In situ and ex situ bioremediation and rhizoremediation of PAH and PCB polluted sites Laura Bardi laura.bardi@entecra.it, Mario Marzona, Roberto Ricci, Raffaella Fabbian C.R.A. Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Istituto Sperimentale per la Nutrizione delle Piante, Torino Riassunto La bonifica di suoli ed acque per uso agrario costituisce una problematica di rilevante importanza ai fini della sicurezza e salubrità dei prodotti alimentari; difatti alcuni xenobioti organici, come alcuni idrocarburi poliaromatici (PAH) ed i policlorobifenili (PCB), sono recalcitranti alla biodegradazione e passano nella catena alimentare. La degradazione può essere migliorata ed accelerata con opportuni interventi tecnologici. La bonifica delle acque è drasticamente migliorata ex situ utilizzando consorzi microbici selezionati, aumentando la biodisponibilità degli xenobioti mediante l’uso di βciclodestrina e controllando la temperatura. La bonifica dei suoli può essere ottenuta in situ mediante rhizoremediation, associando una coltivazione non-food alla biodegradazione ad opera di microrganismi rizosferici. Summary The use of polluted lands for food crops is a main problem for human safety because several organic xenobiotics, such as polycyclic aromatic hydrocarbons (PAH) and polychlorinated biphenyls (PCB) are recalcitrant and pass through the soil and water into the food chain. Biodegradation can be improved and accelerated by technological processes. Water bioremediation is strongly improved with ex situ processes by selected microbial consortia, by increasing bioavailability with β-cyclodestrin and by temperature control. Soil bioremediation can be reached in situ by rhizoremediation, that associates a non-food crop to degradative activity of rhizospheric microorganisms. 1. Introduction Organic xenobiotics are widespread in soils and water and through the food chain they become a main health risk for humans. In fact, some organic xenobiotics are rapidly degraded by microorganisms in soil, but the most recalcitrant among them remain for a longtime in soil, pass into the plant roots and accumulate in different vegetable tissues. The polluted agricultural soils should then be devoted to other uses for long periods, but this can be a social significant problem. The cultivation of industrial crops, such as biomassproducing plants as a source of renewable energy, as an alternative to food-producing crops, can become an important tool to contemporarily reach an efficient bioremediation process and get income. Most organic xenobiotics can be degraded by microorganisms that use them as energy and carbon sources. Several studies have been carried out to accelerate and improve their bioremediation by low-impact technologies, mainly aimed at maintaining the natural characteristics of soil, by stimulating the activity of autoctonous microorganisms; also the rhizoremediation, where the plant roots aid and increase the degradation activity of soil microorganisms, can improve biodegradation.
Polycyclic aromatic hydrocarbons (PAH) are widely spread as pollutants of soils and waters. Hydrocarbons can be degraded by heterothrophic microorganisms [1], but as they are hydrophobic compounds the main factor affecting their degradation is low bioavailability [2]). The recalcitrance of PAHs is related to the ring number; among low molecular weight PAHs, phenanthrene is the most recalcitrant one [3]: following the natural attenuation of petroleum-polluted soils, it is found at high residual concentration levels. Hydrocarbon bioavailability can be increased by cyclodextrins (CD), water-soluble cyclic oligosaccharides with a toroidal hydrophobic cavities with a hydrophilic shell, forming inclusion complexes with hydrophobic molecules of a size compatible with their hydrophobic core [4]. As cyclodextrins are natural, non-toxic compounds, they are suitable for low-impact both ex situ and in situ bioremediation processes. Polychlorinated biphenyls (PCB) are recalcitrant organic compounds extensively used in industrial applications until the mid 1970s. They are extremely unreactive and heat stable chemicals. Their widespread presence in the environment constitutes a great risk to plants, animals and humans and the removal of PCBs represents a great ecological and economical problem [5]. Many microorganisms able to degrade or transform the lower-chlorinated PCB congeners under aerobic conditions have been isolated; the majority of degrading bacteria does not mineralize the molecule but release chlorobenzoate as by-product; thus, to complete the decontamination process, it is necessary to combine chlorinated biphenyls-transforming and chlorobenzoate-degrading bacteria [6]. Because of the contaminant hydrophobicity and complexity the aerobic degradation in soil is very slow: to overcome the problem, a combined remediation strategy has been adopted, including PCB mobilization through surfactants, PCB-degrading soil microrganisms and plants contribution [7]; several plant species have showed ability to release root phenolics into the rhizosphere and thus contribute to the growth of PCB-degrading bacteria in situ [8]. However the problem of an efficient, rapid, non-destructive remediation process for PCB-polluted soils as not yet been solved. In this case the rhizoremediation with non-food crops could be the sole way to remediate PCB polluted sites without risks for human health and with a low impact on territorial socio-economic status. 