AGGREGATE FORMATION OF SULFATE-REDUCING BACTERIAAND METHANOGENS DURING INTERSPECIES HYDROGEN TRANSFER

Gerard Muyzer, Microbial Diversity Course 1993, Woods Hole, USA

Sulfate-reducing bacteria can grow on different substrates in sulfate-depletedmedium, forming acetate and hydrogen as products (Table 1, equation B). But growthis slow, because accumulating hydrogen in the medium inhibits an abundant growth.if hydrogen levels are suppressed, for instance by a hydrogen-utilizing methanogen(Table 1, equation A), then the sulfate-reducing bacterium can grow as good as in asuifate-rich medium (Table 1, equation D). This process is known as interspecieshydrogen transfer (Table 1, equation C)(Bryant et al., 1977). It has been found thatduring interspecies hydrogen transfer most of the hydrogen is transferred over shortdistances within bacterial aggregates. Therefore, two possible models of aggregateformation have been suggested (Thiele et iii., 1988). One, whereby both partners arehomogeneously mixed (lattice model”) and another, whereby the sulfate-reducingbacteria and the methanogenic bacteria are spatially separated in dusters (“dustermodel”). It was the aim of this project to obtain additional insight in the formation ofaggregates of sulfate-reducing bacteria and methanogens during interspecieshydrogen transfer. For this purpose aggregate formation was studied in situ as well asin batch cultures.

Table 1. Equations and free-energy changesG’0

(A)4H2+HCO3-+H+<>CH4+3H20 -32.4

(B) 2 ethanol + 2 H20 <> 2 acetate +2 Ht + 4 H2 ÷ 4.6

(C) 2 ethanol + HC03 <>2 acetate ÷Ht+ CH4 + 1420 - 27.8

(13) 2 ethanol + S04- <>2 acetate + H2S + 21420 -31.8

(E) 3 ethanol + 2 HCO3-<> 2 propionate + acetate + H-t 3H20 -41.5

Sulfate-reducing bacteria grown on butyrate or ethanol were obtained from StevenGoodwin (Dept. of Microbiology, Univ. of Massachusetts). An enrichment ofmethanogens from the Cedar Swamp was a gift of Jill Kreiling (MDC93, WoodsHole). The bacteria were grown in carbonate-buffered, sulfide-reduced mineral saltsfreshwater medium with different substrates, gas mixtures and in the presence orabsence of sulfate (Table 2). The growth experiments were carried out at 30°C inbottles or tubes, sealed with rubber stoppers. Alcohols and volatile fatty adds wereassayed by HPLC analysis. Methane production was measured by a standard gaschromatography procedure. Sulfide was determined by the Schnell test.To evaluate aggregate formation, bacteria were mixed with 0.2% (wt/vol.) agar,poured onto microscope slides, covered and sealed with ‘vaspar’ (1 vol. vaseline and 1vol. paraffin). The slides were incubated at room temperature in a Gas Pack Jar underN2/C02atmosphere. They were observed microscopically every 2 days with phasecontrast as well as with UV-illumination at 420 nm.

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Table 2. Substrate utilization and product hrmation during interspecies hydrogen transfer a

culture substrate S04 gas substr. b acetate prop. C CR4 H2S d

#1 lomMbutyrate - N2/COZ 9.8 0 0 0 -

#2 lomMbutyrate + N2/COz 9.8 0 0 0 -

#3 lomMbutyrate - H2/C02 10 0 0 5.6 -

#4 l7mMethanol - N2/C02 5.1 12.5 5.5 9 -

#5 l7mMethanol + N2/C02 0 16 3 1.1 +++

#6 l7mMethanol - H2/C02 8.5 4.5 4.4 0.6 -

a Overall products (in mM) formed during a period of 7 days, b Substrate (in mM) left after 7 days, C propionate, d-= no color, ÷÷÷ = dark bmwn color

