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I -‘- ' (“'3 ‘4‘“ ‘Rflx ' 1.: “mm “A 3' If I "u, “"1“ 1 "$4 ’1']? 1h HEM“! will}, [fit "‘1’”. .l "p: Jilin"? llfllfllflllflillimflllllmfllllll L 3 1293 00989 5727 This is to certify that the thesis entitled SULFATE REDUCTION IN THE SEDIMENTS OF A EUI‘ROPHIC LAKE presented by RI CHARD LAWRENCE SMITH has been accepted towards fulfillment of the requirements for Ph .D . degree in MiCI‘ObiOlOEy Date jjf’ 67 0-7 539 —_k LIEMRY madman. mate University ‘ l OVERDUE FINES: 25¢ per dey per i ten RETURNING LIBRARY MATERIALS: Place in bookre returnt to rename charge from circulation records -' 53;" H995 Iv 24.1 SULFATE REDUCTION IN THE SEDIMENTS OF A EUTROPHIC LAKE By Richard Lawrence Smith A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1981 Cepyright by RICHARD LAWRENCE SMITH 1981 ABSTRACT SULFATE REDUCTION IN THE SEDIMENTS OF A EUTROPHIC LAKE by Richard L. Smith Concentrations of various sulfur compounds (804', H28, 8°, acid volatile sulfide (AVS), and total sulfur) were determined in the profundal sediments and overlying water column of a shallow eutrophic lake. Low concentrations of sulfate relative to those of AVS and total sulfur and a decrease in total sulfur with sediment depth implied that the contribution of dissimilatory sulfate reduction to H28 production was relatively minor. However addition of 1.0 mm Na235804 to upper sediments in laboratory experiments resulted in the production of H2358 with no apparent lag time. 35S-‘I‘racer experiments indicated an average turnover time of the sediment sulfate pool of 1.5 hours. Total sulfate reduction in a sediment depth profile to 15 cm was 15.3 mmoles sulfate reduced mfz day'l, which corresponds to a mineralization of 30% of the particulate organic matter entering the sediment despite low sediment sulfate concentrations. Reduction of 358° occurred at a lower rate. Most probable number estimates of sediment sulfate-reducing bacteria grown on a variety of sole carbon sources were between 4.5 x 103 and 3.3 x 106 cells/gram dry weight sediment. This represented up to 602 of the total anaerobic heterotrophic bacterial population Richard Lawrence Smith present in the lake sediments. Isolates of sulfate-reducing bacteria obtained from similar enrichments were able to utilize a variety of carbon sources for growth. Included among these were acetate, propionate, and butyrate while lactate, pyruvate, and casamino acids supported growth of all strains isolated. Each substrate tested supported growth of at least one isolate, establishing that a number of carbon sources can potentially serve as electron donors for sulfate reduction in lake sediments. Sediment reactor systems were designed and constructed within which a continual low-level input of sulfate was maintained, thereby simulating sulfate diffusion into freshwater sediments. Potential rates of sulfate reduction in sediments contained within reactors dropped with time and with increasing sulfate additions while effluent acetate concentrations increased in sulfate amended reactors. Addition of R2, lactate, and glucose decreased the effluent acetate concentration in sulfate amended reactors relative to controls and stimulated potential rates of sulfate reduction. Acetate production was attributed to sulfate reduction, suggesting that substrates which can be oxidized to acetate must be included among the primary electron donors for sediment sulfate reduction. Decreases in the effluent acetate concentration coupled with increases in potential rates of sulfate reduction indicated that acetate may also be an electron donor for sulfate reduction at increased sulfate concentrations. Mineralization rates of 14C-substrates were determined in the presence and absence of NazMoO4, an inhibitor of sulfate reduction. Na2M004 inhibited sulfate reduction at all concentrations tested (0.2 mM-ZOOmM), while methane production was inhibited at Na2M004 Richard Lawrence Smith concentrations greater than 2 mM. Initial mineralization rates of glucose were unaffected by Na2M004, however the presence of Na2M004 decreased the mineralization rates of lactate (58% inhibition), propionate (52% inhibition), an amino acid mixture (85% inhibition) and acetate (14% inhibition). These decreases were attributed to the heterotrophic activity of sulfate-reducing bacteria. Hydrogen stimulated the reduction of 35804' 2.5-2.8 fold, demonstrating potential hydrogen oxidation by sulfate-reducing bacteria. These results indicate that sulfate reducers in freshwater sediments utilize an array of substrates as electron donors and are of potential significance to the in gi£g_mineralization of lactate, propionate, and free amino acids in these sediments. Thus sulfate-reducing bacteria appear to be involved with several major steps of anaerobic carbon metabolism in freshwater sediments. To Elaine, for dancing with every mountain she meets. ii ACKNOWLEDGEMENTS I would like to extend my sincere appreciation to Dr. Michael Klug for his guidance, support, and enthusiasm throughout the course of this project. His influence has profoundly affected the emergence of my scientific endeavor from the laboratory to the ”real world". Thanks also to the members of my guidance committee, Dr. J. M. Tiedje, Dr. J. A. Breznak, and Dr. C. A. Reddy and to the faculty and staff of the Kellogg Biological Station. I would also like to recognize John Molongoski, John Dacey, Gary King, Derek Lovley, and Joe Robinson for pertinent contributions and provocative discussions, which have helped improve the quality of this dissertation. Technical help was provided by Ronda Snider, whose assistance was invaluable, and by Debbie Bartel, Karen Dacey, and Rick Greening. I would certainly be remiss without expressing my heartfelt appreciation to Elaine, my wife, whose devotion, sacrifice, and willing assistance were the mainstay of this effort, and to my parents for exemplifying the continual need to grow and understand. iii TABLE OF CONTENTS LIST OF TABLESOOOOOOO0.0....0.0..O00.0.0000...OOOOOOOOOOOOIOOO... LIST OF FIGURESOOOOO0.00.0.0...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO INTRODUCTIONOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.... CHAPTER I. CHAPTER II. CHAPTER III. LITERATURE REVIEW AND OBJECTIVES................... DISSIMILATORY SULFATE REDUCTION.................... SULFATE REDUCTION IN NATURAL HABITATS.............. ELEN‘ENTAIJ SULFUR REDUCTIONOOOOOOOOOOOOOOOOOOOOOOOOO OVERVIEW AND ONECTIVESOO00.00.000.000.000000000000 LITERATURE CITED.0..CO...O...OIOOOOOOOOOOOOOOOOOOOO REDUCTION OF SULFUR COMPOUNDS IN THE SEDIMENTS OF A EUTROPHIC LAKE BASIN.OOOOOOOIOOOOOOOO000...... INTRODUCTION.eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee MATERIALS AND METHODSeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Sampling Siteseeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Sample COlleCtioneeeeeeeeeeeeeeeeeeeeeeeeeeeee Chemical anaIYSBSeeeeeeeeeeeeeeeeeeeeeeeeeeeee Sulfur redUCtioneeeeeeeeeeeeeeeeeeeeeeeeeeeeee RESULTSeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Distribution Of SUIfur compounds.............. SUlfate rEdUCtioneeeeeeeeeeeeeeeeeeeeeeeeeeeee Elemental SUlfur rEdUCtioneeeeeeeeeeeeeeeeeeee DISCIJSSIONOOOOOOOOOOOOOO00......OOOOOOOOOOOOOOOOOOO LITERATURE CITED.OOOOOOOOOOOOOOOOOOOOOOOOOOIOOOOOOO ELECTRON DONORS UTILIZED BY SULFATE-REDUCING BACTERIA ISOLATED FROM EUTROPHIC LAKE SEDIMENTS.... INTRODUCTION.eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee MATERIALS AND METHODSeeeeeeeeeeeeeeeeeeeeeeeeeeeeee SCdiment BamPIQSOeeeeeeeeeeeeeeeeeeeeeeeeeeeee MPN €8t1mateseeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee IBOlationseeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Carbon survey................................. GrOWth With HYdrogeneeeeeeeeeeeeeeeeeeeeeeeeeo RESULTSeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee DISCIJSSIONOOO0.0...OOOOOOOOIOOOOOOOOOOOOOOO00...... LITERATIJRE CITED.O...O0.00.00...00.00.000.000....0. iv page vi vii 12 13 18 23 23 24 24 24 25 26 27 27 34 39 47 53 55 55 56 56 56 57 57 58 58 61 69 CHAPTER IV. CHAPTER V. CHAPTER VI. ELECTRON DONORS UTILIZED BY SULFATE-REDUCING BACTERIA IN SEDIMENT CONTAINING FLOW-THROUGH FLASKSOOOOOOOOOOO0.0.0.0...OOOOOOOOOOOOIOOOOOOO0.0. INTRODUCTIONeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee MATERIALS AND HETHODSeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Sediment reaCtOESeeeeeeeeeeeeeeeeeeeeeeeeeeeee Sulfate redUCtioneeeeeeeeeeeeeeeeeeeeeeeeeeeee Chemical anEIYSESeeeeeeeeeeeeeeeeeeeeeeeeeeeee RESULTSeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee DISCUSSIONOOOIIOOOOOOOOOOO...OOOOOIOOOOOOOOOOOOOCOO LITERA'I'URE CITED.0......O00......OOOOOOOOOOIOIOOOOO ELECTRON DONORS UTILIZED BY SULFATE-REDUCING BACTERIA IN EUTROPHIC LAKE SEDIMENTS............... INTRODUCTIONeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee MATERIALS AND METHODSeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Sediment COlleCtioneeeeeeeeeeeeeeeeeeeeeeeeeee SUlfate redUCtioneeeeeeeeeeeeeeeeeeeeeeeeeeeee Effect of molybdate upon sediment aCtIVICIESeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Mineralization Of lac-BUbStrateseeeeeeeeeeeeee RESULTSeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Effect of NazMoO4 upon sediment processes..... Mineralization Of 1 C‘BUbStrateseeeeeeeeeeeeee EffECt Of H2 upon BUIfate radUCtioneeeeeeeeeee DISCIJSSIONOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO LITERATURE CITED.O.I...O0....OOOOOOOOOOOOOCOOOOO... SIJMMARY AND CONCLUSIONS.OOOOOOOOOOOOOOOOOOOO0.00... REFERENCESOOOOOOOOOO0...0...OOOOOOOOOOCOOOOOOOOOOOO page 71 71 72 72 76 76 77 87 98 100 100 101 101 101 102 103 104 104 106 113 113 118 121 128 LIST OF TABLES Table CHAPTER I 1 Standard free energies for the reduction of selected eleCtron acceptors...................................... CHAPTER II 1 Reduction of 35304' with depth in Wintergreen Lake profundal sediments.oooooooooooooooo00000000000000.0000. CHAPTER III 1 MPH estimates of sulfate-reducing bacteria in Wintergreen Lake profundal surface sediments............ 