JOURNAL OF BACTrIEOLOGY, Feb. 1969, p. 594-602Copyright @ 1969 American Society for Microbiology

Vol. 97, No. 2Printed In U.S.A.

Microbial Dissimilatory Sulfur Cycle in AcidMine Water

JON H. TUTTLE, PATRICK R. DUGAN, CAROL B. MACMILLAN, AmD CHESTER. I. RANDLESAcademic Faculty ofMicrobial and Cellular Biology, Ohio State University, Columbus, Ohio 43210

Received for publication 6 November 1968

Ferric, sulfate, and hydrogen ions are produced from pyritic minerals associatedwith coal as a result of autotrophic bacterial metabolism. Water carrying these ionsaccumulated behind a porous dam composed of wood dust originating at a log-cut-ting mill. As water seeped through the porous dam, it was enriched in organic nu-trients which then supported growth and metabolism of heterotrophic bacteria inthe water downstream from the dam. The heterotrophic microflora within and belowthe sawdust dam included dissimilatory sulfate-reducing anaerobic bacteria whichreduce sulfate to sulfide. The sulfide produced caused the chemical reduction offerric to ferrous ion, and black FeS precipitate was deposited on the pond bottom.A net increase in the pH of the lower pond water was observed when compared tothe upper pond water. Microbial activity in the wood dust was demonstrated, anda sequence of cellulose degradation processes was inferred on the basis of sugar ac-cumulation in mixed cultures in the laboratory, ultimately yielding fermentationproducts which serve as nutrients for sulfate-reducing bacteria. Some of the micro-organisms were isolated and characterized. The biochemical and growth charac-teristics of pure culture isolates were generally consistent with observed reactionsin the acidic environment, with the exception of sulfate-reducing bacteria. Mixedcultures which contained sulfate-reducing bacteria reduced sulfate at pH 3.0 inthe laboratory with sawdust as the only nutrient. Pure cultures of sulfate-reduc-ing bacteria isolated from the mixed cultures did not reduce sulfate below pH5.5.

Autotrophic bacteria in the Thiobacillus-Ferro-bacillus group are responsible for the enzymaticoxidation of ferrous sulfide minerals (e.g., pyriteand marcasite) which are often found associatedwith coal in nature (4). The net result of microbialoxidation of pyritic minerals is an accumulation offerric, sulfate, and hydrogen ions. Drainage waterin regions where such minerals are mined becomehighly acidic and contain relatively high concen-trations of iron, sulfate, and other ions. A markedalteration in the microbial ecology can be ob-served under these conditions, and certain aspectsof this ecosystem have been described (20). Theinhibitory effects of acid mine water on hetero-trophic bacteria of neutral streams were reportedto result from hydrogen ions and, to a lesserextent, sulfate ions, but not from iron ions (10).

In this report, we consider the metabolic activi-ties of microorganisms involved in the reduc-tion of iron and sulfate ions and in the neutraliza-tion of acid in naturally occurring acidic minewater. To our knowledge, the only report of sul-

fate-reducing bacteria in acidic mine drainage isthat of Leathan (8), and this report attributes nosignificance to the presence of sulfate reducers inacid mine water.

Figure 1 is a schematic representation of theacidic stream studied. The stream flow wasimpeded by a dam composed primarily of wooddust, which was a waste product from a small log-cutting mill in the hills of southeastern Ohio. Theretarded flow of water resulted in a pond behindthe dam which is hereafter referred to as the upperpond. Uneven terrain downstream from the damresulted in the formation of the lower pond. Theporous quality of wood dust allowed the acidicwater to permeate through the wood at a low rateand to enter the lower pond. The flow of waterfrom the system at location 7 was slightly lessthan the flow entering the upper pond. Presum-ably, some water was lost via leaching andevaporation. Wood dust was sampled at location3, whereas water was examined at the other sixlocations. Although samples from all locations

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DISSIMILATORY SULFUR CYCLE IN ACID WATER

DIAGRAMOF A WOOD DUST IMPEDED ACID STREAM

Water Flow

Fio. 1. Schematic outline ofan acid stream which is impeded by a dam composed of wood dust, showing theupper and lower ponds and sample locations.

were required for the evaluation, the greatestemphasis was given to samples from the upperand lower ponds.

