial process to obtain active dry yeast. When no changes in molecular markers and ploidy were observed after this process, no changes in genetic composition were confirmed by comparative genome hybridization, considering the clone as a stable hybrid. According to our results, under these conditions, fermentation steps 3 and 5 (30–50 generations) would suffice to obtain genetically stable interspecific and intraspecific hybrids, respectively. [Int Microbiol 2014; 17(4):213-224] Keywords: Saccharomyces cerevisiae · Saccharomyces kudriavzevii · rare-mating in yeast · molecular markers · DNA content evaluation · stabilization of genomes

Introduction The detection of “natural” Saccharomyces hybrid strains in different fermentations [22,29,35], and among the starter cultures used for wine inoculation [9,22,23,33], led to pay attention to the relevance of hybrids in these processes. These hybrids contain an almost complete set of chromosomes from partners in the form of allodiploid or allotetraploid genomes Corresponding author: A. Querol Food Biotechnology Department Institute of Agrochemistry and Food Technology (IATA-CSIC) Av. Agustín Escardino, 7 46980 Paterna, Valencia, Spain E-mail: aquerol@iata.csic.es *

or only portions of the partner’s genomes resulting in alloaneuploids, or strains with chimerical chromosomes [5,17,45,48]. The physiological advantage of hybrids has been proposed to be related to their better fitness than parental strains under intermediate or fluctuating conditions [44]. For this reason, the artificial generation of hybrids has become an interesting strategy in recent years to improve industrial yeast strains. Construction of hybrids in the Saccharomyces genus has been reported between wine strains of Saccharomyces uvarum and various strains of Saccharomyces cerevisiae (for a review, see [48]). The artificial hybrids between S. cerevisiae and other Saccharomyces species, including S. paradoxus and S. kudriavzevii, have also been reported [6,8,39]. Different procedures, including protoplast fusion, mass-mating, spore-to-

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spore mating and rare-mating, have been used for hybrids generation [48]. However, only those methods occurring naturally by mating or natural recombination can be used for the generation of non-genetically modified organisms (nonGMO), in accordance with Directive 2001/18/EC of the European Parliament and the European Council. Protoplast fusion is thus excluded from the group of non-GMOs-producing techniques [11]. Commercialized wine strains have been selected because of their fermentation qualities and stress adaptability during alcoholic fermentation, and also because these strains ensure the production of consistent wines in successive vintages [42]. This means that the strains developed for industrial processes must have stable genomes. In a previous work carried out in our laboratory, different inter- and intraspecific hybrid strains were obtained by employing several hybridization methodologies [39]. However, we observed that instable hybrids showing high DNA content were generally obtained. In other works, polyploid genomes were known to be unstable in S. cerevisiae [20,50] or in hybrids of the Saccharomyces genus [2,26,48]. Similarly, many newly formed polyploids in plants have unstable genomes that undergo rapid repatterning during the first generations, which is particularly important for allopolyploids [49,52]. Because of this trend to the reorganization of the genome and the genetic heterogeneity of the new hybrids [26], the development of a method to ensure proper genetic stability of strains used in industrial applications was necessary. Wine yeast should be adapted to several stress conditions, such as low pH and high sugar content of grape must. The selectivity of fermenting must be further strengthened once anaerobic conditions are established; certain nutrients become depleted and the ethanol level increases [42]. During the process of active dry yeast (ADY) production and the posterior rehidratation, yeast cells are exposed to stressing conditions, such as osmotic, oxidative and thermic stress, and starvation [3,4,14,36,38]. All these stresses exert a strong selective pressure on the microorganisms and could induce changes in unstable genomes. Loss of the type (i.e., parental origin) and content of DNA in the genetic stabilization process during hybrids formation can strongly influence future physiological features and the adaptation of a hybrid to industrial processes. Several examples correlate the influence of genome size differences with phenotypic variations, including cell size [31], generation time [41], and ecological tolerance [19]. Genomic changes such as insertions, deletions and translocations have also been related to yeasts adapting to novel environments [7,16,19].Variations in the number of gene copies occurring in

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polyploids or aneuploids have also been associated with altered gene expression patterns and metabolic activity [18,51]. Genome reduction and rearrangements occurring during the stabilization process might lead to loss of industrially important traits in hybrids, and can be avoided if a selective pressure, mimicking the desired industrial process, is applied during the stabilization. Understanding the stabilization process can help us to design the experimental conditions to develop a new lab-made hybrid for industrial purposes.This work aimed to validate a fast genetic stabilization method for newly generated Saccharomyces hybrids under selective enological conditions, to know how many rounds (or generations) suffice to obtain stable hybrids and to study the changes during the process. The whole stabilization processes in intra- and interspecific hybrids showing different ploidy levels, as a result of using different hybridization methodologies, were also compared. As far as we know, this is the first work that deeply studies the stabilization procedure under enological conditions.

Materials and methods Yeast strains. Four interspecific Saccharomyces cerevisiae  Saccharomyces kudriavzevii hybrids, two obtained from rare-mating (R2 and R8) and two from spore-to-spore mating (S2 and S7), and four intraspecific S. cerevisiae hybrids, two obtained from rare-mating (R1 and R3) and two from sporeto-spore mating (S5 and S8) were selected from a previous work [39] to undergo a genetic stabilization procedure (see hybrid and parental characterization in Table 1). Genetic stabilization procedure. A single colony of each hybrid strain was individually inoculated into 15-ml screw-cap tubes containing 10 ml of synthetic must [46] with 50% glucose and 50% fructose, sterilized by filtration. The samples were incubated at 20ºC without shaking. After fermentation (approximately 15–20 days), an aliquot of approximately 107 cells was used to inoculate a new tube containing the same sterile medium (synthetic must) and was incubated under the same conditions, while a second aliquot was seeded on glucose-peptone-yeast agar (GPY-agar) plates and incubated at 20°C. Ten yeast colonies were randomly picked and characterized by inter- sequences, random amplified polymorphic DNA–PCR (RAPD–PCR) analyses and mtDNA-restriction fragment length polymorphism (mtDNARFLP) patterns. The total DNA content was also measured for each colony showing a different molecular pattern. All the yeast colonies displaying different molecular profiles, regardless the fermentation step at which they were obtained, were individually inoculated in the same synthetic must and, after these individual fermentations, ten colonies from each one were analyzed by the same methods. When one pattern was recovered more than once, we selected this pattern for the last round in which it appeared. We put the original pattern, selected in the fifth round, in an individual fermentation too. We considered that a clone was genetically stable when the colonies recovered after individual fermentations maintained the same molecular profile ( elements, RAPD–PCR and mtDNA-RFLP patterns) and the same ploidy level as the previously inoculated culture.

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Table 1. Molecular and genetic characterization of hybrids and parental strains used in this study (extracted from [39]) Molecular patterns¶ Cross

Methodology

Parental strains

Sc1xSc2

Rare-mating

Spore-to-spore mating

Sc1xSk

Rare-mating

Spore to spore mating

Name

mtDNA

δ-PCR

RAPD-R3

DNA content§

Sc1

δ-Sc1

R3-Sc1

2.7 ± 0.2 a-c

Sc2

δ-Sc2

R3-Sc2

2.5 ± 0.3 a

δ-Sk

R3-Sk

2.2 ± 0.1 a

Sc2

δ-5

R3-8

5.0 ± 0.1 j

Sc1

δ-4a&

R3-7

4.7 ± 0.3 ij

Sc2

δ-10

R3-9

2.7 ± 0.1 a-d

Sc1

δ-14

R3-10

2.8 ± 0.2 a-e

δ-4b*

R3-2

3.2 ± 0.2 a-e

Sc1

δ-4b*

R3-4

4.8 ± 0.1 i-l

δ-9

R3-11

3.4 ± 0.1 c-f

Sc1

δ-8

R3-12

3.2 ± 0.2 a-d

Molecular patterns obtained by mtDNA-RFLP (mtDNA), interdelta sequence DNA polymorphisms (δ-PCR) and RAPD analysis using primer R3 (RAPD-R3). § Values expressed as mean ± standard deviation. Values not shearing the same superscript letter within the column are significantly different (ANOVA and Tukey HSD test, α = 0.05, n = 2). & Patterns δ4 in Pérez-Través et al. [39], both of them are different. ¶

Random amplified polymorphic DNA (RAPD–PCR) analysis. Primer R3 (5´-ATGCAGCCAC-3´) was used for the RAPD–PCR analysis. This primer showed the highest degree of variability among the hybrid strains—including those analyzed in this work—of the 11 primers described in a previous work [39].The patterns obtained from the RAPD–PCR analysis were codified with lowercase letters. Amplified elements DNA polymorphism analysis. Primers delta 12 (5´-TCAACAATGGAATCCCAAC-3´) and delta 21 (5´-CATCTA ACACCGTATATGA-3´), as well as the procedures proposed by Legras and Karst [27], were used to amplify yeast genomic DNA. The patterns obtained from the δ elements analysis were codified with Roman numerals. Mitochondrial DNA-restriction fragment length polymorphism (mtDNA-RFLP) analysis. A mitochondrial DNA restriction analysis was performed by the method of Querol et al. [43] using the endonuclease HinfI (Roche Molecular Biochemicals, Mannheim, Germany). The patterns obtained from the mtDNA-RFLP analysis were codified using capital letters. Irrespective of the molecular marker used, pattern “o” corresponds to the original pattern found in the hybrid prior to the stabilization process. DNA content evaluation. The DNA content of both hybrid and control strains was assessed by flow cytometry using a FACScan cytometer (Becton Dickinson Immunocytometry Systems, Palo Alto, CA, USA) following the methodology described in Lopes et al. [30]. Previously, yeast cells had been grown in GPY during 24 h until stationary phase. DNA content values were scored on the basis of fluorescence intensity compared with haploid (S288c) and diploid (FY1679) reference strains. The value reported for each strain was the result of three independent measures. The results were tested by one-way ANOVA and a Tukey HSD test (α = 0.05, n = 2).

Active dry yeast (ADY) production. Industrial cultivation and drying were performed according to the Laboratory of Research and Development standard protocols (Lallemand Inc. protocols; Lallemand S.A.S., Montreal, Canada) (not provided). A rehydration step, previous to the use of these yeasts in winemaking, is needed. Comparative genome hybridization analysis. Array competitive genomic hybridization (aCGH) was performed using a hybrid clone before and after processing as ADY by following the methodology described in Peris et al. [40]. Experiments were carried out in duplicate and the Cy5-dCTP and Cy3-dCTP dye-swap assays were done to reduce the dye-specific bias. Microarray scanning was carried out using a GenePix Personal 4100A scanner (Axon Instruments/Molecular Devices, USA). Microarray images and raw data were produced using the GenePix Pro 6.1 software (Axon Instruments/Molecular Devices) and the background was subtracted by applying the local feature background median option. M-A plots (M = log2 ratios; A = log2 of the product of intensities) were represented in order to evaluate if the ratio data were intensity-dependent. The normalization process and filtering were done with Acuity 4.0 (Axon Instruments/Molecular Devices Corp.). Raw data were normalized by the ratio-based option. Features with artifacts or flagged as bad were removed from the analysis. Replicates were averaged after filtering. The data from this study are available from GEO [http://www. ncbi.nlm.nih.gov/geo/]; the accession number is GSE46192. Natural must fermentation, HPLC analysis of wines and kinetic analysis. The must employed was Albariño. Fermentable sugars were measured using the HPLC (see below), that gave a value of 213.96 g/l. Yeast assimilable nitrogen was determined by the ammonia assay kit (Boehringer Mannheim,Mannehim, Germany), for the inorganic nitrogen (40% of the total nitrogen amount) and nitrogen content was adjusted to a total of

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220 mg/l by addition of a nitrogen supplement consisting in NH4Cl. Prior to the fermentation, dimethyl dicarbamate (DMDC) at 1 ml/l was added for sterilization purposes. Fermentations were carried out in 100-ml bottles containing 80 ml of Albariño must. Must was inoculated independently with the different yeast strains to reach an initial population of 2 × 106 CFU/ml, and maintained at 22°C. Flasks were closed with Müller valves and monitored by weight loss until a constant weight was obtained. Immediately after the end of fermentation, yeast cells were removed by centrifugation and the supernatants analyzed immediately or stored at –20ºC until use. Each fermentation method was carried out by duplicate. Supernatants were analyzed by HPLC in order to determine the amounts of residual sugars (glucose and fructose), glycerol, and ethanol. A Thermo chromatograph (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a refraction index detector was used. The column was a HyperREZTM XP Carbohydrate H+ 8m (Thermo Fisher Scientific) which was protected by a HyperREZTM XP Carbohydrate Guard (Thermo Fisher Scientific). The conditions used in the analysis were as follows: eluent, 1.5 mM H2SO4; flux, 0.6 ml/min; and oven temperature, 50°C. Samples were diluted 5-fold, filtered through a 0.22-m nylon filter (Symta, Madrid, España) and injected by duplicate. Before curve fitting, weight loss data were corrected to % of consumed sugar according to the formula: C={(m*[S-R])/(mf*S)}*100 were C is the % of sugar consumed at each sample time, m is the weight loss value at this sampling time, S is the sugar concentration in the must at the beginning of experiment (g/l), R is the final sugar concentration in the fermented must (residual sugar, g/l) and mf is the total weight loss value at the end of the fermentation (g). Curve fitting was carried out using the reparametrized Gompertz equation proposed by Zwietering et al. [53]: y = D* exp{−exp[((μmax *e)/D)*(λ – t)+ 1]} where y is the % of consumed sugar; D is the maximum sugar consumption value reached (the asymptotic maximum, %); max is the maximum sugar consumption rate (h–1), and  is the lag phase period during which sugar consumption was not observed (h). Data were fitted using the nonlinear regression module of Statistica 7.0 software package (StatSoft, Tulsa, OK, USA), minimizing the sum of squares of the difference between experimental data and the fitted model. Fit adequacy was checked by the proportion of variance explained by the model (R2) respect to experimental data. Kinetic parameters and HPLC data were analyzed using Statistica 7.0 software package (StatSoft) by one way ANOVA and Tukey test for means comparison.

Results Significant differences were observed not only in the stabilization process of the intraspecific (Saccharomyces cerevisiae  S. cerevisiae) and interspecific (S. cerevisiae  S. kudriavzevii) hybrids, but also in the stabilization of those strains obtained by different procedures (rare-mating and spore-tospore mating). Stabilization of intraspecific hybrids. Different  elements and RAPD–PCR patterns were detected in the colo-

PÉREZ-TRAVÉS ET AL.

nies isolated during the successive fermentations inoculated with each particular hybrid strain. Table 2 provides the frequencies in which each particular combined  elementsRAPD–PCR-mtDNA RFLP pattern appeared. The genetic variability observed during the stabilization of hybrids generated by rare-mating (R2 and R8) was higher than that obtained by spore-to-spore mating (S2 and S7) for both nuclear and mitochondrial genomes. Six new  elements patterns were found among the colonies derived from hybrid R2 (patterns I to VI), and eight patterns were obtained among the colonies derived from R8 (patterns I to VIII). Apart from the aforementioned patterns, the  elements patterns exhibited by the original unstable hybrids R2 and R8 were recovered in the derived colonies isolated from all the successive fermentation steps (Table 2). Low variations were detected among derived colonies by the RAPD–PCR method using primer R3. Only one different pattern was observed in one colony obtained in fermentation step 4 of hybrid R2 (named pattern a) and two (named patterns a and b) were obtained in the colonies derived from hybrid R8 after fermentation steps 4 and 5 (Table 2). No variations in RAPD–PCR patterns were detected among the colonies isolated during the five successive fermentation steps inoculated with hybrids S2 and S7 generated by spore-to-spore mating. Only two δ elements patterns, which differed from that present in the original hybrid, were detected during the stabilization of S2 (patterns I and II) (Table 2). Variations in the mtDNA-RFLP patterns were detected only during the stabilization of hybrid R8 obtained after raremating. Five different mtDNA-RFLP patterns were identified during the process. Individual colonies (clones), representative of each hybrid and molecular pattern detected after the complete set of consecutive fermentations, were used to inoculate fresh synthetic must in order to confirm their genetic stability. Of those colonies showing a same molecular pattern, only those from the last fermentation steps were evaluated individually (i.e., the R2ooo “original pattern” was taken from the fifth fermentation, R2Ioo, R2IIIao, R2IVoo and R2Voo from the fourth, and R2IIoo, R2IIIoo and R2VIoo from the fifth). We followed the same methodology used during the stabilization process: after fermentation, ten colonies were isolated and molecularly characterized. As a result of this evaluation, most clones conserved the same molecular patterns as before, except for clones R2Voo, R8ooA, R8ooB, R8ooC, R8IoB and R8IoD and the original R2 and R8 (data not shown). In order to evaluate if the changes detected between the molecular markers were also coincident with the changes in

– – – – – –

–

δ–14 (o)

R–10 (o)

–

R–9 (o)

–

δ–10 (o)

–

R–7 (o)

–

δ–4 (o)

R–8 (o)

–

δ–5 (o)

Originalc

Sc1(o)

–

Sc2(o)

–

Sc1(o)

–

Sc2(o)

100

–

100

–

100

–

100

–

1st step

100

–

100

–

2nd step

100

–

100

–

3rd step

100

–

100

–

III

–

III

–

4th step

100

–

VIII

VII

–

III

–

5th step

100

–

100

–

Hybrid names R2, R8, S2 and S7 correspond to intraspecific hybrids in Pérez–Través et al. [39]. b δ: patterns obtained by δ elements characterization (identified with roman numbers, patterns exhibited by the original hybrids were designed as “o”); R3: patterns obtained by RAPD–PCR with primer R3 (identified with lowercase letters, patterns exhibited by the original hybrids was designed as “o”). mt: patterns obtained by mtDNA–RFLP analysis (identified with capital letters, patterns exhibited by the original hybrids were designed as “o”). %: percentage of detection of a particular combination of δ elements and RAPD–PCR patterns after a particular fermentation step. c Molecular patterns characterized by Pérez-Través et al. [39]. These patterns were identified as “original patterns (o)” in this work.

