Microbiological Beneficiation of Low Grade Manganese Ores: A Review

www.mwe‐journal.org Journal of Mining World Express (MWE) Volume 4, 2015 doi: 10.14355/mwe.2015.04.005

Microbiological Beneficiation of Low Grade Manganese Ores: A Review Jai S. Ghosh1* and Arvind D. Agate2 1*

Department of Microbiology, Shivaji University, Kolhapur 416006, India

Ex Director, Agharkar Research Institute, Pune 411004. India

2

ghoshjai@gmail.com

Abstract Manganese is a transition element like iron. However, it exists in different valencies like 2, 3, 4 and 7. Of these, Mn2+ is highly water soluble, but is rarely found in natural conditions. Mn4+ is the least water soluble and under different pH and redox conditions it can slowly become colloidal in natural water. It exists naturally as a very common mineral called pyrolusite and is used for pyrometallurgical processes in ferro‐alloys industries. However, in order to have such beneficial application, the manganese content should be more than 80% in the ore. Anything less than this is treated as low grade ore and is usually dumped on land with cultivable soil, in order to reach deeper for the high grade ores. This is highly ecotoxic to the environment and over a period of time can be very damaging to agriculture and the biodiversity which includes human health as it finds its way to ground water table like wells and ponds. As time went by and high grade ore started depleting, miners started going deeper in the ground, with the result of dumping lower grade ores over surface soils. In this study, such low grade ores were examined and found to contain a mixture of Mn3+ and Mn4+ oxides. When such ores were subjected to microbial oxidation, it was found that under certain conditions of growth, Arthrobacter sp could not only convert the Mn3+ to Mn4+ and 67% of the Mn4+ oxides had the ramsdellite γ‐ crystalline structure, which could be used as a depolarizer in the dry cell batteries. This was considered as a very important biotechnological discovery which not only could save the biodiverse environment but also gave a value added product for commercial exploitation.

Keywords Arthrobacter; Pyrolusite; Beneficiation; Depolarizer

Introduction It is reported that the existence of manganese was known to the Indians since time immemorial (Fermor, 1909).

They regarded it as a kind of iron ore and used it in iron smelting, for coloring of glasses and enamels and for making ‘surma’ used for blackening of eye brows (Fermor 1909).

Early Sources in the World Till 1865, when the first ferromanganese alloys production started, manganese was chiefly used as a decolorizer in glass industries and was used to make bleaching powder. The requirement for manganese for such purposes in Europe was met from sources at Tavistock, in Devon, Launceston in Cornwell, Harz in Germany and Piedmont in Italy (Fermor, 1909). Soon these deposits got exhausted and were replaced by Spanish deposits, which also got exhausted. However, the ferromanganese industry was spreading fast in Europe and USA and there was a large demand of manganese. The suppliers of manganese ore which emerged at this time were mainly Russia (815070 tonnes in 1900), Turkey (49468 tonnes in 1899) and Brazil (111000 tonnes in 1900) (Ghose, 1967).

Emergence of Manganese Mines in India During the 1890s, the collector of Vishakhapatnam, Andhra Pradesh, detected that some contractors were breaking manganese ore boulders to prepare ballast for the railways and thus the deposits of Kodur were detected first. The Collector formed a syndicate and produced 685 tonnes from this district and exported the entire amount in 1892. In 1895 Garbham deposits were discovered and in 1898 the production rose to 228951 tonnes, of which 215181 tonnes were exported (80% to England and 20% to USA).

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The Rise and Fall of Indian Manganese Industries Due to the increasing demand in the International market, other companies were formed and new finds in Madhya Pradesh, Mysore and Bihar were discovered. The annual production reached 58500 tonnes by the end of 1906 and India was ahead of Russia which produced 53800 tonnes during that year. Soon there was a demand for Indian manganese ores by many developed nations in the next 15 years and India dominated the market as a major producing nation and exporter of manganese ore. This bright period for India lasted till 1912when she was relegated into the second position by Russia. This was obvious because barring Russia and China, almost all manganese ore producing areas in the world fall in countries, which had practically little or no domestic use of this ore. Hence they had to depend totally, on export of this ore. This could be one of the reasons for slow growth of manganese industry in India. Then the First World War came to India’s rescue, when Russia was completely paralyzed and India was reinstated to the top by 1916. India maintained the lead during 1919 to 1923, with an average production of 634600 tonnes of manganese ores, which was about 44% of the world’s average. Then the export trade witnessed the entry of new competitors from Egypt, Cuba and the Gold Coast (Ghana) (Coggin, Brown and Dey, 1955). By 1932, Russia had recovered and again displaced India from the top. However, another formidable competitor entered the market and it was South Africa. By the end of 1934, South Africa was in the third position followed by Ghana. India maintained her second position till 1958. The second World War did not have a serious impact on India’s market share. Later Russia withdrew from the western market and India showed signs of improvement. In late 1950s, though there was increased demand of manganese ores, India’s export did not increase till 1971, when average annual production touched 1.5 million tones. From here onward, there was a steady decline of Indian manganese ore industry till date. For example the all time low export was recorded in 1970 when India exported only 82000 tonnes. Table 1 summarizes the rise and fall of Indian Manganese ore industry over the years.

TABLE 1: RISE AND FALL OF INDIAN MANGANESE ORE INDUSTRY.

Period

Production (Tonnes)

Percentage of World production

1892‐1898

228,951

‐‐

1899‐1903

715,060

‐‐

1904‐1908

2,586,449

‐‐

1909‐1913

3,621,008

40.7

1914‐1918

2,933,481

34.1

1919‐1923

3,173,147

44.1

1924‐1928

4,841,440

33.6

1929‐1933

2,837,668

‐‐

1934‐1938

3,942,846

16.0

1939‐1943

3,919,075

‐‐

1944‐1948

1,840,392

‐‐

1949‐1953

6,284,601

13.2

1954‐1958

7,767,165

12.2

1959‐1963

7,105,562

9.8

1964‐1968

7,991,703

8.8

1969‐1971

5,028,340

8.4

This decline was stated to be not solely due to the low production values, but also due to the inferior variety of manganese ores that were being mined.

At the end of Second World War manganese ores were found to have many other uses. The two other two important uses were: 1.

As a depolarizer in dry cell batteries

2.

As a lixivium in leaching minerals such as uranium.

Beside these two uses manganese ores were also used to manufacture chemicals like iodine and certain organic and

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inorganic chemicals. Previously, it was used as a drier in manufacture of paints and varnishes, due to its virtue of absorbing oxygen from air. High grade pyrolusite is also used to manufacture different manganese salts like the manganates, as decolorizer in glass and ceramic industry and a pigment in glazes. It was also used in making good quality black bricks. The addition of manganese ores produces tints varying from purple to brown and black, which are used in pottery and enameled iron wares (Johnstone, 1954).

In spite of these limited uses, since the manganese industry in India, was still thriving as export dependent economy, and a concerted effort was needed to concentrate on improving the quality of the ores. The information (since 1979) from one of the foremost manganese producing province of India, Goa, was that the quantity of manganese deposits was diminishing fast in the explored mines and some of the mines were not expected to be producing after 10‐15 years hence. Moreover, the quality of manganese was suffering badly due to the presence of impurities like iron, silica, phosphorous etc. These had made the manganese ores to be a very low paying export item or not fit for export. For instance, presence of excess of phosphorous (more than 0.1%) makes the ore unacceptable for use in manufacture of ferromanganese alloys, as the alloys develop micro cracks and makes the finished product unacceptable.

The classical metallurgical beneficiation processes such as crushing, grinding, sieving, passing over magnetic platforms or chemical treatments like froth floatation either do not succeed or if they do, only partially do so. For instance for an ore found in Andhra Pradesh, Ferro Alloys Corporation of India, provided a list of their attempts to improve the ore at Rai Bahadur Shreeram Mines at Shreeramnagar (Table 2).