2 Isolates of sulfate-reducing bacteria from Wintergreen Lake profundal sediments.................... 3 Sole carbon substrates utilized for growth by sulfate-reducing bacteria isolated from Wintergreen Lake profundal sediments. o o o o o o o o o o o o o o o o o o o 4 H2 utilization by sulfate-reducing bacteria isolated from Wintergreen Lake profundal sediments............... CHAPTER V 1 Effect of NaZMoO4 upon methane production and sulfate reduction in Wintergreen Lake profundal sediments....... 2 Effect of Na2M004 upon mineralization of 2-14C-acetate in Wintergreen Lake profundal sediments................. 3 Effect of Na2M004 upon mineralization rates of 14C substrates in Wintergreen Lake profundal sediments...... 4 Effect of H2 upon sulfate reduction in Wintergreen Lake profundal sedimentsooooooooooooooooooooooooooooooso CHAPTER VI 1 Free energy released by electron donor/sulfate reduction couples demonstrated in Wintergreen Lake profundal sediments.00000000000000.0000.000.000.000.000. vi page 44 59 60 62 63 105 107 112 114 126 LIST OF FIGURES Figure CHAPTER I 1 The relationship between sulfate-reducing bacteria and methanogenic bacteria in freshwater sediments....... CHAPTER II 1 Distribution of sulfur compounds in Wintergreen Lake profundal sediments................................ 2 Distribution of 804' and H28 in Wintergreen Lake profundal sediments and overlying water column on A) 8-17-78 and B)11-7-780oooooooooooooooooooooooooooooo 3 Average annual sulfate concentration in Wintergreen Lake profundal sedimentsoooooooooooooooooooooooooooooooo 4 Distribution of H28 and total acid volatile sulfide (AVS) in Wintergreen Lake profundal sediments on 11-29-79..OOOOOOOOOOOOOOOOOOO0.000000000000000.0.0000... 5 Time course of sulfate reduction at 10°C in profundal sediments amended with 35804' to a final concentration Of 1.0 Mooooooooooooooooooooooooooo 6 Kinetics of sulfate reduction at 10°C in profundal sediments collected in March amended with 3SSO4'........ 7 Reduction of 35804' at 10°C in profundal sediments collected in March at in situ 804' concentrations....... 8 Time course of elemental sulfur reduction at 10°C in profundal sediments amended with 30 mg 35S°/l sediment................................................ CHAPTER IV 1 Diagram of the sediment reactor system.................. 2 Methane concentration in sediment reactor flask headspace............................................... 3 Effluent acetate concentration from sediment reactor flasks receiving varying concentrations Of BUlfateoooooooooooooooooooooooooooooooooooooooooooooo vii page 15 29 31 33 36 38 41 43 46 74 79 81 CHAPTER IV (con't) 4 Potential rates of sulfate reduction in sediment reactor flasks receiving H2, glucose, and/or varying concentrations Of sulfate............................... 5 Potential rates of sulfate reduction in sediment reHCtor flasks receiving laCtateoooooooooooooooooooooooo 6 Effluent acetate, propionate, and lactate concentrations from sediment reactor flasks rece1v1n8 laCtateoso0.0000000000000000...ooooooooooooooo 7 Ratio of the effluent acetate concentrations from reactors receiving no sulfate relative to those rece1V1ng 8u1fateo00000000000000.0000oooooooooo CHAPTER V 1 Effect of Na2M004 upon the mineralization of U'laC-glucose at10°C..ooooooooooooooooooooooooooooooooo 2 Effect of Na2M004 upon the mineralization of 1-14C-pr0p10n8te at10°C..00000000000000.0000.oooooooooo CHAPTER VI 1 A generalized scheme of the role of sulfate reduction in anaerobic carbon metabolism in anOX1C sediments........................................ viii page 84 86 89 91 109 111 124 INTRODUCTION The decomposition of organic matter in the sediments of productive freshwater lakes is typically anaerobic and is characterized by the production of reduced metabolic endproducts. These include both organic and inorganic compounds such as ammonia, hydrogen sulfide, volatile fatty acids, and methane (33). Mineralization of initial fermentation endproducts in these sediments, and in other anaerobic habitats, depends upon the activity of various terminal microbial processes, which serve to complete the metabolism of sedimenting organic matter. These processes include nitrate reduction, sulfate reduction, and methanogenesis. The relative role of each of these \ \w—v—~ terminal processes is often inferred from the concentrations of the various electron acceptors involved (e.g. 10, 33, 54), though this may be misleading if the turnover time of the natural pool is rapid (52). Much of the understanding of sulfate reduction comes from studies in the marine environment where sulfate levels are relatively high. In such systems sulfate reduction appears to dominate anaerobic carbon mineralization in the sediments (21). Sulfate concentratiggs in freshwater lake sediments are very lgwrinucgmparison‘to marine systems and consequently the contribution of sulfate reduction to total carbon mineralization has been inferred to be insignificant (33, S4). The rate of sulfate reduction is dependent upon both electron donor and electron acceptor availability (18, 32). The sulfate concentration is the limiting factor for sulfate reduction in eutrophic lake sediments since carbon is readily available and sediment sulfate concentrations are low (33). However if the rate of sulfate diffusing into the sediments is the rate limiting step, significant rates of sulfate reduction may occur even when the concentration of sulfate in the sediments is virtually undetectable. Other oxidized sulfur compounds may also be utilized for dissimilatory reduction in freshwater sediments. Elemental sulfur reduction has been implicated (39, 55), though as yet has not been demonstrated despite reports that elemental sulfur concentrations can exceed sulfate concentrations in freshwater sediments (36). Hence although the potential exists in eutrophic freshwater sediments, the mineralization of organic carbon coupled to sulfur reduction has been poorly characterized. CHAPTER I LITERATURE REVIEW AND OBJECTIVES Dissimilatory Sulfate Reduction. The utilization of sulfate as a terminal electron acceptor is called dissimilatory sulfate reduction. It is an obligately anaerobic process carried out by bacteria of two distinct genera. Desulfotomaculum sp. are sporeforming, heterotrophic, motile rods (9) while Desulfovibrio sp. are nonsporulating, heterotrophic, motile, spiral-shaped organisms (42). Other reported genera, though not widely recognized, include Desulfomonas (34), Desulfobulbus (26), Desulfobacter, Desulfococcus, Desulfosarcina, and Desulfonema (see 35). As a group these organisms can tolerate a wide range of environmental conditions including temperature, pressure, and salinity (41). They are present in many habitats, including freshwater and marine sediments, sewage, rumen, and human intestinal tracts (19, 27, 28). ’ While this group of bacteria is characterized by the ability to reduce sulfate, some organisms possess the capability to utilize sulfite, thiosulfate, or tetrathionate as electron acceptors (50). Recently Biebl and Pfennig (5) demonstrated that certain strains can also reduce elemental sulfur. Pure cultures of Desulfovibrio desulfuricans andWDL gigas are able to grow in the absence of oxidized inorganic sulfur compounds by reducing fumarate to succinate (28). Sulfate-reducing bacteria were also shown to produce H2 as an electron sink when grown with ethanol or lactate in the absence of sulfate, provided a low partial pressure of hydrogen was maintained (7). The energy obtained by the reduction of these various electron acceptors as compared with the energy obtained from electron acceptors utilized by other organisms is shown in Table 1. While sulfate is not the most energetically favorable electron acceptor for sulfate-reducing bacteria it is the species most frequently encountered in nature. The significance of these other electron acceptors to sulfate-reducing bacteria in sediments has yet to be demonstrated. Carbon metabolism of sulfate-reducing bacteria is generally incomplete, with the production of fatty acids and C02 (41). In general the range of substrates which these organisms can utilize as electron donors is limited (8) with lactate, pyruvate, ethanol, and formate reported to be the preferred electron donors (50). Hydrogen and sulfate can also be utilized as the sole energy source (4); however, an organic carbon source is required for growth (3, 45). This latter reaction may allow the sulfate reducers to more effectively compete for electron donors in sediments if they can participate in interspecies hydrogen transfer similar to that proposed for methanogens (48). Such a transfer was