MATERIAILS AND METHODSSamples. Water samples taken in the field were

placed in sterile 8-oz (0.236 liter) bottles, held on icein a Styrofoam cabinet until reaching the laboratory,and then refrigerated at 8 C. All water samples wereplated on bacteriological culture media within 24 hrafter they were taken from the stream.

Chemical detrtions. Total dissolved iron wasmeasured colorimetrically by the phenanthrolinemethod, according to the procedure described for aHach Field Kit (Hach Chemical Co., Des Moines,Iowa).

Sulfate was determined turbidimetrically as BaSO4precipitate, as described by the Hach procedure, andthe pH was determined with a Beckman pH meter.H2S was semiquantitatively determined by the Hach

procedure, which involved comparison of the amountof black PbS precipitate formed on a lead acetate-impregnated filter paper with papers blackened by aknown concentration of H2S. This procedure wassensitive to 0.1 pg of H2S per ml of water.Pond water was analyzed directly for aldehydes by

the Tollens method (15) and after 20-fold and 40-foldconcentration by evaporation at 100 C.

Chromatography of soluble sugars in wood dustextracts. Extracts were prepared by adding 200 ml ofdistilled water to 100 g of wood dust which had beenobtained from either the top 6 inches (15.24 cm; freshwood dust) or from a depth of 3 ft (91.44 cm) into thepile (partially decomposed wood dust). The mixtureswere allowed to stand at 25 2 C for 2 hr with fre-quent shaking. Wood dust particulates were thenremoved by filtration through Whatman no. 1 paper.The resulting ifitrates were filtered through sterilemembranes (0.45 pAm pore size; Millipore Corp.,Bedford, Mass.) to remove small suspended particlesand to sterilize the solution.A 100-ml amount of each extract was evaporated

under vacuum to dryness over KOH pellets at 22 i

2 C. The dried residues were prepared for chromatog-raphy by resuspending in 1.0 ml of distilled water.Each solution (0.01 ml) was applied separately to

Whatman no. 1 paper which had been previouslyspotted with glucose, xylose, cellobiose, and galactosemarker solutions. Descending chromatograms weredeveloped in isopropanol-water (1:4) at 25 -1: 2 C.After development for 24 to 27 hr, sugars weredetected by the Trevelyan silver nitrate method (21).A 40-fold concentration of lower pond water was alsochromatographed in n-butyl alcohol-acetic acid-water(40:10:22). The concentrated sample was dialyzedagainst distilled water (8 C) for 12 hr in an attempt toremove inorganic ions which interfere with chromato-graphic separation and then were chromatographed inthe same solvent system.Mixed cultu flask systems. Wood dust (400 g)

and water (1 liter) were added to 2-liter Erlenmeyerflasks in the following combinations of wood dust andwater: acid mine water plus partially decomposedwood dust, acid mine water plus fresh wood dust,distilled water plus partially decomposed wood dust,distilled water plus fresh wood dust. Separate flaskscontaining each of the four combinations were incu-bated at 22 4 2 C, 37 C, and 50 C, respectively. Por-tions of liquid from flasks containing acid mine waterwere routinely assayed for iron, sulfate, and pH asdescribed above. All flasks were assayed for carbohy-drates by the anthrone method of Steinecher andRheins (18) and by the Nelson reducing sugar test (3).

Media and growth conditions. Portions (1 ml orappropriate dilutions) of each water sample wereplated on Tryptone Glucose Extract (TGE) Agar(Difco), which was supplemented with 0.5 g of yeastextract per liter (TGYE), and on Sabouraud's Dex-trose (SD) Agar (Difco). The cultures were incubatedat 25 2 C in the air for 3 days.

