SULFATE REDUCTION BY AN IRON(ll1) REDUCING ISOLATE?AND

CAN SULFATE REDUCERS RESPIRE FEWI)?

Ute MUh, Philipps UniversitAt Marburg. FB Biologie, Mikrobiologie, Karl-von-Frisch-Str.35032 Marburg, Germany

ABSTRACT

The sequence of degradation in anaerobic sediments has been described to be Fe(ffl)reduction, followed by sulfate reduction and lastly methanogenesis (Loveley, D.R. andPhillips, E.J.P. 1986, AppI. Environ. Microbiol. 51, 683 - 689). It was also shown thatsome marine sulfate-reducing bacteria are able to use Fe(ffl) as a terminal electron acceptor,thereby causing the accumulation of siderite (FeCO3) in salt-marsh sediments (Coleman,M.L. et al. 1993, Nature 361: 436- 438). In view of this succession of mineralizationevents in anaerobic sediments, it was of interest to test the capacity of further sulfatereducers to utilize iron and iron reducers to utilize sulfate as alternate electron acceptors.For this, enrichment cultures were inoculated with various freshwater and marine samplesand selected for either sulfate or Fe(Ill) reduction. Upon isolation of pure cultures, theisolates were transferred to medium containing the alternate electron acceptor. Due to thetime constraint, the iron reducing organisms had to be tested as mixed cultures for theirability to grow on sulfate. In general the enrichment medium for iron reducers containedacetate as the carbon source. Considering the potential competition of iron reducingorganisms with acetogens, it was also attempted to select for Fe(IJl) reduction ontrimethoxybenzoate.Preliminary results indicate that the isolated sulfate reducers are able to reduce Fe(Ill),albeit at a slow rate. None of the enrichments of iron reducing organisms were found togenerate sulfide from sulfate. Possibly this was due to the short time available. Several ofthe enrichments for iron reduction on trimethoxybenzoate led to significant microbialgrowth.

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INTRODUCTION

Anaerobic degradation in sediments has been described as the succession of three majorphysiological redox reactions: the oxidation of organic matter with reduction of Fe(Ill), themineralization of organic matter under reduction of sulfate and lastly methanogenesis(Reeburgh. W. 5. 1983). In general it is assumed that these processes are mutuallyexclusive and strictly in the above order of events. This observation can be explained bycompetitive advantages of the various organisms: in the presence of non-limiting sulfate,sulfate reducing bacteria perform a thermodynamically more favored reaction thanmethanogens. In addition, it has recently been shown that Fe(ffl) reducing bacteria canoutcompete both sulfate-reducers and methanogens (Lovley and Philipps, 1987).

It is possible then, that the degradation events are characterized by a succession of differentbacterial specialists. However, it has been shown that some marine sulfate-reducers areable to grow on Fe(Ill) as a terminal electron acceptor (Coleman et al., 1993) therebycausing the accumulation of siderite (FeCO3) in salt-marsh sediments. Desulfovibriadesulfuricans was identified as being able to reduce Fe(flI), whereas Desulfobacterpostgatei and Desulfobacter curvatus did not It was therefore found interesting to testfurther sulfate-reducers for their ability to reduce Fe(lIl) and iron-reducing bacteria for theirability to utilize sulfate.

To date there have only been two bacterial strains identified that completely mineralizeorganic compounds to CO2 under reduction of Fe(ffl). The only well studied strain of theseis Geobacter metallireducens (Lovley et al., 1993). The 165 rRNA sequence analysis of 0.metallireducens places it in the delta proteobacteria, with the closest relationship toDesutfuromonas acetoxidans. Other iron reducing organisms, such as Shewanettaputrefaciens, only perform an incomplete oxidation of organic compounds to acetate andCO2.Therefore it also seemed of interest to enrich for further iron reducing strains from avariety of habitats. It was also attempted to isolate cultures that are able to utilize anaromatic carbon source, such as trimethoxybenzoate.

The ability to reduce iron was found in enrichment cultures both from fresh water samplesites and from saline sources. Several of the isolated sulfate-reducers were able to reduceFe(ffl), but tentative results indicate no sulfide production from iron reducing cultures.

MATERIALS AND METHODS

Sample Sites. The desired goal was to find organisms that reduce both sulfate and Fe(lll),possibly at different adaptations. Consequently the samples were picked mainly from siteswhere it was assumed that both elements were present Iron is usually found in anindustrial iron seep on school street. Both the seepage on the road was used (iron seep) aswell as a sample of the sediment, where the seepage enters school street marsh (iron seepsediment). Samples from both school street marsh (on the brackish side of the road) andoyster pond were grown in fresh water1 medium. The oyster pond sample was collectedwhen the tide had risen to approximately half-maximum. So, a salt-water enrichment wasinoculated also.Several samples were collected near the outlet of salt pond: sand that was mixed with rustparticles (sand/rust); moist sand that was baked with rust from an overlaying pipe (rusty

C- ‘Abbreviations: FW (fresh water), SW (sea water), TMB (trimethoxy>enzoate)

pipe); an iron/rust spot that is permanently under water on the concrete of salt pond bridge(concrete) and a black layer that was underneath the rust layer of that spot (concrete, black).