demonstrated in mixed cultures of Desulfovibrio with Hz-producing, acetogenic bacteria (6, 31). The narrow range of known carbon substrates may be the ramification of isolating most sulfate reducers using lactate as the electron donor. Widdel and Pfennig (53) recently isolated a sulfate-reducing, acetate-oxidizing strain of Desulfotomaculum from acetate enrichments. Other strains of this genus are described as lacking the capability to oxidize acetate (9). Acetate-oxidizing, sulfate-reducing activity has Table 1. Standard free energies for the reduction of selected electron acceptorsl. Electron Acceptor Product AGO' (kcal/mole H2) 02 H20 -56.7 no," N2 -53.6 fumarate succinate -20.6 803' H28 -13.8 $203"3 H23 -10.4 504' H28 - 9.1 HCO3’ CH4 - 8.1 So H23 - 6.7 H" H2 + 4.6 1Values from Thauer et a1. (50). also been reported to be present in sewage (17, 32), which indicates the natural occurrence of acetate-oxidizing sulfate reducers. Other investigators have reported isolating sulfate-reducing bacteria that can oxidize propionate and long chain fatty acids (26, see also 35). Sulfate Reduction £2 Natural Habitats. Sulfate reduction plays an important role in carbon metabolism in anaerobic marine habitats due to the high concentrations of sulfate in seawater (21). Since the rate of sulfate reduction is independent of the sulfate concentration at concentrations greater than approximately 10 mM (18), sulfate concentrations are not likely rate limiting in the water column and upper sediments of most marine habitats. This implies that electron donor availability is the rate dependent factor for sulfate reduction (18, 43). Goldhaber and Kaplan (18) conclude that sulfate-reducing bacteria rely on a complex community of fermentative bacteria to supply the small range of organic molecules utilized by sulfate reducers. They also demonstrated that the rate of sulfate reduction was related to the sedimentation rate of organic material. The importance of sulfate reduction to carbon metabolism in marine sediments is supported by studies that report that sulfate reduction catalyzed over 50% of the carbon mineralized in a marine sediment model system (24) and 532 of the mineralization in a coastal marine sediment (21). In the latter case the sulfate reduction rate ranged from 25-200 nmole 804"2 cm'3 clay'l over an annual cycle, which corresponds to a turnover time of the sulfate pool of 4-5 months. Stratified freshwater systems have much lower concentrations of sulfate present in the hypolimnion and the sediments, which limits sulfate reduction (43). As a result the activity of sulfate-reducing bacteria in freshwater sediments has frequently been overlooked. Stuiver (49) found that sulfur transport by sedimentation of organic material was neglible relative to the sulfate concentration, as most of the sulfur present in the sediments was in the form of 804-2, H28, and 8'2. Loss of sulfur from the hypolimnion was neglible as very little sulfate was exchanged between the hypolimnion and epilimnion by eddy diffusion, while the loss of H28 to the epilimnion by both diffusion and ebullition was also small (49). Although sulfate reduction occurs in the water column, the maximum rate occurs in the sediments (47, 49, 52). Dunnette (16) found that H28 production from (358) sulfate displayed saturation kinetics, while cysteine decomposition contributed between 5.1 and 53 percent of the total H28 produced. Although sulfate values may be low in freshwater sediments, drawing conclusions about reaction rates from pool sizes may be in error (21). van Gemerden (52) demonstrated that in mixed cultures of Desulfovibrio desulfuricans and Chromatium vinosum both organisms were able to grow exponentially in the presence of very low total sulfur concentrations. The turnover time of the total amount of sulfur was as short as 15 minutes. Thus a significant amount of sulfate reduction can occur even when sulfate concentrations are low, if a rapid turnover time is evident. Cappenberg (10) found up to 1x107 sulfate-reducing bacteria per liter of wet mud in lake sediments where no sulfate was detectable. Generally in a sediment depth profile significantly greater rates of sulfate reduction are found near the sediment surface and decrease exponentially with depth (2, 21, 35). In marine sediments an estimated 65% of the total sulfate-reducing activity occurred in the top 10 cm 8 (21), while sulfate concentrations (21) and the number of sulfate-reducing organisms (10, 21) displayed similar trends. The exact nature of the sulfate-reducing profile depends (at least in part) on the total electron donors and total electron acceptors available. Current evidence indicates that sulfate reduction and methanogenesis are not compatible. The maximum number of methanogens are reported at sediment depths below that of the maximum number of sulfate reducers in both marine and freshwater sediments (10, 14). In laboratory experiments with marine sediments methane production did not occur until 90% of the sulfate was depleted (29, 30), while negligible methane production was observed in sewage sludge when high levels of sulfate were added (17). Winfrey and Zeikus (54) report that the addition of 0.2 mM sulfate to Lake Mendota sediments inhibited methanogenesis, though sulfate itself is not toxic to methanogenic bacteria (7). Cappenberg (12) concluded that methanogenic bacteria are inhibited by H28 produced by sulfate reducers. He suggests a commensalistic relationship between the two bacterial groups whereby the sulfate reducers produce acetate, which diffuses down and is utilized by methanogenic bacteria (11, 12, 13). The inhibition of methanogenesis by sulfide appears unlikely however at natural sulfide concentrations (7, 14, 30, 54). While acetate production by sulfate-reducing bacteria and subsequent utilization by methanogenic bacteria is still a feasible hypothesis it must be reexamined in light of reports that sulfate-reducing bacteria can oxidize acetate (53). Recent evidence indicates acetate mineralization by both methanogens and sulfate reducers in freshwater and marine sediments (35, 53, 55). Thermodynamically the utilization of acetate by sulfate reducers is 9 favored, yielding 11.3 kcal/mole versus 6.8 kcal/mole yielded by the conversion of acetate to CO2 and CH4 (54). This compares with 38.2 kcal/mole available to classical sulfate-reducing bacteria when oxidizing lactate (50). Since the acetate-oxidizing, sulfate-reducing bacteria isolated to date can utilize only acetate, butyrate, butanol, or ethanol as electron donors (53) they face a competitive disadvantage when competing with lactate-oxidizing, sulfate-reducing bacteria for depleting sulfate concentrations. This appeared to be the case in New Zealand intertidal sediments where acetate oxidation by sulfate-reducing organisms and interstial sulfate values both decreased with depth despite elevated rates of sulfate reduction in the deeper sediments (35). Studies by several investigators have indicated that sulfate-reducing bacteria in marine sediments can oxidize hydrogen (1, 35, 37). Oremland and Taylor (37) demonstrated that sulfate-respiring bacteria were primarily responsible for H2 oxidation while additions of H2 stimulated sulfate reduction with very little increase in methane production (1, 35). It appears that sulfate reducers can compete for hydrogen as well as acetate in marine sediments. However, hydrogen contributed little as an energy source when sulfate-reducing bacteria were grown on lactate and sulfate in culture (25), though active hydrogenase is produced (51). In addition the strain of Desulfovibrio which reportedly oxidizes hydrogen as the sole energy source also oxidizes lactate (3). Thus it appears that sulfate-reducing bacteria oxidize hydrogen only when electron donor availability is rate limiting, though hydrogen oxidation in sediments by sulfate-reducing bacteria at £3 situ hydrogen concentrations has not yet been 10 demonstrated. The hydrogen concentration in eutrophic freshwater sediments is less than 0.7 umoles 1"1 of wet sediment (48). Methanogenic bacteria and sulfate-reducing bacteria can also interact by means of interspecies hydrogen transfer. Winfrey and Zeikus (54) demonstrated that the sulfate inhibition of methanogenesis in freshwater sediments could be reversed by additions of hydrogen. They concluded that sulfate altered the normal carbon and electron flow, which involved H2 transfer to the methanogens in sulfate depleted Lake Mendota sediments. Desulfovibrio sp. can grow in sulfate-free media containing either lactate or ethanol in the presence of a methanogenic bacteria (7). In the absence of sulfate the Desulfovibrio produced H2, which the methanogen utilized to produce methane. The methane-producing bacteria maintain a low partial pressure of H2 in such a system, which allows the reaction to be thermodynamically favorable. Apparently the flow of electrons to methane via H2 does not compete with their flow to sulfate as their data indicates that methane production was reduced by addition of sulfate in stoichiometric amounts. Bryant et al. (7) suggest that sulfate reducers may be important to carbon metabolism in anaerobic habitats containing little or no sulfate if their metabolism is coupled with Hz-utilizing methanogenic bacteria. Several investigators have reported that sulfate-reducing