TABLE 2: EFFECT OF PHYSICAL AND CHEMICAL TREATMENTS ON BENEFICIATION OF MANGANESE ORES (DESHPANDE, 1979)

Treatment

Analysis of components (%) Mn

Fe

SiO2

P

Original sample (no treatment)

44.6

6.1

6.6

0.6

Crushing and sieving (‐3 to ‐12 and 18 mm)

44.6

6.1

6.6

0.6

Grinding in Rod Mill to obtain concentrate

58.6

6.1

4.2

0.6

58‐60

3‐5.8

2‐3

0.6

58‐60

3‐5.8

2‐3

0.6

Ground to ‐100 mesh with Duplex Classifier and passing through immersion magnetic seperator Passing through a batch of floatation cells to (chemically) remove phosphorous.

As is evident from the Table, although there was some slight improvement regarding beneficiation as far as removal of iron and silica were concerned, the quantity of phosphorous was not removed at all as result of these physical and chemical treatments.

The different beneficiation processes that can be tried on manganese ore are: 1.

Removal of excess iron

2.

Removal of excess phosphorous

3.

Oxidize the lower oxides to the MnO2 form

4.

Removal of either metallic or non‐metallic impurities which otherwise may interfere with the ore quality.

Out of these processes, for some, use of microorganisms to beneficiate the ore was attempted or recommended.

This was not conventional method in the strictest sense of the word. Rather it was effective removal of impurities or microbial conversion. Again it must be noted that not all these processes could be tried using microorganisms.

For instance, there existed sketchy information on removal of iron through the use of iron reducing microorganisms (DeSa, 1978) and rather no information on topic 4 from the above list. Out of the 2 beneficial processes (topic 2 and topic 3) that have been tried out in our laboratory (Deshpande, 1979; Bhole, 1979), the microbial conversion of manganese (lower oxides to the higher state of oxidation by using microorganisms) was selected, due to its topical nature and its ease of handling by single person; even though both these processes are being attempted under a Council of Scientific and Industrial Research – Govt. of India (CSIR) scheme. The process was discovered and illustrated in detail from this laboratory as result of work for seven years of work (Agate, 1972; Agate and Deshpande, 1974; Agate, 1975; Agate and Deshpande, 1977 and 1978). This process was claimed to be

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inexpensive, energy conserving and with least pollution potential and had a relevance to the national need.

Although beneficial, this type of a conversion was restricted to monopolize practices in India. Moreover, the electrolytic process could only work profitably in a country with cheap source of electricity.

Manufacture of Dry Cell Batteries Properties of manganese dioxide like the chemical composition, the physical specifications, crystal structure and particle morphology etc., were important for deciding the requirement of an ore employed as depolarizer for the manufacture of dry cell batteries. Although the chemical analysis provides a useful guide in selection of an ore, the final decision rests on the actual results of the tests on cells made under works conditions, particularly with regard to their shelf life (Anon, 1962). A British specification of the chemical composition of the type of ore that would be best suited for dry cell batteries is that the ore should contain 84% MnO2, with a maximum of 2% to 5% iron and carbon dioxide. This ore is required to be porous and lumpy and the size should not exceed 25cm or 8 inch. Table 3 gives the ore specifications as required by different dry cell industries in India (Anon, 1970): TABLE 3: MANGANESE ORE SPECIFICATIONS REQUIRED BY DIFFERENT DRY CELL MANUFACTURING INDUSTRIES (ANON, 1970).

Particulars

Union Carbide India Ltd, India

Havero Industries, India

Estrella Batteries, India

Geep Flash Light, India

MnO2

84‐87%

85‐95%

70‐85%

70‐85%

Iron

2% (maximum)

1% (maximum)

‐‐

‐‐

Copper

0.03%

‐‐

‐‐

‐‐

As and Sb

0.05%

‐‐

‐‐

‐‐

Fe2O3 and Al2O3

‐‐

‐‐

10% (maximum)

‐‐

SiO2 and insolubles

‐‐

1% (maximum)

‐‐

‐‐

Acid insolubles

‐‐

‐‐

10% (maximum)

‐‐

Structure and variety

Battery grade ore, gamma type, size should be in lump form not exceeding 8 inch in any dimensions.

Crystal structure gamma type

Crystal structure gamma type

Crystal structure gamma type

It appears that the primary requirement of an ore to be battery grade type is that it should have more than 80% MnO2 and that it should have crystal structure gamma type as shown schematically in Figure 1. It differs from the beta variety in that the beta variety is more tetragonal in structure, where as the gamma variety is orthorhombic ramsdellite in structure (DeWolff, 1959).

FIGURE 1, CRYSTAL LATTICE OF GAMMA VARIETY OF MNO2 DUE TO MICRO‐TWINNING AND DE WOLFF DISORDER (SIMON, MORTON AND GISLASON, 2004)

Due to the structure of the gamma variety, it makes the MnO2 a good depolarizer which is used in dry cell batteries.

Therefore, the battery grade ores are classified based on the content of gamma variety MnO2 and then be used suitably in manufacturing of different batteries.

Ores of Manganese Manganese occurs in minutest amounts in almost all rocks like granite, limestone and clay, but its mere presence is not of any commercial value unless manganese rich ores are present in sufficient quantities of mineable size and shape.

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The commercially worked manganese ores are the oxides and the commonest ones are Pyrolusite and psilomelane.

Beside these ores, the other types are braunite and jacobsite which are usually the oxysilicate types. In combination with these are the hausmanite type of ore which occurs abundantly in many parts of the world including western Australia. The carbonate type of ore is the rhodochrosite. In some parts of Ghana and South Africa, a very valuable ore is found, which is called as ramsdellite. This is the pure form of gamma variety of pyrolusite, ideal for use in any dry battery manufacturing process. Small quantities of these ores are found in some parts of India, such as Dongri Buzurg mines of Manganese Ores India Limited (MOIL) in Bhandara district of Maharashtra, India. The list of ores of manganese is presented in Table 4.

TABLE 4: DIFFERENT TYPES OF MANGANESE ORES

Ores

Chemical nature

Manganosite

MnO

Pyrochroite

Mn(OH)2

Manganite

Mn2O3, H2O

Hausmanite

Mn3O4

Polianite, Pyrolusite

MnO2

Taphroite

Mn2(SiO4)

Braunite

3Mn2O3MnSiO2

Rhodonite

(Mn, Fe, Ca,)(SiO2)

Pyromanganite

(Mn, Fe)(SiO2)

Spessarite

Mn3Al2(SiO4)

Psilomelane

BeMn, Mn8O16(OH)4

Albendite

MnS

Rhodochrosite

MnCO3

Jacobsite

MnFeO4

1.

Pyrolusite: It is a black oxide of manganese (MnO2). It occurs as an earthy material or as radiating fibres (Barnowitz, 1943). It is usually in the beta form of crystal structure. It’s harder variety is called Polianite, which occurs as needle shaped crystals and is of wide spread occurrence. The manganese content is about 80% or above.

2.

Psilomelane: After Pyrolusite, this is the most important oxide mineral of manganese. It usually occurs in a massive botryoidally stalactite form. Depending on the quantity of admixture its manganese quantity varies widely from 45‐60%.

3.

Braunite: It is an oxysilicate of manganese usually having above 60% manganese.

4.

Hausmanite: It generally occurs with braunite and is higher in manganese content (above 60%) It is brown in color when occurring freely or blackish brown in color when it is with other types of ores.

These are some important ores of manganese of commercial importance. The other ores are listed in Table 4. Out of these ores, the manganese ores of relevance to the present study are the hausmanite and braunite type of ores, which occurs in southern and central parts of India. Generally a typical Indian Manganese ore is a mixture of psilomelane and braunite.

Geomicrobiology of Manganese Manganese is present in earth’s crust at 0.1% level but its distribution is not uniform. The element can exist in oxidation states of 0, +2, +3, +4, +6 and +7. However, it is most commonly found in +2 and +4 states. Rarely +3 is seen in nature. Of all these +2 is the most water soluble form while +3 and +4 are water insoluble oxides.

Manganese ions are more stable than the ferrous ions under different conditions of pH and Eh. Based on the equilibrium computation manganese should exist predominantly as Mn2+ below pH 5.5 and as Mn4+ above pH 5.5.

At Eh around 0.8V Mn2+ is 100 ppm in presence of HCO3 ions. At Eh below 0.5V Mn2+ may dominate upto pH 7.8 to 8.0.