Anaerobic microorganisms, other than Desulfo-vibrio, were determined by adding 1.0-ml portions of asingle tube dilution series to 9.0 ml of ThioglycollateMedium (Difco). The reciprocal of the highest dilu-tion that showed growth after 7 days at 25 1 2 C, asdetermined by the appearance of turbidity in theanaerobic zone of the tubes, was taken as the numberof organisms.

Sulfate-reducing bacteria were enumerated with astandard three-tube most probable number (MPN)method (1), according to the tube culture techniquedescribed by Postgate (13) with Desulfovibrio desul-

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56TITLE Er AL.

furicans medium no. 3. Positive tubes were black afterincubation at 25 :1 2 C for 6 to. 21 days.

Cellulose-digesting microorganisms were isolatedand cultured in the cellulose enrichment medium ofMcBee (9). Cellulose was obtained from rope cottonwhich was hydrolyzed in 11.8 N HCI for 48 hr at 25 412 C, washed seven times with distilled water, andground to a paste with a mortar and pestle. Plateswere incubated at 25 - 2 C for 14 days. Cellulosedigesters were identified by the appearance of a clearzone around the colony.

Iron-oxidizing chemoautotrophic bacteria werecounted by a five-tube MPN technique (1) in the saltsmedium of Silverman and Lundgren (16). Positivetubes were determined after 15 days of incubation at25 i 2 C by the presence of a dark red-brown precipi-tate. Uninoculated control tubes and those in whichgrowth did not occur contained a light yellow-brownprecipitate at the end of the incubation period due toautooxidation of ferrous iron.

Sulfur-oxidizing chemoautotrophic bacteria wereenumerated in the same manner as were the iron oxi-dizers. Elemental sulfur (0.1 g per 10 ml) replaced theFeSO4.7H20 in the culture medium, and positivetubes were taken as those in which sufficient acid wasproduced to cause five drops of a 1% thymol bluesolution to turn red (red, pH 1.2, to yellow, pH 2.8).Control tubes and negative growth tubes gave a yellowindicator reaction.

Aerobic heterotrophic and facultatively aerobicheterotrophic isolates other than Streptomyces wereexamined for the ability to ferment or assimilate glu-cose, lactose, adonitol, arabinose, dulcitol, galactose,inositol, inulin, levulose, maltose, mannitol, mannose,melibiose, raffinose, rhamnose, salicin, sorbitol,trehalose, and xylose. Glucose and lactose utilizationwas determined in tubes of Purple Broth Base (PBB;Difco) which contained 1.0% (w/v) sugar. All othercarbohydrates were added as sterile differential discs(Difco) to plates of PBB containing 1.5% agar, whichhad been spread with 0.1 ml of a 24-hr Nutrient Brothculture of the isolate. All cultures were incubated at25 2 C for 48 hr. A color change of purple to yellow(bromocresol purple, yellow, pH 5.2, to purple, pH6.8) was taken as an indication of fermentation. En-hancement of growth around the disc was consideredas an indication of oxidative utilization. All othersugar fermentation determinations were conductedwith standard microbiological methods (18).

Oxidase production was determined by the methodof Kovacs (7).

Nitrate reduction was determined according to themethod described in the Difco Manual (9th ed., DifcoLabs., Inc., 1953). Cultures were incubated at 25 42 C for 18 hr. An 0.1-g amount of zinc dust was addedto each negative tube, and the presence of a pink colorassured that the nitrate had been reduced only tonitrite and not to N2 gas, which may have resulted in afalse negative observation.

Gelatin liquefaction was determined by the methoddescribed in the Difco Manual after incubation at 25

2 C for 5 days.Streptomyces species isolated from wood dust were

tested for their ability to ferment cellulose, glucose,

and xylose at 22 1 2 C, 37 C, and 50 C. Cells wereinoculated into PBB containing 1% (w/v) glucose,xylose, or cellulose (Whatman no. 1 paper powder)and were incubated for 48 hr at the respective tempera-tures.