organisms can oxidize methane while producing sulfide. Much of the evidence comes from the observed distribution of methane in marine environments (30) where a concave up curve is viewed as indicative of methane consumption in surface sediments (30, 44). Evidence indicates that Desulfovibrio desulfuricans can oxidize methane, but at a very 11 slow rate (15). Anaerobic methane oxidation was observed in hypolimnetic water samples from Lake Mendota (38), though additions of sulfate did not stimulate methane oxidation; acetate or lactate and sulfate were required for growth of enrichments. Zehnder and Brock (56) reported anoxic methane oxidation in the sediments of the same lake, attributing the activity to methanogens rather than sulfate-reducing bacteria. Apparently methane oxidation by sulfate reducers is relatively unimportant in sediments that have high amounts of readily utilizable organic carbon sources (30, 44). Measurement of sulfate-reducing activity in natural sediments has been attempted using several different procedures. One of the most common is enumeration of sulfate-reducing bacteria. However, the number of sulfate-reducing organisms does not reflect the activity of sulfate reduction measured with Na235804 in either freshwater or marine sediments (16, 21). Jbrgensen (23) reports that colony counts underestimate the true numbers of sulfate reducers by 1000 fold. As previously indicated pool sizes of sulfate are not reliable estimators of sulfate reduction. Dunnette (16) found that the contribution to total sulfide by sulfate reduction and organic putrefaction varied, while Sorokin (46) indicated that the maximum H28 concentration and the rates of sulfate reduction rarely correspond. The accumulation of sulfide is also not considered to be a useful measurement of sulfate reduction. The accumulation of reduced sulfur compounds was found to underestimate the $3 3153 metabolism of coastal marine sediments IO-fold because it neglected diffusional losses of sulfide (23). A portion of the sulfide produced is also precipitated by heavy metals, of which iron is the most important (18). Initially 12 iron combines with free sulfide to form the intermediate iron sulfides of mackinawite (£88) and greigite (F8384), however these are not stable under sedimentary conditions and slowly transform to pyrite (FeSZ). Pyrite is acid insoluable and difficult to quantify in sediments. Jorgensen (21) estimates that 102 of the sulfide produced in coastal marine sediments is transformed to pyrite, however pyrite formation can be very rapid in salt marsh sediments (20). Determinations of stable sulfur isotope ratios have proven to be an important aid in examining the origin of the various sulfur pools and estimating the rates of bacterial sulfate reduction (21). The stable isotope 328 reacts more rapidly than does 34S during bacterial reduction. This results in sulfide, acid volatile sulfide, and pyrite pools enriched in 323, while the sulfate pool is enriched in 34S (18). Rates of sulfate reduction may be calculated by measuring the depletion of total sulfate in pore water with depth, and hence with time (18). It is difficult to establish the dynamics of the sulfur cycle from stable isotope determinations (21) and therefore radiotracer techniques using 358 have been developed to increase sensitivity when measuring sulfate reduction (22, 24, 46). This technique takes advantage of the availability of carrier-free Na235804, which can be amended to sediments without altering the 1£;§$£g_sulfate concentration. Elemental Sulfur Reduction. Although it is found in many environments, very little is known about the reduction of elemental sulfur to sulfide. Postgate (40) was unable to demonstrate elemental sulfur reduction using six strains of Desulfovibrio. However cultures of Desulfovibrio growing on formate and reducing sulfate displayed large increases in the production of sulfide when elemental sulfur was 13 added (52), indicating that So may have been reduced to H28. As indicated previously Biebl and Pfennig (5) demonstrated 8° reduction to sulfide by several sulfate-reducing organisms. They have also isolated and described Desulfuromonas acetoxidans, which is capable of reducing 'elemental sulfur through acetate, ethanol, or propanol oxidation to C02. This organism is unable to utilize other inorganic sulfur compounds as electron acceptors. Previously the oxidation of acetate coupled to the reduction of elemental sulfur was not thought to yield enough energy to produce ATP (AGO' - -4.0 kcal/mole). However, accounting for physiological conditions (HS‘ - lO‘ZM) the free energy change can be sufficient to produce ATP (AGO' - -14.8 kcal/mole) (50). The organism does not possess hydrogenase and can not use hydrogen as an electron donor. The distribution of this organism in sediments and other natural habitats is unknown. Overview and Objectives 2: Research. The proposed role of sulfate reducing bacteria in eutrophic freshwater lake sediments is depicted in Figure 1. The sulfate reducers compete with other organisms (for example methanogens) for low molecular weight organic carbon sources or hydrogen. These are utilized primarily as electron donors, and are either completely oxidized to C02 or only partially oxidized to CO2 and a second lower energy containing carbon substrate. Alternatively if the sulfate concentrations are very low the hydrogenase containing sulfate-reducers may produce molecular hydrogen, which would be rapidly utilized by methanogens. This would make the process energetically favorable for the sulfate-reducers and allow them to remain metabolically active in the absence of sulfate. 14 Figure 1. The relationship between sulfate-reducing bacteria and methanogenic bacteria in freshwater sediments. woo woo \fi mm: AA oAfi naofl on_H mmmuzgmm mmmezmzmmn mzmwozomasmann .wawuaamu mm: :usouw :awcs you maumaomd mo panaac mnu a>aw mmauucmm o — a o a o o H 0 ~ 0 o g o a I nnmnn uoa< o o c o o o a q o a c N o o c a mcoz o H N o N H N N o N N H N N N N mumumo< o a m N m N a w m w o o w a w w mum>=uhm o o mg a m w 0 AN N NH a 0 HA o - Nu mumuomd N D a 3 .d V .d 1n 3 N In "N S n1 m m w m n m a a m m. m o m. m C., .3... .23... a w a a .m. a m m a m m w p a w m w m a w m m. w a a w a .32.: s. .35; a 14 9 a a T. 14 a a a mo Hanaaz aumuumnam :onumo maumuumnam :uaouu .mucaafivam Hmvcsmoua axmq coauwuaucaz aouw vaumaoma muuauamp mafiasvaulaumMHam Np £u3ouw you vanwaaua maumuumnsm conuma aHam .m manna 63 Table 4. H2 utilization by sulfate-reducing bacteria isolated from Wintergreen Lake profundal sediments. Colony Diameter (mm)8 Strain H2 ATM N2 ATM 13002 0.81 (0.10;6) 0.38 (0.03;6) 13004 0.97 (0.06;6) 0.42 (0.03;6) 13006 0.68 (0.09;6) 0.43 (0.05;7) 13007 0.76 (0.05;6) 0.40 (0.06;6) 1F008 0.70 (0.07;6) 0.43 (0.03;6) 13010 0.42 (0.0S;6) 0.45 (0.0S;6) 13011 0.95 (0.03;6) 0.41 (0.02;6) 13012 0.93 (0.10;6) 0.48 (0.04;6) 13013 0.51 (0.08;6) 0.36 (0.02;6) 83002 1.07 (0.16;6) 0.61 (0.02;6) 8F003 1.40 (0;2) 0.61 (0.04;4) 8F006 0.71 (0.16;6) 0.37 (0.04;6) 83008 1.27 (0.19;3) 0.70 (0;1) 83011 0.81 (0.07;6) 0.45 (0.05;6) 8F012 0.58 (0.13;6) 0.35 (0.05;6) 93001 1.06 (0.17;6) 0.57 (0.04;6) 93002 0.97 (0.21;6) 0.57 (0.03;6) 153001 0.62 (0.09;6) 0.32 (0.03;6) 153002 0.64 (0.08;6) 0.42 (0.05;6) 153003 0.77 (0.14;6) 0.58 (0.03;5) ATCC 7757b 0.61 (0.10;6) 0.45 (0.08;6) IBrackets enclose 95? confidence interval estimator and n. bDesulfovibrio desulfuricans. 64 Wintergreen Lake profundal surface sediments. The average estimate when lactate was utilized as a carbon substrate (1.6 x 104 cell/ cm53 of sediment) is within the range of 9.3 x 104 sulfate-reducing organisms/cm?"3 of sediment reported for a coastal marine sediment (8) and the 2'10 x 103 sulfate-reducing organisms/cm,-3 of sediment reported for freshwater surface sediments (3). The range of estimates obtained with lactate as a carbon source was nearly an order of magnitude, which was probably due to both sampling and sediment heterogeneity since populations of sulfate-reducing bacteria typically vary with sediment depth (3, 8). Lactate is the preferred carbon source of most cultures of sulfate-reducing bacteria and therefore most frequently utilized for enrichments and isolations of these organisms. Lactate provided the highest MPN estimates of sulfate reducers present in Wintergreen Lake sediments. However a number of other carbon sources also enriched for sulfate-reducing organisms (Table 1). The electron donors utilized by sulfate reducers growing in control enrichments were probably natural sediment substrates, the yeast extract present in the medium, or the H2 present in the glovebox atmosphere. These results do not imply that sulfate-reducing bacteria are capable of directly utilizing any given one of these substrates for either carbon or energy, for an intermediate group(s) of organisms may be involved in the initial catabolism of the substrate while the sulfate reducers are utilizing subsequent metabolites. The results do however indicate that the heterotrophic utilization of these carbon substrates in the sediments potentially involves sulfate-reducing organisms. Many strains of sulfate-reducing bacteria require growth factors, which typically are provided as yeast extract in growth media (16, 17). 