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FIGURE 2. THE STABILITY FIELDS OF MANGANESE SPECIES IN AQUEOUS SOLUTE ION (HEM, 1963).

Although in theory, 0.1ppm of Mn2+ ions in aqueous solution should readily autooxidize when exposed to air at pH value above 4.0. However, this will not happen until pH exceeds 8.0. This fact is extremely important when manganese oxidation by microorganisms is considered.

The most import geomicrobiological interaction with manganese is: 1. Reduction; 2. Oxidation and 3. Precipitation/concentration.

The 2 steps 1 and 2 are enzymatic processes and the last effect is non ‐ enzymatic, initiated by mechanical adsorption. Out of these processes microbial oxidation of manganese is detailed as follows: The enzymatic reduction of manganese has been thoroughly studied which involves a lot of fundamental studies.

These are dissimilatory reduction process, to detoxify the metal.

Dissimilatory Reduction of Mn(IV) Tetravalent manganese (Mn4+) and trivalent iron (Fe3+) have been studied in detail with respect to microbial physiology. Both these ions are good alternative electron acceptors for many microorganisms. There are similar ions like nitrate, sulphate etc. which too also accept electrons beside oxygen. It has been proved by growing strict autotrophs like Acidthiobacillus thiooxidans and Acidthiobacillus ferrooxidans in presence of Mn4+ in 9K medium, where these organisms had been found to carry out oxidation of elemental sulfur or sulfides to sulfates or ferrous to ferric ions and reducing Mn4+ to Mn2+ (Ghosh and Imai, 1985). Apparently, Mn4+ was preferred over ferric ions to be used as electron acceptor. Similarly many heterotrophic organisms have also been reported to use Mn4+. In one instance, Bacillus subterraneus has been isolated from an Australian deep thermal aquifer and was found to be growing well in presence of yeast extract mostly as carbon source and utilizing Mn4+ as electron acceptor, naturally (Kanso, Greene and Patel, 2002). Similarly, Shewanella oneidensis has been reported to carry out MnO2 reduction under CO2 stress conditions (Wu et al., 2010). In Indonesia in Matano lake it has been found that anoxygenic microbial methane oxidation takes place while Mn4+ reduction occurs (Crowe et al., 2011). This sort of reduction is found to be highly essential in the environments like benthic deposits, where microorganisms need such electron acceptors to oxidize organic carbon (which is usually present as either natural or anthropogenic pollutants), essential for their survival (Thamdrup, Rossello‐Mora and Amann, 2000).

It must be remembered that both manganese and iron are toxic to the environment in macro concentrations i.e. above 10‐15μM level. However, iron can be immobilized and its toxicity can be reduced by converting it to its sulfide form, which is not the case with manganese (Stumm and Morgan, 1981). Infact manganese rarely forms its

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sulfide compound. This has been further conceptualized with the help of Shewanella putrefaciens during hydrogen and formate oxidation (Nealson and Myers, 1992). Over a period of time if this reduced manganese is not removed by artificial chelation, then it can suppress the growth of the organism. This has been observed with the Mn (IV) and Fe(III) reducer‐ Geobacter sulfurreducens (Mehta et al., 2005) especially when c‐ type membrane bound cytochrome was required to be reduced.

The enzymatic oxidation of manganese is studied in case of Arthrobacter sp. (formerly Corynebacterium strain B), which was isolated by Bromfield in 1956. The other organisms studied in this respect were Sphaerotilus discophorous (now Leptothrix discophora) (Ali and Stokes, 1977), which is doubted by other workers (Hogan, 1973; Haji and Makenson, 1976), Citrobacter freundii (Douka, 1977 and 1980) and Metallogenium sp along with the organisms mentioned such as Leptothrix pseudoochracea and Arthrobacter siderocapsulatus (Dubinina, 1978). Among the fungi that have been found to oxidize Mn(II) to either Mn(III) or Mn(IV) are Acremonium, Penicillium, Cladosporium, Phoma, Verticellium etc. Most of the time these organisms use polyphenol oxidases like laccase which is a multi copper oxidases and carries out oxidation of manganese by one electron transfer to oxygen (Miyata et al., 2007).

The Oxidation of manganese by bacteria proceeds by 3 different mechanisms as: 1.

The oxidation of free Mn++ ions is catalyzed by a manganese oxidase that conveys electrons to oxygen by a cytochrome pathway.

Mn++ + ½ O2 + H2O → MnO2 + 2H+

2.

The oxidation of free Mn++ ions may also be catalyzed by catalase in a reaction with metabolically produced H2O2.

Mn++ + H2O2 → MnO2 + 2H+

3.

The manganese oxidation is prebound to Mn++++ oxidase that shunts electrons to oxygen by a cytochrome pathway.

MnMnO3 + ½ O2 +H2O → 2H2MnO3

In Marine bacteria a different kind of constitutive Mn++ oxidizing system is detected and detailed studies on Arthrobacter 37 by Ehrlich (1964) showed that the Mn++ is oxidized by a 2‐step reaction: Mn++ + H2MnO3 → MnMnO3 + 2H+ MnMnO3 +½ O2 + 2H2O → 2H2MnO3 This is the system which is responsible for formation of ferromanganese nodules in deep sea sediments. This is because the poor crystalline structure of oxidized δ‐MnO2:

FIGURE 3. THE STRUCTURAL MODEL OF BIOGENIC MnO2 (Miyata et al., 2007).

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It can be seen from the above Figure‐ 3, that due to gaps in the crystalline structure of biogenic MnO2, toxic elements like lead (Pb) is trapped and this is one of the mechanisms of forming polymetallic nodules in the ocean bed.

In the cultural manifestation it may be stressed that deposition of manganese in any liquid medium or in colonies is no proof of enzymatic manganese oxidation, because no enzymatically oxidized can also accumulate as brown manganese oxide in their colonies. In this group many bacteria are included as listed by Ehrlich (1981) which oxidizes Mn++ by indirect means or by an unknown mechanism. Such bacteria seemed to be present in fresh water and other water distribution system. The microorganisms causing precipitation of preformed oxidized manganese through adsorption can do so by production of slime as in the case of budding sheathed bacteria belonging to genera Metallogenium, Pedomicrobium, Hyphomicrobium and Siderocapsa.

Manganese reducing and oxidizing microorganisms play an important role in mobilization and immobilization of manganese in soil. Fixed manganese in soil may be concentrated in nodules or in arid environments as desert varnishes. Conversely manganese reducing microorganisms may mobilize the oxidized or fixed manganese, releasing it into the water column. This reduced manganese often leads to manganese carbonate formation, a different form of fixed manganese.

Out of the processes mentioned above, the microbial oxidation of manganese is very interesting, particularly the bacterial oxidation of manganese yields economically important products like battery grade MnO2. Hence, this process of bacterial conversion of manganese was selected for further studies, since the basic data on the process had accumulated in our laboratory and hence the optimization of the process was studied here.

Attempts to Obtain Manganese Oxidizing Bacteria Used for Beneficiation Purposes Bacterial oxidation of manganese in soil was first observed by Beijerinck as early as 1913. Since in the early days, the pure culture techniques to study microorganisms were not developed, the role of the microorganism responsible for bringing about such changes was not clearly understood. One of the first efforts to establish the importance of systematic study of manganese oxidizing microorganisms in soil was initiated by Bromfield and Skerman (1950), when the role played by this element in plant growth was speculated upon (Ottow, 1969). It was reported that Chromobacterium and Corynebacterium spp from soil carried out manganese oxidation (Bromfield and Skerman, 1950). Now various genera of bacteria have been implicated in oxidation of manganese, such as Arthrobacter, Bacillus (Ehrlich, 1966), Pseudomonas (Dubinina, 1970), Sphhaerotilus (Rouf and Stokes, 1964), Hyphomicrobium (Tyler and Marshall, 1967) and Citrobacter (Douka, 1980). The ecosystems, which were studied for isolation of manganese oxidizing bacteria included soils (Bromfield, 1956), fresh water pipeline deposits (Tyler and Marshall, 1967) sea water (Douka, 1980) and manganese ores (Deshpande, 1979).