RESULTSTable 1 is a compilation of the average chemical

and biological conditions (data) obtained fromsamples taken from the upper and lower pondsover a 1-year period. Each average represents amiinimum of four determinations. The sulfate-reducing bacteria from the lower pond representtwo different types. These have been tentativelyidentified as a Desulfovibrio and a Desulfotomacu-lum species. The Desulfotomaculum isolate wasa peritrichously flagellated sporeforming anaero-bic rod when observed under the electron micro-scope. It resisted heat (80 C for 15 min). TheDesulfovibrio isolate was a polarly flagellated,slightly curved or straight anaerobic rod andproduced considerably more sulfide than thesporeforming isolate.Data obtained from samples taken on the same

day are summarized in Table 2. The heterotrophicaerobes listed in Table 1 consist of 10 differentyeasts and 12 different bacteria.Ten different yeasts were isolated from the

upper and lower ponds, and were examined forsugar-fermenting capacity because of their poten-tial production of alcohols and organic acidswhich could serve as nutrients for the dissimila-tory sulfate-reducing bacteria found in both thewood dust pile and the lower pond. Three of theisolates have tentatively been characterized asstrains of Rhodotorula glutinis. None of the iso-lates fermented or utilized the following com-pounds: dulcitol, inositol, inulin, melibiose,rhamnose, salicin, sorbitol, and trehalose.

Fourgram-positiveisolates, tentativelyclassifiedas Bacillus species on the basis of aerobic sporeformation, were obtained from the upper andlower ponds.Seven physiological types or groups of gram-

negative rod-shaped aerobic bacteria were iso-lated from the stream system. The bacteria werepartially characterized with standard biochemicalreactions (17). One type was isolated from bothupper and lower ponds. This organism was tenta-tively identified as an Aerobacter or closely relatedgenus on the basis of glucose and lactose fermen-tation, facultative aerobic growth on Thioglycol-late Medium (Difco), and colony characteristicsin Eosin Methylene Blue Agar (EMB; Difco).The remaining gram-negative rods were con-

sidered to be pseudomonads on the basis of lackof fermentation of adonitol, arabinose, dulcitol,fructose, galactose, glucose, inositol, inulin, lac-

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TABLE 1. Chemical and biological changes which occurred in acid mine water upon passagethrough wood dust"

Determination Upper pond (I)b Lower pond (5)

pH 2.84 3.38(3.90-2.40) (4.85-2.70)

S04 concn (jumoles/ml) 8.765 6.100(5.205-12.492) (3.277-10.306)

Total Fe concn (jumoles/ml) 1.067 0.313(0.788-1.325) (0.064-0.681)

Sulfur-oxidizing bacteria (MPN/100 ml) 9,580 1,820,000(130-33,000) (23,000-7,000,000)

Fe-oxidizing bacteria (MPN/100 ml) 9,520 426,000(490-33,000) (33,000-1,400,000)

Anaerobes/ml on Thioglycollate Medium 2.5 528(0-10) (10-1,000)

So42- reducers (MPN/100 ml) 0 876(0-2,400)

Heterotrophic aerobes/ml onSD Agar 15.4 82,1000

(2-44) (49-290,000)TGYE Agar 47.4 350,000

(2.5-110) (470-1,700,000)

aValues are averages calculated from a minimum of four readings. Ranges of values are given inparentheses beneath the average values.bThe numbers in parentheses after upper pond and lower pond refer to the schematic outline shown in

Fig. 1.