Spore to spore mating

Rare-mating

Hybrid

Hybridization method

Molecular patterns and frequency (%)b

Table 2. Molecular characterization of yeast colonies after successive fermentation steps of intraspecific hybrids and frequency

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Fig. 1. Changes in DNA content of hybrid cultures during stabilization process of intraspecific (spore-to-spore hybrids S2 and S7, and rare-mating hybrids R2 and R8) and interspecific (spore-to-spore hybrids S5 and S8, and rare-mating hybrids R1 and R3) hybrids. Circles: spore-to-spore hybrids S2 (intraspecific) and S5 (interspecific). Triangles: spore-to-spore hybrids S7 (intraspecific) and S8 (interspecific). Squares: Rare-mating hybrids. Diamonds: stable rare-mating hybrids. Solid line indicate the ploidy value showed by the parental Sc1. Dotted line indicate the ploidy value showed by the parental Sc2 (intraspecific hybrids stabilization) and parent Sk (interspecific hybrids stabilization). Filled symbols indicate cultures with the same molecular pattern found in the original hybrid inoculated in the first fermentation step. Empty symbols indicate cultures with molecular patterns different from the original. Symbols with different letters among cultures derived from a same original hybrid, indicate statistically significant differences (ANOVA and HSD Tukey test, α = 0.05).

total DNA content, the clones having each different molecular pattern were subjected to measuring DNA content by flow cytometry (see supplementary Table 1, ST1; it can be requested to authors). Figure 1 shows the evolution in the total DNA content values obtained for all analyzed clones derived from each original hybrid strain during the stabilization process. After this analysis, we observed that all the clones obtained after the consecutive fermentation steps of the spore-to-sporegenerated hybrids conserved the same ploidy values found in original hybrids S2 and S7, including those showing different δ elements patterns (Fig. 1, ST1). Among the clones derived from rare-mating-generated hybrids R2 and R8, the DNA content values varied from 5n (n being the DNA content of a haploid laboratory strain) in the original inoculated hybrids to approximately 2.5n in the clones (Fig. 1, ST1). Most of the clones derived from original hybrid R2 (obtained from fermentations steps 3, 4 and 5) had significantly different DNA content values from the value obtained in the original hybrid (close to 2.5n). An exception was observed for clone R2Ioo and clone R2Voo from fermentation

steps 1 and 4, respectively, whose values came close to 5n (Fig. 1, ST1). Finally, all the clones isolated from the different fermentation steps, but showing the original molecular pattern, also conserved the same ploidy value of around 5n (Fig. 1, ST1). Three different situations were observed for the ploidy values shown by the clones derived from original hybrid R8. All the clones having an original molecular pattern in the nuclear genome (R8ooo, R8ooA, R8ooB and R8ooC) conserved high ploidy values ranging from 4.5n to 5n (Fig. 1, ST1). The DNA content of clones R8IoB and R8IoD, bearing  elements pattern I, which emerged in fermentation step 1, was near 3.5n. The remaining clones, isolated from fermentations 4 and 5, exhibited ploidy values which came close to 2.5n (Fig. 1, ST1). The DNA content analysis carried out in the colonies obtained after individual clone fermentation revealed high ploidy variability among the colonies derived from the clones with high DNA contents (R2ooo, R2Voo, R8ooo, R8ooA, R8ooB, R8ooC, R8IoB and R8IoD). In their δ pattern, R8IoB and R8IoD also changed. The clones whose DNA content came close to 2.5n maintained

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Fig. 2. Analysis of total DNA content (as fluorescence intensity) carried out by flow cytometry in the stable hybrid R2IIIo (A) and in the instable hybrid R2Vo (B) before (left) and after (right) individual inoculation of synthetic must. Shadowed areas indicate the total DNA content of the cultures before inoculation. Lines in color indicate fluorescence intensity of colonies recovered after individual fermentations. Arrows indicate picks considered for DNA content determinations.

the same values after individual fermentation. An example about the variation or the maintenance in the ploidy levels after individual clone fermentation is shown in Fig. 2.

process (Fig. 1, Table 4). After inoculating fresh media with individual clones, no changes were observed in molecular patterns and ploidy levels (data not shown).

Stabilization of interspecific hybrids. For interspecific hybrid R3 (generated by rare-mating), and S5 and S8 (generated by spore-to-spore mating), all the clones obtained during the five fermentation steps showed the same molecular pattern at both the nuclear and mitochondrial levels, as detected in the original hybrid strains (data not shown). The stabilization process of hybrid R1 evidenced no variation in genomic DNA patterns, event though new mtDNA patterns appeared, particularly in early process stages (fermentation step1 and 2; Table 3). The emergence of new mtDNA patterns could indicate that rearrangements have occurred. One of the new patterns was present until the end of the stabilization assay. In all cases, significantly different ploidy values were observed between the originally inoculated hybrid strains and all the clones recovered after each fermentation step, irrespective of the hybridization method employed for hybrid generation (rare-mating or spore-to-spore mating). After the first step, clones maintained the same ploidy value until the end of the

Stability evaluation after active dry yeast (ADY) production. A decision was made to evaluate if clones, obtained by the methodology proposed in this work, maintained their genetic stability after undergoing the ADY production process (Lallemand Inc. protocols). For this purpose, stable intraspecific hybrid clones were selected to undergo the ADY preparation process. These clones were selected because intraspecific hybrids were more variable during the stabilization process than interspecific ones. Furthermore, our approach based on employing an S. cerevisiaebased microarray is not useful for detecting genes from S. kudriavzevii, which greatly diverge with S. cerevisiae. Stabilized clones R2IVoo and R8IIaE were used for ADY production under the habitual conditions (Lallemand Inc. protocols). After the process, the produced ADY samples were rehydrated and seeded in the complete medium. Ten colonies of each sample, obtained after incubation, were evaluated by the same genetic markers and ploidy previously employed

– – – –

–

δ–9 (o)

δ–8 (o)

S8 R–12 (o)

R–11 (o)

R–4 (o)

–

δ–4 (o)

–

R3 R–2 (o)

δ–4 (o)

–

Sc1(o)

Sk (o)

Sc1(o)

–

100

–

100

–

Sk (o)

Originalc δ

–

1st step

100

–

2nd step

100

–

3rd step

Molecular patterns and frequency (%)b

100

–

4th step

100

–

100

–

5th step

100

–

100

–

Original hybrid

4.8 ± 0,1b

3.4 ± 0,1b

3.2 ± 0,2b

–

ooo

ooo ooo

2.6 ± 0.2a

ooo

ooF

ooE

–

2.7 ± 0,1a

2.4 ± 0.2a

3.5 ± 0.1a

ooo

2.8 ± 0.2a

2.5 ± 0.3a

3.4 ± 0.3a

– a

ooo

–

– 2.5 ± 0.2a 2.6 ± 0.2

2.52 ± 0.22

–

2.65 ± 0.13a

DNA content&

–

ooC

–

ooA

Pattern#

3rd step

–

2.6 ± 0.1

–

ooC

–

2.7 ± 0.1a

DNA content&

–

ooA

Pattern#

2.5 ± 0.2a

3.4 ± 0.1a

–

– ooo

2.8 ± 0.3 2.9 ± 0.2 a

ooC ooD

3.1 ± 0.1 b

ooB a

2.8 ± 0.2a

DNA content&

ooA

Pattern#

2nd step

ooo

–

ooA

Pattern#

2.8 ± 0,3a

2.5 ± 0.1a

3.5 ± 0.1a

–

2.50± 0.09a

DNA content&

4th step

ooo

–

ooA

Pattern#

2.7 ± 0.2a

2.4 ± 0.2a

3.6 ± 0.1a

–

2.64 ± 0.03a

DNA content&

5th step

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Hybrid names R1, R3, S5 and S8 correspond to interspecific hybrids in Pérez-Través et al. [39]. Values expressed as mean ± standard deviation. Values not sharing the same superscript letter within the column are significantly different (ANOVA and Tukey HSD test, α = 0.05, n=2). # Molecular patterns obtained by combination of interdelta, R3 and mtDNA-RFLP profiles. All colonies were considered as stable when both molecular patterns and DNA content did not change after individual colony fermentation.

3.2 ± 0.2b

DNA content

1st step

Table 4. DNA content of hybrids showing different combined molecular patterns during the whole process of interspecific hybrids stabilization

Hybrid names R1, R3, S5 and S8 correspond to interspecific hybrids in Pérez–Través et al. [39]. δ: patterns obtained by δ elements characterization (identified with roman numbers, patterns exhibited by the original hybrids were designed as “o”); R3: patterns obtained by RAPD–PCR with primer R3 (identified with lowercase letters, patterns exhibited by the original hybrids was designed as “o”). mt: patterns obtained by mtDNA–RFLP analysis (identified with capital letters, patterns exhibited by the original hybrids were designed as “o”). %: percentage of detection of a particular combination of δ elements and RAPD–PCR patterns after a particular fermentation step. c Molecular patterns characterized by Pérez-Través et al. [39]. These patterns were identified as “original patterns (o)” in this work.

Spore-to-spore mating

Rare–mating

Hybrida

Hybridization method

Table 3. Molecular characterization of yeast colonies after successive fermentation steps of interspecific hybrids and frequency

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Table 5. Main kinetic parameters of the fermentations carried out with both parental and hybrid strains on Albariño must at 22°C and chemical analysis of the final fermented products Chemical parametersa

Kinetic parametersa Strain

K (h–1)b

l (h)

t95(h)c

Glucose (g/l)d

Fructose (g/l)

Glycerol (g/l)

Ethanol (% v/v)

R2IVo

1.57 ± 0.02

19.38 ± 0.92

125.20 ± 1.20

bdl

1.09 ± 0.11

5.35 ± 0.06

11.81 ± 0.01

R2IVo LSA

1.54 ± 0.01

20.39 ± 0.50

125.97 ± 0.95

bdl

0.89 ± 0.02

5.38 ± 0.03

11.79 ± 0.03

Values expressed as mean ± standard deviation. Values not sharing the same superscript letter within the column are significantly different (ANOVA and Tukey HSD test, α = 0.05, n = 2). b K: kinetic constant. c t95: time necessary to consume 95% of fermented sugars. d bdl: value below detection limit (0.05 g/l). a

during stabilization. No changes were observed in the evaluated parameters of the obtained colonies in relation to the clone R2IVoo before the dryness process, otherwise their happened for the clone R8IIaE, which changed in its δ profile (data not shown). Additionally, in order to ensure that no changes in genomic constitution—including variation in genes copy number— occurred during ADY production for R2IVoo clone, the rehydrated culture was compared at a single gene resolution with the same strain without being subjected to the dryness process. For this comparison, genomic DNA isolated from the clone before dryness and labelled with one fluorescent dye was mixed with the DNA from the colonies obtained after ADY production and rehydration, which was labelled with a different dye. This mixture was then co-hybridized in a S. cerevisiae DNA microarray (see Materials and methods). Differences in the log2 of the Cy5/Cy3 signal ratio obtained for each open reading frame (ORF) probably indicated variations in the relative copy number of S. cerevisiae genes present in the hybrid strain before and after the dryness process. Log2 ratios close to zero for a particular ORF indicated the presence of the same number of DNA copies in both genomes, while higher or lower log2 ratios than zero might indicate more or less copies, or even depleted genes (ORF deletions). Our results do not evidenced changes in the gene copy numbers between the two analyzed genomes, suggesting that no changes in the DNA composition of clone R2IVoo had occurred in the industrial dry yeast generation process (data not shown). Finally we decided to carry out a fermentation in natural must with the hybrid clone before and after ADY production. No differences were found in residual sugars content, glycerol and ethanol production, neither in parameters analysis (latency, maximum fermentation rate and time necessary to consume 95% of fermentable sugars) (Table 5).

Discussion Interspecific hybrids have been isolated from different fermented beverages, including wine, cider and beer [45,48]. Even one of the most popular beverages, lager beer, is prepared by hybrid yeast S. pastorianus containing both the S. cerevisiae and S. eubayanus subgenomes [28]. In most cases, hybrids acquire interesting combinations of physiological features from parental strains, and prove to be promising tools for specific technological uses. For this reason, many artificial hybrid yeasts have been constructed in recent decades to improve different industrial processes such as winemaking [6,12,39], brewing [47] and bakery [25,47], and also for basic studies [13,34]. However, only a few works mention and evaluate the necessary genetic stabilization process occurring immediately after hybridization [2,6,26,39], an important aspect when the strains are going to be used in industrial processes, where the product homogeneity is desired because starters ensures the production of consistent products in successive vintages [42]. Genome reduction and rearrangements occurring during the stabilization of newly formed hybrids have been reported, and these processes seemed to be different in unstressed or in a salt-stressed media [19,20]. These phenomena might lead to loss of industrially important traits in hybrids, and could be avoided if a selective pressure, mimicking the desired industrial process, were applied during the stabilization. For this reason, a major aspect in the hybrids study is the careful selection of stabilization conditions. In a previous work carried out in our laboratory [39], intraspecific S. cerevisiae  S. cerevisiae and interspecific S. cerevisiae  S. kudriavzevii hybrids were successfully obtained by means of different hybridization methods, which

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included protoplast fusion, rare-mating and spore-to-spore mating. Here we present the changes observed in some interspecific and intraspecific hybrid strains generated in that previous work throughout the genetic stabilization process carried out in selective media (in this case, by successive fermentation steps in synthetic must). We compared the stabilization process in the inter- and intraspecific hybrids showing high ploidy values (resulting from the rare-mating of two parental strains close to diploidy) and the stabilization of hybrids close to diploidy (most hybrids resulted from spore-to-spore mating). Flow cytometry identified large-scale (ploidy level) changes in genome size throughout the stabilization process in most hybrids. This reduction was significant, particularly for the hybrids generated by rare-mating, which originally had two diploid parental sets of chromosomes. Genome reduction in intraspecific rare-mating hybrids R2 and R8 seemed to occur drastically in fermentation steps 3 and 4 (Fig. 1), although an intermediate reduction occurred in hybrid R8 in fermentation step 1. According to the results obtained after fermentation with the individual R2 and R8 derived colonies, stable clones corresponded only to those having the same ploidy values found in parental strains Sc1 (2.7n) and Sc2 (2.3n). The genome reduction in interspecific hybrids was faster than that observed for intraspecific ones. This reduction occurred in fermentation step1 (Fig. 1). All the colonies recovered in fermentation steps 2 to 5 had the same ploidy values. The ploidy values at which hybrids became stable were similar to the parental strains ploidy (in S5, S8 and R1) or to a higher one (R3). A similar genome reduction process has been also evidenced by Antunovics et al. [2] after the stabilization of S. cerevisiae  S. uvarum hybrids by successive sporulation events, and also by Marinoni et al. [32] after interspecific hybridization by mass-mating. In experimental evolution studies, Gerstein et al. [19] observed that the DNA content of triploid and tetraploid cultures of S. cerevisiae diminished. This reduction occurs in the first generations and all the clones show a tendency to stabilize, with ploidy values close to 2n (historical ploidy values, the ploidy shown by the original strain). The authors also observed that cultures maintain a higher ploidy under stress conditions. Chromosomal instability in artificial polyploid S. cerevisiae strains has been previously observed by several authors [1,19,50], together with high mutation and recombination levels. In this work, apart from a reduced ploidy, variation in nuclear (evidenced in new  elements and RAPD–PCR profiles) and mitochondrial (evidenced in new mtDNA-RFLP patterns) genomes was observed during the stabilization process. All

PÉREZ-TRAVÉS ET AL.

these changes resulted in a large number of clones derived from an individual hybrid. Thus, the stabilization process generated a genetic variability among the recovered colonies. These new molecular patterns were observed mainly during the stabilization of the intraspecific hybrids obtained by raremating, which evidenced the existence of extensive genetic rearrangements among genetically similar genomes. This phenomenon was not observed for interspecific hybrids, irrespective of the hybridization method used for their generation; only hybrid R1 showed mitochondrial genome variability after fermentation step 1, but only one pattern consecutively appeared until the end of the process (R1ooA). Contrarily to our results, Bellon et al. [6] have not detected changes in DNA molecular patterns in recently generated interspecific hybrid strains after 50 generations in the model medium and grape juice. However, those authors reported neither changes in ploidy values nor having monitoring these changes throughout the stabilization process. To sum up, different situations emerged throughout the process after analyzing hybrids: (i) stabilization by gradual loss of genetic material with no detectable changes in nuclear or mitochondrial DNA patterns (interspecific hybrids R3, S5 and S8); (ii) stabilization after nuclear genetic rearrangements and ploidy reduction until historical values in parental strains (rare-mating intraspecific hybrids) with (R8) or without (R2) mitochondrial genome changes; (iii) stabilization after rapid loss of genetic material with no changes in genomic markers, but in the mtDNA-RFLP patterns (interspecific hybrid R1). From our results, we could conclude that both nuclear and mitochondrial genomes could undergo changes during the stabilization process of newly generated intra- and interspecific hybrids in the genus Saccharomyces. Intraspecific hybrids seemed to require more generations to produce genetically stable cells, while interspecific hybrids underwent a faster stabilization process and were active mainly in early stages. ADY production is an essential step to prepare a wine yeast starter culture, during which yeast is affected by a number of different stresses [3,4,14,21,36]. As changes in the ploidy level, genes copy number, and chromosomal rearrangements have been observed in Saccharomyces strains subjected to different stress [15,19,37] or culture conditions [10,16,24], we evaluated the genomic stability of two representative hybrids strains by molecular markers and ploidy analyses before and after ADY production. Two clones were selected, as representative of the set of hybrids obtained from intraspecific rare-mating, because the stabilization of such hybrids shows the highest variability in ploidy and molecular patterns. We observed no changes in DNA content of both

STABILIZATION PROCESS IN SACCHAROMYCES

strains, but molecular patterns changed in one of them (R8IIaE hybrid strain). We observed no large amplification or deletion in the genome of R2IVoo clone after the process, and no differences were found when we compared, in fermentation, the hybrid before and after ADY production. These results evidenced the success of both the stabilization protocol and the selection of stable hybrids proposed in this work. Our results suggest that molecular markers such as  elements and mtDNA-RFLP patterns, as well as ploidy evaluation, would allow the quick assessment of the genotypic stability of recently generated inter- and intraspecific Saccharomyces hybrid strains, and that the evaluation of these parameters should be done before and after ADY production. According to our results, and by considering that a stable hybrid must maintain the same molecular pattern and the same ploidy level during successive cell divisions, we found that fermentation steps 3 and 5 (30 and 50 generations) sufficed to obtain genetically stable interspecific and intraspecific hybrids, respectively, regardless of the hybridization methodology used for their generation.

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10.

11.

12.

13.