Organisms belonging to genera Arthrobacter, Bacillus and Hyphomicrobium spp were isolated and their manganese oxidizing capacities were studied (Agate, 1972; Agate and Deshpande, 1977, 1978). It was found that manganese oxidizing cultures of Hyphomicrobium and Arthrobacter spp could oxidize hausmanite variety of manganese ore ( containing preponderance of Mn3O4 form) to the pyrolusite type (containing MnO2 predominantly) (Deshpande, 1979). It was revealed that Hyphomicrobium spp could concomitantly release phosphorous as an impurity in some of the Goa manganese ores, while probably oxidizing the manganese moiety from the crystal lattice structure of the ore (Bhole, 1979). Since both these processes were commercially important – the former in the manufacture of MnO2 for dry cell batteries (bioconversion) and the latter for removal of impurities from the ores (beneficiation), it was proposed to optimize such beneficial activities of bacteria at an expanded scale in the laboratory, so that the possible parameters could be worked out for extrapolation to large scale operations.

Out of the 2 processes available, the process of microbial conversion of manganese was taken for optimization purposes during these studies. Even though the process was carried out equally and efficiently by Arthrobacter and Hyphomicrobium spp (Deshpande, 1979), it was considered worthwhile to develop only the process using Arthrobacter sp for the following reasons: 1.

Hyphomicrobium are temperature sensitive i.e. these organisms will grow only at around 25oC and, hence, these organisms (or processes based on these organisms) will be difficult to use in the field, where there are usually temperature fluctuations exceeding 25oC.

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2.

There are very slow growing heterotrophic organisms which needs a solid surface to adhere to, which other contaminating heterotrophic bacteria can overgrow and hinder the process or make the process ineffective.

Therefore, the process of oxidation/conversion using Arthrobacter sp was preferred and it was decided to optimize the conditions for such a process. Arthrobacter sp are sturdy, fast growing organism and they can tolerate fluctuating field temperatures (from virtually 10oC to around 40oC in India).

Isolation and Identification of Cultures As the first step, the available cultures were screened and it was found that either they have lost their manganese oxidizing activity or it is present on a greatly diminished scale. While investigating the reasons for such a phenomenon, it becomes apparent that even though regular culture preservation program did exist at that time, the activity checks were rarely carried out, at the time of passing on these cultures from one person to another in the laboratory. Moreover, the cultures were mostly preserved by serial transfers and it was possible that under the circumstances periodic selections might have taken place and the cultures could have genetically selected out so that they failed to retain their activity conditions of the tests in this investigation. Rather than attempting to rejuvenate these cultures or find out a cause for their reported failure, it was decided to carry out fresh isolation of manganese oxidizing bacteria and later institute a plan for culture preservation coupled with activity check, so that the new cultures do not suffer the same fate.

Eco System Used The isolation of manganese oxidizing bacteria was carried out from the ecosystems that were used before viz. soils and water. Soils used were primarily from mining regions. Water samples used were from wells and streams and pile deposits on the river flowing through the city. The important environmental factors, which influence the microflora of these ecosystems, viz. the pile deposits were: (i) the quantity of manganese in the flowing water and (ii) the temperature effect. It was reported that the organism had the ability to concentrate manganese from such a system where the manganese content was usually low (Agate, 1972). The temperature in such a system was found to be 35oC in summer and about 15oC‐20oC in winter. On microbiological examination of such deposits during summer and winter months, it was reported that during the summer months, the Arthrobacter sp were predominant and during the winter months, thread like filamentous, budding bacteria viz. the Hyphomicrobium sp were predominant (Agate and Deshpande, 1977). Therefore, isolation of Arthrobacter sp was carried out during the summer months, as these organisms were reported to bring about conversion of hausmanite form of the ore to pyrolusite form (Deshpande, 1979).

Isolation The isolation protocol that was followed was based on the earlier work of Deshpande (1979) and Bhole (1979).

Briefly it may be recorded as follows: Using an enrichment culture methodology, the manganese oxidizing organisms were enriched in Bromfield’s liquid medium (Bromfield, 1958) containing 0.5% MnSO4, H2O. The medium was modified with the following composition (KH2PO4‐0.005%; MgSO4‐0.002%; (NH4)2SO4‐0.01%; Yeast Extract‐0.005% and MnSO4‐0.5%). Since no acid was expected to be produced, Ca3(PO4)2 was omitted from the original medium along with the readily assimilable carbon source, sucrose as the organism would utilize sucrose as an alternative energy source and would stop oxidizing manganese. The yeast extract was sufficient to develop the required biomass for the manganese oxidation process.

A set of 200 ml of sterile medium were inoculated with different soil and water samples. The flasks were then incubated on a rotary shaker at 140 rpm at 28oC for 4 days. The growth in the flasks was checked and it was noted that different types of organisms predominantly consisted of gram positive non‐sporulating rods. After 4 days of incubation, 1% of this was used as inoculum and was transferred to a series of flasks containing fresh medium for the second enrichment. The growth from these flasks was reconfirmed to be of the same variety as before. These were then cultured on ‐ plates of modified Bromfield’s medium solidified with 2.9% agar‐agar. The plates were incubated at 28oC for 4 days. Isolated colonies, showing a zone of brown coloration around the colonies were

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picked up, in preference to colonies which remained colorless. The brown coloration indicated manganese oxidation, which was confirmed by Benzidine test (Bromfield, 1956), where in 1% solution of benzidine HCl 100 ml of 2N acetic acid was poured on plates and it gave a dark blue color with oxides of manganese in the brown colored zones. The pure cultures were taken on slants of modified Bromfield’s agar.

Identifications The bacterial isolates were further characterized as per the key, constructed out of the eighth edition of Bergey’s Manual of Determinative Bacteriology (1978) upto the generic level.

Preservation of Cultures There was a need for an ongoing culture preservation program to preserve these isolates and a systematic program was instituted as follows: 1) Maintenance The cultures were routinely maintained by serial transfers on agar slants. Earlier, the manganese oxidizers were maintained on Bromfield’s medium. It was felt that the medium may be too rich for the growth of these organisms for a prolonged period of time. Hence an attempt was made to slow down their metabolism by reducing the nutrients and compounding a modified medium, as discussed before. Such a method of maintaining cultures of manganese oxidizers on this modified medium couple with routine monthly activity checks, it was found that the manganese oxidizing activity of the cultures could be maintained for a period of 150 days without appreciable decrease, instead of 90 days on original Bromfield’s medium as shown in Figure 4a, 4b 4c and 4d.

FIGURE 4A. ACTIVITY CHECK AFTER 60 DAYS, OF CULTURES PRESERVED ON BROMFIELD’S MEDIUM AND MODIFIED BROMFIELD’S MEDIUM. ■‐ACTIVITY ON BROMFIELD’S MEDIUM AND –ON MODIFIED BROMFIELD’S MEDIUM.

FIGURE 4B. ACTIVITY CHECK AFTER 90 DAYS, OF CULTURES PRESERVED ON BROMFIELD’S MEDIUM AND MODIFIED BROMFIELD’S MEDIUM. ■‐ ACTIVITY ON BROMFIELD’S MEDIUM AND–ON MODIFIED BROMFIELD’S MEDIUM.

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FIGURE 4C. ACTIVITY CHECK AFTER 120 DAYS, OF CULTURES PRESERVED ON BROMFIELD’S MEDIUM AND MODIFIED BROMFIELD’S MEDIUM. ■‐ACTIVITY ON BROMFIELD’S MEDIUM AND –ON MODIFIED BROMFIELD’S MEDIUM.

FIGURE 4D. ACTIVITY CHECK AFTER 150 DAYS, OF CULTURES PRESERVED ON BROMFIELD’S MEDIUM AND MODIFIED BROMFIELD’S MEDIUM. ■‐ACTIVITY ON BROMFIELD’S MEDIUM AND –ON MODIFIED BROMFIELD’S MEDIUM.