TABLE 2. Chemical, biological, and temperature differences in samples collected on the same date

Sample Air temp Sample pH 5Q conc Total Fe concn Sulfur oxidizers Fe oxidizers SO42 reducerslocationa (C) temp (C) P (umoles/ml) (umoles/ml) (MPN/100 ml) (MPN!100 ml) (MPN/100 ml)

1 -6 6 3.1 10.098 1.325 1.3 X 102 4.9 X 102 02 -6 6 2.8 9.890 1.468 1.1 X 102 8.0 X 101 03 -6 334 -6 13 4.3 3.825 0.681 107 4.9 X 105 7.0 X 1025 -6 6 3.4 3.825 0.663 7.0 X 106 1.4 X 106 1.4 X 10'

4.Ob 4.268b 0.734b 7.9 X 10"b 1.0 X 107b 1.9 X lOb6 -6 6 3.35 4.268 0.573 2.2 X 104 2.1 X 10' 2.0 X 10'7 -6 6 3.2 5.309 0.645 4.9 X 104 4.6 X 10' 0

a As shown in Fig. 1.b Samples taken at bottom-water interface.

tose, maltose, mannitol, mannose, melibiose,raffinose, rhamnose, salicin, sorbitol, sucrose,trehalose, and xylose. Six different pseudomonadtypes were differentiated on the basis of the pres-ence or absence of nmotility, ability to reducenitrate to nitrite, gelatin hydrolysis, presence orabsence of oxidase (7), and pigmentation. Noattempt was made to study flagellation or toclassify these organisms as to genus. The organ-isms were presumed to be Pseudomonas, Xan-thomonas, Acetomonas, or closely related bacteria.

Microbiological examination of the wood dustyielded seven different isolates which were tenta-tively identified as Streptomyces species on the

basis of filamentous growth habit and colonycharacteristics. A summary of cellulose, glucose,and xylose utilization by these isolates after 48 hrof incubation is shown in Table 3. In general,the isolates grew much better at 37 C than ateither 50 or 22 C. Isolates 6 and 7 had littleactivity on the substrates tested at any of theincubation temperatures employed.One white-rot type of Basidiomycete was ob-

served to have extensive mycelial growth in thetop several inches of the wood dust pile. In addi-tion, one Clostridium strain was isolated. Gram-negative rod-shaped bacteria were also present inthe wood dust. No attempt was made to isolate

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and identify these species, except one, which wassimilar to the Aerobacter species isolated fromthe upper pond.

Aldehydes were present in both upper andlower ponds (Tollens method; 15), but the inten-sity of the reaction indicated semiquantitativelythat a greater concentration of aldehydes waspresent in the lower pond.Chromatographic examination of concentrated

lower pond water indicated the presence of sugars.The components could not be resolved by thetechniques employed, and much tailing occurredbecause of the high salt content of the water.Dialyzed concentrates did not contain sugars,and the procedure could not be used to remove thetailing due to salts. Fresh wood dust extracts alsocontained sugars. Two components co-chromato-graphed with glucose and xylose markers. Par-tially degraded wood dust extracts did not con-tain these components. Both extracts containedinseparable components which did not migrate asfar as the glucose, xylose, cellobiose, and galactosemarkers.

Figures 2 and 3 show the effect of temperatureand wood dust condition on sulfate reduction.Maximal sulfate reduction occurred in flaskscontaining partially degraded wood dust.

Figures 4 and 5 show the changes in pH in thesame flask cultures. Note that the rise in pHcorrelates with the removal of sulfate.Table 4 is a compilation of the anthrone and

reducing sugar concentrations in culture superna-tant fluids which contained a mixed microflorataken from wood dust. Data from flask cultures inwhich distilled water was substituted for acid minewater are also included. The anthrone proceduremeasures total soluble sugars, providing that themonomeric units are able to form furfurals in thepresence of concentrated sulfuric acid. In contrast

TABLE 3. Utilization ofglucose, cellulose, and xyloseby Streptomyces isolates at three different

temperaturesa

Glucose Cellulose XyloseIsolateno.