14. Acknowledgements. This work has been supported by grants AGL2009-12673-CO2-01 and AGL2009-12673-CO2-02 and AGL201239937-C02-01 and AGL2012-39937-C02-02 from the Spanish Government to A. Querol and E. Barrio, respectively. L. P-T. and C. L. wish to acknowledge the CSIC and the Spanish Ministry of Education and Science for an I3P fellowship and a postdoctoral contract, respectively. The authors acknowledge the Laboratory of Research and Development (Lallemand S.A.S.) for the industrial production and drying of hybrid yeast.

15.

16.

17. Competing interests. None declared. 18. 19.

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39. Pérez-Través L, Lopes CA, Barrio E, Querol A (2012) Evaluation of different genetic procedures for the generation of artificial hybrids in Saccharomyces genus for winemaking. Int J Food Microbiol 156:102-111 40. Peris D, Lopes C, Belloch C, Querol A, Barrio E (2012) Comparative genomics among Saccharomyces cerevisiae  Saccharomyces kudriavzevii natural hybrid strains isolated from wine and beer reveals different origins. BMC Genomics 13:407 41. Petrov DA (2001) Evolution of genome size: new approaches to an old problem. Trends Genet 17:23-28 42. Pretorius IS (2000) Tailoring wine yeast for the new millennium: novel approaches to the ancient art of winemaking. Yeast 16:675-729 43. Querol A, Barrio E, Huerta T, Ramón D (1992) Molecular monitoring of wine fermentations conducted by active dry yeast strains. Appl Environ Microbiol 58:2948-2953 44. Querol A, Fernández-Espinar MT, del Olmo M, Barrio E (2003) Adaptative evolution of wine yeast. Int J Food Microbiol 86:3-10 45. Querol A, Bond U (2009) The complex and dynamic genomes of industrial yeasts. FEMS Microbiol Lett 293:1-10 46. Rossignol T, Dulau L, Julien A, Blondin B (2003) Genome-wide monitoring of wine yeast gene expression during alcoholic fermentation. Yeast 20:1369-1385 47. Sato M, Kishimoto M, Watari J, Takashio M (2002) Breeding of brewer’s yeast by hybridization between a top-fermenting yeast Saccharomyces cerevisiae and a cryophilic yeast Saccharomyces bayanus. J Biosci Bioeng 93:509-511 48. Sipiczki M (2008) Interspecies hybridization and recombination in Saccharomyces wine yeasts. FEMS Yeast Res 8:996-1007 49. Song K, Lu P, Tang K, Osborn TC (1995) Rapid genome change in synthetic polyploids of Brassica and its implications for polyploid evolution. Proc Natl Acad Sci USA 92:7719-7723 50. Storchova Z, Breneman A, Cande J, Dunn J, Burbank K, O’Toole E, Pellman D (2006) Genome-wide genetic analysis of polyploidy in yeast. Nature 443:541-547 51. Weiss RL, Kukora JR, Adams J (1975) The relationship between enzyme activity, cell geometry, and fitness in Saccharomyces cerevisiae. Proc Natll Acad Sci USA 72:794-798 52. Wendel JF (2000) Genome evolution in polyploids. Plant Mol Biol 42:225-249 53. Zwietering MH, Jongerburger I, Rombouts FM, Van’t Riet K (1990) Modeling of the bacteria growth curve. Appl Environ Microbiol 56:1875-1881

RESEARCH ARTICLE IÄã ÙÄ ã®ÊÄ ½ M® ÙÊ ®Ê½Ê¦ù (2014) 17:225-233 doi:10.2436/20.1501.01.225. ISSN (print): 1139-6709. e-ISSN: 1618-1095

www.im.microbios.org

Comparative microbial ecology of the water column of an extreme acidic pit lake, Nuestra Señora del Carmen, and the Río Tinto basin (Iberian Pyrite Belt) Elena González-Toril1*, Esther Santofimia2, Enrique López-Pamo2, Antonio García-Moyano3, Ángeles Aguilera1, Ricardo Amils1,4 Center for Astrobiology (INTA-CSIC), Torrejón de Ardoz, Spain. 2Geological Survey of Spain, Madrid, Spain. 3 Institute of Biology, University of Bergen, Bergen, Norway. 4Center for Molecular Biology Severo Ochoa, Autonomous University of Madrid-CSIC, Cantoblanco, Spain

Received 10 September 2014 · 15 December 2014

Summary. The Iberian Pyrite Belt, located in Southwestern Spain, represents one of the world’s largest accumulations of mine wastes and acid mine drainages. This study reports the comparative microbial ecology of the water column of Nuestra Señora del Carmen acid pit lake with the extreme acidic Río Tinto basin. The canonical correspondence analysis identified members of the Leptospirillum, Acidiphilium, Metallibacterium, Acidithiobacillus, Ferrimicrobium and Acidisphaera genera as the most representative microorganisms of both ecosystems. The presence of archaeal members is scarce in both systems. Only sequences clustering with the Thermoplasmata have been retrieved in the bottom layer of Nuestra Señora del Carmen and one station of Río Tinto. Although the photosynthetically active radiation values measured in this lake upper layer were low, they were sufficient to activate photosynthesis in acidophilic microorganisms. All identified photosynthetic microorganisms in Nuestra Señora del Carmen (members of the Chlamydomonas, Zygnemopsis and Klebsormidium genera) are major members of the photosynthetic eukaryotic community characterized in Río Tinto basin. This study demonstrates a close relationship between the microbial diversity of Nuestra Señora del Carmen pit lake and the diversity detected in the Río Tinto basin, which underlain the influence of the shared mineral substrates in the microbial ecology of these ecosystems. [Int Microbiol 2014; 17(4):225233] Keywords: iron cycle · acidic pit lakes · acidophilic microorganisms · Río Tinto · Iberian Pyrite Belt

Introduction Extreme acidic environments are rather scarce and mainly associated with volcanic and metal mining activities [16,24]. The peculiar ecology and physiology of acidophilic microorCorresponding author: E. González-Toril Centro de Astrobiología (INTA-CSIC) Carretera de Ajalvir, Km 4 28850 Torrejón de Ardoz, Spain. Tel. +34-915206412 Fax +34-915201074 E-mail: gonzalezte@cab.inta-csic.es

ganisms have interested microbiologists since their discovery [12] because the extreme acidic conditions in which they develop are the product of microbial metabolism. In mining areas oxidation of metal sulfides by acidophilic chemolithotrophic microorganisms leads to the formation of highly acidic, metal-laden waters [34] known as acid mine drainage (AMD). The Iberian Pyrite Belt (IPB) represents one of the world’s largest accumulations of mine wastes and AMDs [40]. Currently, there are more than 25 pit lakes between the provinces of Huelva and Seville (SW Spain) [31]. IPB pit lakes are nor-

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Fig. 1. Río Tinto basin and Nuestra Señora del Carmen (NSC) pit lake locations in the IPB.

mally acidic and present a wide range of sizes, depths, and physico-chemical characteristics. Their low pH and high concentrations of toxic heavy metals have limited the biological diversity of these lakes [42]. Nuestra Señora del Carmen (NSC) is a 110-m long and 80-m wide pit lake from the IPB, with a depth of 34 m and a volume of ca. 79,500 m3 [44], an acidic pH (between 2.1 and 2.5), and high concentrations of sulfate and toxic heavy metals (Fig. 1 and Table 1). The geomicrobiology of the IPB is mainly known from studies of the Río Tinto basin [2,3,4,19,20,21,33,47] or Río Odiel basin [30]. However, our knowledge of microbial diversity in pit lakes from this area is far from complete since very few studies have been reported [17,42,48]. In addition, most studies of the geomicrobiology of the IPB have focused on the biological oxidation of iron and sulfur, due to their importance in biohydrometallurgical processes, whereas little attention has been paid to the biological reduction of these elements under the anoxic conditions existing at the bottom of these lakes. To improve the efficiency of biohydrometallurgical operations, especially those in which control of environ-

mental conditions is difficult such as heap leaching, it is necessary to gain information on the microbial ecology operating in these ecosystems. In this work we compared the microbial ecology of the water column of an extreme acidic pit lake, NSC, with the diversity detected along the course of Río Tinto, a well-established model of AMD of the Iberian Pyrite Belt (Fig. 1).

Materials and methods Sampling. Field measurements and water and sediments sampling in the Río Tinto basin were carried out in May 2005 [20] and those in the NSC pit lake were carried out in May 2009 [42]. The Río Tinto environmental conditions were those described by García-Moyano et al. [20]. Environmental conditions for the water column of the pit lake were measured with a Hydrolab Datasonde S5 probe (Hach, USA). Water samples were collected from different depths using an opaque, 2.2-l PVC bottle (Beta Plus Wildlife Supply). All samples were filtered on site with 0.45-μm membrane filters from Millipore, stored in 125-ml polyethylene bottles, acidified with HNO3 (1 ml), and kept at 4ºC during transport. One-liter water samples for DNA extraction were collected and immediately filtered in situ through a Millex-GS Millipore filter (pore size, 0.22 μm; diameter, 50 mm). Filters were stored at –20°C

MICROBIAL ECOLOGY IN RÍO TINTO

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227

Table 1. Water chemical composition and physicochemical parameters measured at Río Tinto and Nuestra Señora del Carmen. The table shows the average values for pH, redox potential (ORP), electric conductivity (EC), dissolved oxygen (DO) and major elements of Río Tinto basin and NSC pit lake. (b.d.l.): below detection limit of the technique. Nuestra Señora del Carmen

Río Tinto

NSC 0.1 m

NSC 5m

NSC 15 m

NSC 25 m

RT1

RT2

RT3

RT5

RT6

RT8

RT9

RT10

RT11

2.4

2.1

2.0

3.7

2.7

2.6

2.8

2.7

3.2

ORP (mV)

534

375

465

329

637

413

490

387

530

519

EC (mS/Cm)

8.11

9.09

9.10

30.50

8.68

19.40

11.10

5.16

6.55

7.80

6.32

2.53

DO (mg/l)

8.12

0.09

3.70

6.10

5.00

5.32

7.40

7.82

4.00

6.40

6.70

SO42– (g/l)

6.36

6.40

6.52

6.19

15.04

1.65

0.79

2.77

0.79

1.79

1.69

1.39

0.36

Fe(II) (mg/l)

0.90

338

353

12837

176

1286

1527

431

1505

572

768

164

Fe(III) (mg/l)

596

368

388

350

2369

750

4194

891

306

273

867

Cu (mg/l)

27.4

28.6

29.6

28.8

20.4

b.d.l.

6.7

2.1

8.3

104.8

93.5

Co (mg/l)

0.65

0.67

164.30

6.70

8.01

13.81

2.22

8.80

7.80

6.00

0.70

Zn (mg/l)

8.20

8.99

8.90

8.84

20.40

31.51

29.70

28.00

43.32

111.41

118.63

103.71

20.40

Mn (mg/l)

77.8

82.2

84.1

83.5

49.8

179.8

151.2

143.8

30.6

55.8

42.0

34.4

7.7

Ni (mg/l)

0.78

0.77

0.76

0.74

11.31

1.51

3.31

1.42

0.41

1.00

1.81

1.30

0.30

until further processing. Sediment samples were obtained as described in García-Moyano et al. [20]. Sediment samples were collected at 1 cm deep. Samples for microscopic observation were collected in sterile tubes, fixed with 2% of formaldehyde and kept at 4°C until further analysis. Analytical procedures. Water samples were analyzed by atomic absorption spectrometry (AAS, Varian SpectrAA 220FS), inductively coupled plasma-atomic emission spectrometry (ICP-AES, Varian Vista MPX), and inductively coupled plasma-mass-spectrometry (ICP-MS, Leco Renaissance). The accuracy of the analytical methods was verified against certified water references (TM-27.3 and TMDA-51.3 from the National Water Research Institute). Fe(II) concentration was measured by reflectance photometry with a Merck RQflex10 reflectometer and Reflectoquant analytical strips. Dissolved organic carbon (DOC) was analyzed by a Shimadzu TOC-V CPH analyzer and nitrogen and phosphorus by absorption UV-Vis spectrophotometry with an Alliance Integral Plus continuous flow autoanalyzer. Microscopy and morphotype identification. Identification of algae and heterotrophic protists was carried out down to the lowest possible taxonomic level by direct microscopic observation of different morphological features based on previous studies of the eukaryotic communities in acidic environments [2–4]. A Zeiss Axioscope 2 microscope equipped with phasecontrast was used in this work. DNA extraction, PCR amplification and sequencing. Fast DNA Spin kit for soil (MP Biomedicals, CA, USA) was used for DNA extraction according to the manufacturer’s instructions. Samples were washed five times with TE buffer (10mM TrisHCl, 1mM EDTA, pH 8.0) prior to DNA extraction. DNA was purified by passage through a GeneClean Turbo column (MP Biomedicals, CA, USA). The 16S and 18S rRNA genes were amplified according to previously described methodologies [2,21] using the universal Bacteria-specific primers 27f and 1492r [29], Archaea-specific primers 21f

and 1492r [1,14], and Eukarya-specific 20f and 1800r primers [2]. PCR amplified genes were purified by GeneClean Turbo Column (MP Biomedicals, CA, USA) and cloned using the Topo TA Cloning Kit (Invitrogen, CA, USA). M13f and M13r primers were used for sequencing. PCR products were directly sequenced using a Big-Dye sequencing kit (Applied Biosystem) according to manufacturer’s instructions. Phylogenetic analysis. Sequences were analyzed using BLAST at the NCBI database (http://ncbi.nlim.nih.gov/BLAST) and added together with the most important BLAST hits to create a database of over 50,000 homologous prokaryotic 16S rRNA primary sequences by using the ARB software package aligning tool [32]. Phylogenetic trees were generated using parsimony and neighbor-joining with a subset of 100 nearly full-length sequences (>1,400 bp). Filters which excluded highly variable positions were used. An ARB-generated distance matrix was used as the input file to DOTUR program [45] and sequences were clustered into operational taxonomic units (OTUs) based on 100% and 97% sequence of similarity. Rarefaction analysis and the Chao1 non-parametric diversity estimator [9] were applied to the clone library in order to estimate how completely the library had been sampled and to extrapolate to total sequence diversity. Community diversity was studied by estimating the similarity between communities based on members and structure [45]. Sequences used in this study can be found in the EMBL sequence database under accession numbers JF737859–JF737929, JF807634–JF807641 and KC619546–KC619624 [19,42]. Multivariate analysis. Data were analyzed using a combination of constrained and unconstrained multivariate statistical methods in order to account for both total variation in the data and variations explainable by environmental data. Of the analyzed elements, we retained 12 environment variables that had no missing values, 8 chemical elements (SO42-, Fe(II), Fe(III), Cu, Co, Zn, Mn and Ni) and pH, redox potential, electric conductivity, and

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dissolved oxygen values (Table 1). Environmental data were transformed using ln(x+0.1) and normalized to zero mean and unit variance. Canonical correspondence analysis (CCA) was used to relate the microbial community data with environmental data. It was conducted using OTUs of 16S rRNA genes acquired at different sampling stations (18 samples from 9 different stations for the river) and 2 depths for the pit lake. Samples from the river were collected from sediments (S) or water column (W) for every station. The analyzed pit lake samples were from the surface (NSC01) and from 15-meter depth (NSC15). The significances of the first CCA axis and of all CCA axes combined were tested using Monte Carlo permutation tests. CCA tests were performed by using the multivariate data analysis software CANOCO 4.5 [7]. The program CANODRAW 4.0 in the Canoco package was used for graphical presentation of ordination results [7].

Results and Discussion Nuestra Señora del Carmen (NSC) was an open cast exploitation abandoned in 1976 [26]. The NSC pit lake is acidic with high concentrations of sulfate and metals (Mg, Fe, Al, Mn and Cu) (Table 1). It is usually a meromictic lake, showing chemical stratification [43,44]. However, at the beginning of some dry winters, a period of mixing and total homogenization could be observed, temporarily transforming it into a holomictic lake. This process ended after intense episodes of rain

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[44]. In May 2009, the pit lake showed a two-layer chemical and thermal stratification: a 2-meter thin upper layer and a denser homogeneous bottom layer (from 3 m to 35 m depth) (Fig 2). High redox potentials values corresponding to oxygen-saturated conditions were measured in the upper layer, where Fe was predominantly oxidized (Table 1). The bottom layers of acidic meromictic lakes are usually anoxic, which favour the microbial reduction of iron, generating low redox potentials [6,8,15,22,36,37,49]. By contrast, during this study the bottom layer of the NSC pit lake was in a slightly reducing condition with similar concentrations of reduced and oxidized iron (Table 1). This peculiar distribution of the iron redox pair has been previously detected in other acidic pit lakes [23,37]. Preliminary studies in the NSC pit lake [43,44] revealed variable Fe(II)/Fe(III) ratios in the bottom layer as a consequence of the lake dynamics. During the mixing period, dissolved oxygen increases in the bottom layers promoting biological iron oxidation processes, thus reducing the Fe(II)/ Fe(III) ratio. When stratification was restored, an increase in the Fe(II)/Fe(III) ratio due to microbial anaerobic respiration was detected [43,44]. Photosynthetically active radiation (PAR) was 4000 mol/s.m2 in the surface and practically zero from 2 meters deep up to the bottom (Fig. 2).

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Fig. 2. Physicochemical variables of the NSC pit lake. Temperature (T), electric conductivity (EC), dissolved oxygen (DO), redox potential (ORP), chlorophyll-a (CHL-a) and photosynthetically active radiation (PAR).

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Fig. 3. Bacterial abundance comparison between NSC pit lake and Río Tinto. Relative abundance of OTUs in bacterial genera (in percent) is shown for each depth (0m and 15m) for the NSC pit lake and for the water column (RT-W) and sediments (RT-S) for the Río Tinto basin.

