The influence of sulfate and nitrate on the methane formation by methanogenic archaea in freshwater sediments

In this thesis the effect of inorganic electron acceptors (sulfate and nitrate) on methane emission from freshwater sediments in the Netherlands was investigated. The chosen study area was a polder located between Leiden and Utrecht, and is representative for similar polders in The Netherlands (Chapter 3). The polder contains peat grasslands in which ditches are lying used for maintaining stable water levels. The ditches contain sediment which is a potential source of CH 4 . In freshwater environments, sulfate can be introduced by infiltration water, supply water or due to the oxidation of S-rich organic matter and iron sulfide. Also high nitrate concentrations can occur in the groundwater as a result of intensive agricultural activities. Therefore, in The Netherlands, sulfate and nitrate concentrations in the water may control the methane emission from methanogenic environments.The influence of sulfate and nitrate on methanogenesisMethane is produced by methanogenic archaea (methanogenesis) living in syntrophic association with fermentative and acetogenic bacteria. In presence of sulfate and nitrate, sulfate- and nitrate-reducing populations may successfully compete with these methanogenic consortia. In Chapter 4 the sediment was investigated for its potential methanogenic and syntrophic activity and the influence of sulfate and nitrate on these potential activities. Addition of acetate stimulated both methane formation and sulfate reduction, indicating that an active acetate-utilizing population of methanogens and sulfate reducers was present in the sediment. When inorganic electron acceptors were absent, substrates like propionate and butyrate were converted by syntrophic methanogenic consortia. However, addition of sulfate or nitrate resulted in the complete inhibition of these consortia. Our results showed that propionate and butyrate were directly used by the sulfate and nitrate reducers. This indicated that the syntrophic methanogenic consortia could not compete with nitrate and sulfate reducers.Acetate, a key intermediate in the anaerobic degradation of organic matterIn Chapter 5 the importance of methanogenesis and sulfate reduction in a freshwater sediment was investigated by using (non) specific inhibitors. Only the combined inhibition of methanogenesis and sulfate reduction resulted in the accumulation of intermediates (acetate, propionate and valerate). Acetate was the most important compound in the accumulation (93 mole %) and thereby confirming its role as a key intermediate in the terminal step of organic matter mineralization. Furthermore, the inhibition studies showed that about 70-80% of the total carbon flow to CH 4 was through acetate. This clearly demonstrated that acetate was quantitatively the most important substrate for methanogens in the sediment. Addition of chloroform (CHCl 3 ) inhibited methanogens and acetate-utilizing sulfate reducers in the sediment. Pure culture studies showed that CHCl 3 was an inhibitor of growth and product formation by methanogenic archaea, homoacetogenic bacteria, a syntrophic bacterium ( Syntrophobacter fumaroxidans ) and the sulfate-reducing bacterium ( Desulfotomaculum acetoxidans ) operating the acetylCoA-pathway.In the sediment acetate is quantitatively the most important substrate for methanogens (chapter 5). Therefore, the anaerobic conversion of [2- 13C] acetate in the presence of sulfate or nitrate was investigated (Chapter 6). Aceticlastic methanogenesis was the dominant acetate-utilizing process when the sulfate concentration was below 70 m M. At higher sulfate concentrations the formation of 13C-labeled CH 4 decreased significantly, indicating that methanogens and sulfate reducers were competing for the same substrate. When sufficient sulfate (&gt;500 m M) was present the outcome of the competition was in favor of the sulfate reducers. Unexpectedly, nitrate-reducing bacteria hardly competed with methanogens and sulfate reducers for the available acetate. The electron-acceptor/acetate ratio indicated that denitrification was coupled to the oxidation of reduced sulfur compounds or other electron donors rather than to the oxidation of acetate. Furthermore, nitrate reduction seemed to have a direct inhibitory effect on methanogenesis, and an indirect effect as a consequence of the oxidation of reduced sulfur-compounds to sulfate. The inhibition of methanogenesis by nitrate was probably not the result of competition for substrate but was due to the formation of toxic intermediates of the denitrification processes. The fact that acetate-utilizing nitrate reducers were outnumbered by the methanogens and sulfate reducers and hardly competed with these types of microorganisms for the available acetate indicated that acetate-utilizing nitrate reducers played a minor role in the degradation of acetate in the sediment (Chapter 6).Anaerobic acetate-utilizing microorganismsEnumeration of acetate-utilizing anaerobes gave insight into the different groups of microorganisms involved in the acetate metabolism in the sediment. In Chapter 7 the physiological properties of the acetate-utilizing anaerobes obtained by direct serial dilution of freshwater sediment are described. An acetate-utilizing methanogen (culture AMPB-Zg) was enriched and appeared to be closely related to Methanosaeta concilii . The most dominant acetate-utilizing sulfate reducer (strain ASRB-Zg) in the sediment was related to Desulfotomaculum nigrificans and Desulfotomaculum thermosapovorans . This result supported our hypothesis that acetate is a competitive substrate for methanogens and sulfate reducers in the sediment (Chapter 5 and 6). An acetate-utilizing nitrate reducer (strain ANRB-Zg) was isolated which showed to be related to Variovorax paradoxus . In the presence of acetate and nitrate, strain ANRB-Zg was capable of oxidizing reduced sulfur compounds to sulfate. Strain ANRB-Zg may have been involved in the oxidation of reduced sulfur compounds to sulfate in the sediment (Chapter 6). However, at this moment too little information is available to understand the exact role of strain ANRB-Zg in the sulfur and carbon cycle of the sediment. The degradation of acetate in the absence and presence of SO 42-and NO 3-is depicted in Fig. 1. The dominant acetate-utilizing anaerobes and their metabolic interactions are given as well.Figure 1: The influence of sulfate and nitrate on aceticlastic methanogenesis in freshwater sediment. AMPB: aceticlastic methanogen, ASRB: acetate-utilizing sulfate reducer, ANRB: acetate-utilizing nitrate reducer. Thick stripped lines represent competition for acetate between AMPB and ASRB. Thick dotted lines represent inhibition caused by toxic intermediates.Finally, the conversion of acetate by methanogenic and sulfidogenic communities under acetate-limited conditions was studied in Chapter 8. Our results showed that the acetate-utilizing methanogens were able to compete efficiently with the sulfate reducers for the available acetate in an acetate-limited chemostat with sulfate in excess during a long-term experiment (1 year). From the chemostat studies it became clear that the kinetic properties of the acetate-utilizing methanogen and sulfate reducer were almost similar. Unfortunately, exact values for these kinetic properties are still lacking. Therefore predictions based on these parameters about the outcome of the competition of methanogens and sulfate reducers for acetate could not be made. In Chapter 2 a review of the physiological, ecological and biochemical aspects of acetate-utilizing anaerobes and their metabolic interactions are presented.Concluding remarksThe results which are presented in this thesis advanced our knowledge of the effect of sulfate and nitrate on methane formation in sediments which are found in a typical Dutch polder. The sediment is a potential source of methane but it remains unclear if the sediment emits high quantities of methane. It was assumed that the methane emission is in the same order of magnitude (42-225 kg CH 4 ha -1yr -1) as reported for another sediment. The presence of sulfate appeared to be a major factor in controlling the formation of methane. This is due to the competition between acetate-utilizing methanogens and sulfate reducers. Nevertheless, the origin of sulfate and its effect on methane emission on the long-term is not fully understood. The inhibitory effect of nitrate on methanogenesis appears to be the result of the formation of toxic intermediates of the denitrification processes but tangible proof is still lacking at this moment. Also the physiology and ecophysiology of some of the dominant acetate-utilizing anaerobes, and the metabolic interactions among them are not completely resolved. Further investigations of these topics are needed to get a better understanding of the environment as a source of methane and the emission from it. Intriguingly, measurements of CH 4 emissions from grasslands near the location of the sediments have shown that a net methane consumption in the area is possible. This indicates that methane produced in the ditches and originating from other sources may be oxidized again by the grassland soils. To determine a methane budget for Dutch polders the potential sink and/or source capacity of the grasslands should be included to get insight in the contribution to the emission of methane to the atmosphere.</p