22 C 37 C SO C 22 C 37 C SO C 22 C 37 C SO C

2 + ++ -+ ++ -+ ++-3 A A -++++-++++-4 ++-+ +-+++-5 -+--+--++-7

"Reactions are indicated as follows:-, negativeresponse; -, slight utilization; +, utilization;++, greater utilization than +; A, acid produced.

to the anthrone procedure, Nelson's test does nothydrolyze glucosidic linkages. Therefore, anincrease in reducing power over the amount ofanthrone measurable sugar is interpreted as adecrease in the average chain length of solublecellulose polymers.

1000

u

'SE.

t

E

x12

800

600

400

200

-200

0 4 8 12 16 20 24

Time hI Days

Fio. 2. Influence oftemperature on sulfate reductionin flasks containing a mixed microflora and partiallydecomposed wood dust.

000

LEGEND

a 22 ±2"C370C

~~~~~501,C

600

400

0

-200

0 4 a 12 16 20 24

Time in Days

FJG. 3. Influence oftemperature on sulfate reductionin flasks containing a mixed microflora andfresh wooddust.

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VOL. 97, 1969 DISSIMILATORY SULFUR CYCLE IN ACID WATER 599

TABLE 4. Values showing change (.ug/ml) in concentration of total and reducing sugars as result ofmixedfermentation of wood dust at three different temperatures

22 C 37 C 50 CDetermination Sugar

0 Time 14 Days Net gain 0 Time 14 Days Net gain 0 Time 14 Days Net gain

Distilled water + fresh AG 275 360 +85 275 215 -60 185 440 +225wood dust Rb 70 250 +180 60 200 +140 175 350 +175

Acid mine water + fresh A 190 240 +50 170 235 +65 130 460 +330wood dust R 45 130 +85 15 350 +335 95 275 +180

Distilled water + par- A 65 55 -10 65 55 -10 30 85 +55tially degraded wood R 50 45 -5 40 50 +10 40 245 +205dust

Acid mine water + par- A 45 50 +5 40 45 +5 5 95 +90tially degraded wood R 0 5 +5 20 40 +20 10 235 +225dust

a Anthrone total soluble sugars.b Nelson's reducing sugars.

a lDI)SCUSSION- .- Water entering the upper pond was typical of6 ---- acidic mine drainage as regards low pH, high

iron and sulfate ion concentrations, numbers of.z >- _ _iron- and sulfur-oxidizing autotrophic bacteria,

and low numbers of heterotrophic bacteria, par-4 -_.. ticularly anaerobes. No sulfate-reducing bacteria

LE_E could be found in the upper pond. Four gram-9t272i2-C | positive species were isolated from the upper pond

A50*CsO-cand were tentatively identified as Bacillus species.3 This is of interest because we had previously0 2 4 6 a lo 12 14 16s 20 22 24 reported that gram-positive bacteria of neutral

streams were extremely susceptible to acid mineTime h Days water (20).

FIG. 4. Influence of temperature on pH change in In contrast, the lower pond contained a vastlyflasks containing a mixed microflora and partially differentmicroflora.Aerobicheterotrophicmicrodecomposed wood dust. organisms were present in markedly higher

numbers. The predominant aerobes or facultativeaerobes were yeasts and higher fungi and 12

- . different physiological types of bacteria. Most7 - ..were tentatively classified as pseudomonads.6 - - -- _ - - - - - Higher plate counts on TGYE agar than on the

more acid SD agar indicate that, although many5 /: | of the organisms in the lower pond were acid-

tolerant and viable, they grew more favorably onthe neutral TGYE growth medium. This suggests

4 - -, - - - - that they were entering the lower pond from thewood dust pile and were probably not growing in

a* 22+-C I II Ithe lower pond.50st c | A 200-fold increase in numbers of anaerobic

3- - - - bacteria was observed in the lower pond as com-

0 2 4 6 e 10 12 14 16 Is 20 22 24 pared to the upper pond. This is undoubtedly dueTime h Day to organic materials from the wood dust which

Fio. 5. Influence of temperature on pH change in serve as nutrient sources, and the effect of theseflasks containing a mixed microflora and fresh wood nutrients on lowering the oxidation-reductiondust. (O/R) potential.