2. Report 2.1 PAH pollution 2.1.1 In vitro biodegradation assays In vitro biodegradation assays were carried out in order to focus the best environmental and technological conditions to promote microbial degradative activity. Several parameters were tested oriented to different technical application, such as ex situ or in situ bioremediation of aqueous, slurry or soil matrix. Phenanthrene was chosen as a model molecule as one of the most recalcitrant among PAHs. 2.1.1.1 Degradation in aqueous phase Assays in aqueous phase were mainly aimed to focus the physiological behaviour of microbial consortia in controlled conditions and to check the best conditions for ex situ water bioremediation.
Nine phenanthrene-degrading microbial consortia were selected from different polluted soils and waters. The most active microbial consortium for degradation of phenanthrene was used in following assays, in which different incubation temperatures (15°C, 28°C and 37°C), different commercial forms (α−, β−, γ- and HP-β-) CD and different phenanthrene concentrations (500 ppm and 1000 ppm) were tested evaluating the residual phenanthrene concentration after one month by PAH extraction and HPLC analysis. At 15°C there was no degradation. At 28°C, 21.3% phenanthrene was degraded; β-CD did not improve degradation (21%); α-CD induced an increase (26.4%), while γ-CD (17.4%) and HP-β-CD (15.4%) caused a decrease of degradation. The formation of the inclusion complex, even if increases water solubility of phenanthrene, could cause a shield that reduces its bioavailability when the diameter of the internal cavity of CD is higher (γ-CD), while when the cavity is smaller (α-CD) part of the molecule could be exposed, making it available to the microbial attack. The decrease with β-HP-CD could be explained by its higher polarity, that could be an obstacle for the formation of inclusion complexes. At 37°C the behaviour was completely different: a lower degradation (16.4%) than at 28°C was observed, while CD increased it (β-CD: 56.8%; α-CD: 36,8%; γ-CD: 18.9%). This could mean that higher temperature decreased the microbial metabolic activity, but contemporarily modified the inclusion constant of the phenanthrene complex: then the greater degradation with CDs was not due to biological, but probably to physico-chemical effects of higher temperature. Moreover, the higher increase observed with β-CD could be due to its increased solubility [4]. The initial concentrations of phenanthrene did not significantly modified the degradation rate, that is therefore influenced by the sole bioavailable PAH fraction that can be considered constant during early phases of the process. 2.1.1.2 Degradation in slurry and solid phase Assays in slurry phase were aimed to evaluate if ex situ soil bioremediation, that allow to control several physiological conditions (i.e. temperature), could significantly improve and accelerate PAH degradation, even in spite of the higher costs. Microcosms were set up wit sterile soil to which 80% water, 1000 ppm phenanthrene, 1% β-CD and degradative microbial consortium were added; C:N:P=100:15:1 was reached with urea and diammonium phosphate, after 3 months incubation on a shaker at 150 rpm at 37°C the residual phenanthrene was analysed and compared to microcosms set up and incubated in the same conditions, but in solid phase (soil moist with 25 % water, static incubation). Despite in slurry phase, due to shaking, the contact between hydrocarbon and microbial cells and the temperature control are improved, it was observed that the phenanthrene biodegradation was significantly higher in solid phase (88%) than in slurry phase (17%). As in aqueous phase assays, β-CD increased the degradation in both solid (96%) and slurry (42%) phase, confirming the crucial role of bioavailability in biodegradation processes. The better performance obtained in solid phase can be explained by the higher oxygen availability, as in slurry phase soil macropores are filled with water; so oxygenation showed to be more important than temperature and bioavailability for soil bioremediation.