We took advantage of the recent characterization of the microbial diversity analyzed in two layers (surface and 15 m depth) of the NSC pit lake [42] for its comparison with the diversity reported for different sampling stations along the Río Tinto basin [20]. In the upper layer of the NSC pit lake, almost 80% of the identified sequences belonged to the Alphaproteobacteria class, being Acidiphilium the most abundant phylotype. Only 20% of the sequences detected at the surface were related to the Nitrospirae phylum, all clustering within the genus Leptospirillum. Additionally, sequences belonging to the Planctomycetes phylum (2%), the Gammaproteobacteria class (2%) and the Acidisphaera genus (2%)

were detected in this layer (Fig. 3). No sequences belonging to the archaeal domain could be retrieved from the surface sample. By contrast, at 15 m depth, 39% of the sequences all related to the Gammaproteobacteria class, clustered within the species Acidithiobacillus ferrooxidans. A 28% of the sequences were related to the Nitrospirae phylum, with Leptospirillum being the most abundant genus and 26% of the sequences clustered within the Actinobacteria class. Sequences related to the Chloroflexi (4%) and Acidisphaera (1%) genera were also detected (Fig. 3). Sequences belonging to the archaeal domain, all of them related to the Euryarchaeota

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phylum, specifically to the Thermoplasmata class, were identified in this layer. As expected, Fe(II) concentrations in the upper layer of the NSC pit lake and the water column of Río Tinto were extremely low (Table 1). Even though at the surface Acidiphilium spp. together with photoreduction of iron could generate Fe(II), the reduced form of iron does not accumulate because is rapidly oxidized by iron oxidizing bacteria, like Leptospirillum [5,11,18,41,42]. When compared to the diversity reported for Rio Tinto’s water column the most important difference was the absence of the iron oxidizing bacteria A. ferrooxidans in the upper oxidized layer of the pit lake (Fig. 3). Considering the physicochemical characteristics of both systems (Table 1 and Fig. 2) and the fact that A. ferrooxidans is present at the 15-m deep layer, a possible interpretation is that A. ferrooxidans is mainly involved in the reduction of Fe rather than its oxidation in this ecosystem. The bottom layer of the NSC pit lake showed a higher level of diversity, which correlates with the higher diversity observed in the sediments of the Río Tinto basin [20]. In this case, the detected Acidithiobacillus spp. and members of the Acidimicrobiaceae family, both facultative iron reducing bacteria under anoxic conditions [13], could be responsible of the increase in the Fe(II)/Fe(III) ratio in this layer when the pit lake was chemically stratified, while Leptospirillum and some Acidimicrobiaceae members could oxidize iron when stratification broke down and the mixing introduced oxygen in the bottom layer. A similar situation might be operating in the sediments of Río Tinto, in which iron reducers of the Acidithiobacillus, Acidiphilium and Acidimicrobium genera can be found together with iron oxidizers of the Leptospirillum, Ferrovum like and Ferrimicrobium genera, although in this case the influence of an effective compartmentalization facilitated by the semisolid matrix of the sediments, inexistent in the water column, has to be considered. A sulfur cycle is also operating at the bottom layer of the NSC similarly to the Río Tinto basin. Reduced sulfur compounds are the energy source used by A. ferrooxidans during anaerobic respiration [25]. In addition, some strains of A. ferrooxidans and Thermoplasmata could grow anaerobically using hydrogen as electron donor and sulfate as an electron acceptor [25,35]. Sulfate-reducing bacteria were not detected in the NSC pit lake while microorganisms capable of this anaerobic respiration were identified in the Río Tinto sediments [38,39] (Fig. 3). This is probably due to the environmental compartmentalization that can be generated in the sediments. Although microbial sulfate reduction can proceed in environ-

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ments with a low pH, it is well established that inhibition processes are present in AMD systems. Possible inhibitory factors include the metabolites and organic acids, which can be toxic depending on pH. Metal sulfide precipitation and competition with other bacteria, namely iron-reducing bacteria, can inhibit sulfate reduction [28]. Based on this, it is easier to find SRB in Río Tinto sediments than in the NSC water column. Sediment microniches are suitable for the SRB activity, while in the water column the SRB activity is easily inhibited. Sequences closely related to Chloroflexi have been detected in both the NSC pit lake and the water column of one Río Tinto sampling site (RT8) [19,42]. This type of sequences have been retrieved from different mine drainage environments [20,46,50]. Although the role of these bacteria in extreme acidic environments has to be clarified, bacteria belonging to this phylum have been isolated and related with the iron cycle [27]. No archaeal sequences could be retrieved neither in the upper layer of NSC nor in the Río Tinto water column. However, sequences related to the Euryarchaeota phylum of Archaea, specifically to the Thermoplasmata class, were detected in the bottom layer of the pit lake and the sediments of one sampling station from the Río Tinto basin (RT9). The eukaryotic 18S rRNA gene clone library for NSC only showed positive results for the surface layer. In this layer, measured PAR values were low (Fig. 2), but sufficient to activate photosynthesis in acidophilic species [10]. All identified sequences were related to the genus Chlamydomonas, although microscopic observations also showed the presence of filamentous algae related to the Zygnemopsis and Klebsormidium genera as well as diatoms belonging to the genus Pinnularia, which could not be detected by molecular techniques [2]. Chlamydomonas is one of the most abundant algae detected in the Río Tinto basin, followed by the filamentous algae belonging to the Zygnemopsis and Klebsormidium genera [3]. In the river, diatoms have been detected mainly associated to the most extreme conditions [3]. Although the level of eukaryotic diversity detected in the Río Tinto basin is much higher than in the NSC pite lake due to its size (92 km) and the existence of very productive biofilms covering the rocks along the entire course of the river [2,3,4] the similarities existing between both ecosystems are noteworthy. Canonical correspondence analysis (CCA) was used to correlate microbial community data with environmental data in both ecosystems. Fig. 4 shows the results of this analysis as a three biplots, to facilitate its interpretation. The CCA was conducted using OTUs of 16S rRNA genes acquired at different depths for the NSC pit lake and different sampling stati-

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Fig. 4. Canonical correspondence analysis based on variance of species (OTUs) with respect to environmental data. Results were graphed in three biplots. The eigenvalues for each axis generated by CCA indicate how much of the variation seen in species data can be explained by that canonical axis. A 51% of the correlation between OTUs, sample sites and environmental data was explained by two axes. The presence or absence of data was used for bacterial and archaeal OTUs. Different OTUs are represented by genera names and triangles. Environmental variables used in the analysis are shown by arrows. Sampling sites are indicated by dots and the station name (NSC01, NSC15, RT1w, RT1s, RT2w, RT2s, RT3w, RT3s, RT5w, RT5s, RT6w, RT6s, RT8w, RT8s, RT9w, RT9s, RT10w, RT10s, RT11w and RT11s). Samples from the river were from sediments (S) or water column (W). Pit lake samples analyzed were from the surface (NSC01) and from 15-m depth (NSC15).Units of the elements were mg/l. Units of sulfate was g/l. Units of environmental data were mV for redox potential (ORP), mS/cm for electric conductivity (EC) and mg/l for dissolved oxygen (DO).

ons for the Río Tinto basin. Samples are plotted in different areas of the diagram depending on their environmental characteristics. The CCA generates an ordinate diagram in which axes are created by a combination of environmental variables [7] The eigenvalues for each axis generated by CCA indicate how much of the variation seen in the species data can be explained by that canonical axis. In this case, 51% of the correlation between OTUs sampling sites and environmental data could be explained by two axes (p-value < 0.05). Metals (with the exception of Mn), sulfate, redox potential (ORP) and electric conductivity (EC) correlate inversely with pH and dissolved oxygen. The most important conclusion is that there is an important homogeneity in most of the samples, both at the physico-chemical and microbiological level. This is the reason why so many data occupy the centre of the graph, and the size of the arrows are rather small. The microorganisms appearing in most sampling stations were Leptospirillum, followed by Acidiphilium, Metallibacterium, Acidithiobacillus, Ferrimicrobium and Acidisphaera, all of them present in NSC and Río Tinto samples (Fig. 4). Río Tinto sam-

pling stations RT1, RT3, and RT8 appear at the centre of the graphs. They are very similar, so they can be considered the most representative samples of the river from an environmental and microbiological point of view (Fig. 4). These samples show the highest concentrations on sulfate, Ni and Co. These stations are the closest to the deep layer of the NSC pit lake. The dissolved oxygen concentration is the only difference. From this analysis, it is clear that the NSC acidic pit lake has many common geomicrobiological features with the Río Tinto basin that underlie the influence of the shared substrates of the IPB in the microbial ecology of both ecosystems. This study demonstrates a close relationship between the microbial diversity of NSC acidic pit lake and the diversity detected in the Río Tinto basin. The canonical correspondence analysis identified members of the Leptospirillum, Acidiphilium, Metallibacterium, Acidithiobacillus, Ferrimicrobium and Acidisphaera as the most representative microorganisms of both ecosystems. The presence of archaeal members was scarce in both systems. Only sequences clustering with the Thermoplasmata were retrieved in the bottom layer of NSC

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and one sampling station of Río Tinto. Although the measured PAR values were very low in the upper NSC layer, they were sufficient to activate photosynthesis in acidophilic microorganisms. All identified photosynthetic microorganisms in the NSC (members of the Chlamydomonas, Zygnemopsis and Klebsormidium genera) were important members of the characterized photosynthetic eukaryotic community identified in the Río Tinto basin, which underlines the commonality between both ecosystems also at the eukaryotic level. Acknowledgements. This work has been supported by grants CGL2011-22540 and CGL2009-11059 from the MINECO, Grant 478 from IGME and grant 250-350-IPBSL from ERC. Competing interests. None declared.

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RESEARCH ARTICLE IÄã ÙÄ ã®ÊÄ ½ M® ÙÊ ®Ê½Ê¦ù (2014) 17:235-247 doi:10.2436/20.1501.01.226 ISSN (print): 1139-6709. e-ISSN: 1618-1095 www.im.microbios.org

Ignimbrite textural properties as determinants of endolithic colonization patterns from hyper-arid Atacama Desert Beatriz Cámara1*, Shino Suzuki2, Kenneth H Nealson2,3, Jacek Wierzchos1, Carmen Ascaso1, Octavio Artieda4, Asunción de los Ríos1 1 National Museum of Natural Sciences-CSIC, Madrid, Spain. 2J. Craig Venter Institute, La Jolla, CA, USA. University of Southern California, Los Angeles, CA, USA. 4Department of Plant Biology, Ecology and Earth Sciences. University of Extremadura, Plasencia, Spain

Received 23 September 2014 · 15 December 2014 Summary. This study explores the photosynthetic microbial colonization of rhyolitic ignimbrites in Lomas de Tilocalar, a hyper-arid region of the Atacama Desert, Chile. Colonization appeared in the form of a green layer a few millimeters beneath the ignimbrite surface. Some ignimbrite rocks revealed two distinct micromorphological areas of identical mineralogical and chemical composition but different textural properties. According to texture, colonization patterns varied in terms of the extension and depth of colonization. The diversity of photosynthetic microorganisms was assessed by denaturing gradient gel electrophoresis (DGGE) of the 23S rRNA gene and by generating clone libraries of the 16S rRNA gene. We observed a low diversity of photosynthetic microorganisms colonizing the ignimbrite microhabitat. Most rRNA gene sequences recovered greatly resembled those of Chroococcidiopsis hypolith clones from arid deserts. These results point to highly restrictive conditions of the hyper-arid Atacama Desert conditioning the diversity of cyanobacteria, and suggest that microbial colonization and composition patterns might be determined by the microscale physico-chemical properties of the ignimbrite rocks. [Int Microbiol 2014; 17(4):235-247] Keywords: Chroococcidiopsis sp. · endoliths · ignimbrite · rock porosity · volcanic rock · Atacama Desert

Introduction Microbial communities in terrestrial volcanic environments have been scarcely investigated despite the wide distribution of highly colonized volcanic rocks on Earth (approximately 95% of the Earth’s crust) [2,13,23,30]. Most microbiological studies have been conducted almost exclusively in volcanic regions of Iceland where temperatures are low and snow cov* Corresponding author: B. Cámara Museo Nacional de Ciencias Naturales-CSIC Serrano, 115 bis 28006 Madrid, Spain Tel. +91-7452500. Fax: +91-5640800 E-mail: beacamara@gmail.com

ers the ground during most of the year [12,31,32,36–38]. Many such studies have linked the diversity of microbial communities in volcanic environments to rock geochemistry and have centered on identifying the effects of diversity on weathering and soil neogenesis [12,38]. This type of characterization study is essential to understand the potential roles of these microorganisms in the weathering processes that contribute to the global carbonate-silicate cycle [11,12,32,36]. The geochemical (chemical and mineralogical composition) and textural (size, crystal shape and organization, mineral and matrix porosity) properties of rocks determine how susceptible, or bioreceptive, a rock is to colonization by different microorganisms and organisms [27,44]. In particular, the movement of water through the rock matrix is conditioned

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Fig. 1 (A) Map showing the geomorphic units of central Atacama Desert and the study area (arrow), south of the Salar de Atacama basin. (B) Landscape of the sampling site in Lomas de Tilocalar showing the location of the microweather station and Pliocene Tucucaro ignimbrite rocks on a typical N-S trending ridge.

by the physical properties of the rock (e.g., pore size, pore connectivity, permeability, water absorption kinetics, or water retention capacity) and this, in turn, will determine its microbial colonization capacity [44,58]. Other factors such as pH, level of exposure to climate and nutrient sources are also known to influence the bioreceptivity of rocks [16,46]. So far, various groups of microorganisms have been observed to inhabit volcanic rocks featuring a variety of mineralogical, chemical and textural properties [2,12,23,31,32, 36–38]. Among these, cyanobacteria play an important role in rock weathering via their capacity to extract bio-essential elements from rocks by increasing the pH of their surroundings [7]. Further, in a controlled laboratory experiment, both cyanobacterial growth and weathering rates of colonized volcanic rocks were found to be affected by the chemical and mineralogical composition of the rock. Thus, basalt rocks (highly essential element levels) showed higher rates of cyanobacterial growth and substrate dissolution than rhyolitic rocks (high silica levels) [45]. These observations have revealed that the physico-chemical properties of rocks determine the metabolic activity of microorganisms, and that, vice-versa, rocks can also be modified by the activities of their colonizing microorganisms [31,59]. The Atacama Desert features extensive areas covered with felsic lava flows arising from volcanic activity in the Andes [4,26]. Ignimbrite structures dominate the Preandean Depression and Altiplano areas of this desert and are thought to have formed in the Pliocene and Upper Miocene as a result of pyroclastic flows erupted from large boiler systems in the Alti-

plano area, east of the Salar de Atacama [17,21]. Recently, Wierzchos et al. [62] detected microbial communities inhabiting ignimbrites in the Preandean Depression area. These authors have proposed that the volcanic rock interior protects endolithic cyanobacteria and associated heterotrophic bacteria against the intense UV radiation and high visible light levels that prevail in this region. This study served to broaden the spectrum of rock substrates known to act as lithic microhabitats for microorganisms in the hyper-arid region of the Atacama Desert, namely ca-sulfate crusts [63], halite [15,52], quartz rocks [40] rock-varnish [39], rhyolite-gypsum, and calcite rocks [18], and finally, ignimbrite rocks [62]. This study examines the diversity of photosynthetic bacteria that colonize two texturally different areas of rhyolitic ignimbrite rocks in the hyper-arid Atacama Desert. Its objective was understanding why these two habitats show different patterns of microbial diversity given their similar chemistry, yet different microstructure. Microbial diversity was examined through DGGE and clone libraries of 23S rRNA and 16S rRNA gene sequences. The textural properties of rocks were determined using several microscopy and analytical procedures.

Materials and methods Site description and sampling. The study site was Lomas de Tilocalar (23º57′25″S, 68º10′12″W and 2986 m asl), south of the Salar de Atacama basin in the eastern region of the Atacama Desert (Chile) (Fig. 1A). This area shows a N-S trending depression facing eastward towards the Cordon de Lila range, along with several subparallel N-S trending ridges topped with Plio-

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cene Tucucaro ignimbrite (3.2 ± 0.3 Ma, Fig. 1B) [24,26,51]. A detailed description of the area and its microclimate (for the period 20 March 2010 to 23 September 2011) may be found in Wierzchos et al. [62]. The site, featuring extremely low rainfall and a high evapotranspiration rate, is classified as hyper-arid (aridity index ~ 0.0075). Over the period specified above, mean annual temperature was 14.4ºC with a maximum of 37.5 ºC and minimum of –7.4 ºC. Relative humidity was exceptionally low, the annual mean being 16.2%, and ranging from 1 to 95.3%. Precipitations consisted of occasional rainfall events (22 mm/yr in 4 separate episodes) as the only source of liquid water in this area. In 2010, samples of ignimbrite rocks were randomly collected with the help of a hammer and a chisel 3–10 m from the microclimate station Onset HOBO Weather Station Data Logger (Onset Computed Company, Bourne, MA, USA). Fracturing exposed a visible green band of endolithic colonization. The ignimbrites sampled originated from pyroclastic flows consisting of gas, ash and lava deposited chaotically while still hot. This means that postdepositional flow likely gave rise to the typical texture of ignimbrite containing flat pumice fragments. The Tucucaro ignimbrite [51] collected for this study is beige or dark (indicated as zone IgD in Fig. 2A,B), and features flat pumice fragments (indicated as IgW-white zone in Fig. 2A,B). Note that the green band of endolithic colonization appears continuous from one zone to the other in ignimbrite rocks showing this IgD/IgW interface. Thus, we searched for this feature by fracturing rocks, though the task was challenging because of the scarcity of rocks showing this continuous green band. The collected rock fragments were sealed in sterile zip-lock bags (Whirlpark, Fisher Scientific) avoiding any hand contact. Samples were transported stored dry in the dark at ambient temperature. Once at the laboratory (within 20 days), they were immediately processed. X-ray diffraction (XRD) and X-ray fluorescence spectroscopy (XRF). The mineralogical composition of IgD and IgW was determined by X-ray powder diffraction, using a Philips X′ Pert diffractometer PW 1830 for polycrystalline samples with a graphite-monochromated CuKα radiation source (Cu cathode of wavelength Κα = 1.54051). Samples were ground to a particle size ≤40 μm and this powder was used to obtain the XRD diffractograms. For qualitative analysis of the crystalline phases present in the samples, the Power Diffraction File (PDF-2, 1999) of the International Centre for Diffraction Data (ICDD) was used. A semi-quantitative analysis of these phases was performed using the normalized reference intensity ratio (RIR) method [10] using RIR values for each phase from the powder diffraction database (ICDD). Quantitative chemical composition in terms of major and minor elements was determined by X-ray fluorescence (XRF) spectroscopy. XRF spectra for the ignimbrite rock fragments were acquired using a Philips PW-1404 spectrometer with Sc-Mo X-ray tube and scintillation gas (PR- 10) detector, after milling in an agate mortar. For analytical data treatment, we used Super-Q Manager Geostandards software (CRNS, France). Polarized light microscopy. The petrographical study of the two differentiated areas of ignimbrite rocks was conducted on thin sections (30 μm thick) examined using a Nikon Eclipse LV100 Pol polarized light microscope (PLM) equipped with a Nikon DS-Fi1 digital camera. Scanning electron microscopy in back-scattered electron mode (SEM-BSE). Ignimbrite rocks showing the IgD/IgW interface were processed for their examination by SEM-BSE following Wierzchos & Ascaso [61]. Briefly, samples were dehydrated and embedded in LR-White resin, finely polished and carbon coated. Finally, the polished surfaces of rock-embedded samples were observed in a Zeiss DMS 960 SEM microscope equipped with a four-diode, semiconductor BSE detector and ISIS Link EDS (Energy-Dispersive X ray Spectroscopy) microanalytical system under the conditions: 0º input angle, 35º output angle, 15 kV acceleration potential, 6–15 mm working distance and 5–10 nA probe current.