During the incubation period, no substrate was used by cultures #1, #2 and #3.However, methane was formed in culture #3, which was produced by themethanogens growing on externally provided hydrogen (Table 1, equation A).Interspecies hydrogen transfer was observed in culture #4 (Table 1, equation C).Ethanol was converted into acetate and hydrogen, the latter used by the methanogensto produce methane. In addition to acetate and methane formation, propionate wasformed. In culture #5, ethanol was completely converted into acetate and propionate,while sulfate was converted into sulfide. In contrast to culture #3, culture #6 onlyevolved a small amount of methane, while ethanol was converted into equal amountsof acetate and propionate. It has been known that propionate can be formed inmethanogenic co-cultures either by the ethanol-fermenting bacterium Pelobacterpropionicus or by Desulfobulbus propionicus (Table 1, equation E). Further study isneeded to identify the microorganism which is responsible for the propionateproduction in this experiment.Microscopical observation of slide #4 showed many motile vibrio-like bacteria, aswell as motile spirila. The latter is likely Methanospirillum, as it fluorescent under liv-illumination at 420 nm. In the control slide (with ethanol and sulfate) only vibrio-likebacteria were observed. Rhodamine-labeled oligonucleotide probes specific formethanogens, for SRB, and for other phylogenetic bacterial groups gave no additionalinformation of the species composition. All bacteria seem to stain more or less withthe red-fluorescent probes. Despite the observed interspecies hydrogen transfer, noaggregate formation between sulfate-reducing bacteria and methanogenic bacteriacould observed, neither on the slides nor in the batch cultures. This is probable due toH2 accumulation in the sealed environments to a level whereby the methanogens donot need the close proximity of the sulfate-reducing bacteria for the hydrogentransfer. Future experiments, therefore, have to be performed with pure culturesgrown in flow cells or fluidized bed reactors to prevent hydrogen accumulation in themedium. The spatial distribution of the different partners within these aggregates canthen be studied in more detail by the combined application of fluorescentoligonuleotide probes and confocal laser microscopy.

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ReferencesThiele, J. H., M. Chartrain and J. C. Zeikus. 1998. Control of interspecies electron flow

during anaerobic digestion: role of floc formation in syntrophic methanogenesis.App!. Environ. MicrobioL 54, 10-19.

Bryant, M.P., L.L. Campbell, C. A. Reddy, and M. R. Crabill, 1977. Growth ofDesulfovibrio in lactate or ethanol media low in sulfate in association with P12-utilizing methanogenic bacteria. App!. Environ. Microbiol. 33, 1162-1169.

ISOLATION OF BACTERIA FROM THE GUT OF A SPIDER CRAB

Gerard Muyzer, Microbial Diversity Course 1993, Woods Hole, USA

Goal of the project was to isolate different anaerobic bacteria from the gut of a spidercrab, Libinia dubia (phylum: Arthropoda, class: Crustacea, order: Decapoda).Emphasis was put on the isolation of chitine-degrading bacteria, cellulolytic bacteria,sulfate-reducing bacteria and methanogenic bacteria. The gut from a spider crab wasremoved aseptically and transferred directly to a carbonate-buffered, sulfide-reducedmineral salts marine medium. After homogenation of the gut in the anaerobic hood,0.8 ml was transferred to 8 ml of fresh medium. A serial dilution was made from thistube. Subsequently, 0.8 ml aliquots of the iO to 10’ dilutions were transferred totubes containing medium plus one of the following substrates: (1) 10 mM acetate, (2)10 mM lactate, (3) hydrogen, (4) chitine (1 mg/mi), and (5) cellulose (1 mg/ml). After2 weeks of incubation at 30°C no visible growth could be observed. One of thefollowing conclusions could be drawn from these observations: (1) an incubation timeof 2 weeks is too short to observe visible growth, (2) the microflora grows aerobically,or (3) the gut of the spider crab is sterile. Aithough the last conclusion seems to beunrealistic, Boyle and Mitchelle (1978) have described the absence of microorganismsin the entire digestive tract from other crustaceans, viz. isopod and amphipod species.Also during this course, Andreas Brune and Lee Hughes were not able to visualizebacteria in the gut of an isopod (pers. comm.) In addition to these observations,attempts to amplify the 16S rRNA genes from gut contents of a marine shrimp, whichwere done by the author at the Max-Planck-Institute for Marine Microbiology inBremen (Germany) failed.

ReferencesBoyle, P. J., and R. Mitchell. 1978. Absence of microorganisms in crustacean digestive

tracts. Science 200, 1157-1159.

AcknowledgmentsThe author is greatly indebted to Steven Goodwin and Jill Kreiling for providingbacterial cultures, to Sandra Nierzwicki-Bauer for providing oligonucleotides probes,to the Marine Resource Center (MBL, Woods Hole) for providing a spider crab, toBob Bullis (Marine Resource Center, MBL, Woods Hole) for ‘de-guting’ the animal,and to Andreas Brune and Jørg Overmann for helpful discussion and advice.

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