2.1.2 In situ biodegradation assay In an in situ assay the increase of PAH bioavailability, reached by the addition of β-CD, was associated to rhizoremediation in a diesel fuel-polluted site. Two plots were set up, impermeabilized with polyethylene sheets and filled with agrarian soil. Diesel fuel was spread on the surface (250 g/mq), and 50 g/mq urea were added to provide the nitrogen necessary to allow the microbial growth. A commercial microbial consortium containing rhizospheric microorganisms was spreaded in one plot (B). Soybean (Glycine max), chosen as a potential non-food crop for biodiesel production, was seeded in both plots A and B. Soil samples were taken from surface to 60 cm depth after seeding (time 1) and after harvesting (time 2). β-CD (170 g/mq) was spreaded after the first sampling in plot B. Residual total PAHs and microbial populations (total heterothrophic and PAH-degrading microorganisms) were analysed in soil samples. The soybean seeds were also analyzed to determine the residual PAH [9]. The rhizoremediation showed to be very effective on PAH remediation: after 6 weeks from seeding the residual total PAHs in soil were about 1,4% of the initial concentration. The addition of β-CD
at this phase significantly increased the degradation of residual PAH in the following 4 months (degradation 46% in comparison with 3% without β-CD). As a higher risk of groundwater pollution could be supposed as a consequence of increased PAH water solubility, due to the in situ addition of β-CD, soil was separately sampled at different depths. The mobility of PAH in soil is influenced by organic matter, and each fraction of dissolved organic matter differently interact with hydrocarbons: i.e. humic acids favour the adsorption of hydrocarbons in the superficial soil layers, while fulvic acids cause an increased mobility [10]. The percent distribution of residual PAH found along the soil profile showed that β-CD can reduce PAH leaching, retaining a higher percent of them in superficial layers (Fig. 1). PAH leaching - time 2
PAH leaching - time 1
0-20 CD
20-40
no CD
depth (cm)
depth (cm)
0-20
CD
20-40
no CD
40-60
40-60 0
20
40
60
PAH (%)
80
100
0
20
40
60
80
PAH (%)
Fig. 1 – Percent distribution of residual PAH along the soil profile during (time 1) and at the end (time 2) of bioremediation process with (CD) and without (no CD) the addition of βcyclodextrin
Soybeans were analysed for PAH content to reach informations on both health risk from cultivation of crops on contaminated soils and possible PAH phytoextraction as a tool to further improve the soil bioremediation. Traces of PAH were found in seeds, indicating that a root uptake happened and that bioremediation of PAH contaminated soils is necessary to prevent the health risk of the presence of PAH in foods. PAH content of seeds
was higher and qualitatively different in plot B: this means that β-CD also influenced PAH root uptake and xylematic translocation. 2.2 PCB pollution A three years project aimed to remediate a PCB-polluted agricultural site has been carried out (LIFE FREEPCB) with co-financement of UE, as a Life-environment project, and Regione Lombardia. 2.2.1 In vitro biodegradation assays Microcosms were prepared with 800 g soil from a PCB-polluted site (5-10 µg/kg). Different variables and their combination effects on biodegradation were tested in triplicate. Monitoring during 5 months for PCB concentration, heterothrophic and PCBdegrading microbial populations, water content and pH was carried out. PCB degradation results are shown in table 1. 3g/kg β-cyclodextrin appears to be the most effective action, with 30-50% PCB degraded in 4 months. Cyclodextrin effect on PCB degradation has already been proved [11] [12]; this work highlights the efficacy of CD also at very low and old contamination levels. 