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Mercury intrusion porosimetry (MIP). The pore systems of the IgD and IgW zones were characterized by mercury intrusion porosimetry in terms of their connected porosity, pore size distribution and mean pore size according to Benavente et al. [5]. The equipment used was a PoreMaster 60/ Quantachrome instrument to determine porosity (connected porosity) of the rock samples in the range of pore diameters 0.00036 μm to 190.58 μm. In the Washburn equation, a mercury surface tension of 0.48N/m was used, and a rock-mercury contact angle of 140 dag was used in the Laplace equation. Porosity (total porosity %) was then determined as the weight-normalized volume of mercury intruded in the sample. DNA extraction. Sample preparation for denaturing gradient gel electrophoresis (DGGE). Ten colonized fragments of ignimbrite rock showing the IgD/IgW interface were used for this study. Given the low volume of colonized material in each ignimbrite rock, material from several rocks was mixed to provide 100-mg samples for DNA extraction. Three different pooled samples of each texture (IgD-I, IgD-II, IgD-III and IgW-I, IgW-II, IgW-III) were processed for DGGE analysis. The colonized material was collected using a sterile blade from the fresh green band of ten rock fragments. Prior to DNA extraction, the rock material was frozen in liquid nitrogen and then pulverized with the aid of a sterile micropestle (Eppendorf micropestle, Sigma-Aldrich). Total genomic DNA was extracted using the UltraClean Microbial DNA Isolation kit (Mobio Laboratories, Solana Beach, CA, USA) according to the manufacturer’s instructions. Sample preparation for the construction of clone libraries. Two pooled rock samples were prepared to construct a clone library representative of each ignimbrite zone (IgD and IgW) as described above for the DGGE samples. Total genomic DNA was extracted using the Ultraclean® Soil DNA Isolation Kit (Mobio Laboratories Inc., Carlsbad, CA, USA) following the manufacturer’s instructions. For both analyses, the quality and quantity of the total genomic DNA extracted was determined in a NanoDrop spectrophotometer 1000 (Thermo Scientific, Waltham, MA, USA). Genomic DNA (ca. 10–50 ng) was used for PCR amplification with different genetic markers. Denaturing gradient gel electrophoresis (DGGE) of the 23S rRNA gene. To obtain a quick overview of photosynthetic microbial composition, the endolithic colonization of IgD and IgW in the prepared samples was analyzed by denaturing gradient gel electrophoresis (DGGE). Total genomic DNA from samples IgD-I, IgD-II, IgD-III and IgW-I, IgW-II, IgW-III was used for PCR amplification using specific primers for phototrophs: p23SrV_f1_GC and p23SrV_r1. These primers flank domain V of the 23S rRNA gene, a hypervariable region of this gene (~400 bp) only present in cyanobacteria and plastids, and can discriminate among taxa at the species levels [55]. The PCR and thermal cycling conditions used were as described by Sherwood & Presting [55]. The PCR products obtained from zones IgD and IgW were separated on a D-Code Universal Mutation Detection System (BioRad, Hercules, CA, USA) using 6% acrylamide/bisacrylamide gel (37.5:1) on a 30–70% denaturing gradient of urea-formamide (where 100% is defined as 7 M urea and 40% v/v). Electrophoresis was carried out in 1X TAE buffer at 100 V and 60°C for 16 h. The predominant DGGE bands (in terms of intensity and frequency) were excised using a sterile scalpel, and incubated in sterile distilled water overnight at 4°C. Next, the DGGE bands were reamplified under the same PCR conditions using the same primer pair as described above, but this time without the GC clamp. The identity of the new PCR products was verified by comparing their electrophoretic positions with the original positions of the other bands in the DGGE gel. PCR products were sequenced at the Macrogen Company sequencing service (Seoul, South Korea). Phylotypes were determined based on sequence similarity ≥99%. All DGGE band sequences obtained were deposited in GenBank (KP238376–81).

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Construction of 16S rRNA gene clone libraries.Total genomic DNA from IgD and IgW samples was used for PCR amplification of the 16S ribosomal RNA gene using the following set of primers for eubacteria: specific primer U27f and universal primer U1492r [41]. Each 25 l reaction mixture contained 0.625 U of Taq polymerase TaKaRa Ex Taq 5U/l (Takara Bio Inc.), 0.2 mM of each of the four dNTPs, 0.2 M of each primer (10 mM), 2.5 l Ex Taq Buffer (10X) (Mg2+ free), 2 l of DNA template (10–50 ng) and sterile bidistilled water up to a volume of 25 l. The PCR conditions were an initial denaturation step at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min and 30 s, and a final extension phase of 72°C for 7 min. The purified PCR products (1400 bp) were then used to construct one clone library for each sample (IgD and IgW). To this end, the purified products were ligated into the pGEM-T vector (pGEM®-T Easy Vector System kit, Promega®) according to the supplier’s instructions, and subsequently introduced into Escherichia coli JM109 competent cells (Promega, USA). After incubation at 37°C for 18 h, approximately 50 white colonies were randomly selected from each genomic library, purified and used as DNA template in the insert PCR reaction using primers M13F and M13R. For this, a reaction volume of 25 l was prepared following the protocol described above. The PCR products containing the insert (1700 bp) were purified and sequenced on an ABI DNA sequencer 3730xl (Applied Biosystems, Carlsbad, California, USA) and the resulting chromatograms analyzed using the software FinchTV 1.4.0 [www. geospiza.com/FinchTV]. Chimeras were identified using the Bellerophon server [http://comp-bio.anu.edu.au/Bellerophon/doc/doc.html] [33] with the Huber-Hugenholtz correction and a window size of 300 bp as selected parameters. Remaining sequences were clustered in operational taxonomic units (OTUs) using MOTHUR software [http://www.mothur.org/] [54]. OTUs were defined as showing 95% sequence similarity (cutoff 0.05) for the genus level and 97% sequence similarity (cutoff 0.03) for the species level [53], using the furthest-neighbor algorithm at an accuracy of 0.01. The resulting 16S rRNA gene sequences were deposited in GenBank (KP238382-411). Finally, library coverage was estimated as a measure of the sampling effort. Also calculated were the variables Shannon’s index (H’) and Simpson’s index of diversity (1-D), Pielou’s evenness index (E), and the richness estimator SChao1. As a measure of similarity between the two clone libraries, the Sørensen index (Cs) was also calculated. Phylogenetic analysis. Representative sequences for each OTU (selected using the “Get.oturep” option in MOTHUR) and DGGE phylotype were used for the 23S rRNA and 16S rRNA gene sequence alignments respectively. In both alignments, similar sequences identified in a BLAST search and sequences from representative species of cyanobacteria available at GenBank were also included. Sequence alignments were performed using MUSCLE 3.8 [19] and manually checked and corrected in BIOEDIT 7.0.52 [29]. To exclude sequence ends and ambiguously aligned regions, Gblocks v.091b [9] was used. To generate a phylogenetic tree, nucleotide substitution models were statistically selected by JMODELTEST [49] [available at http:// darwin.uvigo.es]. According to the Akaike information criterion (AIC) [1], the best fitting model of sequence evolution was the General Time Reversible (GTR) substitution model [57] with estimation of invariant sites (+ I) assuming a gamma distribution with six rate categories (+ G). All data sets were analyzed using maximum likelihood and Bayesian inference approaches. Bayesian analyses were performed using the Metropolis coupled Bayesian Markov chain Monte Carlo algorithm (MC)3 implemented in the software MRBAYES v.3.1.2 [34] [http://morphbank.ebc.uu.se/mrbayes]. Analyses (MC)3 for 16S and 23S rRNA gene sequence alignments were run for 8,000,000 and 5,000,000 generations respectively, using a random tree as starting point, 12 simultaneous channels, a temperature of 0.1 and a sampling frequency of 100 generations. The first 20,000 trees for the 16S rRNA align-

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ment and 12,500 trees for the 23S alignment were discarded as burn-in, after verification of the likelihood parameters in TRACER v.1.4 [50]. 50% majority-rule consensus trees were obtained from the remaining trees using the sumt option. In addition, maximum likelihood (ML) tests were run in the PhyML 3.0 server [28] [http://www.atgc-montpellier.fr/phyml/] using a nonparametric test with 1000 bootstrap replications to asses branch support [20]. Finally, the resultant phylogenetic trees were visualized with FigTree v.1.3.1 [http://tree.bio.ed.ac.uk/software/figtree/]. Chloroflexus aggregans was used as outgroup for the 23S rRNA gene phylogenetic tree while Deinococcus radiodurans and Thermus sp. were used as outgroups for the 16S rRNA gene phylogenetic tree.

Results Physico-chemical characterization of ignimbrite rocks. Zones IgW and IgD displayed a similar mineralogical composition as revealed by semiquantitative X-ray analyses. The major mineral phases identified were andesine (48%), sodium disordered anorthite (44%) and biotite (8%). According to the XRF spectra, zones IgW and IgD were practically identical in composition as previously reported for IgD by Wierzchos et al. [62] (Table 1). Based on its chemical composition and total alkali-silica (TAS) diagram [42], which represents the relationship between alkali (wt% of Na2O + K2O) and silicate (wt% SiO2) mineral contents, Table 1. Chemical composition determined by XRF in terms of major elements (%) of zones with different color, IgD and IgW, in ignimbrite rocks (LOI: Loss of Ignition) Elements

IgD*

IgW

SiO2

71.05

72.21

Al2O3

14.56

13.66

Fe2O3 (total)

1.64

1.49

MnO

0.06

MgO

0.58

0.54

CaO

1.50

1.60

Na2O

3.66

2.99

SO3

0.00

0.39

K2O

5.12

5.80

TiO2

0.40

0.38

P 2O 5

0.08

0.07

LOI

1.37

0.81

*Data obtained from Wierzchos et al. [62].

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Fig. 2 (A) Cross-section of an ignimbrite rock showing the interface between the two defined zones designated IgD (dark ignimbrite) and IgW (white ignimbrite), scale 1 cm. (B) Detailed view of Fig. 2(A) (square) showing the IgD/IgW interface and endolithic colonization patterns specific to each zone, scale 1 mm. (C) SEM-BSE image of the IgD/IgW interface comprised of a matrix of glass shards (asterisks), plagioclase phenocrysts (triangles) and vesicular pores (p), scale 500 μm. (D–F) Plane-polarized light micrographs of IgD and IgW revealing their different textures. (E) Matrix of welded glass shards (asterisk) in IgD. And (F) matrix of glass shards containing elongated vesicles (arrowheads) in IgW. Note the intense exfoliation pattern defined by the elongated vesicles (arrows). Scales 100 μm from D and E, and 200 μm from F.

the material collected in this study was classified as weakly welded rhyolitic ignimbrite. The interface between zones IgD and IgW was examined by SEM-BSE and petrographic microscopy (Fig. 2C,D). In the SEM-BSE image (Fig. 2C), both IgD and IgW showed the presence of plagioclase crystals, yet vesicular pores of the matrix of glass fragments in which these were embedded varied in size. This was confirmed by petrographic microscopy observation of the same interface (Fig. 2D). A detailed view of each zone was obtained by petrographic

microscopy (Fig. 2E,F). In these images, it is possible to discern a matrix of glass fragments weakly welded in the beige colored zone (IgD, Fig. 2E) and fragments of glass with small elongated vesicles in the white zone (IgW). This white zone showed intense exfoliation with the exception of areas around the phenocrystals (Fig. 2F). Zones IgD and IgW were also compared in terms of their porosity and mean and median pore diameters determined by mercury intrusion porosimetry (MIP) (Table 2). These data confirmed our microscopy observations, with zone IgD showing a greater

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Table 2. Total porosity, mean and median pore diameter distribution and volume of pores (%) with diameter <1 m and >1 m determined by IPM in IgD and IgW IgD

IgW

Total porosity (%, volume)

14.4 (± 0.33)

9.65 (± 0.34)

Mean pore diameter (m)

1.14 (± 0.52)

0.62 (± 0.12)

Median pore diameter (m)

11.27 (± 1.31)

2.46 (± 0.31)

% Pores with diameter < 1 m

15.3

29.9

% Pores with diameter > 1 m

84.7

70.1

nº samples

total porosity (14%) and mean pore diameter (1.14 ± 0.52 m). According to Pittman’s definition of microporosity [47], the volume of micropores of diameter <1 m expressed relative to total porosity was higher in IgW (29.9%) than in IgD (15.3%).

f. Four different phylotypes (I, II, III and IV) were defined based on ≥99% sequence similarity. Three of these phylotypes were detected in both ignimbrite textures, while phylotype IV only appeared in one IgD profile (Table 3). Based on 97% and

Photosynthetic microbial composition. Analysis of the 23S rRNA gene. DGGE profiles of the 23S rRNA gene (Fig. 3) contained a small number of bands, and differences were detected between IgD and IgW textures. Eleven DGGE bands were excised, reamplified and sequenced, seven corresponding to IgD (bands a–g) and four to IgW (bands h–k) (Fig. 3 and Table 3). Two of the IgD profiles were identical, while IgD-I showed a unique DGGE profile. Of note, we observed a DGGE band common to all three IgD profiles (a, c and f). High variation was observed in the IgW sample profiles, with the exception of one DGGE band shared between IgW-I (band h) and IgW-III (band k). When comparing profiles according to texture, DGGE profiles for samples IgD-I and IgW-III emerged as identical. IgW showed bands common to the IgD profiles. Similar electrophoretic positions were observed for IgD-I band b, IgW-I band h and IgW-III band k; IgW-II band i, IgD-II band d and IgD-III band g; and IgW-III band j, IgD-I band a, IgD-II band c and IgD-III band

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Patterns of endolithic colonization. Upon fracturing the ignimbrite rocks, microbial endolithic colonization appeared as a readily discernible green band a few millimeters below the rock surface (zone indicated in Fig. 2A,B). These endolithic communities showed distinct colonization patterns in the zones of different textural properties. In the beige zone IgD, this pattern was a narrow green band (0.5–1 mm thick) at a depth of 1 mm (Fig. 2B), while in IgW, the green band was slightly thicker and more diffuse and dispersed such that its depth ranged from 1 to 2 mm (Fig. 2B).

Fig. 3. DGGE profiles of PCR-amplified 23S rRNA gene fragments (404 bp) derived from representative samples of the green bands of endolithic colonization in zones IgD and IgW (IgD dark ignimbrite, IgW white ignimbrite). Three samples of each texture (zone) were analyzed (I-III). Letters a-k indicate DGGE bands that were excised, reamplified, purified and sequenced for analysis (see Table 3).

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. Table 3. DGGE bands of 23S rRNA gen from the IgD and IgW textures of ignimbrite rocks indicated in Fig. 3, and BLAST analysis of the obtained phylotypes (based on sequence similarity ≥99%) DGGE Bands

Phylotype

IgD

IgW

Closest GenBank sequences, (accession number), % similarity

Location

Closest sequecens of identified organisms in GenBank (accession number), % similarity

a, c, f, j



Uncultured bacterium isolate DGGE gel band A (JQ700570), 99% ; band B (JQ700571), 98% Uncultured Chroococcidiopsis clone A1.1, NA4.3 (FJ805915, FJ805885), 98%

Atacama Desert, Chile Lybian Desert, Egypt Death Valley, USA

Microcoleous chthonoplastes (AM709630), 91%

b, h, k



Uncultured Chroococcidiopsis clone AS5.13-8-5 (FJ805955-50, 47), 99%

Turpan Depression, China

Lyngbya aestuarii PCC 7419 (AY584522), 91%

d, g, i

III



Uncultured bacterium isolate DGGE gel band B (JQ700571), 100%; band A, (JQ700570), 98% Uncultured Chroococcidiopsis clone A1.1, NA4.3 (FJ805915, FJ805885), 97%

Atacama Desert, Chile Lybian Desert, Egypt Death Valley, USA

Thermosynechococcus sp. NK55 (CP006735), 93%



Uncultured bacterium isolate DGGE gel band B (JQ700571) 99%; band A (JQ100570), 99% Uncultured Chroococcidiopsis clone A1.1, NA4.3 (FJ805915, FJ805885) 98%

Atacama Desert, Chile Lybian Desert, Egypt Death Valley, USA

Thermosynechococcus sp. NK55 (CP006735), 92%

95% sequence similarities, two potential species and two potential genera (Genera 1 and 2) were differentiated in IgD and IgW, one species included in Genera 1 (Phylotypes I, III and IV), and the remaining species in Genera 2 (Phylotype II). BLAST analysis of the recovered phylotypes indicated they were all cyanobacteria showing most sequence similarity with uncultured bacterial isolates from the Atacama Desert (Chile) and uncultured hypolithic members of the Chroococcidiopsis genus from other extreme hyper-arid environments (Lybian Desert, Egypt, and Death Valley, USA). Sequences closest to organisms from the GenBank database were those of Microcoleus chthonoplastes, Thermosynechococcus sp. and Lyngbya aestuarii (showing 91–93% similarity, Table 3). Our 23S rRNA gene sequence alignment showed a final size of 406 nucleotide positions, including gaps and a total of 54 sequences. The phylogenetic positions of the phylotypes corresponding to the DGGE bands were resolved by Bayesian inference and maximum likelihood methods, yielding consensus trees with the same topology (Fig. 4). The 50% majorityrule consensus tree showed that the DGGE phylotypes (pink

color in Fig. 4) clustered with two uncultured bacteria isolated in a previous investigation from IgD ignimbrite rocks [62] and with different uncultured hypolithic clones of Chroococcidiopsis cyanobacteria from hot arid deserts (Turpan Depression, China; Desert Dubai, United Arab Emirates; Desert Death Valley, USA; Atacama Desert, Chile in Bahl et al. [3]). The latter sequences correspond to the “HOT DESERT I” and “HOT DESERT II” groups designated by Bahl et al. [3]. Phylotype II was detected in “HOT DESERT II” with a posterior probability (PP) = 99% and bootstrap probability (BP) = 94.9%; remaining phylotypes (I, III and IV) clustered with sequences belonging to the “HOT DESERT I” group. Analysis of 16S rRNA gene. In total, 96 clones were obtained from both libraries (IgD and IgW), of which 73 clones provided high quality sequences, 37 from IgD and 36 from IgW. According to the criteria that sequence similarities >97% and >95% correspond to similar species or genera, respectively, the sequences obtained in this study indicated three OTUs in each library. At the species level, two OTUs were

