Project report

Isolation of Methanogenic bacteria from the Cedar swampsediments

R.Mohanraju,Centre of advanced study in Marine biologyPortonovo 608502 India.

Introduction

Methane a clean fuel is produced by a group of bacteria

referred to as the xnethanogenic bacteria which are

classifed under the Archaebacteria. This group of bacteria

are known to utilise a wide variety of substrates likeAcetate, Formate, 112:C02, methanol and in the marine

environments methylamines and dimethylsulphide are the major

substrates. The methanogens are strict anaerobes and are

known to be present in many different anoxic environments

including the guts of fishes and termites.Most of the

methanogens are mesophilic and grow at a ph range of 6 to

8.Very few studies have been carried out in high acidic

environments.Detail studies on methanogens from high acidic

environments is to be undertaken for better understanding

the methanogenic process in these environments.

The present study was focussed mainly to isolate a

methanogenic strain from an acidic environment and

characterize it. Other microcosm studies like substrate

specificity, ph and temperature optimum levels with the rate

of methane production is also planned.

Description of the study area:

Studies were carried out from the Cedar swamp

sediments. The Volta experiment carried out in this

environment showed a high production of methane . The in

situ water ph was 4.8 and that of sediment was 5.6.

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Experimental procedures;

Sediment samples were collected and enrichment weremade with different substrates viz. H2 C02, Forinate,

Acetate, Methanol and Trimethylamine at 30 C The anaerobic

techniques and medium composition were followed as detailed

in the summer course handouts. Methane production was foundto be fairly good in the H2 CO2 and formate enrichments.

Microscopic observation showed long rods which showed

fluorescence under UV excitation due to the presence of thecoenyme F420. This is a unique character of the methanogens.

The H2 CO2 and Formate enrichments were further transferred

to medium with streptomycin (final concentration of

2.5ug/ml) and were incubated at 30 C. It was found that theenrichment under H2:CO2 produced more methane than with

forinate. This enrichment was used as the inoculum for

isolating a methanogenic strain. The Reoux bottles(Hermann

et al 1986) were used for isolation and streaking were

carried out inside the glove bag. The bottles werepressurized with H2 CO2 and were incubated at 30 C. The

bottles were then observed for appearance of colonies . Once

the colonies were found to be about 0.5mm in diameter it

was removed and observed for fluoroescence. The headspace of

the bottles were also analysed for the methane.

RESULTS:

It was found that the appearance of colonies took

nearly 10 days and a colony of 0.5mm in diameter took about

20 days to grow . The colony showed fluorescence and methane

was also recorded in the head space. The morphology of the

colony was long rods. The colonies were restreked onto fresh

plates with some streptomcin with it. Due to lack of time

the work could not be completed here.

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Simultaneously another experiment was planned andcarried out. The 112 Co2 enrichment was successfully

transferred to medium with streptomycin. It was observed

that intially growth in the first tube with streptomycin was

inhibited for quite some time but later the methanogens were

able to grow well. The microscopic observation revealed that

the cells were composed of both short and long rods and

produced methane to a level of 8.6%. So this enrichment was

selected to study methanogenesis under different substrateand temperature. So substrates like Methanol(5OmN), 112 CO2

and Formate(5OmM) were added into separate bottles in

triplicates and were incubated at 30 and 37 C. Thisexperiment was carried out for 10 days. The results obtainedare shown in Fig 1 &2 .The enrichment under H2C02 growing at

a temperature of 37 C produced a better percentage ofmethane than with the other substrates at 37 or 30 C. Theenrichment did not show any growth with methanol and with

Formate the methane production was about 6—6.5% whencompared to H2 CO21 It is concluded that at 37 C themethane production was more and H2 CO2 was a better

substrate than the other substrates. The cells were long andshort rods and showed very good fluorescence.

COMMENT

Due to lack of time and slow growth of these

methanogens not much work could be carried out and so the

cultures are being taken with me and further studies will becarried out in my lab in India and would take all steps topublish this work

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Literature cited

Balch,W.E., C.E.Fox, L.J.Magrum, C.R.Woese 1979 Nicrobiol.

Rev.43:260—296.

Doddema,H.J. and G.D.Vogels 1978. Appi. Environ. Microbiol.,

36:752—754.

Edwards, T., B.C.McBride 1975. Appi. l4icrobiol.29: 540—545.

Jones,W.J., D.P.Nagle and W.B.Whitiuan 1987. Microbiol. Rev:

51: 135—177.

Oremland, R.S. 1979.Liiuno].. Oceanogr.24:1136—1141.

Patel,G.B., G.D.Sprott and J.E. Fein 1990. Int.J.Syst.

Bacteriol. 40;12—18.

Zeikus, J.G and N.Winfrey 1976. Appi. Environ. Microbiol.

31:99—107.

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SECOND PROJECT

In regard to get myself acquainted with the techniques

in sequencing the 165 ribosomal RNA . I carried out studies

with E.Coli and Kentrophorous DNA and carried out sequencing

studies.

Inorder to sequence the 16S rRNA, few colonies of

E.Coli and Rentrophorous were used as a tempelate for

Polymerase chain reaction (PCR), with a universal primer

(RP1492) and a specific primer for eubacteria(FPL8) for the

corresponding gene. The next step of ligation into the phaqe

Ml3 was carried out. The phaqe particles were transformed

into competant E.Coli JM1O1 cells and these cells were grown

over night and plated on LB plates with X-gal. As the insert

would impair galactosidae activity of the phaqe, white

phages indicated the positive phaqes and phaqes with no

insert £on blue phaqes. The white phages were transferred

into fresh cells of E.Coli and grown overnight. The

bacterial cells were then harvesteand removed, The viral

particles lysed and DNA was extracted. This DNA was used for

a single stranded DNA sequencing gel.

As we not could complete the work as scheduled for the

course. So further study on the sequence and building of

the phylogenetic tree of the organism would be completed

later.

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