3g/kg cyclodextrin seems to be the optimal surfactant concentration: at 1g/kg the degradation percentage is lower (23%) and at 6g/kg is comparable to 3g/kg. The other tested parameters did not show effects significant enough to justify a field scale trial: neither selected PCB-degrading microbial consortium (MICRO), nor the addition of nutrients (NUT) to stimulate microbial growth and activity, nor structuring agent to improve the soil oxygenation (STRUT), alone or in combination with β-CD (MIX), reached high degradation percentage. PCB concentration trend (ug/kg, month) assay
description
0
1
2
3
4
5
% PCB degradation
B
untreated soil
11,07
9,8
10,6
10,3
10,3
−
7-11%
CD 1g/kg
β-CD, 1g/kg
10,33
8,8
9
7,9
8
−
23%
CD 3g/kg
β-CD, 3g/kg
11,07
11,4
5,5
7,3
7,8
−
30-50%
CD 6g/kg
β-CD, 6g/kg
10,33
7,8
8
7,1
7,2
−
30%
NUT
nutrients addition (N,P)
11,07
10,3
17,2
10,7
11,4
−
0-7%
STRUT
structuring agent
11,07
10,3
10,5
9,7
10,7
−
3-12%
MIX
CD 3g/kg, nut, Strut
10,79
9,1
10,5
10,7
−
9,7
13-15%
MICRO
selected consortium
10,79
9,8
9,4
9,9
−
9,7
10-13%
Tab. 1 – In vitro PCB biodegradation assays As with PAHs, CD form water-soluble inclusion complexes with PCB, making them more available to the specialized microorganisms [14]. In a field application, PCB solubilization
could promote contaminant migration to the groundwater; then assays to evaluate leaching and mobility through the soil profile were conducted in order to evaluate cyclodextrin field application feasibility. For leaching test, 90 g of soil were mixed with water in 10 l/kg ratio and placed on a shaker for 24 h; after settling, eluate was analyzed for PCB concentration. To evaluate PCB/cyclodextrin mobility through the soil profile simulating an abundant rainfall, soil microcosms were prepared as described previously and flooded with 1:1 (w/w) demineralised water. After 24 h contact, water was let flow through the soil and analysed for PCB content. In both tests soil samples treated with 3g/kg cyclodextrin and untreated were analyzed during 3 months. PCBs were not detected (<1 µg/l) in eluates from both tests, neither in untreated nor in CD-treated soil during 3 months monitoring. Moreover, in order to verify the absence of toxic by-products deriving from PCB biodegradation, soil microcosms with 3g/kg cyclodextrin were monitored for chlorobenzoate presence during 3 months: no chlorobenzoates were detected following PCB degradation. 2.2.2 In situ biodegradation assay In situ assays were carried out in an agricultural site with a 30 ha surface contaminated by PCBs (5-10 µg/kg); during three years, the action of plants, CD and microorganisms alone and in association were evaluated. 7 200 mq experimental plots were set up. One plot was considered as control, seeded with crops on untreated soil. Plots were monitored for PCB concentration and microbiological parameters. Plants chosen among traditional crops of the area were potentially non-food, renewable energy producing crops. Four species have been tested: maize (Zea mays), hemp (Cannabis sativa), soybean (Glycine max) and potato (Solanum tuberosum). It has been demonstrated that hemp (that was not a present typical crop of the area, but potentially suitable), because of its fast growth and wide root apparatus, can be used to extract pollutants from soil [13], applying a rhizoremediation approach. In addition to different crops, β-CD addition and inoculum with commercial fertilizer based on mixed rhizospheric microorganisms were also tested. Plant tissues were sampled in order to evaluate PCB accumulation in roots, shoots, leaves, seeds and tubers. Results obtained during three cultural cycles showed a great variability, that made it