65 Yeast extract was included in the media employed in these studies to facilitate isolation of as many groups of sulfate-reducing bacteria as possible. Controls containing yeast extract along with impurities in agar, but no added carbon substrate (i.e. none), supported growth of several strains of sulfate-reducing organisms. Growth was relatively slow and these organisms were unable to grow when the enrichments were streaked upon media containing many of the tested carbon substrates. The yeast extract concentration was lowered to 0.052 and liquid media used to survey carbon substrates utilized by the sulfate-reducing isolates to avoid subjective determinations relative to control results. As a group the sulfate-reducing bacterial isolates utilized all of the sole carbon substrates tested for growth (Table 3). Included among these substrates were acetate, propionate, and butyrate. Acetate-oxidizing, sulfate-reducing bacteria have been reported in only three instances (see 18, 10, 21), yet 52% of these isolates were able to grow in an acetate containing medium. These acetate-oxidizing isolates also oxidized a number of other carbon substrates, including lactate, which distinguishes them from Desulfotomaculum acetoxidans (21) and suggests that acetate-oxidizing, sulfate-reducing bacteria in natural sediments are not restricted to acetate as the sole electron donor. To the author's knowledge Desulfovibrio rebentschikii is the only sulfate-reducing bacteria described capable of oxidizing butyrate (1, see 18), though it was last long ago, while propionate oxidation by sulfate reducers has only been recently reported (10). Thus a significant fraction of these isolates oxidize carbon substrates which are not considered to be characteristic substrates for sulfate-reducing 66 bacteria. Lactate, pyruvate, malate, succinate, and ethanol also supported growth of most isolates and are commonly oxidized by many strains of sulfate-reducing bacteria (16). All strains tested were capable of good growth with casamino acids as a carbon source, though this substrate is not normally included in the list of carbon substrates utilized by sulfate-reducing bacteria. This may be a characteristic common to many sulfate-reducing bacteria since a number of sulfate-reducing isolates grow with casamino acids as a substrate, including standard laboratory strains (19). The Desulfovibrio reference strain used here (ATCC 7757) also utilized casamino acids (Table 3). The ability to utilize amino acids as electron donors for sulfate reduction may be significant in Wintergreen Lake sediments which receive large inputs of proteinaceous seston (14). Differences were evident in the carbon sources utilized by the sulfate-reducing isolates relative to the carbon substrate with which they were isolated (Table 3). This is particularly true for growth with formate or alanine by lactate isolated organisms versus pyruvate isolated organisms and succinate and malate utilization by the control isolates as compared to the other isolates. These differences illustrate the hazard of making general inferences as to the carbon substrates utilized by sulfate-reducing bacteria in natural habitats when most of the standard strains of sulfate-reducing bacteria have been isolated with a single substrate (i.e. lactate). The wide range of carbon sources utilized by the sulfate-reducing isolates confirms the MPN results and reemphasizes the role of sulfate-reducing bacteria in the heterotrophic utilization of these carbon sources in the sediments. For example MPN estimates with 67 succinate, malate, formate, and methanol as carbon sources were high (Table 1) and many isolates were able to oxidize these substrates (Table 3), although attempts to isolate sulfate-reducing bacteria with media containing these carbon sources were unsuccessful. All the isolates utilized lactate, pyruvate, ethanol, and casamino acids as growth substrates, yet MPN results for pyruvate and casamino acid containing media were significantly lower than MPN results with lactate. This may have been due to other organisms successfully outcompeting the sulfate-reducing populations for pyruvate and casamino acids in the MPN enrichments. It is also possible that lactate-oxidizing sulfate reducers were present in the sediment which could not oxidize casamino acids or pyruvate, but such organisms were not isolated. Included among the list of electron donors utilized by sulfate-reducing bacteria is molecular H2 (2, 16), provided an organic carbon source, such as yeast extract or acetate, is present in the medium (2). The MPN enrichments (Table 1) were incubated in an atmosphere containing 10% H2. The results indicate that Hz was not the principle electron donor since the control estimate was low, while several carbon sources displayed MPN estimates significantly above that of the control. The sulfate-reducing bacterial isolates were able to utilize H2 as a supplemental electron donor, however, as evidenced by a stimulation of growth. A solid medium was employed for these tests to maximize exposure to H2, and as noted previously growth was evident from agar impurities. Thus controls incubated with a N2 atmosphere exhibited growth as well, though significantly less. Only one strain was apparently unable to oxidize H2 while most of the others were able 68 to do so to a greater extent than the Desulfovibrio reference strain. These results indicate that hydrogen can be a potential electron donor for sulfate reduction in Wintergreen lake sediments. The significance of H2 oxidation by sulfate-reducing bacteria in eutrophic sediments is ‘unclear however, in light of the report that H2 contributes little to the energy metabolism of sulfate-reducing bacteria grown on lactate plus sulfate (9). If this is the case H2 oxidation by sulfate reducers in eutrophic sediments may be a relatively minor process. In summary these results indicate that sulfate-reducing bacteria from freshwater sediments can be enriched with an array of carbon sources, while a wide range of carbon sources were utilized for growth by sulfate-reducing bacteria isolated from these enrichments. These included several strains capable of oxidizing acetate and propionate. The substrates tested represent carbon sources available in eutrophic freshwater lake sediments. The results do not establish the natural electron donors for sulfate-reducing bacteria, however, they do establish that a number of carbon sources can potentially serve as electron donors for sulfate reduction in these sediments. l. 2. 3. 4. 5. 6. 7. 8. 9. 10. LITERATURE CITED Baars, J. K. 1930. Over sulfaatreductie door bacterién. Dissertation. Delft. Badziang, W., R. K. Thauer, and J. G. Zeikus. 1978. Isolation and characterization of Desulfovibrio growing on hydrogen plus sulfate as the sole energy source. Arch. Microbiol. 116:41-49. Cappenberg, T. E. 1974. Interrelations between sulfate-reducing and methane producing bacteria in bottom deposits of a freshwater lake. I. Field Observations. Antonie van Leeuwenhoek'42:285-295. Cappenberg, T. E. 1974. Interrelations between sulfate-reducing and methane-producing bacteria in bottom deposits of a freshwater lake. II. Inhibition Experiments. Antonie van Leeuwenhoek‘49:297-306. Cappenberg, T. E., and R. A. Prins. 1974. Interrelations between sulfate-reducing and methane-producing bacteria in bottom deposits of a freshwater lake. III. Experiments with 1l‘C-labeled substrates. Antonie van Leeuwenhoek 49:457-469. Howard, B. H., and R. E. Hungate. 1976. Desulfovibrio of the sheep rumen. Appl. and Environ. Microbiol. 22:598-602. Jones, H. E. 1971. Sulfate-reducing bacterium with unusual morphology and pigment content. J. Bacteriol. 106:339-346. Jérgensen, B. B. 1977. The sulfur cycle of a coastal marine sediment (Limfjorden, Denmark). Limnol. and Oceanogr. 33814-832 o Khosrovi, B., R. MacPherson, J. D. A. Miller. 1971. Some observations on growth and hydrogen uptake by Desulfovibrio vulgaris. Arch. Microbial..§2}324-337. Laanbroek, H. J. 1980. Oxidation of short chain fatty acids by sulfate-reducing bacteria in freshwater and marine sediments. Abstracts of the Second International Symp. on Microbial ECOlogYO p. 2060 69 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 70 Leclerc, H., C. Oger, H. Beerens, and D. A. A. Mossel. 1980. Occurrence of sulphate reducing bacteria in the human intestinal flora and in the aquatic environment. Water Research 1_4_: 2 53-256 . LeGall, J., and J. R. Postgate. 1973. The physiology of sulfate reducing bacteria. Adv. Microbial Physiol. 19:81-133. Molongoski, J. J., and M. J. Klug. 1976. Characterization of anaerobic heterotrophic bacteria isolated from freshwater sediments. Appl. Environ. Microbiol. 21:83-90. Molongoski, J. J., and M. J. Klug. 1980. Quantification and characterization of sedimenting particulate organic matter in a shallow hypereutrophic lake. Freshwater Biol. 19:497-506. Pfennig, N. 1974. Rhodopseudomonas globiformis sp. n., a new species of Rhodospirilleceace. Arch. Microbiol. 100:197-206. Postgate, J. R. 1965. Recent advances in the study of the sulfate-reducing bacteria. Bacterial. Rev. 22:425-441. Postgage, J. R. 1965. Enrichment and isolation of sulfate-reducing bacteria. p. 190-197. '22: H. G. Schlegel and E. Kroger (ed.). Anreicherungskultur und Mutantenauslese. Fisher Verlag. Stuttgart, Germany. Selwyn, S. C. and J. R. Postgate. 1959. A search for the Rubentschikii group of Desulfovibrio. Antonie van Leeuwenhoek 22:465-472. Skyring, G. W., H. E. Jones, and D. Goodchild. 