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Fig. 4.Consensus phylogenetic tree (50%-majority rule) based on the 23S rRNA gene region (406 pb) reconstructed using Bayesian inference and maximum likelihood to infer the phylogenetic positions of the DGGE phylotypes recovered from zones IgD and IgW (indicated in pink). Branches in bold are supported by both methods with Bayesian posterior probability (PP) ≥ 95% and bootstrap probability (BP) > 70%. One asterisk indicates a branch supported only by PP, and two asterisks a branch supported only by BP. Chloroflexus aggregans was used as outgroup. In the right margin of the figure, we provide the groups designated by Bahl et al. [3] for uncultured Chroococcidiopsis hypolithic clones from hot and cold arid deserts.

common to IgD and IgW (OTU-1 and OTU-2), while the remaining OTUs of each library were unique to one or other zone (OTU-3: IgD and OTU-4: IgW). At the genus level, three OTUs were also recovered (OTU-A, OTU-B, OTU-C) in both ignimbrite zones: OTU-A including OTU-1, OTU-B including OTU-2, and OTU-C including OTU-3 and OTU-4. In terms of relative abundances of each OTU at the species level, the most abundant OTUs were OTU-1 (59%) and OTU-3 (38%) in the IgD library and OTU-1 (61%) and OTU-4 (36 %) in the IgW library, OTU-2 being in both cases the OTU with the lowest number of sequences (2%). Our statistical analysis revealed 97% coverage and similar indices of diversity and evenness in both libraries. The Shannon index (H’) and Simpson index of diversity (1-D) were low in terms of richness and abundance (H′IgD= 0.774 and H′IgW =0.768; 1-DIgD= 0.517 and 1-DIgW= 0.509) in both libraries. Pielou’s in-

dex revealed a tendency towards evenness in both libraries (0.705 in IgD and 0.699 in IgW). In addition, the estimator SChao1 indicated low, identical species richness for IgW and IgD (3 in both cases). This similarity between the two libraries was also supported by the Sorensen index (Cs = 0.67). BLAST analysis of the most representative sequences of each OTU obtained at the species level revealed their greater similarity with sequences of uncultured hypolithic bacterium clones, most related to the Chroococcidiopsis genus. Further, sequences also displayed 93–98% similarity to that of the closest identified organism from the GenBank database (Table 4). The alignment of 16S rRNA gene sequences resulted in a final size of 604 nucleotide positions, including gaps, with a total of 50 sequences. In the consensus tree (Fig. 5), common OTUs to IgD and IgW appeared in two distinct clades (OTU-1 supported by PP = 98%, OTU-2 supported by PP = 99% and

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Table 4. OTUs identity obtained from clone libraries (16S rRNA gene) from the IgD and IgW ignimbrite textures OTUsa

IgD

IgW

OTU-1

(22) b

(22)

Uncultured bacterium clone 151 (HM241016) 97%

Chroococcidiopsis CC1 (DQ914863) 93%

OTU-2

(1)

Uncultured bacterium clone 225 (HM241090) 99%

Chroococcidiopsis CC3 (DQ914865) 98%

OTU-3

(14)

Uncultured bacterium clone 157 (HM241022) 98%

Chroococcidiopsis CC3 (DQ914865) 95%

Uncultured bacterium clone 186 (HM241051) 98%

Chroococcidiopsis CC3 (DQ914865) 94%

OTU-4

(13)

Closest GenBank sequences, (accession number), % similarity

Closest sequecens of identified organisms in GenBank (accession number), % similarity

The definition of OTUs (at the species level) was based on a sequence similarity > 97% for phylogenetic analysis. Numbers indicate the number of sequences included in each OTU.

a b

BP = 93.9%), clustering cyanobacterial sequences from the Chroococcidiopsis genus and from hypolithic cyanobacteria isolated from arid extreme environments, including sequences corresponding to the Yungay area of the Atacama Desert. The taxonomic affiliations of OTU-3 and OTU-4, unique to each library, were not completely resolved. Both OTUs clustered in the same clade, along with sequences of hypolithic microorganisms from deserts and sequences of the Oscillatoriales order, but without statistical support.

Discussion Volcanic rocks from the Atacama Desert are ideal models to explore microbial adaptation strategies in extreme arid environments. In a previous work it was shown that the ignimbrite rock interior provides protection from damaging UV radiation and excessive visible light [62]. This work is extended to assess the textural properties of ignimbrite as a determining factor for endolithic microbial colonization patterns. Cyanobacteria were detected as the main colonizers of the interior of weakly welded rhyolitic ignimbrite rocks collected from the Preandean Depression area of the Atacama Desert, where environmental conditions are stubbornly dry. Note that many rock fragments showed two well-differentiated micromorphological zones of different texture yet identical chemical and mineralogical composition (IgD and IgW). The novelty of the present work lies in the differences detected in patterns of cyanobacterial colonization and composition between these two zones. The different microbial colonization patterns observed in IgD (beige zone) and IgW (white zone) could be correlated with the porosity and color of each zone given their practi-

cally identical chemistry. Endolithic colonization patterns have been related to the intrinsic properties of building materials such as dolostone and granite [8,22,27,44]. The textural differences detected here between IgD and IgW were mainly due to their petrophysical properties, total porosity, pore size distribution and shape and orientation of pores. These features are known to influence water movement through the rock matrix along with water retention. In our study, the greater proportion of micropores detected in IgW (29.9%), their tubular shape and parallel orientation to the rock surface could be responsible for the more diffuse and deeper green band of microbial colonization observed compared to the thinner band observed for IgD. Micropores could have a greater water retention capacity and reduced evaporation rate, because, after a wetting event they can trap and more easily retain water than larger pores [44,46,58,60,62]. According to Omelon et al. [46], the presence of water in micropores, especially those with translucent walls such as ignimbrite pores, can enhance light penetration and therefore increase the light available for photosynthesis in the cryptoendolithic habitat [46]. In addition, the lighter color of the IgW zone, probably attributable to its smaller proportions of total Fe (Table 1), could also lead to higher light transmittance through this texture [43]. Factors such as porosity and light distribution have been shown to play a key role in microbial community structure in polar deserts [46,48] and other environments [31,43,56], and may differ at the microscale level. Both clone libraries and DGGE gels revealed a low number of cyanobacterial phylotypes in our rock samples, most of which were related to the genus Chroococcidiopsis, suggesting a very low diversity of photosynthetic microorganisms. Three cyanobacterial phylotypes (I, II and III) common to both ignimbrite zones and one phylotype (IV) only present in IgD

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Fig. 5. Consensus phylogenetic tree (50%-majority rule) based on the 16S rRNA gene region (604 pb) reconstructed using Bayesian inference and maximum likelihood to infer the phylogenetic positions of the OTUs recovered from the Ig clone libraries (IgD pink, IgW blue). Branches in bold are supported by both methods with Bayesian posterior probability (PP) â&#x2030;Ľ 95% and bootstrap probability (BP) > 70%. One asterisk indicates a branch supported only by PP, and two asterisks a branch supported only by BP. Chloroflexus aggregans, Deinococcus radiodurans and Thermus sp. sequences were used as outgroups. In the right margin of the figure, we provide sequences generated in previous studies on microbial communities of the Atacama Desert.

were detected by DGGE and led to the identification of two potential species from distinct genera. In addition, our clone libraries revealed four OTUs of three different genera, one more than those detected through DGGE. Two of these taxonomic units (OTU-1 and OTU-2) were common to both textures and the other two were unique to IgD (OTU-3) or IgW (OTU-4). The latter represented a high proportion of clones in their corresponding textures and clustered in the same clade in the 16S rRNA phylogenetic tree, indicating their same genus but different species. The taxonomic affiliations of OTU-3 and OTU-4 could not be resolved though they seemed to be related to two Oscillatoria strains. In a phylogenetic study of Chroococcidiopsis taxa, two oscillatorian strains have been found to cluster with statistical support within Chroccoccidiopsis (Donne 2013, PhD dissertation). According to the authors of this study, during phylogenetic analysis hyper-variable regions important for the separation of Chroococcidiopsis and these oscillatorian taxa could have been removed in the multi-

sequence alignment. In effect, the 16S rRNA gene is commonly used as genetic marker for the phylogenetic analysis of cyanobacteria, but this marker is sometimes not able to resolve relationships among very closely related organisms [35]. The detection of different phylotypes in the two micromorphological ignimbrite zones suggests cyanobacterial composition variations across a small spatial scale associated with different textures. Such microvariations in cyanobacterial diversity could be attributable to slightly different conditions in terms of availability of water and light. Further work is needed, including light transmittance measurements, to address this hypothesis. To confirm the idea that cyanobacteria were the only phototrophic microorganisms comprising the microbial communities detected, we also assessed the presence of eukaryotic microorganisms using different primer set and PCR condition combinations (data not shown), though no amplification products were obtained. Moreover, neither could we detect DGGE

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bands corresponding to algae. Wierzchos et al. [62] reached a similar conclusion after their microscopy analysis of IgD rock samples from the same region. Cyanobacteria are ubiquitous in the volcanic rocks of many terrestrial environments [11,23,31,36,37]. However, in early successional volcanic communities such as those found in lava flows following the recent eruption of a volcano in southern Iceland, phototrophy is not thought to be a dominant biogeochemical process [38]. Considering the Pliocene and Upper Miocene [17,21] age of the ignimbrite rocks of the Preandean Depression area of the Atacama Desert, an early successional stage for the endolithic communities examined here can be ruled out especially since cyanobacteria are the main photosynthetic members of these microbial communities. The presence of the genus Chroococcidiopsis as a component of the photosynthetic endolithic community in Lomas de Tilocalar is not surprising. Several studies have identified periodic desiccation as one of the most determining factors for microbial colonization of terrestrial volcanic rocks mainly comprising desiccation resistant or spore forming microorganisms [11]. Chroococcidiopsis spp. are known for their ability to withstand long desiccation periods [25] and cell damage by UV radiation [6], which makes this group of prokaryotes among the best-adapted to desert conditions [6,25]. The present findings broaden the spectrum of Atacama Desert rock substrates in which the presence of Chroococcidiopsis has been detected [18,39,40, 62]. The cyanobacterial phylotypes detected in our ignimbrite samples (IgD and IgW) were unrelated to endolithic and epilithic phylotypes recovered from other volcanic rocks [12,23,31,32,36–38]. In fact, most of the sequences obtained in this study showed most similarity with sequences of Chroococcidiopsis hypolithic clones from arid hot deserts including the Atacama Desert, Death Valley, Turpan Depression and Lybian Desert [3]. This suggests that microorganisms inhabiting the cryptoendolithic microhabitat of ignimbrite and hypolithic microhabitat of quartz are subjected to similar selective pressures giving rise to highly specialized microorganisms able to withstand long periods of desiccation. In effect, the potential for endolithic behavior of Antarctic hypolithic microorganisms has been recently shown [14]. In conclusion, the present findings contribute to the current understanding of the microbial diversity of both hot arid deserts and volcanic rocks, and identify subtle physico-chemical rock properties as determinants of microbial colonization patterns. Our findings provide direction for future studies, in-

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volving a greater sampling effort and high-throughput technologies, designed to gain further insight into endolithic microbial diversity and the impacts of textural features on this diversity and ecosystem functioning. Acknowledgments. The authors thank F. Pinto, V. Souza-Egipsy, and T. Carnota for help with the SEM-BSE observations, M.J. Malo for help with the molecular biology techniques, R. Gonzalez and M. Juanco for help with the MIP work, D. Herrera for help with sample collection in the Atacama Desert, Ana Burton for editorial assistance and reviewers for their constructive comments. This study was supported by grants CGL2010-16004 and CTM2012-38222-C02-02 and CGL2013-42509 from the Spanish Ministry of Economy and Competitiveness and by a predoctoral FPI fellowship program (BES-2007-15145).

Competing interests. None declared.

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PERSPECTIVES IÄã ÙÄ ã®ÊÄ ½ M® ÙÊ ®Ê½Ê¦ù (2014) 17:247-251 doi:10.2436/20.1501.01.227 ISSN (print): 1139-6709. e-ISSN: 1618-1095 www.im.microbios.org

ALLEA Statement on Enhancement of Open Access to Scientific Publications in Europe* . ALLEA Permanent Working Group on Intellectual Property Rights

Open Access to scientific publications is one among several other policies that will accelerate the move towards Open Science In its April 2012 declaration entitled “Open Science for the 21st century”, ALLEA stressed the need to promote (i) access to scientific publications as soon and as freely as possible (hereafter “Open Access” or “OA”), (ii) the development of open platforms allowing access to research data that are discoverable and re-usable (hereafter “Open Data”), (iii) support for interoperable e-infrastructures to manage the scale of future data flows (hereafter “Open e-Infrastructure”), (iv) the culture of open science based on online collaborations and high standards of quality and integrity (hereafter “Open Scientific Culture”). OA is a crucial element in reaching an Open Science model that will flourish rapidly. But the transition to Open Science requires more than just a fine-tuned policy on OA to scientific publications. While Open Data and Open Infrastructure mainly require the support of, and funding by, public authorities, OA to scientific publications requires a redesign of how scientific researchers, editors of learned journals, research funding bodies, libraries and archiving institutions interact with the publishing industry. In contrast to policies geared towards Open Data, Open e-Infrastructure or Open Scientific Culture, an OA policy can conflict with the copyright-based claims made by the publishers who, in general, are by assignment the owners of copyright on journal articles [1]. There is a need to

respond to some demands of journal publishers [2], since their views on the publication process and on the legacy of the past cannot simply be disregarded. Ignoring them may help to explain why the implementation of the OA model has been somewhat delayed. ALLEA urges public authorities and funding institutions to adopt concrete steps towards an OA model [3].

The traditional system for the publication and dissemination of scientific journals has shown some limits The revenues of the scientific, technical and medical (hereafter the “scientific”) publishers amounted to €24.9 billion for 2010, with a growth of 4.3% compared to 2009, not with standing the difficult economic situation [4]. The scientific publishing sector is now quite concentrated with big players such as Elsevier (2200 journals, including Cell and The Lancet), Springer (around 2000 journals), Wiley-Blackwell (1500) and the Nature Publishing Group [5]. Scientific publishing still appears to be a profitable business. At the same time, the cost of journals for libraries has risen dramatically. According to the libraries, the payments for journals quadrupled between 1986 and 2011, with an average annual increase of 3.5% above inflation. “This increase cannot only be explained by the increased number of scientific articles published” [see COM(2012) 410 final, p. 4]. This leads to the conclusion that public bodies which sub-

*This statement was prepared on October 2013 by the ALLEA Permanent Working Group on Intellectual Property Rights. ALLEA, the federaƟon of All European Academies, works to contribute to improving the framework condiƟons under which science and scholarship can excel. The working group was chaired by Prof. Joseph Straus (Union of the German Academies of Sciences and HumaniƟes) and Prof. Alain Strowel (Saint-Louis University, Brussels) is the lead author of the statement. It can be accessed at hƩp://www.allea.org/Content/ALLEA/Statement_ALLEA_Open_Access_2013-11.pdf. This arƟcle is published here with the permission of ALLEA.

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sidise research have also to pay for permitting other researchers to access published research results. But scientific publishers also include smaller players, for instance many University presses and learned societies, whose economic model might substantially differ. Not all academic publishers operate solely for commercial gain and the implementation of OA should be rolled out in such a way as to preserve the best of existing publishing practices. It is useful to note that many not-for-profit organisations such as academies, learned societies and professional associations raise a substantial part of their income from their publishing activities and this is then used to cross-subsidise other parts of the research system such as early career fellowships, mobility grants, etc. Any OA policy has to take into account the varying situations of publishers. In particular, large publishers may enhance revenue by offering electronic (and/or paper) journals in packages, with the result that libraries may be obliged to subscribe to the whole bundle, although they are only interested in some parts of it. In contrast, small publishers may well not have the stock to engage in such a practice; and so may be free from any objection of this kind. Some members of the scientific community have quite properly voiced their concern about the rising cost of accessing knowledge. Others have even called for the boycott certain publishers. The objections are particularly acute in the field of natural and medical sciences, probably less for journals in the humanities and social sciences, such as economics, politics, history and law reviews. A new compact between the different parties involved in the financing of research, the production of scientific articles, their assessment through peer-review, their dissemination and their preservation appears necessary. The tensions with commercial publishers and some entrenched practices in journal publishing probably slow down the indispensable move towards an OA model.

Open Access relies on fundamental legal principles and is rightly supported by authorities, in particular the European Commission i) Fundamental legal principles OA is supported by the right “to share in scientific advancement and its benefits” that is enshrined in Article 27(1) 01 the 1948 Universal Declaration of Human Rights, a principle that has become a binding norm as Article 15 of the International Covenant on Economic, Social and Cultural Rights (1966). At

ALLEA

the same time, Article 27(2) recognises “the right to the protection the moral and material interests resulting from any scientific, (...) production of which he is the author”. In Europe, the freedom of scientific research is recognized by Article 13 of the Charter of Fundamental Rights, while “intellectual property” is equally protected under Article 17(2) of the Charter. ii) Towards OA in Europe The Berlin Declaration on OA of 2003 was a landmark in the drive towards better access to scientific materials. Since then, several national and international bodies have pleaded in favour of OA. For many years, the European Commission has supported the move to OA. In its “Horizon 2020” which follows the previous Framework Programs, the Commission envisages that all research results should be made freely accessible online. In a July 2012 Communication entitled “Towards better access to scientific information: Boosting the benefits public investments in research” (COM(2012) 401 final), the Commission has identified some barriers hindering the transition to OA. The lack of coordination between universities, research institutions and libraries, the absence of a transparent path for moving out of the standard publishing model, the lack of information and infrastructure that will allow researchers to comply easily with OA via self-archiving, the fear of contractual disagreements with their existing publisher and the absence of mechanisms for enforcing OA policies, all help to explain why the transition to OA is slow. In its July 2012 Recommendation on access to and preservation of scientific information (C(2012) 4890 final), the Commission distinguished several issues that require action: on top of recommending “open access to scientific publications”, the Commission advocates the “open access to research data” (e.g. searchable and linked datasets), the “preservation and re-use of scientific information” (e.g. system of electronic deposit), the development of “e-infrastructures” (the electronic systems for underpinning the dissemination of scientific information), the multi-stakeholder dialogue at different levels and the coordination between Member States. iii) Towards OA in the U.S. On February 22, 2013, President Obama’s Executive Office issued a memorandum on “Increasing Access to the Results of Federally Funded Scientific Research”. Under the Name “Public Access to Scientific Publications”, this document stresses that the results of unclassified research that are published in peer-reviewed publications directly arising from

ALLEA ON OPEN ACCESS.