difficult to deduce undoubted conclusions; anyway, some important informations were reached: seeds (soybean and maize) do not accumulate PCBs, as well as tubers (< 1µg/kg), while PCB were found in shoots and leaves. Hemp accumulated PCB in roots much more efficiently than other species: at the end of the cultural cycle 30-40 µg/kg (dry weight) were detected in hemp radical apparatus against 20-27 µg/kg of maize. The differences due to the effect of β-CD on PCB translocation to plant tissues, even if very significant in some trial, resulted to be not repeatable in different years. 3. Conclusions Rhizoremediation experiments carried out on both PAH and PCB polluted soils allowed to confirm the very high health risk caused by crops cultivation on polluted sites: in fact, contaminants are translocated into the plant tissues and by this way they pass into the food chain. Remediation of agricultural soils then become crucial for human safety, and
rhizoremediation with non-food crops is suitable as it allows to contemporarily reach soil decontamination and a yield for farmers. Moreover, soil in situ remediation resulted to be suitable more than ex situ processing: in fact, the technological additional actions that can be carried out ex situ did not show an additional effect on bioremediation efficacy significant enough to make allowance for the additional costs and the higher environmental impact due to soil removal and handling. Just one factor can be crucial to improve bioremediation process: the addition of β-cyclodextrin, that increase bioavailability accelerating the degradative processes as well as it decreases the risk of groundwater pollution due to contaminants leaching and migration. Aknowledgements - The UE-financed PCB Life project has been carried out with cofinancement and cooperation of ARPA Lombardia, U.O. Risorse naturali e paesaggistiche, Milano and Dipartimento provinciale di Bergamo; Consorzio Interuniversitario Nazionale per la Biologia Molecolare delle Piante di Siena and University of Pavia-DET, Sez. di Micologia; CCS Aosta srl. The PAH bioremediation project is carried out with cooperation of CRA-ISZ, University of Turin (Department of General and Organic Chemistry and Department of Exploitation and Protection of Agricultural and Forestry Resources), Biosearch Italia s.r.l. (Turin) and CCS Aosta s.r.l.. 4. References [1] Cookson, J.T. (ed.) 1995, Bioremediation Engineering, McGraw-Hill, Inc., USA, pp.95-164. [2] Tabak H.H., Govind R. 1997 Ann. N. Y. Acad. Sci. 829:36--61. [3] Cerniglia C.E. and Heitkamp M.A. 1989, in U. Varanasi (ed.), Metabolism of polycyclic aromatic hydrocarbons in the aquatic environment, CRC Press, Boca Raton, FL, USA, pp. 41-68. [4] Szejtli J. 1988 Cyclodextrin technology, Kluwer Academic Publishers, Dordrecht, The Netherland, 1-393 pp. [5] Demnerova K., Stiborova H., Leigh M.B., Pieper D., Pazlarova J., Brenner V., Macek T., Macova M. 2002 Water, Air, Soil Pollutiont: Focus 3, pp.47-55 [6] Abraham W-R., Nogales B., Golyshin P.N., Pieper D.H., Timmis K.N. 2002 Curr. Opinion. Microbiol. 5, 246-253 [7] Schnabel W.E. and White D.M. 2001 Int. J. Phytoremediation 3(2), pp. 203-220 [8] Fletcher J. and Hedge R. 1995 Chemosphere 31, 3009-3016 [9] Bardi L., Martini C., Opsi F., Bertolone E., Belviso S., Masoero G., Marzona M. Ajmone Marsan F. 2006 Journal of Inclusion Phenomena and Microcyclic Chemistry, 57:439-444. [10] Petruzzelli L., Celi L., Cignetti A. and Ajmone Marsan F. 2002 J. of Environmental Science and Health. [11] Fava F. and Ciccotosto V.F. 2002 Appl. Microbiol. Biotechnol. 58, pp. 393-399 [12] Fava F., Di Gioia D., Marchetti L. 1998 Biotechnol. Bioeng. 58 (4), pp. 345-355 [13] Campbell S., Paquin D., Awaya J.D., Li Q.X. 2002 Int. J. Phytorem. 4(2): 157-168.