2) Preservation by Lyophilization In addition to this it was also decided to lyophilize the cultures for longer preservation. The cultures were grown in modified Bromfield’s liquid medium for 48hrs on a rotary shaker (120 rpm) at 28oC. The cells were then centrifuged out in cold (4oC) in sterile conditions at 22000 x g. and then washed by centrifuging 3 times with sterile 0.1N phosphate buffer (pH 6.0). The cell pellet thus obtained was resuspended in (10%w/v) sterile skimmed milk. Aliquots of 0.5 ml of this suspension were frozen in shell freezing bath at ‐80oC, containing methanol and then dried under vacuum on a VerTis Lyophilizer. Six vials of each culture was sealed and tested under vacuum for leaks. One vial from each set was opened for viability (in modified Bromfield’s liquid medium) purity check and activity in the same medium. The efficacy of conversion of ore containing hausmanite was tested by the method outlined later (op.cit). The results are shown in Figure 5.

FIGURE 5, ACTIVITY CHECK OF CULTURES AFTER LYOPHILIZATION. AND

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– 3 MONTHS;

‐24 MONTHS

‐ 6 MONTHS;

‐12 MONTHS

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It can be seen from the figure 5, that the activity of thr cultures tested did not decrease singnificantly even after a period of 2 years, indicating that the preservation by lyophilization was perhaps one of the best methods of maintenance without the loss of activity to a considerable extent.

Preparation of Inoculum by Use of Inert Carriers Eventhough the cultures lyophilized by the method outlined above were shown to retain their beneficial activity, there were certain limitations on the amount of cultures generated in this way. The most important limitation would be to use these cultures on a large heap under the field conditions. Here, a certain amount of inoculum would be necessary, which would definitely be more thatn the amount of lyphilized cultures in small ampoules.

Again opening and rehydrating the lyophilized ampoules or sealed materials in the field would be extremely difficult. Hence, it was thought that if such materials were to be mixed with an inert carriers, not only would it solve these problems, but the carrier material, if chosen judiciously, would also be able afford some protection to the cell mass on exposure to field conditions.

A parallel analogy of such preparation of bacterial cultures could be taken with respect to nitrogen fixing bacteria.

In the early days, pure cultures of nitrogen fixers in agar medium, were used to spread on field prior to sowing. In early 1920s, these cultures were replaced by sterile soil and then to peat and lignite (Date and Rougheley, 1977).

The apparent advantage of these treatment resulted in better survival, longer shelf life, mass of manufacture and distribution. Since these were the exact points, which we had in mind, the following experiments were carried out.

Types of Carriers The successful inoculum preparation is dependent on selection of suitable carrier material and its pretreatment, various materials such as soil and ore were tried for this purpose. The ores are quite an unconventional sunstrate to be used in this fashion, but the previous results had indicated that Acidthiobacillus ferroxidans cultures when mixed with sterile chalcopyrite ores retained their activity for a prolonged period of time (Kulkarni, 1983). This has apparent justification, in the sense that the microorganisms were found to have survived prolonged period of time in the ore itself, as shown by earlier results on copper and manganese ores (Agate and Deshpande,1974 and 1977; Agate, 1980 and 1981) made it logical to think that this material devoid of any external nutrients would be able to sustain the microbial cultutres. There is another advantage and that is the ore from which the cultures were isolated or the material on which the microorganisms were going to react, is added as carrier, would do away with problems of the microorganisms not coming in contact with the concentration of materials which would be their substrate for bioconversion.

The cultures of Arthrobacter (A9) was grown in modified Bromfield’s liquid medium on rotary shaker 120 r.p.m. at 28oC for 48 hrs. The cells were centrifuged, washed and the density was adjusted to more tha 109 cells /ml as confirmed by counting in Neuber’s chamber. The two carriers used were soil and ore. The carriers were sterilised by autoclaving at 121oC for 20 minutes for 3 days successively. These carriers were checked for sterility. The cultures were mixed with the carriers and the activity was checked for 3 months.

Out of these 2 carriers tried, ore was found to be better in that, it allowed the microorganisms to not only survive but retain their activity. It is shown in Figure 6.

However, when experiments were carried out to mix large amounts of cultures with the ores, it was found that during mixing of such cultures with ore or materials to be converted resulted in growth of other organisms also. At times it was difficult to restarin other organisms from growing as the conversion experiments utilized nutrients for the heterotrophs.Incorporation of antimicrobial antibiotics had a debilitating effect on the beneficial heterotrophic bacteria, although in latter parts of the experiment, the fungal growth was checked using antifungal antibiotics like nystatin (50ppm).

It was not known whether the mixing with carrier promotes a shock to the cultures, so that there is a significant lag in growth of the desired organisms which allowed these to be overgrown by other heterotrophs. The possibility of contamination due to insufficient sterilization of carrier was ruled out through the use of proper controls.

Therefore, it is concluded that unless a better choice of substarte precludes the possibility of contamination, mixing with ores might not work in the case of manganese converting bacteria. That it had worked with Acidthiobacillus

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ferrooxidans could be largely attributed to the nature of these organisms (chemolithotrophic), which automatically prevents the heterotrophic bacteria from growing in the acidic inorganic salts medium used for growing and spraying these organisms on the material to be leached (Kulkarni, 1983).

All these cultures were maintained with a MCM Number series starting with B. These will be referred accordingly in the following optimization studies unless otherwise specified. The cultures will be mostly of Arthrobacter and Bacillus spp.

FIGURE 6, ACTIVITY CHECK ON 2 CARRIERS VIZ. SOIL AND ORE. SOIL AND

– 60 DAYS IN SOIL;

‐ 60 DAYS IN ORE;

‐90 DAYS IN

‐90 DAYS IN ORE.

Optimization Studies One of the beneficial activities of manganese oxidizing bacteria is the bioconversion of manganese from a lower variety – hausmanite to a better variety predominantly containing MnO2 form. This type of activity was reported for the first time from this laboratory (Agate and Deshpande, 1974; Agate, 1975), while studying the ecology of freshwater pile deposits. This is the activity, which is loosely termed as ‘beneficiation’ as it could be used for upgrading the quality of manganese minerals present in our country (Agate and Deshpande, 1977). It might be noted that such a possibility was hinted at by Deshpande and Agate (1978) in their work on another organism viz. the Hyphomicrobium sp. It was also confirmed that manganese obtained after microbial interaction, contained 80% of crystals of the gamma‐variety, used in the manufacture of dry cell batteries (Deshpande 1979).

As mentioned earlier, the gamma variety of MnO2 used in the manufacture of dry cell batteries is mined only in few countries in the world like Ghana and there has been a constant effort in Western Australia to upgrade their low grade manganese ore deposits, through biological beneficiation. Since in India the presence of manganese conversion through electrochemical means is a monopolized process and the alternate means suggested by National Metallurgical Laboratory, Jamshedpur (India) is energy intensive process, such a microbiological process involving minimum energy consumption needs to be developed.

This investigation was decided to be limited only to the process of manganese oxidation, carried out by manganese oxidizing bacteria other than Hyphomicrobium sp. Therefore, it was decided to check the manganese oxidation by cultures isolated during the studies, viz. Arthrobacter, Bacillus and Pseudomonas sp and this was carried out by using 2 methods as follows: Qualitative Detection The colonies appearing on Bromfield’s medium showed brown coloration around them, which was an indication that soluble manganese ins in the medium have probably got transformed into manganic oxide. This brown coloration around the colonies was observed, when the plates were incubated at 28oC for 48 hrs. The colonies themselves did not show the brown coloration. The oxidation of manganese was further confirmed by adding Benzidine HCl reagent on to the plates, when the brown zones gave blue coloration with this reagent.

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During the primary screening only those colonies which showed brown zones around them were isolated and maintained.

Quantitative Estimation In order to select and quantify the manganese oxidized, 2 methods were used. The organisms were grown in liquid modified Bromfield’s medium.

When using a soluble form of Mn++ such as MnSO4 as substrate, the changes in the redox potential and the subsequent changes in pH values were recorded. As a result, the redox potential, also termed as Eh, changes with the changes in the oxidative state of manganese and also with the soluble manganese ion concentration. The pH check was carried out to see that pH did not go towards alkalinity, which leads to autooxidation of manganese.