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The increase in pH and the decrease in iron andsulfate concentrations (Table 1) in the lowerpond, in comparison to the upper pond, can beattributed to the influence of the wood dust dam.This can be seen by comparing the chemicalparameters at station 4, where the water leaves thewood dust, with the same parameters at station 5(Table 2). Although some activity with respect tosulfate reduction occurs in the lower pond, most ofthe sulfate reduction process undoubtedly occurs inthe wood dust. The discrepancy between chemicalparameters at the surface and at the bottom of thelower pond probably results from the ability ofthe chemical assays used to detect ions present ininsoluble form as precipitates in the pond bottomsamples [e.g., Fe2(SO4)3J.Stream flow rates which are influenced by en-

vironmental conditions caused fluctuations inmeasured parameters in the field. The parametersappeared to coincide with observed water condi-tions. For example, when the lower pond waterhad a reddish color, the sulfate concentration andpH were nearly equal to the upper pond water.Under these conditions, low numbers of sulfatereducers were recovered. When the lower pondwas greenish-black, the acidity and sulfate con-centration were lower than in the upper pond andsulfate-reducers were always recovered in highernumbers. Free H2S was detected in the water onlywhen the color was greenish-black. The color wasundoubtedly due to precipitation of FeS, which isin agreement with the lower iron concentrationfound in the lower pond.

It is interesting to note that dissimilatory sulfatereducers are active in a highly acidic environment.The two cultures which were isolated did notreduce sulfate in artificial media in the laboratorywhen the pH was below 5.5. However, sulfate wasreduced in the laboratory when sawdust was usedas the substrate in a mixed culture system at apH of 2.8. It is possible that a microenvironmentof higher pH is set up around wood or other sus-pended particulates in a system having a low pH.

It must be emphasized that sulfate reductioncannot occur in the absence of organic materials,since sulfate-reducing bacteria are generally re-garded as heterotrophic (11, 12, 14). The sub-strate range for these bacteria consists chiefly offermentation products and not sugars or polysac-charides. Recalling that the lower pond containssoluble sugar and that sugars believed to be glu-cose and xylose disappeared in the depths of thesawdust, it seems clear that a cellulose degrada-tion process involving several physiological typesof microorganisms is required to provide sub-strate for the sulfate-reducing bacteria. The datain Table 4 give some insight into this process.

In the 22 or 37 C cultures containing fresh wood

dust and either distilled water or acid mine water,reducingpower (Nelson's method) increased morethan anthrone measurable sugar. This suggeststhat sugars in relatively long chain lengths aredegraded to smaller fragments more rapidly thaninsoluble wood dust is converted to sugars, other-wise one would expect a greater increase in non-reducing as compared to reducing sugar. This isespecially true of the 37 C culture which con-tained acid mine water. At 14 days, the ratio ofreducing sugar to anthrone sugar is greater than1. It also suggests activity of mesophilic ferment-ing microorganisms at 37 C in fresh wood dust.These organisms are probably anaerobic, sinceaerobic bacteria would be expected to utilizereducing sugars either fermentatively or oxida-tively. It must be emphasized that acid had noeffect upon cellulose degradation in this system,and that traces of H2S present had no effect uponthe determination of reducing sugars. Then, thedifference in reducing power between the distilledwater and acid mine water, fresh wood dust cul-tures can be attributed to a lowering of the O/Rpotential by H2S and to the effect of the loweredO/R potential on the bacteria responsible forthe fermentation process. The ratio of reducingsugars to anthrone sugars (greater than 1 :1)indicates the presence of aliphatic aldehydes andketones in units of four carbons or less as opposedto compounds of five carbons or more, which arerequired to react with anthrone reagent.

In contrast to fresh wood dust cultures at 37 C,the supernatant fluids of fresh wood dust culturesat 50 C show a greater buildup of anthrone sugarsthan of reducing sugars. This suggests the activityof thermophilic cellulose-splitting microorganismsupon the insoluble wood dust cellulose. Thegreater buildup of anthrone sugars in the acidmine water culture, in which sulfate reductionwas occurring, also indicates that this may be ananaerobic process.The lack of change in anthrone or reducing