1977. The taxonomy of some new isolates of dissimilatory sulfate-reducing bacteria. Can. J. Microbiol. 2;:1415-1425. Toerien, D. P., P. G. Thiel, and M. M. Hattingh. 1968. Enumeration, isolation, and identification of sulfate-reducing bacteria of anaerobic digestion. Water Res. 2:505-513. Tsuji, K., and T. Yagi. 1980. Significance of hydrogen burst from growing cultures of Desulfovibrio vulggris, Miyazaki, and the role of hydrogenase and cytochrome c3 in energy production system Arch. Microbial..l2§:3S-42. Widdel, F., and N. Pfennig. 1977. A new anaerobic, sparing, acetate-oxidizing, sulfate-reducing bacterium, Desulfotomaculum (emed.) acetoxidans. Arch. Microbiol. 112:119-122. CHAPTER IV ELECTRON DONORS UTILIZED BY SULFATE-REDUCING BACTERIA IN SEDIMENT CONTAINING FLOW-THROUGH FLASKS INTRODUCTION Previous studies have indicated that significant rates of sulfate reduction occur in eutrophic lake sediments in spite of low interstitial sulfate concentrations (Chapter II). Although methane production is the predominant terminal metabolic process in these sediments, sulfate reduction can account for up to 302 of the total anaerobic carbon mineralized. A comprehensive view of the role of sulfate reduction in these sediments must include a description of the electron donors utilized for sulfate reduction. Few studies, however, have investigated natural electron donors for sulfate reduction. Even in marine sediments, where sulfate reduction is the primary terminal electron acceptor, specific electron donors and the contribution of each to total sulfate reduction have not been fully established. The response of a microbial community as it is subjected to environmental perturbations often yields valuable information regarding the activities of that community. Therefore anaerobic microbial ecosystems have been frequently subjected to artificial manipulations such as physical changes (5, 12) or substrate additions (3, 6, 16, 19) to characterize the prevalent metabolic pathways. Such an approach has been utilized to investigate proposed electron donors for sulfate reduction in marine sediments by measuring the response of sulfate 71 72 reduction to additions of lactate, acetate, pyruvate, hydrogen, or formate (1, 2, 13, 14). Metabolic activities in freshwater sediments, where sulfate concentrations are low, offer an excellent opportunity to examine the competition between sulfate-reducing organisms and other heterotrophs for electron donors. Investigation of these activities with perturbation experiments, however, must maintain the low levels of sulfate present in the sediment since the rate of sulfate reduction, and to a certain extent other sediment activities, depends directly on the sulfate concentration available. This investigation describes the use of sediment containing flow-through flasks designed to maintain a continuous low level input of sulfate, which simulates sulfate diffusion into freshwater sediments. Changes in potential rates of sulfate reduction as a function of added carbon sources and varying sulfate concentrations were examined in this system to investigate natural electron donors for sulfate reduction in eutrophic lake sediments. MATERIALS AND METHODS Sediment reactors: Profundal surface sediment from.Wintergreen Lake, a shallow (zm-6.5 m) hypereutrophic lake located in Southwestern Michigan (9), were sampled with an Eckman dredge. Jars were completely filled with sediment, sealed, and stored at 10°C; experiments were initiated the day of collection. Sediment was homogenized in the jars with a paint shaker, transferred to an anaerobic glovebox, and the sediment pooled. Subsamples (700 ml) were transferred to flow-through flasks (called reactors for brevity, Fig. 1), stoppered with a butyl rubber stopper, and sealed with pressure sensitive tape. The reactors were removed from the glovebox and placed on stirring platforms in a Figure 1. 73 Diagram of the sediment reactor system. 1) bottomless 1 L reagent bottle with sidearm; 2) 4" to 3" PVC reducing union; 3) 4” PVC closet connector with 1/8” inner Plexiglasab sleeve; 4) milled Plexiglass® insert; 5) filter sandwich, a) outer layers of porous polyethylene sheet (35 um pore size) and b) inner layer of Nitex (10 pm pore size); 6) butyl rubber O-ring; 7) magnetic stir bar; 8) threaded rod; 9) needle guide; 10) butyl rubber stopper with crimp seal. heck valve "i 6 9 Peristaltic Sterile glass wool Reservoir .\ 0 l 0 Filter sandwich d O ‘\\\\\\\ \\\\\\\\\\\\\\\\‘ ‘ \\\\1} M . WGM‘OJUIMH! \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ Stirrer .9 Peristaltic pump Effluent trap Ice bath GUTH 75 10°C incubator. Each reactor was connected in series to a check valve (1/3 psi) and a water displacement gas trap by 3/16" O.D. Tygon tubing connected to a syringe needle, which was inserted through the reactor stopper. Headspace gas was flushed 10 minutes with prereduced N2 through a syringe needle and exhausted via the gas trap. Input and output flow rates were maintained at 2 ml/min by a Technicon proportioning pump (Technicon Corp.); residence time of the effluent was less than one hour in the effluent lines. Effluent was collected in traps maintained in an ice bath and frozen until analysis. Analysis of interstial water obtained directly from the reactor flasks indicated that the concentration of volatile fatty acids and sulfate in the effluent were the same as those in the reactor sediment. The reactor input solution contained (per liter): a) 10 ml of a trace element solution (15); b) Na2SO4, 0.53g; c) NaHCO3, 0.5g; d) K2HPO4, 0.2g. A solution containing items a and b was adjusted to pH 7.2 in the resevoir flasks, autoclaved, and flushed with prereduced N2 while cooling. Filter sterilized stock solutions of items c and d, pH 7.4, were added when cool. The flasks were stoppered and sealed to preclude the entrance of 02 and connected to the reactors with sterile 0.03” I.D. Tygon microbore tubing. A constant pressure of N2, 3 psi, was maintained in each resevoir headspace. The reactors initially received the input solution for 3 or 4 days followed by addition of a second input solution containing the appropriate sulfate concentration and carbon substrate. This was designated as To. When added with another substrate the sulfate concentration was 0.53 g/l, which corresponds to an input rate of 0.18 mmoles reactor '1 day'l. Carbon sources included were (mmoles/l): glucose, 15 and lactate, 15. When 76 Hz was included as a substrate, the gas trap was not connected to the reactor except briefly prior to each H2 addition to vent the excess pressure. Twice daily H2 (50 m1, 1 atm) was added to the appropriate reactors through a syringe and needle. Sulfate reduction: Triplicate subsamples (5.0 ml) of sediment were taken by syringe and needle through the reactor sidearms and transferred to 30 ml Wheaton serum bottles previously flushed with prereduced 32(93z)/002(7z) and sealed with Teflon lined rubber septa (Supleco, Inc.). To each sample 0.5 ml of prereduced 10 mM N3235804 (New England Nuclear, 3-6 uCi/ml) was added through a syringe and needle. The samples were blended with a Vortex mixer and incubated at 10°C for 1 hour. The reaction was terminated by the addition of 0.5 ml of prereduced 10% zinc acetate and the samples frozen until analysis. The H2358 produced was distilled into 22 CdC12 as previously described (Chapter II), 10 m1 of Aqueous Counting Scintillant (Amersham Searle) added to each trap (scintillation vials), and the radioactivity determined with a Beckman L88000 liquid scintillation counter (Beckman Instruments). Since the sulfate-reducing activity was measured at sulfate concentrations demonstrated to be saturating for sulfate reduction at $3 3253 sulfate concentrations (Chapter II), the rates measured here will hereafter be referred to as potential rates of sulfate reduction. Chemical analyses: The concentration of methane in the headspace of each reactor was subsampled daily by syringe and needle. Methane was analyzed gas chromatographically as previously described (10). Effluent sulfate concentrations were determined turbidimetrically (18); alkalinity was monitored with a Hach Chemical Corporation test kit. 77 Volatile fatty acids were assayed using a Varian 3700 gas chromatograph equipped with a flame ionization detector. Analysis was made with a glass column (1.8 m x 2 mm ID) packed with 15% SP 1220 plus 12 H3PO4 on a solid support of Chromosorb W AW (100/120 mesh) at 110°C. Effluent lactate concentrations were analyzed by adding 0.1 ml of 0.4 M tetraethylammonium hydroxide to 5 ml of reactor effluent. The samples were dried and redissolved in 1 ml of an acetone benzylbromide solution, 400:1 (V/V), and incubated 2 hours at room temperature. Benzyl lactate was analyzed on a stainless steel column (2 m x 2 mm ID) packed with 10% butanediol succinate on Supelcoport (100/120 mesh) at 180°C using the chromatograph described above. RESULTS Two distinct rates of methane production were observed in the sediment reactors (Fig. 2); an initial rate of 0.57 mmoles CH4 1'1 of sediment day"1 was observed for 2-3 days (day -1 to day 1) and a second, lower rate of 0.04 mmoles CH4 1"1 of sediment day"1 observed for the duration of the experiment. The second rate corresponded with an increase in the effluent acetate concentration (Fig. 3). The effluent pH and alkalinity did not change over the same period. The final rate of methane production was inhibited by 402, 751, and 95% when the sulfate addition after T0 was 0.18, 1.4 (not shown), and 14 mmoles 804' reactor“l day”1 respectively, as compared to a control receiving no sulfate. Addition of H2 to the reactor headspace stimulated methane production when the sulfate input after T0 was 0.18 mmole sulfate reactor'l day'1 (Fig. 2). This rate of sulfate input, 0.18 mmoles reactor"l day-1, simulates the rate of sulfate diffusing into Wintergreen Lake profundal surface sediments (Chapter II). 