Federal funding should be stored for preservation in the long term. Also those publications should be made “publicly accessible to search, retrieve, and analyse in ways that maximize the impact and accountability of the Federal research investment”. In developing this Public Access policy, the U.S. agencies are asked to “maximiz(e) the potential to create new business opportunities” and to “prevent the unauthorized mass redistribution of scholarly publications”. iv) Positive impact of OA Similarly, ALLEA believes that, on top of the obvious gains in terms of improved access, the development of OA could create new business opportunities and reduce the level of unauthorised dissemination of publications. Publishers might play a new and important role in an OA model that would reduce the financial burden for libraries, research organisations, universities and, ultimately, the funding institutions. At the same time, the move towards OA does not mean that copyright has norole to play in the open environment: rather than ensuring revenues directly commensurate to the number of copies distributed, copyright, and in particular its principles on attribution of authorship and integrity of works, should govern the Open Scientific Culture that goes along with OpenScience. However, it would be naïve to think that OA will automatically reduce the financial burden for the funding institutions. It might even grow initially when the OA infrastructures are being established.

ALLEA supports the European and U.S. policy objectives for OA relating to scientific publications, and urges that steps towards implementation be set in train ALLEA fully supports the European Commission’s recommendations of July 2012. In particular, ALLEA wants to stress the need to: In general: • “Define clear policies for the dissemination of and OA to scientific publications resulting from publicly funded research”; beyond general policies, concrete objectives and indicators should be used, based on implementation plans and awareness programs; • Put in place much needed financial planning for the move to OA; For the funding institutions: • Ensure that they define clear policies for OA to the publications resulting from the funded projects;

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• Include in the career evaluation of researchers not only traditional publications in (peer-reviewed) journals, but also publications in open mode; For the timing of OA implementation and the embargo periods: • Require OA to be implemented as soon as possible. Some flexibility is needed; in certain areas of research, shorter embargos make sense; For the public institutions involved in the negotiation with publishers: • Improve transparency about the terms and conditions negotiated between publishers and public institutions which foster research; • Promote partnerships between public institutions (in particular libraries) at national and European level; For the researchers: • Give guidance to researchers on how to comply with OA policies and make them more aware of what the standard publishing contracts allow them to do(for example authors tend to underestimate what they can do with pre-publication versions, e.g. self-archiving, use in course packs, etc.); • Foster the awareness among researchers of the copyright licences needed for OA to be quickly implemented and “encourage researchers to retain their copyright while granting licences to publishers”; • Support the academic careers of researchers who actively share the results of their research; For entrepreneurs who directly need access to scientific knowledge: • Allow unaffiliated persons and SMEs to access scientific publications under reasonable conditions. ALLEA also supports the adoption by European funding agencies of objectives similar to those outlined in the February 2013 memorandum of the Obama administration: • “Ensure that the public can read, download, and analyse in digital form final peer-reviewed manuscripts or final published documents”; • “Ensure full public access to the metadata of publications without charge upon first publication in a data format that ensures interoperability with current and future search technology”; • “Ensure that attribution to authors, journals, and original publishers is maintained”;

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ALLEA

In its July 2012 Communication, the Commission retains the usual distinction between “Gold” OA and “Green” OA: while Gold OA shifts the payment publication costs from readers (vi a subscriptions) to researchers and their institution, Green OA is synonymous with self-archiving [6].

Some disciplines (e.g. astrophysics) have a long-standing, researcher driven commitment to use of OA tools to drive scholarly communication, while others have yet to embark in a meaningful way upon an OA pathway. The implementation of a Gold model must allow for different pace and level of engagement across the disciplines. Funding institutions should be encouraged to outline clearly how they will support and fund meaningful OA. A key element of this should be a commitment to resource OA as a specific item within research grants made by public research funders. The implementation of a retrospective requirement for OA should be avoided. A worrying feature of any author-pays model is that it could inhibit publication by independent or under-funded researchers, for instance coming from less wealthy countries. This is another reason for not favouring a Gold model across the board. ALLEA is opposed to a research assessment system that would only take Gold publications into account: the adoption of such an assessment system would very probably lead to an increase of the price to be paid for Gold publications, as researchers and institutions will be locked in the Gold OA model.

i) Gold OA Gold OA is favoured by scientific publishers and sometimes supported by public authorities. In the UK for instance, the government considers that the results of all publicly funded research should preferably be published in the Gold mode. However, the government did not indicate how it would be financed [7]. In the Commission’s FP7 and under Horizon 2020, Gold OA is eligible for funding as part of research grants. The Gold OA might present some advantages, but ALLEA stresses that the price for a publication under the Gold OA must remain reasonable. It appears that the price to be paid for a Gold publication is usually between €1500 and €5000 [8]. According to some experts, a fee between €500 and €1000 would appear reasonable [9]. The publishers should remain reasonable in setting the price for the Gold model. This price should cover the costs resulting from publishing and be as transparent as possible. Public authorities should ensure that the price asked by publishers remains commensurate with the overall funding of the project. For large scientific projects, it is easier to allocate a reasonable amount for Gold publication; for research projects supported by smaller grants, such as in the humanities and social sciences, the payment of the same fee might not appear adequate. Thus the Gold model could be favoured in certain fields and for large projects.

ii) Green OA In the “Green” model, the published and peer-reviewed article “is archived by the researcher in an online repository before, after or alongside its publication” [COM(2012) 410 final, p. 5]. Publishers can recoup their investment by selling subscriptions and charging pay-per-download/view fees during the embargo period and after. ALLEA tends to favour the Green model for humanities and social sciences. But the Green model could also apply to small research projects in other disciplines. This model supports the long-standing scholarly principle of “freedom to publish” by ensuring that researchers retain ultimate authority as to where and how they publish their scholarly outputs. A short embargo should apply. The embargo could vary depending on the discipline. In last moving research fields, the embargo could be for six months; some fields like physics and maths are relatively slow moving, and a longer embargo thus appears adequate. Efforts should also be made to ensure that a draft version can be archived before the publication (but after peer review clears the way) and that, more importantly, the final version is archived alongside the publication in the journal. To maintain the high quality of scientific literature is of utmost importance. There are indications of an increasing

• “Ensure that publications and metadata are stored in an archive that i) provides for long-term preservation and access to the content without charge (and) ii) uses standards, widely available and, to the extent possible, non-proprietary archival formats for texts and associated content”. Now that there is a broad consensus with regard to the policy orientations in Europe and in the U.S., all measures supporting OA should be implemented within a strict time frame.

ALLEA in particular supports the Green OA model, but invites funding institutions and public authorities to help the scientific community to put in place self-archiving solutions

ALLEA ON OPEN ACCESS.

number of cases of misconduct in research, and therefore high quality peer review is more important than ever. In a model where the researcher pays for publication, it may be tempting for publishers to accept contributions of questionable scientific quality. Therefore, it appears necessary to define standards to be applied by the publishers for high quality peer review. iii) In General Although ALLEA supports an OA policy, both the Gold and the Green models may create problems. It is essential to address those problems. ALLEA encourages the European Commission to assess OA policies so as to enable policymakers and the scientific and scholarly community to understand better the costs, savings and benefits arising from OA. Various licence models could be adopted for the Gold and Green OA models. ALLEA believes that most researchers would favour a model of open licence that requires the author to be named (attribution), but prohibits commercial re-use (model of the Creative Commons - BY - NC). Further consultation with the research communities is needed before a model is agreed upon for this element of OA practice. The best solution may be to leave some choice as to the type of open licence to adopt. ALLEA also considers that OA, which allows short-term access to publications, should be complemented by a system ensuring the long-term preservation of publications (and research data). This could be done by an effective system of deposit, but also through the preservation of the hardware and software needed to read the publications (and data) in the future. It is also essential that the universities and research institutions put in place a repository system. The European Commission should fund the development of those institutional repositories. It should also define the standards for online repositories (this also relates to the need to invest in e-Infrastructure; see above on the factors that promote Open Science). A ranking of repositories might be a way to indicate quality standards. More should be done to assess the quality of OA repositories. It is probably not useful to have OA repositories containing pre-prints, working papers and postprints all together in the same spot. The lack of quality standards for repositories is a disincentive for scientists to publish under an OA model. ALLEA hopes that moving to OA will help scientific insti-

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tutions to save money, but it is important to realize that an OA model might impose new burdens on researchers and their employers. New tasks for the researchers should in any case be kept to a minimum. As stressed by five leading UK learned societies: “Implementing OA policies will require a substantial shift in community altitudes and behaviour in some disciplines, and all stakeholders need to increase their efforts to communicate more effectively with researchers” [10]. This is also an important element to be taken into account by the European authorities before embarking on a possibly far-reaching reform of the practices of scientific publication. The policy and guidelines to be adopted should in any case take into account the important differences which exist between the interests of scientists and publishers in the area of natural sciences, on one side, and in the area of humanities and social science, on the other. Notes 1.

Within the bread issue of open access to scientific information, it is thus important to distinguish the issue of open access to peer- reviewed research articles (referred to as Open Access or OA) and the issue of access to scientific research data (referred to as Open Data). 2. In its July 2012 Recommendation (C(2012) 4890 final), the Commission mentions that “(15) Given the transitional state of the publishing sector, stakeholders need to come together to accompany the transition process and look for sustainable solutions for the scientific publishing process”. 3. For example, in September 2012, the UK announced a ₤ 10 million investment to help universities with the transition to open access to publiclyfunded research findings and to kick-start the process of developing policies and setting up funds to meet the costs of article processing charges (see: http://www.stm -assoc.org/industry-news/uk-government-invests10-million-gbp-to-help-unversities-move-to-open-access/). 4. See: http://www.stm-assec.org/wp-content/uploads/STMStatOct2011. jpg. 5. Le Monde, March 2, 2013, p. 4 Supplement. 6. According to the Commission’s Communication (p. 5), “currently some 20 % of all scientific articles are available in open access form,60 % of which follow the “Green’ model”. 7. More clearly, the Wellcome Trust has said the Gold OA should be paid out of the research grant which would be adjusted accordingly. 8. Le Monde, March 2, 2013, p. 5 Supplement. 9. B. Rentier, President of the University of Liège, quoted in Le Monde, March 2, 2013, p.5 Supplement. 10. Open Access in the UK and what it means for scientific research. A joint statement from The Academy of Medical Sciences, the Institute of Physics, the Royal Society, the Royal Society of Chemistry, and the Society of Biology, February 2013, p. 2. Accessed at: https://royalsociety.org/uploadedFiles/Royal_Society_Content/z_events/2013/scientific-discussion/oa-workshop/2013-Open-Access-Joint-Statement.pdf.

INDEX VOLUME 17 INTERNATIONAL MICROBIOLOGY (2014) www.im.microbios.org

Contents Volume 17 · 2014 Aguilera A  González-Toril E ALLEA Working Group  ALLEA Statement on Enhancement of Open Access to Scientific Publications in Europe, 247 doi:10.2436/20.1501.01.227 Alippi AM  Tetracycline-resistance encoding plasmids from different Paenibacillus larvae, the causal agent of american foulbrood disease, isolated from commerical honeys, 49 Álvarez JR  López-García MT Amils R  González-Toril E Aragon V  Manrique-Ramírez P Ariza-Miguel J  Molecular epidemiology of methicillin-resistant Staphylococcus aureus in a university hospital in northwestern Spain, 149 doi:10.2436/20.1501.01.217 Artieda O  Cámara B Ascaso C  Cámara B Badosa E  Bonaterra A Baixeras J  Carrasco P Bañeras L  Ramió-Pujol S Barbé J  Spricigo DA Barrio E  Pérez-Través L Becerra A  A phylogenetic approach to the early evolution of autotrophy: the case of the reverse TCA and the reductive acetyl-CoA pathways, 91 doi:10.2436/20.1501.01.211 Bengoechea JA  Spricigo DA Berlanga M  Biofilm formation on polystyrene in detached vs. planktonic cells of polyhydroxyalkanoate-accumulating Halomonas venusta, 205 doi:10.2436/20.1501.01.223 Bevilacqua N  Rizzo A Biffi S  Chiellini C Bonaterra A  Phenotypic comparison of clinical and plant-beneficial strains of Pantoea agglomerans, 81 doi:10.2436/20.1501.01.210 Borrull A  López-Martínez G Buommino E Rizzo A Cámara B  Ignimbrite textural properties as determinants of endolithic colonization patterns from hyper-arid Atacama Desert, 235 doi:10.2436/20.1501.01.226 Carrasco P  Succession of the gut microbiota in the cockroach Blattella germanica, 99 doi:10.2436/20.1501.01.212 Carratelli CR  Rizzo A Castro JA  Matas M

Castro N  A multiplex PCR for the simultaneous detection of Tenacibaculum maritimum and Edwardsiella tarda in aquaculture, 111 doi:10.2436/20.1501.01.213 Chaillou S  Lhomme E Champomier-Vergès MC  Lhomme E Chiellini C  Endophytic and rhizospheric bacterial communities isolated from the medicinal plants Echinacea purpurea and Echinacea angustifolia, 165 doi:10.2436/20.1501.01.219 Chiron H  Lhomme E Cifuentes C  Matas M Colprim J  Ramió-Pujol S Cordero-Otero R  López-Martínez G Cortés P  Spricigo DA Crognale S  Luziatelli F D’Annibale A  Luziatelli F de Araújo DAM  Padilha IQM De Filippis A  Rizzo A de los Ríos A  Cámara B Diaz P  Padilha IQM Domènech Ò  Berlanga M Dousset X  Lhomme E Ducasse MB  Lhomme E Duffy B  Bonaterra A Emiliani G  Chiellini C Esteban GF  Yeates AM Fabiani A  Chiellini C Fani R  Chiellini C Fernández-Natal I  Ariza-Miguel J Firenzuoli F  Chiellini C Gallo E  Chiellini C Galofré-Milà N  Manrique-Ramírez P Ganigué R  Ramió-Pujol S García-Ferris C  Becerra A García-Moyano A  González-Toril E González-Candelas  Matas M González-Toril E  Comparative microbial ecology of the water column of an extreme acidic pit lake, Nuestra Señora del Carmen, and the Río Tinto basin (Iberian Pyrite Belt), 225 doi:10.2436/20.1501.01.225 Gori L  Chiellini C Grisi TCSL  Padilha IQM Guerrero R  Berlanga M Hernández M  Ariza-Miguel J Homar  Matas M

Kolter R  Romero D Latorre A  Carrasco P Lazcano A  Becerra A León IE  Alippi AM Lhomme E  A polyphasic approach to study the dynamics of microbial population of an organic wheat sourdough during its conversion to gluten-free sourdough, 1 doi:10.2436/20.1501.01.202 Llagostera M  Spricigo DA Lopes CA  Pérez-Través L López AC  Alippi AM López-García MT  Cell immobilization of Streptomyces coelicolor: effect on differentiation anad actinorhodin production, 75 doi:10.2436/20.1501.01.209 López-Martínez G  Metabolomic characterization of yeast cells after dehydration stress, 131 doi:10.2436/20.1501.01.215 López-Labrador FX  Matas M López-Pamo E  González-Toril E Luziatelli F  Screening, isolation, and characterization of glycosyl-hydrolaseproducing fungi from desert halophyte plants, 41 doi:10.2436/20.1501.01.206 Magariños B  Castro N Maggini V  Chiellini C Maida I  Chiellini C Manrique-Ramírez P  Identification of a class B acid phosphatase in Haemophilus parasuis, 141 doi:10.2436/20.1501.01.216 Manteca Á  López-García MT Matas M  Relating the outcome of HCV infection and different host SNP polymorphisms in a Majorcan population coinfected with HCV-HIV and treated with pegIFN-RBV, 11 doi:10.2436/20.1501.01.203 Mengoni A  Chiellini C Mezaize S  Lhomme E Mocali S  Chiellini C Montesinos  Bonaterra A Moragues MD  Sáez-Rosón A Moranta D  Spricigo DA Moresi M  Luziatelli F Moya A  Carrasco P Moya A  Matas M Nealson KH  Cámara B

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Onno B  Lhomme E Padilha IQM  A glucuronoxylan-specific xylanase from a new Paenibacillus favisporus strain isolated from tropical soil of Brazil, 175 doi:10.2436/20.1501.01.220 Pastor FIJ  Padilha IQM Payeras A  Matas M Peretó J  Becerra A Pérez-Cobas AE  Carrasco P Pérez-Través L  Stabilization process in Saccharomyces intra- and interspecific hybrids in fermentative conditions, 213 doi:10.2436/20.1501.01.224 Petruccioli M  Luziatelli F Picornell A  Matas M Poblet M  López-Martínez G Querol A  Pérez-Través L Ramió-Pujol S  Impact of formate on growth and productivity of Clostridium ljungdahlii PETC and Clostridium carboxidivorans P7 grown on syngas, 195 doi:10.2436/20.1501.01.222

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Ramon C  Matas M Rezzonico F  Bonaterra A Rioseras B  López-García MT Rivas M  Becerra A Rizzo A  Transforming activities of Chlamydia pneumoniae in human mesothelial cells, 185 doi:10.2436/20.1501.01.221 Rodríguez-Lázaro D  Ariza-Miguel J Romero D  Functional amyloids in bacteria, 65 Doi:10.2436/20.1501.01.208 Rozès N  López-Martínez G Ruzzi M  Luziatelli F Sáez-Rosón A  Identification of superficial Candida albicans germ tube antigens in a rabbit model of disseminated candidiasis. A proteomic approach, 21 Doi:10.2436/20.1501.01.204 Santofimia E  González-Toril E Serrano M  Manrique-Ramírez P Sevilla MJ  Sáez-Rosón A Spricigo DA  Significance of tagI and mfd genes in the virulence of non-typeable Haemophilus influenzae, 159 doi:10.2436/20.1501.01.218 Suzuki S  Cámara B