If insoluble form of manganese (such as hausmanite ore) is used as a substrate (Figure 3), the oxidized manganese in the ores could be determined chemically by oxalic acid‐ KMnO4 titration (Vogel, 1978), where MnO2 present in the ore oxidizes oxalic acid under hot acidic conditions and the residual oxalic acid is then quantitated using standard KMnO4 solution under the same conditions.

Since the aim of this study was to optimize the manganese oxidizing ability of suitable organism, which could oxidize the inferior variety of ore to the superior pyrolusite variety, it was essential that the efficiency of oxidation of manganese should be calculated in terms of increased MnO2 and the organism giving the highest conversion should be taken up for further studies.

Selection of Cultures Preparation of Inoculum All the bacterial cultures of bacterial cultures Arthrobacter, Bacillus and Pseudomonas sp were grown on modified Bromfield’s agar slants for 48hrs at 28oC. This growth on slants was washed and suspended in sterile 0.85% NaCl solution. The absorbance of the suspension was adjusted to 0.4 at 550nm for the sake of uniformity. The culture suspensions were added at 1% (v/v) level to the ore, unless otherwise specified.

Preparation of Ore Samples The manganese ore containing a preponderance of hausmanite (2MnO, MnO2) was obtained through kind courtesy of Mysore Minerals Limited, Dandeli, India. The chemical analysis of this ore was provided by the organization which is reproduced below: Manganese ‐‐‐‐‐45‐50% Iron‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐8 – 12% Phosphorous‐‐‐0.02‐0.03% It was found that out of the manganese content (40%) the MnO content was about 60%.

The ore was first crushed with a 3 H.P. Lynx jaw crusher (M/s Lawrence and Mayo, New Delhi) to 0.5 cm size and the with help of a 2 H.P. roller crusher (M/s Minerals Processing Equipment Pvt. Ltd., Mumbai) and the required particle size was obtained after sieving through proper sieves (‐30 mesh to ‐200 mesh). The different particle sized ore was added at 10% (w/v) level for the following experiments.

One percent suspension (0.4 O.D.550) of the culture was added to each of the 500 ml Erlenmeyer’s flasks containing the ore at 10% (w/v) and the modified Bromfield’s liquid medium. The flasks were incubated at 28oC on a rotary shaker (120r.p.m) for 15 days. At the end of the incubation period the contents of the flasks were filtered through a gauze cloth and were washed several times with water to remove, as far as possible, all medium components and any other soluble impurities. The washed ore was dried at 100oC in a hot air oven. The same quantity of ore from the control experiment (without the inoculum) was also dried in the same manner. The MnO2 content of the ore to be converted and all samples of washed and dried ores exposed to bacterial action was measured by a quantitative method using oxalic acid‐KMnO4 titration.

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This basic methodology was followed for all subsequent experiments. At the time of selection or optimization only one parameter to be tested, was changed at a time.

Choice of the Medium Here 3 different media were tried, they were: i)

Modified Bromfield’s liquid medium (KH2PO4‐0.005%; MgSO4‐0.002%; (NH4)2SO4‐0.01%; Yeast Extract‐ 0.005% and MnSO4‐0.5%)

ii) 1% peptone water (Peptone‐1%; NaCl‐0.5%), and iii) Glucose yeast extract medium (Glucose‐1%; Yeast Extract‐ 1%).

The results of observation obtained using 3 media are recorded in Figure 7.

FIGURE 7, PERCENTAGE OF MnO2 AFTER THE CULTURES WERE GROWN IN PRESENCE OF ORE IN DIFFERENT MEDIA. IN PRESENCE OF MODIFIED BROMFIELD’S MEDIUM AND

– GROWN

‐ 1% PEPTONE WATER.

It can be seen from this figure that there was no conversion in Glucose yeast Extract medium and the conversion in 1% peptone water was less than that of modified Bromfield’s medium. Therefore, for all conversion experiments only modified Bromfield’s medium was used, unless otherwise specified.

Pulp Density In order to optimize the pulp density of the ore in the medium, experiments were carried out using the above medium, to which ore ground to ‐50 mesh size was added in different concentrations from 10% to 100%. It might be noted that a slurry consisting of 100 g ore in 100 ml medium gave a thick suspension which could be stirred on a shaker. Concentrations above this density posed problems during shaking incubations, as it started forming a thick paste. The results of the experiment to find the optimum pulp density are given in Figure 8.

FIGURE 8, PERCENTAGE CONVERSION TO MnO2 AFTER THE CULTURES WERE GROWN IN DIFFERENT PULP DENSITY OF ORE.

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– 10%,

‐ 50%,

‐ 100% AND

‐ 150%

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It was shown in case of all the cultures tested that upto 100%(w/v) pulp density the conversion was maximum. It was therefore, decided to keep the pulp density of the ore at 100 % level in all experiments unless otherwise stated.

It was evident that out of the 21 types of cultures tested, 10 cultures (8 of Arthrobacter and 2 of Bacillus sp) showed the maximum conversion between 53.3% and 61.59% with Arthrobacter and between 55.43% and 60.25% with Bacillus sp. Hence, these cultures were selected for further studies for optimization of other parameters. It was believed that, if the conversion rates were high enough then these rates could be used for scale up process as the efficiently may slightly fall during scale‐up. There are other obvious reasons, such as economics of the process, which will be dealt later.

Metal Toxicity It was gathered from previous reports, that mercury, cobalt, lead and zinc occur quite frequently with manganese ores (Kuzin and Egorov, 1979). It was, therefore,decided to study the effects of these metal ions by incorporating these in the medium on manganese oxidation using MnSO4 as substrate. This was carried out by measusing the redox potential and the change in pH as the growth proceeded (as described in quantitative estimation of manganese in this chapter).

Preparation of Medium with the Toxicants Modified Bromfield’s medium was used with 0.5% MnSO4, H2O. Four different concentrations (0.001%, 0.005%, 0.01% and 0.02% w/v) of such soluble metal salts, viz. CoCl2, ZnSO4, Pb(NO3)2 and HgCl2 were incorporatedinto the medium. Two controls were kept, one in which these metal salts were not added and also not inoculated and the other, in which there were metal salts were not added but were with the respective cultures. All the 10 selected cultures were tested for metal toxicity using the above medium and the change in Eh and pH was recorded. It was observed that all the metal salts were toxic at 0.001 % level. However, when these were present in the natural sate in the ore no toxic effects were noticed during the conversion experiments, as the manganese oxidation proceeded without any hinderances. This was possibly due to the fact that metals existed in the ore in insoluble forms and, therefore, the metals could not be toxic to the growth and subsequent manganese oxidation process.

Effect of Temperature It was essential to find out the optimum temperature for conversion. Hence, experiments in 5 different sets were carried out as described before. The flasks were incubated at 10oC, 20oC, 30oC andt 35oC. At 10oC and 20oC, the incubation was carried out in refrigerator and BOD incubator. At 30oC and 35oC the incubation was carried out on a metabolic shaker with stirring at 150 r.p.m. (M/s Thermolab Equipments, Mumbai). It was noted that at 10oC and 20oC, there was neither growth or oxidation of manganese. This could be due to 2 reasons: i)

The cultures needed vigorous aeration and hence, they were unable to grow and oxidize manganese.

ii) As noticed in seasonal ecological successions of these organisms, the temperatures were too low for the growth and subsequent osidation of manganese.

Similarly at, temperatures at 35oC and above, no oxidation and growth were observed as the temperature of incubation was too high. The results of manganese oxidation at 30oC and 35oC are recorded in Figure 9.

FIGURE 9, EFFECT OF TEMPERATURE ON MANGANESE CONVERSION.

–AT 30oC AND

‐AT 35oC.

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It was observed that the organisms carried out maximum beneficiation at 30oC. Adaptation to Higher Concentration Of Mn++ The eco systems from which these isolates were obtained were soil, freshwater and freshwater pile deposits. In the former 2 systems, the concentration of manganese was low ( in the range of 0.001% to 0.002%), where as in the latter i.e. freshwater pile deposits the maximum concentration of manganese was 0.01% (w/w). This indicated that the cultures were able to withstand higher concentration of manganese without previously coming in contact with it, since they effected conversion in 100% pulp density, as illustrated above. Inorder to exploit this ability of the organisms further, the organisms were subjected to grow at increasingly higher concentration of manganese i.e. from 3600 ppm to 54000 ppm of Mn++ in modified Bromfield’s medium. It is known that the organisms were known to grow at 1800 ppm of Mn++ right from the time fof isolation as the recommended concentration of Mn++ in Bromfield’s medium.