78 Figure 2. Methane concentration in the sediment reactor flask headspace. Reactor input prior to T0 was 0.18 mmoles 804' reactor"1 day’l. After To sulfate inputs as indicated. MMOLES CH4! 1 SEDIMENT 79 30 r ‘ «0.18 MMOLES .. "’ H2 .1 fONTROL 2.0 ~ ' - / A \018 MMOLES 3. a d \14 MMOLES 1.0 - - 7 4 / - I l J l I ’2 O 2 6 8 80 Figure 3. Effluent acetate concentration from sediment reactor flasks receiving varying concentrations of sulfate. Reactor input prior to T0 was 0.18 mmoles 804' reactor"l day'l. After To sulfate input as indicated. MM ACETATE CONCENTRATION 81 _‘ l .414 MMOLES /CONTRO\ A 0.18 MMOLES I - DAYS 4 6 8 82 Potential rates of sulfate reduction in sediments of control reactors receiving no sulfate after To (Fig. 4a) were reduced nearly 502 in 6 days. When the sulfate addition continued after To the potential rates of sulfate reduction decreased in relation to increasing additions of sulfate, up to an addition rate of 1.4 mmoles 804' reactor"l day"1 (Fig. 4a). The rate of decrease was greater than that displayed by control reactors at all concentrations of amended sulfate. The effluent sulfate concentration from the reactors receiving the two higher sulfate inputs markedly increased throughout the 8-day time course (both were greater than 3 mM after day 2), indicating that the sulfate-reducing activity was saturated with respect to sulfate. An increase in the rate of sulfate reduction was observed in these latter two reactors (Fig. 4a) after day 2; the highest increase (2.5 fold) observed in the reactor receiving the highest sulfate input. Sulfate additions increased the effluent acetate concentration over that of the control (Fig. 3). The reactor receiving the highest sulfate input, 14 mmoles 304' reactor’1 day'l, displayed a significant drop in the effluent acetate concentration, after an initial increase, this decrease corresponded with an observed increase in the potential rate of sulfate reduction (Fig. 4), which subsequently dropped as the acetate concentration continued to decrease. The addition of lactate to sediments contained in a reactor flask maintained the original rate of potential sulfate reduction when sulfate was included in the input (Fig. 5). When added alone lactate decreased the potential rate of sulfate reduction with time. Glucose and H2 additions resulted in a drop in the potential rates of sulfate Figure l! e 83 Potential rates of sulfate reduction in sediment reactor flasks receiving H2, glucose, and/or varying concentrations of sulfate. Reactor input prior to T0 was 0.18 mmoles 804' reactor”l day'l. After To inputs as indicated. When sulfate was included input rates were the same as prior to To, except for A where values indicate rates of sulfate input. m>20 mM) of Na2M004 inhibited both total methane production and 14CH4 production from 2-140-acetate in profundal sediments. A comparison of total methane production (Table 1) and production from acetate (Table 2) indicates that methane production from acetate was inhibited to a greater extent by N32M004 than was methane production from H2 and 002. Both sources of methane production were relatively unaffected at lower concentrations of Na2M004 (i.e. <20mM). Heterotrophic metabolic processes not involving immediate precursors for sulfate reduction or methane production appeared to be unaffected by Na2M004 as rates of glucose mineralization were unaltered in the presence of 20 mM Na2M004 (Figure 1). The RI index for 2-140-acetate mineralization indicates that 20% of the total mineralization of the methyl group of acetate was oxidation to 002. Na2MOO4 inhibited 69% of this oxidation, indicating that 14% of the acetate mineralization in the absence of Na2MoO4 could be attributed to sulfate reduction, while 6% of the acetate was mineralized by some other group of organisms in the sediment. 116 The mineralization rates of acetate, lactate, propionate, and an amino acid mixture in the presence and absence of NaZMoO4 strongly imply that sulfate-reducing bacteria are directly involved in the mineralization of these substrates in Wintergreen Lake profundal sediments (Table 2 and 3). The actual mineralization rates of glucose, lactate, and free amino acids can't be extrapolated from these data since although low concentrations of 14C-substrates were added these exogenous additions are considered to have altered the natural concentration. Additions of 14C-propionate or ll‘C-acetate did not significantly alter the natural sediment concentrations (see 14). Although 52% of the total propionate mineralization could be attributed to sulfate reduction compared to 14% of total acetate mineralization, conversely these data do not indicate the relative contribution of acetate and propionate to sulfate reduction since the sediment propionate concentration is 10-fold lower than the sediment acetate concentration (D. Lovley, personal communication). The inhibition of mineralization by NazMo04 at natural substrate concentrations represents a minimum estimate of mineralization by sulfate-reducing bacteria since inhibition of sulfate reduction can increase a given substrate's availability to other sediment microorganisms. Hydrogen can also serve as an electron donor for sulfate-reducing bacteria (3). Cocultures of Desulfovibrio can participate in interspecies H2 transfer as well by oxidizing H2 and reducing sulfate (1, 4). Oremland and Taylor (17) determined that sulfate-reducing bacteria were primarily responsible for H2 consumption in marine sediments, while a 3-fold stimulation of sulfate reduction by hydrogen addition was reported in salt marsh sediments (2). H2 stimulated 117 sulfate reduction 2.5-2.8 fold in Wintergreen Lake profundal sediments, indicating potential hydrogen oxidation by sulfate reduction in freshwater sediments. However methanogenic bacteria in these sediments are the primary H2 consuming organisms (21). This is supported by a lack of an increase in total methane production in the presence of Na2M004 (Table 1), indicating that hydrogen oxidation by sulfate-reducing bacteria at 12 2152 hydrogen concentrations is relatively minor. Few studies have determined natural electron donors for sulfate reduction in anoxic sediments. Most evidence has been determined indirectly with lactate, acetate, and hydrogen being commonly implicated as electron donors (17, 23). The data presented here indicate that lactate, acetate, free amino acids, and propionate potentially provide electrons for sulfate reduction in the freshwater sediments examined. Each of these carbon substrates can be oxidized directly in the absence of H2 by one or more of several sulfate-reducing isolates from'Wintergreen.Lake profundal sediments (Chapter III). Thus it appears that an array of substrates serve as natural electron donors for sulfate reduction in these sediments and that sulfate reducers are of potential significance to the 12 2122 mineralization of lactate, propionate and free amino acids, though mineralization of any given substrate may represent only a small fraction of the total electron flow through sulfate reduction. 1) 2) 3) 4) 5) 6) 7) 8) 9) LITERATURE CITED Abram, J. W., and D. B. Nedwell. 1978. Inhibition of methanogenesis by sulfate reducing bacteria competing for transferred hydrogen. Arch. Microbiol. 117:89-92. Abram, J. W., and D. B. Nedwell. 1978. Hydrogen as a substrate for methanogenesis and sulfate reduction in anaerobic salt marsh sediment. Arch. Microbiol. 117:93-97. Badziang, W., R. K. Thauer, and J. G. Zeikus. 1978. Isolation and characterization of Desulfovibrio growing on hydrogen plus sulfate as the sole energy source. Arch. Microbiol. 116341-49e Boone, D. R., and M. P. Bryant. 1980. Propionate-degrading bacterium, Syntrophobacter wolinii sp. nov. gen. nov., from methanogenic ecosystems. Appl. Environ. Microbiol. 293626-632. Busev, A. I. 1964. Analytical chemistry of Molybdenum. Israel Program for Scientific Translations p. 6-8. Cappenberg, T. E., and R. A. Prins. 1974. Interrelations between sulfate-reducing and methane-producing bacteria in bottom dzposits of a freshwater lake. III. Experiments with l C-labeled substrates. Antonie van Leeuwenhoek 493457-469. Huisingh, J., and G. Matrone. 1972. Copper-molybdenum interactions with the sulfate-reducing systems in rumen microorganisms. Proc. Soc. Exp. Biol. Med. 139:518—521. Huisingh, J., J. J. McNeill and G. Matrone. 1974. Sulfate reduction by a Desulfovibrig species isolated from sheep rumen. Appl. Microbiol. ‘223489-497. Huisingh, J., D. C. Milholland, and G. Matrone. 1975. Effect of molybdate on sulfide production from methionine and sulfate by ruminal microorganisms of sheep. J. Nutr. 105:1199-1205. 10) J6rgensen, B. B. 1977. The sulfur cycle of a coastal marine sediment (Limfjorden, Denmark). Limnol. and Oceanogr. 