Toranzo AE  Castro N Tufano MA  Rizzo A Valenzuela SV  Padilha IQM van de Pol C  Carrasco P Vannacci A  Chiellini C Wierzchos J  Cámara B Yagüe P  López-García MT Yeates AM  Local ciliate communities associated with aquatic macrophytes, 31 doi:10.2436/20.1501.01.205 Zagorec M  Lhomme E

Authors Index · 2014 Aguilera A  225 Alipppi AM  49 Álvarez JR  75 Amils R  225 Aragon V  141 Ariza-Miguel J  149 Artieda O  235 Ascaso C  235 Badosa E  81 Baixeras J  99 Bañeras L  195 Barbé J  159 Barrio E  213 Becerra A  91 Bengoechea JA  159 Berlanga M  205 Bevilacqua N  185 Biffi S  165 Bonaterra A  81 Borrull A  131 Buommino E  185 Cámara B  235 Carrasco P  99 Carratelli CR  185 Castro JA  11 Castro N  111 Chaillou S  1 Champomier-Vergès MC  1 Chiellini C  165 Chiron H  1 Cifuentes C  11 Colprim J  195 Cordero-Otero R  131 Cortés P  159 Crognale S  41 D’Annibale A  41 de Araújo DAM  175 de Filippis A  185 de los Ríos A  235 Diaz P  175 Domènech Ò  205 Dousset X  1 Ducasse MB  1 Duffy B  81 Emiliani G  165 Esteban GF  31

Fabiani A  165 Fani R  165 Fernández-Natal I  149 Firenzuoli F  165 Gallo E  165 Galofré-Milà N  141 Ganigué R  195 García-Ferris C  91 García-Moyano A  225 González-Candelas F  11 González-Toril E  225 Gori L  165 Grisi TCSL  175 Guerrero R  205 Hernández M  149 Homar F  11 Kolter R  65 Latorre A  99 Lazcano A  91 León IE  49 Lhomme E  1 Llagostera M  159 Lopes CA  213 López AC  49 López-García MT  75 López-Labrador FX  11 López-Martínez G  131 López-Pamo E  225 Luziatelli F  41 Magariños B  111 Maggini V  165 Maida I  165 Manrique-Ramírez P  141 Manteca A  75 Matas M  11 Mengoni A  165 Mezaize S  1 Mocali S  165 Montesinos E  81 Moragues MD  21 Moranta D  159 Moresi M  41 Moya A  11 Moya A  99 Nealson NH  235

Onno B  1 Padilha IQM  175 Payeras A  11 Peretó J  91 Pérez-Cobas AE  99 Pérez-Través L  213 Petruccioli M  41 Picornell A  11 Poblet M  131 Querol A  213 Ramió-Pujol S  195 Ramon C  11 Rezzonico F  81 Rioseras B  75 Rivas M  91 Rizzo A  185 Rodríguez-Lázaro D  149 Romero D  65 Rozès N  131 Ruzzi M  41 Sáez-Rosón A  21 Santofimia E  225 Serrano M  141 Sevilla MJ  21 Spricigo DA  159 Suzuki S  235 Toranzo AE  111 Tufano MA  185 Valenzuela SV  175 van de Pol C  99 Vannacci A  165 Wierzchos J  235 Yagüe P  75 Yeates AM  31 Zagorec M  1

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Keywords Index · 2014 16S rRNA gene 99 Acidic pit lakes 225 Acidophilic microorganisms 225 Adhesion on polystyrene 205 Alginate beads 205 Alkylating agents 159 American foulbrood disease (AFB) 49 Ames test 81 Amylase activity 41 Anthrax 119 Antibiotics 75 Aquaculture 111 Atacama desert 235 Autotrophic pathways 91 Bacillus subtilis 65 Bacterial biofilms 65 Bactericidal index 17(4) Biocontrol 81 Biofilms 31 Biofuels 195 Biosafety 81 Blattella germanica 99 Caenorhabditis elegans 21 Candida 1 Candida albicans 11 Cell death 75 Cell surface physicochemical characteristics 205 Cellular transformation 185 Cellulase activity 41 Chlamydia pneumoniae 185 Chroococcidiopsis 235 Ciliates 31 Clonal population 149 Clostridium carboxidivorans 195 Clostridium ljungdhalii 195 Cockroach gut microbiota 99 Cytotoxicity 185 Dehydration stress 131 Differentiation 75 DNA content evaluation 213 Echinacea angustifolia 165 Echinacea purpurea 165 Ecological succession 99 Edwardsiella tarda 111 Encapsulation 75 Endoliths 235 Endophytes 165 Endosymbionts 99 Erwinia amylovora 81

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Extracellular matrix 65 Fish pathology 111 Formate 195 Genes tagI and mfd 159 Germ tube antibodies 21 Glässer’s disease 141 Gluten-free food 1 Glycosyl hydrolases GH30 175 Haemophilus influenzae 159 Haemophilus parasuis 141 HCV-HIV co-infection 11 Halomonas venusta MAT-28 205 Hemolytic activity 81 Honeybees 49 Human mesothelial cells 185 Hydrophobicity 17(4) Iberian Pyrite Belt 225 Ignimbrite 235 Invasive candidiasis 21 Iron cycle 225 Lactic acid bacteria 1 Lactobacillus spp 1 Last common ancestor (LCA-LUCA) 91 Lewis acid-base properties 17(4) Macrophytes 31 Medicinal plants 165 Meloidogyne javanica 81 Metabolite extraction 131 Methicillin-resistant Staphylococcus aureus (MRSA) 149 Microbial biodiversity 31 Molecular epidemiology 149 Molecular markers 213 mtDNA halogroups 11 Multilocus sequence typing 149 Multiplex PCR 111

Pantoea agglomerans 81 Penicillium expansum 81 PHA 205 Phosphatase activity 141 Plasmids 49 Rabbit model 21 Ranunculus 31 Rare-mating in yeast 213 Reverse Krebs cycle 91 Rhizosphere 165 Rio Tinto 225 Rock porosity 235 Saccharomyces 1 Saccharomyces cerevisiae 131, 213 Saccharomyces kudriavzevii 213 Saccharomyces mikatae 131 Schizophyllum commune 41 SNP polymorphisms 11 Sourdough 1 Stabilization of genomes 213 Streptomyces coelicolor 75 Susceptibility to antibiotics 17(4) Syngas fermentation 195 Tamarix ssp 41 TasA amyloid-like fibers 65 Tenacibaculum maritimum 111 Tetracycline resistance 49 Toxicity 81 Toxin phenethyl isothiocyanate (PEITC) 31 Tumoral markers 185 Viability 131 Virulence 159 Volcanic rock 235 Wild yeast 131 Wood-Ljungdahl pathway 91 Xylanase 175

Nasturtium 31 Naumovia castellii 131 Non-specific acid phosphatases 141 Open Access 247 Organic 1 Origin of life 91 Paenibacillus favisporus 175 Paenibacillus larvae 49

Yeast 1

List of reviewers · 2014 The editorial staff of INTERNATIONAL MICROBIOLOGY thanks the following persons for their invaluable assistance in reviewing manuscripts from January through December 2014. The names of several reviewers have been omitted at their request. Aguirre García, Juan. Univer. of Prince Edward Island, Charlottetown, Canada Amils, Ricardo. Autonomous University of Madrid, Madrid, Spain Antón, Josefa. Alicante, University Alicante, Spain Ayala, Juan. Autonomous University of Madrid, Madrid, Spain Balboa, Sabela. Sheffield University, Sheffield, UK Barberan, Albert. University of Colorado, Boulder, CO, USA Barea, José Miguel. Experimental Station El Zaidín-CSIC, Granada, Spain Beaz, Roxana. University Rovira Virgili, Reus, Spain Bely, Peter. Slovak Academy of Sciences, Bratislava, Slovakia Berlanga, Mercedes. University of Barcelona, Barcelona, Spain Bonaterra, Anna. University of Girona, Girona, Spain Boscia, Donato. Institute Sustainable Plant Protection, CNR, Torino, Italy, Bou, German. Institute for Biomedical Research, A Coruña, Spain Burgos, William D. The Pennsylvania State University, Univer. Park, PA, USA Campoy, Sussana. Autonomous University of Barcelona, Bellaterra, Spain Cantoral, Jesús Manuel. University of Cadiz, Cádiz, Spain Casadesús, Josep. University of Sevilla, Sevilla, Spain Chen, Ding. UT Southwestern Medical Center, Dallas, TX, USA Claus, Harald. Johannes Gutenberg-University of Mainz, Mainz, Germany Compant, Stephane. Austrian Institute of Technology GmbH, Tull, Austria Cubero, Jaime. INIA Plant Protection, Madrid, Spain De la Torre, M. Angeles. University of Lleida Lleida, Spain del Campo, Javier. University of British Columbia, Vancouver, Canada Díaz, Ramón. University of Navarra, Pamplona, Spain Edwards, François. Centre for Ecology and Hydrology, Wallingford, UK Engel, Philipp. University of Lausanne, Lausanne, Switzerland Espinosa, Manuel. Center of Biological Research, CSIC, Madrid, Spain Eulogio, Valentín. University of Valencia, Valencia, Spain Fromm, Hill. University of Tel Aviv, Tel Aviv, Israel Garcìa del Portillo, Francisco. Spanish Center for Biotechnol., Madrid, Spain Garmendia, Juncal. University of Navarra, Pamplona, Spain Giraldo, Rafael. Center for Biological Research, CSIC, Madrid, Spain Goldman, Aaron. Oberlin College Oberlin, OH, USA Gonzalez-Zorn, Bruno. University of Madrid, Madrid, Spain Gottschalk, Marcelo. University of Montreal, Montreal, Canada Guarner, Francisco. Vall d’Hebron Institute of Research, Barcelona, Spain Guarro, Josep. University Rovira Virgili, Reus, Spain Heiss-Blanquet, Senta. IFP New Energies, Rueil-Malmaison, France

Herrero, Enric. University of Lleida, Lleida, Spain Imperial, Juan. Technical University of Madrid, Madrid, Spain Köpke, Michael. Lanza Tech, Inc. Auckland, New Zealand Liras, Paloma. University of Leon, Leon, Spain López Calderón, Isabel. University of Sevilla, Sevilla, Spain Maberly. Stephen. Lancaster Environment Centre, Lancaster, UK McGlynn, Shawn. California Institute of Techology, Pasadena, CA, USA McPhillips, Katrina. Limerick Inst. of Techn., Appl. Sc., Limerick, Ireland Miller, Ana. Instituto de Recursos Naturales y Agroecología, Sevilla, Spain Mingorance, Jesús. Hospital Universitario La Paz, Madrid, Spain Mira, Alex.Fund Health & Biomed Res Oral Microbiome Lab.,Valencia, Spain Montesinos, Emili. University of Girona, Girona, Spain Mrvčić, Jasna. University of Zagreb, Croatia Penadés, José R. Research Institute in Livestock Mountain, Segorbe, Spain Piqueras, Mercè. Catalan Assoc. Science Comm., Barcelona, Spain Randez Gil, Francisca. IATA-CSIC, Paterna, Valencia Rementeria, Aitor. University of the Basque Country, Bilbao, Spain Ribas, Catalina. Center of Molecular Biology, CSIC, Madrid, Span Rivilla, Rafael. Autonomous University of Madrid, Madrid, Spain Rodríguez Navarro, Carlos. University of Granada, Granada, Spain Rosselló Mora, Ramon. University of the Balearic Island, Mallorca, Spain Russell, Michael. Jet Propulsion Laboratory, Pasadena, CA, USA Schereider, Eckart. Robert Koch-Institute, Berlin, Germany Solano, Cristina. Public University of Navarra-CSIC, Pamplona, Spain St John, Franz. University of Maryland, Baltimore, MD, USA Stockwell, Virginia. Oregon State University, OR, USA Tenreiro, Rogério. University of Lisbon, Lisbon, Portugal Vila, Jordi. University of Barcelona, Barcelona, Spain Villaverde, Antoni. Autonomous University of Barcelona, Barcelona, Spain Vindel, Ana. Institute of Health Carlos III, Madrid, Spain Viñas, Miquel. University of Barcelona, Barcelona, Spain Virolle, Marie-Joelle. University of Paris XI, Orsay, France Wang, Gaoyan. Cornell University, Cornell, NY, USA Wilkins, Mark. Oklahoma State University, Stilwater, OK, USA Yuste, José. Institute of Health Carlos III, Madrid, Spain Zaragoza, Óscar. National Center for Microbiology, Madrid, Span Zhao, Ling. University of Tennessee, Knoxville, TN, USA

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Acknowledgement of Institutional Subscriptions INTERNATIONAL MICROBIOLOGY staunchly supports the policy of open access (Open Access Initiative, see Int Microbiol 7:157161). Thus, the journal recognizes the help received from the many institutions and centers that pay for a subscription—in spite of the possibility to download complete and current issues of the journal free of charge. We would therefore like to thank those entities. Their generous contribution, together with the efforts of the many individuals involved in preparing each issue of INTERNATIONAL MICROBIOLOGY, makes publication of the journal possible and plays an important role in improving and expanding the field of microbiology in the world. Some of those institutions and centers are: Area de Microbiología. Departamento de Biología Aplicada. Universidad de Almería / Biblioteca. Institut Químic de Sarrià. Universitat Ramon Llull. Barcelona / Biblioteca. Instituto Nacional de Seguridad e Higiene en el Trabajo-Ministerio de Trabajo y Asuntos Sociales. Barcelona / Ecologia microbiana. Departament de Genètica i de Microbiologia. Universitat Autònoma de Barcelona. Bellaterra (Barcelona) / Biblioteca. Institut de Biotecnologia i Biomedicina. Universitat Autònoma de Barcelona. Bellaterra (Barcelona) / Laboratori d’Ecogenètica. Departament de Microbiologia. Universitat de Barcelona / Departament de Microbiologia i Parasitologia Sanitàries. Facultat de Farmàcia. Universitat de Barcelona / Societat Catalana de Biologia. Institut d’Estudis Catalans. Barcelona / Departamento de Microbiologia. Universidade Federal de Minas Gerais. Belo Horizonte. Brasil / Departamento de Inmunología, Microbiología y Parasitología, Universidad del País Vasco, UPV-EHU. Bilbao / Biblioteca. Universidad de Buenos Aires. Argentina / Biblioteca. Facultad de Ciencias. Universidad de Burgos / Biblioteca. Departamento de Producción Animal CIAM-Centro Mabegondo. Abegondo (Coruña) / Laboratorio de Microbioloxia. Universidade da Coruña. A Coruña / Biblioteca. Divisió

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Alimentària del IRTA-Centre de Tecnologia de la Carn. Generalitat de Catalunya. Monells (Girona) / Biblioteca Montilivi. Facultat de Ciències. Universitat de Girona / Área de Microbiología. Departamento de Ciencias de la Salud. Universidad de Jaén / Microbiologia. Departament de Ciències Mèdiques Bàsiques. Facultat de Medicina. Universitat de Lleida / Laboratorio de Microbiología Aplicada. Centro de Biología Molecular. Universidad Autónoma de Madrid-CSIC. Cantoblanco (Madrid) / Laboratorio de Patógenos Bacterianos Intracelulares. Centro Nacional de BiotecnologíaCSIC. Cantoblanco (Madrid) / Grupo de Investigación de Bioingeniería y Materiales (BIO-MAT). Escuela Técnica Superior de Ingenieros Industriales. Universidad Politécnica de Madrid / Biblioteca. Centro de Investigaciones Biológicas, CSIC. Madrid / Merck Sharp & Dohme de España. Madrid / Departamento de Microbiología. Facultad de Ciencias. Universidad de Málaga / Grupo de Fisiología Microbiana. Depto. de Genética y Microbiología. Universidad de Murcia. Espinardo (Murcia) / Library. Department of Geosciences. University of Massachusetts-Amherst. USA / Biblioteca de Ciencias. Universidad de Navarra. Pamplona / Grupo de Genética y Microbiología. Departamento de Producción Agraria. Universidad Pública de Navarra. Pamplona / Microbiología Ambiental. Departamento de Biología. Universidad de Puerto Rico. Río Piedras. Puerto Rico / Biblioteca General. Universidad San Francisco de Quito. Ecuador / Biblioteca. Facultat de Medicina. Universitat Rovira Virgili. Reus / Instituto de Microbiología Bioquímica-Departamento de Microbiología y Genética. CSIC-Universidad de Salamanca / Departamento de Microbiología y Parasitología. Universidad de Santiago de Compostela. Santiago de Compostela / Laboratorio de Referencia de E. coli (LREC). Facultad de Veterinaria. Universidad de Santiago de Compostela. Lugo / Departamento de Genética. Universidad de Sevilla / Tecnología de los Alimentos. Facultad de Ciencias. Universidad de Vigo / General Library. Marine Biological Laboratory. Woods Hole, Massachusetts, USA.

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S (2005) Architecture of a nascent Sphingomonas sp. biofilm under varied hydrodynamic conditions. Appl Environ Microbiol 71:2677-2686 Books Miller JH (1972) Experiments in molecular genetics. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA Book chapters Lo N, Eggleton P (2011) Termite phylogenetics and co-cladogenesis with symbionts. In: Bignell DE, Yves R, Nathan L (eds) Biology of termites: a modern synthesis, 2nd ed. Springer, Heidelberg, Germany, pp.27-50 Please list the first eight authors and then add “et al.” if there are additional authors. Citation of articles that have appeared in electronic journals is allowed if access to them is unlimited and their URL or DOI number to the full-text article is supplied. Tables and Figures should be restricted to the minimum needed to clarify the text; a total number (F + T) of five is recommended. Neither tables nor figures should be used to present results that can be described with a short statement in the text. They also must not be integrated into the text. Figure legends must be typed double-spaced on a separate page and appended to the text. Photographs should be well contrasted and not exceed the printing area (17.6 × 23.6 cm). Magnification of micrographs should be shown by a bar marker. For color illustrations, the authors will be expected to pay the extra costs of 600.00 € per article. Color figures may be accepted for use on the cover of the issue in which the paper will appear. Tables must be numbered consecutively with Arabic numerals and submitted separately from the text at the end of the paper. Tables may be edited to permit more compact typesetting. The publisher reserves the right to reduce or enlarge figures and tables.

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