-> Two samples were collected in Sippewissett salt marsh, where both iron reducingorganisms and sulfate-reducers had been found previously: near a rusty refrigerator andfrom a ditch with sulfide containing mud.

Generation ofFerric Iron Hydroxide. A 0.4 M solution of FeCI3 was adjusted to pH 7.0with pellets of NaOH. The iron solution turned into a suspension and was allowed to settleovernight To remove the chloride ions, the slurry was washed by centrifugation andresuspension in sterile water. For immediate use, the suspension was microwaved at fullpower for 45 seconds in a sterile flask. For storage, the Fe(OH)3was adjusted to pH 11and autoclaved. A further possibility is to dry the ferric oxide at 125° C. The ensueingprecipitate can then be pulverized and sterilized by further heating. This should giveFe(OH)3which is better suited for quantition. However, a precipate was obtained that didnot resuspend well and gave a purple color rather than rust red, so only the Fe(OH)3suspension was used to make media.

Media. The enrichment medium for iron-reducers contained per liter: 0.5 g NaHCO3, 0.1 gCaCl22H2O,0.1 g KCI, 1.5 g NH4C1, 0.6 g NaH2PO4 0.1 g NaCI, 0.1 g MgC126H20, 0.1 g MgSO4•7H20, 5 mg MnCl24H20, 5 mg Na2MoO4•2H20. NaC1 was

added (20 g) for inoculations from saline sources.The following solutions were added afterautoclaving and cooling: 1 ml SL-10 Trace Elements solution, 1 ml Six Vitamins Solutionand I ml Vit B12. The medium was buffered with 30 ml I M NaHCO3and cooled under80 % N2 and 20 % CO2.The pH was adjusted to be between 6.5 and 6.8. Fe(OH)3wasadded to concentrations of 100 mM and 5 mM. As carbon source, either 30 mM acetate or30 mM trimethoxybenzoate was added prior to inoculation.

When the isolated sulfate reducing organisms were grown on the iron containing medium,5 drops of 0.5 M sulfide were added to 30 ml culture in order to ensure a low redoxpotential in the medium. The medium for growth of sulfate-reducers was preparedaccording to the course protocol, but without suffide.

All the cultures were incubated at 30° C.

Assays. Iron reduction was measured with the ferrozine assay as described by Lovley(Lovley, 1986). For this 0.1 ml of sample is mixed with 5 ml 0.5 N HCI. Culture samplesare removed with a syringe and measured by weighing directly into the dilute acid. After 15minutes, 0.1 ml of the sample-acid mixture is added to 5 ml of ferrozine solution (lgllferrozine in 50 mM HEPES, pH 7.0) and the absorbance is measured at 562 nm. Tocalibrate the assay, a standard curve was made with Fe(NH4)2(504)ifrom 20 mM to 100mM. The absorbance readings were perfectly linear in that range. Most of the samples,however, were too dilute in Fe(ll) concentration. The assay was therefore modified to giveabsorbance values in the range of 0.05 to 1.0. For this, 0.1 ml sample was added to 1 mlof 0.5 N HCI, and 0.1 ml of the latter was added to 2.5 ml of ferrozine solution.Theoretically this should give a 10 fold increase in sensitivity. In fact, the increase is notdirectly proportional. Consequently, the absorbance values obtained were not convertedinto Fe(ll) concentrations, but were used as qualitative comparisons only.

Sulfate reduction was determined with the Schnell test A 0.2 ml sample was added to 2 mlof reagent mix (0.42 ml conc. HCI, 0.13 g CuSO4 5H20 in 100 ml H20) and theformation of a brown precipitate observed.

0 RESULTS

Bacterial growth was found in a variety of the enrichment cultures (Table 1). All freshwater enrichments were grown in the presence of 100 mM Fe(Ill). The SW enrichmentsfrom I through 24 were grown in the presence of 100 mM Fe(ffl), all the higher numberedsamples with 5 mM Fe(m). The lower concentration of iron should be adequate forgrowth. In most cases it was possible to predict Fe(ffl) reduction by visual inspection: therust colored medium turned black. The only exception was seen with the Sippewissettinoculates which turned black even when no significant iron reduction could be detected.Possibly this was due to the presence of sulfide, which was tested by inoculating withautoclaved samples from the salt marsh. In both cases the medium turned black within 2days, but no Fe(ll) could be determined.

Only the secondary cultures were examined for bacterial morphology. A variety of shapeswere found, all of which were rods, some motile and some non-motile,

It was attempted to isolate single colonies by starting a shake series of cultures 8, 35 and38. By the end of the course (5 days after inoculation) no clearing zones of the suspendedFe(1Il) could be seen. Possibly, streaking on plates would have been a faster form ofisolation.

Due to the time restriction, it was decided to test mixed cultures of iron-reducers on theirability to reduce sulfide. FW enrichments 10, 35 and 38 and SW enrichments 8, 13, 26 and27 were used to inoculate sulfate containing medium. No sulfide was added in order toperform the Schnell test for fast identification. This seemed reasonable, since there isevidence for oxygen tolerant sulfate-reducers (Frund and Cohen, 1992). Possibly the ironreducing bacteria, which do not require a low redox potential for growth, would alsoreduce sulfate in an environment that is anoxic, but above -300 mV. The cultures weretested on the last possible day, after 3 days of growth. No sulfide formation or significantturbidity could be detected.