sugars in cultures at 22 or 37 C, which containedpartially degraded wood dust and either distilledwater or acid mine water, suggests a steady deg-radation sequence: insoluble wood dust -* an-throne sugar -* reducing sugar -. fermentationproducts which were utilized by sulfate-reducingbacteria. Comparison to cultures in fresh wooddust suggests that substrates available to themicroflora have been depleted in the degradedwood dust.At 50 C, in both cultures which contained

partially degraded wood dust, reducing sugarsincreased over anthrone sugars. These data sug-gest that the primary activity of thermophiliccellulose-splitting microbes in partially degradedwood dust was the cleavage of soluble degrada-

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DISSIMILATORY SULFUR CYCLE IN ACID WATER

FIG. 6. Schematic outline of the acid stream system depicting the distribution ofchemical changes and micro-organisms along the seven sample locations.

tion products to smaller units. The buildup ofreducing materials at 50 C but not at 37 or at 22C suggests that mesophilic bacteria were respon-sible for their further degradation.

It is clear that the microorganisms isolatedfrom the wood dust do not represent the overallactivity. Any or all of these species, particularlythe Streptomyces species which degraded cellu-lose, may be important to the sulfate reducers,not only as a source of nutrients but also asmetabolic agents for lowering the O/R potential.This is also true of the heterotrophic lower pondisolates.

It is quite surprising to find higher numbers ofaerobic iron- and sulfur-oxidizing bacteria presentin the lower pond under generally anaerobicconditions when compared to the upper pond.We must assume they are increasing in numbers inthe lower pond. This may reflect the availabilityof ferrous and sulfide ions as energy sources.Growth of these bacteria in the lower pondprobably results in reacidification of the water.We have not examined the isolates for the pos-sibility of facultative autotrophic growth onsulfur and organic compounds, although reportsindicate (2, 5; C. Remsen and D. Lundgren,Bacteriol. Proc., p. 83, 1963) that heterotrophicgrowth of these organisms might be possible.Origin of the oxygen requirement for autotrophiciron and sulfide oxidation is a puzzling questionin this environment. We do not understand how02 exchange at the air water interface can satisfythe 02 demand of the organisms in the presenceof detectable H2S. One possibility is a cyclicpresence and absence of 02 which could berelated to photosynthetic cycling. NumerousEuglena cells were observed microscopically in

wet mounts prepared from the lower pond water.These organisms may liberate 02 during daylightand the system would be anaerobic during darkperiods, allowing sulfate reduction to preceed.Our data, however, do not suggest such fluctua-tions since H2S is detected in the light. Anotherpossibility is the simultaneous growth resultingfrom a symbiotic association of sulfate reducers,iron and sulfur oxidizers, and photosyntheticEuglena. Ehrlich reported on the balance ofecology with reference to carbon metabolism inan acidic environment (6) and discussed the roleof algae, protozoa, yeasts, and chemoauto-trophic bacteria.

Figure 6 is a schematic summary of the activi-ties of microorganisms in the upper and lowerpond system as influenced by metabolic activityin the wood dust pile. This represents a completedissimilatory sulfur cycle as well as an Fe2 -

Fe3 -. Fe2 cycle in a rather unusual ecosystem.

ACKNOWLEDGMENTS

We are grateful for the use of the facilities at the Water Re-sources Center of the Ohio State University and for the technicalassistance of Rebecca Olenzak and Kathleen Miller.

This investigation was supported by allotment grant no. 14-01-0001-805, 980 from the Office of Water Resources Research, U.S.Department of Interior.

LmIRATURE CITED

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