Modified Bromfield’s liquid medium was prepared with manganese at 36000 ppm of Mn++. One hundred ml of this medium was dispensed in 250 ml capacity Erlenmeyer’s flasks and the flasks were autoclaved at 121oC for 20 minutes. These flasks were then inoculated with 1% culture suspension (0.4 absorbance550nm) and the flasks were incubated at 28oC on a rotary shaker (140 r.p.m.) for 10 days. When visible indication of growth(brown coloration with turbidity) was observed in the flasks (confirmed microsopically), the cultures were serially transferred to increasing concentrations of MnSO4 (from 0.5% to 15%), corresponding to 1800 ppm to 54000 ppm of Mn++ ions, and were incubated as above.

All the 10 cultures indicated visually good manganese oxidation in 10 days time in 29000 ppm of Mn++, except for the culture of Arthrobacter sp (B26), which was growing very slowly after transferring from 1800 ppm of Mn++ and was not growing at all in 29000 ppm of Mn++. At 36000 ppm of Mn++ only Arthrobacter sp (B21, B23 and B25) and Bacillus sp (B97) gave good growth and manganese oxidation. The results of further adaptation study at 54000 ppm of Mn++ are recorded in Table 5.

TABLE 5: STUDIES ON ADAPTATION TO HIGHER CONCENTRATION OF MN++.

Culture MCM No.

Mn++ concentration in PPM 3600

10,500

18,000

29,000

36,000

54,000

B20

+

+

+

+

‐

‐

B21

+

+

+

+

+

‐

B22

+

+

+

+

‐

‐

B23

+

+

+

+

+

+

B97

+

+

+

+

+

‐

B24

+

+

+

+

‐

‐

B25

+

+

+

+

+

‐

B98

+

+

+

+

‐

‐

B26

+

+

+

v

‐

‐

B27

+

+

+

+

‐

‐

Note: + growth and oxidation of manganese ‐ no growth and no oxidation of manganese v Scanty growth with or without oxidation of manganese

It can be seen from this table that only Arthrobacter sp (B23) gave good growth and Manganese oxidation (67%) at 54000 ppm of Mn++.

Testing of Manganese Oxidizing Activity of the Cultures All the 4 cultures i.e. Arthrobacter (B21, B23 and B25) and Bacillus (B97) were used in this study. The cultures were grown in modified Bromfield’s liquid medium as it was not possible to get a solid agar medium at that concentration of Mn++ (36000 ppm). The washed suspensions of the cultures were prepared in following manner.

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The organisms were sub cultured 4 to 5 times in the above medium with 36000 ppm of Mn++ (B21, B25 and B97) and with 54000 ppm of Mn++ (B23), till the cultures were able to grow in 4 days time instead of 10 days. Such 4 days old cultures were used to test the manganese oxidation as described before. The results are recorded in Figure 10.

FIGURE 10, MANGANESE OXIDIZING ABILITY OF CULTURES AFTER ADAPTATION TO HIGHER CONCENTRATIONS OF Mn++ IN MODIFIED LIQUID BROMFIELD’S MEDIUM.

Effect of Shaking/Static Condition of Incubation Since the organisms to be tested were strict aerobes and the process of oxidation of manganese was an aerobic process, it was essential to have good aeration for optimum oxidation. This could be achieved in 2 ways: we could provide sterile air through an aerator to the flasks when the oxidation is being carried out or the flasks could be incubated on a reciprocatory shaker.

The experiment was carried out in 2 sets of flasks in the way described before. It was found that the shaking condition of incubation gave 61% conversion with cultures: B21 and B25 and 67% conversion with B23, while 60.25% conversion was observed with B97.When the same cultures were incubated at static conditions there was no conversion of Mn++ even after 15 days of incubation at 28oC. Effect of Particle Size of the Ore It was very essential that the effect of particle size on the oxidation of manganese should be determined, as one must obtain an ore with a particle size which should prove to be cost effective for processing, at the same time yield optimum oxidation of the particles, in order to make the process economically feasible on a large scale operation. The other factors that might be playing a crucial role are that the finer the particle size, the toxicants would tend to form colloids in the medium and hence inhibit the process. This could ever hinder proper aeration which also might hamper the process. On the other hand, if the particles are too large then only the surface of the particles would get oxidized and the process would not be economically feasible, as the conversion will not be extensive. For this purpose the ore was crushed in the manner described before so as to conform to: ‐30 mesh equivalent to particle size 650nm ‐50 mesh equivalent to particle size 300nm ‐100 mesh equivalent to particle size of 150nm ‐200 mesh equivalent to particle size of 75nm It was observed that percentage conversion with ‐30 mesh size was as low as 20% and that with ‐100 and ‐200 mesh size was nil. However, using ‐50 mesh particles, it was possible to achieve 61% conversion with B21 and B25, with B23 and B97; it was 67% and 60.25 respectively. One of the most probable reason for very small particle sized ore, that it could be death of a large portion of the biomass due to abrasive effect of the particles.

Effect of pH It may be mentioned that in the composition of modified Bromfield’s medium the pH was around 6 to prevent all possibility of autooxidation, as this is reported to take place at and above pH 8.0 (Hem 1963). In this study,

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different pH values below pH 8.0 (5.2, 6.2 and 7.2) were tested for manganese oxidation by the 4 cultures. The experiment was carried out as mentioned before in 3 sets of media at the pH values stated above. The results are as shown in Figure 11.

FIGURE 11.: EFFECT OF pH ON MANGANESE CONVERSION.

– pH 5.2;

‐ pH 6.2 AND

‐ pH 7.2.

It was observed that B21 and B97 could maximally oxidize manganese at pH 5.2, whereas B23 and B25 did so at pH 6.2, again coroborating the fact that it was mostly microbial oxidation of manganese.

Our effeorts to optimize the different parameters for conversion process could be summarized as follows: Temperature: 30oC pH: 5.2 (for B21 and B97); 6.2 (B23 and B25) Medium for conversion: Modified Bromfield’s liquid medium Pulp Density: 100% Particle size: 300 nm Hence, Arthrobacter (B23) was superior to the other 3 cultures in conversion efficiency from the point of view of economy and it was used in scale‐up process outlined later.

Even though, B23 was better, all the 4 cultures were studied further because of following reasons: In a natural system in the field, it is quite possible that no single culture would do the job alone and therefore, it is always advisable to test more than one culture (in the form of consortium) for such purposes. In fact permutation and combination experiments, using these 4 cultures ought to have been tried and if as a result, one observes the cumulative effect in conversion, it would have been ideal. However, these experiments were not carried out due to paucity of time, but this area is referred for future explorations to workers in this field.

Scale-Up Studies Since the parameters were optimized for conversion of manganese at laboratory level using the strain of Arthrobacter B23, it was decided to use the findings to scale‐up the process, using a vat, if possible.

It was observed that the conventional scale‐up techniques, employing glass columns or air‐lift percolators, would be of limited use due to the impeding contamination problems as it was difficult to get the columns sterilized and the minimum precaution to keep the column free from excessive contamination would be very difficult. The earlier experiments using inverted glass bottles (Deshpande, 1979) were not feasible in this case, as preventing the contamination was a problem through the use of antibacterial agents. It was decided therefore, to carry out the experiments using vat leaching methods.

The scale‐up was first effected at 1.5 Kg level using a 2 liter flask, at 100% pulp density and the experiment was carried out in triplicate on a shaker (120 r.p.m.) at 30oC. The conversion efficiency, as obtained previously without changing any of the parameters, was again 67%.