Course enrichments of sulfate-reducers from oyster pond were subjected to two sets ofshake series. Five isolates were incubated in the Fe(ffl) containing medium (Table 1).Sulfide was added to these cultures, since the bacteria had been enriched under reducedconditions. The same amount of sulfide was added to a control bottle, which did not turnblack or showed any Fe(ll) formation (tested after 4 days).

By visual inspection, four of the obtained colonies were pure and one still showed bacteriaof mixed morphology. Sample 5: long curved rods, non-motile; sample R: short, fat rods,tapered at one end, non-motile; sample B: very thin, long, straight rods, banded, non-motile and in equal amounts: short, fat rods, non-motile; sample 1: medium curved rods;sample 2: medium long, thick, curved rods, non-motile.

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Table 1. Growth of cultures on Fe(Ill) containing medium.

Sample # Source of inoculate Water Carbon Days of growth -

source Fe(ll) reduction

I ironseep FW acetate 16H2 iron seep sediment FW acetate 16 ++4 oyster pond FW acetate 16 +++ 21 ++++13 oyster pond SW acetate 7 +++ 12 -&+-H14 oysterpond SW TMI3 7o11 schoolstniarsh FW acetate 7++ 12+-t-++12 school stmarsh FW TMB 7 ÷15 sand/rust SW acetate 7 o16 sand/rust SW TMB 707 rustypipe SW acetate 16+ 21+-i-17 rustypipe SW acetate 7°18 rustypipe SW TMB 7o19 concrete SW acetate 7 +++20 concrete SW TMB 7021 salt pond SW acetate 7 +++22 saltpond SW 1’MB 7023 concrete, black SW acetate 7 024 concrete, black SW TMB 7 a26 Sippewissett. fridge SW acetate 5 ++++ 10 ++++27 Sippewissett, fridge SW TMB 5 +++ 10 H27 Sippewissett, ditch SW acetate 5 + 10 +29 Sippewissett, ditch SW 1’MB 5 + 10 +++

30 SulfatereducerS SW lactate 3o 8 +31 SulfatereducerR SW acetate 3+ 8+32 Sulfate reducer B SW lactate 3 o/÷ 8 +33 Sulfate reducer 1 SW lactate 3 + 8 ++34 Sulfate reducer 2 SW acetate 3 o/+ 8 +

10(2°) 1 FW acetate 8÷+ 13+-H-25(2°) 1 FW TMB So/+ 13++35(3°) 10 FW acetate 4 +36(2°) 11 FW acetate 4o37(2°) 13 SW acetate 4o38(2°) 4 FW actetate 4+++39(2°) 8 SW acetate 4o40(2°) 26 SW acetate 4 o41(2°) 27 SW TMB 40

Legend to the table:Symbol0 Ot.05+ 0.05-0.1++ 0.1 -0.25

0.25 - 0.40.4 - 0.7

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DISCUSSION

Enrichment of iron reducing organisms from a variety of the examined sample sites wassuccessful. It is possible, despite the difference in source material, that eventually most theisolates will be characterized as being identical strains. Most commonly, enrichments willlead to isolation of either Geobacrer metatlireducens or Shewanella puirefacietzs. Furtherpurification and characterisation have to be made in order to determine whether new ironreducing organisms have been isolated.

No conclusion can be drawn whether any of the iron reducing cultures are able to utilizesulfate as a terminal electron acceptor. The time of incubation was too short to interpret anegative result Some of the inoculates showed a response on the Fe(flI)-containingmedium within 3 days, but it is quite likely that there is a time lag for switching to analternate electron acceptor. Furthermore, a significant amount of Fe(ffl) was transferredwith the inoculate, which most likely was not consumed after the first three days.

Consistent with reports in the literature, the sulfate reducing isolates were able to reduceFe(ffl). The rate of Fe(ll) formation was slow compared to the enrichment cultures, but noattempts were made to correlate the rate to cell densities. The importance of iron reductionfor these isolates, whether it enables them to survive or actually to grow, will still have tobe investigated.

REFERENCES

Coleman, M.L., Hedrick, D.B., Lovley, D.R,, White, D.C. & Pye, K. (1993) Nature361, 436 - 439.

FrUnd, C. & Cohen, Y. (1992) App!. Environ. Microbial. 58, 70 - 77.Lovley, D.R. & Phillips, E.J.P. (1986) App!. Environ. Microbial. 51, 683 - 689.Lovley, D.R. & Phillips, E.J.P. (1987) AppI. Environ. Microbial. 53, 2636-2641.Lovley, D.R., Giovannoni, 5.3., White, D.C., Champine, 3.E., Philipps, E.J.P.,Gorby, Y.A. & Goodwin, S. (1993) Arch. Microbial. 159, 336 - 344.

Reeburgh, W.S. (1983) Annu. Rev. Earth Planet. Sci. 11,269 - 298.

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