223814-832. 118 11) 12) 13) 14) 15) 16) 17) 18) 19) 20) 21) 22) 23) 119 Jdrgensen, B. B., J. G. Kuenen, and Y. Cohen. 1979. Microbial transformations of sulfur compounds in a stratified lake (Solar Lake, Sinai). Limnol. and Oceanogr. 253799-822. King, G. M., and W. J. Wiebe. 1980. Regulation of sulfate concentrations and methanogenesis in salt marsh soils. Estuarine and Coastal Marine Science 193215-223. Molongoski, J. J., and M. J. Klug. 1980. Quantification and characterization of sedimenting particulate organic matter in a shallow hypereutrophic lake. Freshwater Biol. 193497-506. Molongoski, J. J., and M. J. Klug. 1980. Anaerobic metabolism of particulate organic matter in the sediments of a hypereutrophic lake. Freshwater Biol. 193507-518. Mountfort, D. 0., R. A. Asher, E. L. Mays, and J. M. Tiedje. 1980. Carbon and electron flow in mud and sandflat intertidal sediments at Delaware Inlet, Nelson, New Zealand. Appl. Environ. Microbiol. 22:686-694. Oremland, R. S., and M. P. Silverman. 1979. Microbial sulfate reduction measured by an automated electrical impendance technique. Geomicrobiol. J. 13355-372. Oremland, R. 8., and B. F. Taylor. 1978. Sulfate reduction and methanogenesis in marine sediments. Geochim. et Cosmochim. Acta'423209-214. Peck, H. D. 1959. The ATP-dependant reduction of sulfate with hydrogen in extracts of Desulfovibrio desulfuricans. Proc. “ate Acade SCie UeSeAe _45 3701-708e Peck, H. D. 1962. Comparative metabolism of inorganic sulfur compounds in microorganisms. Bacterial. Rev. 22:67-94. Smith, R. L., and M. J. Klug. Reduction of sulfur compounds in eutrophic freshwater sediments. Appl. Environ. Microbiol. (In press). Strayer, R. F., and J. M. Tiedje. 1978. Kinetic parameters of the conversion of methane precursors to methane in a hypereutrophic lake sediment. Appl. Environ. Microbiol. 223330-340. Taylor, B. F. and R. S. Oremland. 1979. Depletion of adenosine triphosphate in Desulfovibrio by oxyanions of Group VI Elements. Current Microbial. 23101-103. Winfrey, M. R., and J. G. Zeikus. 1977. Effect of sulfate on carbon and election flow during microbial methanogenesis in freshwater sediments. Appl. Environ. Microbiol. 22:275-281. 120 24) Winfrey, M. R., and J. G. Zeikus. 1979. Microbial methanogenesis and acetate metabolism in a meromictic lake. Appl. Environ. MicrObiole 31:213-221e 25) Wolin, M. J., and T. L. Miller. 1980. Molybdate and sulfide inhibit H2 and increase formate production from glucose by Ruminococcus albus. Arch. Microbiol. 124:137-142. CHAPTER VI SUMMARY AND CONCLUSIONS This study has demonstrated that high rates of sulfate reduction occur in the sediments of eutrophic lakes despite low interstitial sulfate concentrations. Although profiles of organic sulfur and H28 concentrations suggest otherwise dissimilatory sulfate reduction appears to be the major mechanism for H28 production. The highest rates of sulfate reduction occur in surface sediments and appear to be limited by the rate of sulfate diffusing into the sediments from the overlying water column. These two processes serve to maintain the low interstitial sulfate concentration, which has a rapid turnover time. The significance of sulfate reduction is emphasized by total sulfate reduction in a sediment depth profile, which can account for 30% of the mineralization of the particulate organic matter entering the sediments. Sulfate-reducing bacteria isolated from these sediments can utilize a wide array of substrates for carbon and/or electron donors; substrates that are typically found in eutrophic sediments. The range of substrates supporting growth by isolates and MPN enrichments indicates that electron donors utilized for sulfate reduction in natural habitats has been previously underestimated. Sulfate-reducing bacteria oxidize intermediate molecular weight compounds to acetate in sediment contained in reactor flasks and are stimulated by additions of 121 122 lactate, H2, and glucose. Mineralization eXperiments in the presence and absence of Na2M004, an inhibitor of sulfate reduction, also indicates that sulfate reducers utilize an array of substrates as electron donors and are of potential significance to the 13 2132 mineralization of lactate, propionate and free amino acids. Since the acetate concentration is greater than the sulfate concentration in these sediments the oxidation of acetate by sulfate reduction appears to be saturated with respect to acetate. Based upon these results a scheme conceptualizing sulfate reduction in anoxic sediments as it relates to carbon flow is given in Figure 1. The pathway of carbon flow (double-lined arrows) is the same whether C02 or 804' is the principle electron acceptor. The figure demonstrates that sulfate-reducing bacteria can be involved with each major step of oxidative carbon metabolism (steps 1-4), the magnitude of the involvement is dependent primarily upon the sulfate concentration available. Ample evidence indicates that when the sulfate concentration is sufficient sulfate reduction mediates carbon flow. Marine ecosystems are the classic example of carbon metabolism dominated by sulfate reduction when sulfate concentrations are not limiting. The nature of decompositional processes predicates that the energy available to sulfate-reducing bacteria from each subsequent step of anaerobic mineralization is less than previous steps. Thus based upon thermodynamic considerations succesively increasing concentrations of sulfate are required to favor oxidation by sulfate reduction for steps 1 through 5. The energy available at standard conditions strongly favors those sulfate reducers that can utilize steps 1, 2, or 3 for 123 Figure 1. A generalized scheme of the role of sulfate reduction in anaerobic carbon metabolism in anoxic sediments. (Modified from (2)). 124 ¥~+§ AE_me: $53 E 53338 NE BEBE Sigh—ESQ H.258 E N8 ”I I r 88" EE 4&58 mun—Em GEE ozmrd N8 + my. A mN: 1 N\ M £4 SE: .szSE .mfléggfi V998 125 electron donors (except for fatty acid oxidation). However based upon 111 _s_i£11 sediment concentrations the energy available to sulfate reducers from.H2 oxidation is considerably less (Table 1), making the reaction thermodynamically competitive with only acetate and propionate oxidation. Thermodynamic considerations are not the sole factors determining electron donors utilized by sulfate-reducing bacteria. Electron donor availability and competition with other organisms are determinative factors as well. This study has demonstrated that amino acids and propionate can serve as electron donors for sulfate reduction in eutrophic freshwater sediments, in addition to lactate, acetate, and H2. Amino acid oxidation and propionate oxidation by sulfate-reducing bacteria have not been previously demonstrated in natural habitats. Unique among the organisms isolated is the ability of a large percentage to oxidize acetate, lactate, and amino acids. Since the sediment concentration of lactate and free amino acids is very low, a model is proposed in which sulfate-reducing bacteria oxidize available higher molecular weight intermediates (i.e. lactate and amino acids) and simultaneously oxidize the more energy deficient substrates (i.e. H2, acetate, and in some cases propionate) despite the low sediment sulfate concentrations. If the sulfate-reducing bacteria that were isolated from Wintergreen Lake sediments are representative of freshwater sediment sulfate reducers then individual organisms would be responsible for a multitude of oxidative processes; a novel concept concerning sediment sulfate reduction. Implicit in this scheme is the complete oxidation of the higher molecular weight intermediates to C02 by sulfate reduction. The exact contribution of the higher 126 Table 1. Free energy released by electron donor/sulfate reduction couples demonstrated in Wintergreen Lake profundal sediments. Electron Donor Product AGO' AG' 2 Alanine 2 acetate +NH4+ -32.7a -38.3b 2 Lactate 2 acetate -38.3 -37.0 4/3 Propionate 4/3 acetate -12.0 -12.8 4 H2 4 H20 -36.3 -12.6 Acetate C02 -11.3 -10.4 akcal/mole 804' reduced (6). bkcal/mole 804' reduced. Based upon Wintergreen Lake in situ concentrations of: sulfate, SOuM (Chapter II); sulfide, 200pM (Chapter II); acetate 130uM (2): propionate, lOuM (2); dissolved inorganic carbon, 12.5 mM (1); NH3, ZmM (4); H2, 3.7 x 10'5 atm. (5); and the presupposed concentrations of luM for lactate and alanine. 127 molecular weight electron donors to total sulfate reduction, however, remains to be determined when more sensitive techniques have been developed to measure the concentrations and turnover times of these intermediates. 1. 2. 4. 5. 6. 128 LITERATURE CITED Klug, Me Je, Ge Me King, Re Le Smith, De Le LOVley, and Je We He Dacey. Comparative aspects of anaerobic carbon and electron flow in freshwater sediments. (in prep). Lovley, D. L. personal communication. Molongoski, J. J. 1978. Sedimentation and anaerobic metabolism of particulate organic matter in the sediments of a hypereutrophic lake. Ph.D. Thesis. Michigan State University. Molongoski, J. J., and M. J. Klug. 1980. Anaerobic metabolism of particulate organic matter in the sediments of a hypereutrophic lake. Freshwater Biol. 193507-518. Robinson, J. personal communication. Thauer, R. K., K. Jungermann, and K. Decker. 1977. Energy conservation in chemotrophic anaerobic bacteria. Bacterial. Rev. 31:100-180.