Arthrobacter strain B23 was inoculated at 1% level in a stainless steel vat (18 gauge, 50 cm in height and a diameter

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of 38 cm) provided with a lid. The vat could be surface sterilized, before charging it with 14 kg ore in 14 L of modified Bromfield’s liquid medium. The contents of the vat were stirred periodically with the help of 2 stirrers (at 150 r.p.m,) having variable speed (Remi industries, India). The lid could not be modified during the tenure of our experiment and hence, it was not possible to prevent the entry of other microorganisms from the top, although adequate care was taken to try to prevent such occurrences by closing the top with aluminum foil. It was found at the end of the fourth day of incubation that contamination had occurred and it was not possible to control it thereafter. This affected the conversion efficiency of the culture in such a way that it was not possible to continue the experiment beyond the seventh day, when it was observed that the oxidized manganese was getting reduced and was in solution. Although, this type of leaching efforts was conducive to experiments described by Deshpande (1979), in the present studies it had no value.. Therefore, this conversion experiment had to be stopped.

It is possible, in future, to carry out these experiments in sterile fermenter type of vat or by employing sterilization techniques during the process in a closed vat. If using such a converter, manganese is oxidized to MnO2 form at a scale of 500 kg to 800 kg at a time, even with a reduced efficiency of conversion (say 55%), which is expected with such large volume, it would still be commercially feasible. Further pyrometallurgical processes would require less energy as 50% of the 80% particles would have already got converted.

Commercial Feasibility of Such a Process The cost would be less than the one estimated if electrochemical methods had to be deployed for such low grade ores with the 67% conversion obtained with Arthrobacter than the reported one with Hyphomicrobium (58%) (Deshpande, 1979). It is granted that many other variables like labor, capital outlay, depreciation etc. are not taken into consideration and hence these speculations would remain idle. However, a beginning has to be made, hence, such attempts. It was hoped, that such a cost effective and energy saving process would pave the way for utilizing such methods by the end users of technology i.e. the mining industry (as and when there would be a revival of the manganese mining industry in India) and the dry cell battery manufacturers.

ACKNOWLEDGEMENT

The authors are grateful to the Department of Microbiology and the authorities of Agharkar Research Institute, Pune, India, for extending the laboratory facilities for completion of this study. The work was completed with help of funding from Council of Scientific and Industrial Research, New Delhi, India (1980‐1983).

REFERENCES

[1]

Agate A. D Paper presented at the 13th Annual Conference of Association of Microbiologists of India, Pune, India 92 (1972).

[2]

Agate A.D. Society of Indian Microbiologist News, 25 (1975): 38

[3]

Agate A.D. Paper presented at the Seminar on Recent Trends in Training and Research in Aquatic Biology, Bhavnagar, India 112 (1980).

[4]

Agate A.D. Paper presented at the International Conference on Culture Collection‐IV, Brno, Chechoslovakia. 16 (1981).

[5]

Agate A.D. and Deshpande H.A. Paper presented at the International Round Table Conference on Leaching Braunshweig, Germany, 243 (1977).

[6]

Agate A.D. and Deshpande H.A. Paper presented at the International symposium on Environmental Biogeochemistry, Wolfenbutel, Germany, 9 (1977).

[7]

Agate A.D. and Deshpande H.A. Paper presented at the Industrial Fermentation Symposium, Jammu, India, 111 (1978).

[8]

Agate A.D. and Deshpande H.A. Paper presented at the 15th Annual Conference of Association of Micobiologists of India, Bangalore, India, 15 (1974).

[9]

Ali S.H. and Stokes J.L. Antonie van Leewenhoek Journal of Microbiology and Serology, 37 (1971): 519‐525.

[10] Anon, Commonwealth Economic Committee on Iron and steel and alloying metals, A review, H.M.Stationary office, London (1962).

[11] Anon, Indian Mineral Year Book, Indian Bureau of Mines, Nagpur (1970).

71

www.mwe‐journal.org Journal of Mining World Express (MWE) Volume 4, 2015

[12] Bhole B. D., Microbial beneficiation studies on Manganese ores, M.Sc. Dissertation, University of Poona, Pune, India (1979).

[13] Bromfield S.M. and Skerman V.B.D. Soil Science 69 (1950): 337‐348.

[14] Bromfield S.M. Plant and Soil 9 (1958): 325‐337.

[15] Bromfield S.M. Plant and Soil 10 (1958): 147‐160 [16] Coggin B. J. and Dey A. K. Indian Mineral Wealth, Oxford University Press, London (1955).

[17] Crowe S.A., Katsev S., Leslie K., Sturm A., Magen C., Nomosatryo S., Pack M.A., Kessler J.D., Reeburgh W.S., Roberts J.A., González L., Douglas H. G., Mucci A., Sundby B. and Fowle D.A., Geobiology. 9 (2011): 61‐78.

[18] Date R.A. and Rougheley R.J. A treatise on Dinitrogen Fixation, Section IV, John Wiley and Sons, Toronto. (1977). 243.

[19] DeSa C., Studies on Microorganisms associated with iron ores, M.Sc. dissertation, University of Bombay, Mumbai, India (1978).

[20] Deshpande H.A. Studies on Microbial conversion of Manganese, Ph.D. Thesis, University of Poona, Pune India (1979).

[21] DeWolff P.H. Acta Crystallography, 12 (1959): 341‐347.

[22] Douka C. E., Soil Biology and Biochemistry 9 (1977): 89‐97 [23] Douka C. E. Applied Environmental Microbiology, 39 (1980): 74‐80.

[24] Dubinina G. A. Z. Allg. Mikrobiol. 10 (1970): 309‐320 [25] Dubinina G.A. Mikrobiologiya 47 (1978): 591‐599.

[26] Ehrlich H. L. Journal of Bacteriology, 86 (1964): 350‐356.

[27] Ehrlich H. L. Geomicrobiology, Marcel Dekker, New York (1981).

[28] Fermor L. L. Memoirs of Geological Survey of India, The Manganese ore deposits in India, (1909): 37 [29] Ghosh J. and Imai K., Journal of FermentationTechnology 63 (1985): 259‐263.

[30] Ghose S. K. Minerals and Industries, New Delhi, India. III (3) (1967).

[31] Hajj H. and Makenson J. Applied and Environmental Microbiology 32 (1976): 699.

[32] Hem J.D. Chemical Equilibria and rates of Manganese Oxidation, US Government Printing Office, Washington DC. (1963).

[33] Hogen V.C., Electron transport and Manganese Oxidation in Leptothrix discophora Ph.D. Thesis, Ohio State University, Ohio US (1973).

[34] Johnstone W.D. Academic Brasileira deCiencias Anus., 26 (1954): 87‐100.

[35] Kanso S., Greene A.C. and Patel B.K.C. International Journal of Systematic and Evolutionary Microbiology 52 (2002): 869– 874.

[36] Kulkarni V. M. Studies of Microorganisms associated with Copper, University of Pune, Pune, India (1983).

[37] Mehta T., Coppi M. V., Childers S. E. and Lovley D. R. Applied and Environmental Microbiology, 71 (2005): 8634‐8641 [38] Miyata N., Tani Y., Sakata M. and Iwahori K. Journal of Bioscience and Bioengineering, 104(2007): 1‐8.

[39] Nealson K. H. and Myers C. R. Applied and Environmental Microbiology 58 (1992): 439‐443.

[40] Ottow J.C.G. Zentralbl. Bakteriol. Parasitenk Infektionsker. Hyg. II, 123 (1969): 600‐608.

[41] Rouf M. A. and Stokes J.L. Archives Für Microbiology, 49 (1964):132 [42] Simon D.E., Morton R.W. and Gislason J.J. Advances in X‐Ray Analysis, 47 (2004): 267‐280.

[43] Stumm W., and Morgan J. J. Aquatic chemistry, 2nd ed.Wiley‐Interscience, New York (1981).

[44] Thamdrup B., Rossello‐Mora R. and Amann R. Applied and Environmental Microbiology, 66 (2000): 2888‐2897.

[45] Tyler P.A. and Marshall K.C. Antonie van Leewenhoek Journal of Microbiology and Serology, 33 (1967): 171.

[46] Wu B., Shao H., Wang Z., Hu Y., Tang Y.J. and Jun Y.S. Environmental Science and Technology 44 (2010): 9213‐9218.

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