Microbial processes of CH4 production in a rice paddy soil: model and experimental validation.

The importance of different anaerobic processes leading to CH4 production in rice paddies is quantified by a combination of experiments and model. A mechanistic model is presented that describes competition for acetate and H2/CO2, inhibition effects and chemolithotrophic redox reactions. The model is calibrated with anaerobic incubation experiments with slurried rice soil, monitoring electron donors and electron acceptors influencing CH4 production. Only the values for maximum conversion rates (Vmax) for sulphate and iron reduction and CH4 production are tuned. The model is validated with similar experiments in which extra electron donors or electron acceptors had been added. The differences between model estimates without kinetic parameter adjustments and experiment were not significant, showing that the model contains adequate process descriptions. The model is sensitive to the estimates of Vmax, that are site dependent and to the description of substrate release, that drives all competition processes. For well-shaken systems, the model is less sensitive to chemolithotrophic reactions and inhibitions. Inhibition of sulphate reduction and methanogenesis during iron reduction can however explain acetate accumulation at the start of the incubations. Iron reduction itself is most probably retarded due to manganese reductio

The influence of sulfate and nitrate on the methane formation by methanogenic archaea in freshwater sediments

In this thesis the effect of inorganic electron acceptors (sulfate and nitrate) on methane emission from freshwater sediments in the Netherlands was investigated. The chosen study area was a polder located between Leiden and Utrecht, and is representative for similar polders in The Netherlands (Chapter 3). The polder contains peat grasslands in which ditches are lying used for maintaining stable water levels. The ditches contain sediment which is a potential source of CH 4 . In freshwater environments, sulfate can be introduced by infiltration water, supply water or due to the oxidation of S-rich organic matter and iron sulfide. Also high nitrate concentrations can occur in the groundwater as a result of intensive agricultural activities. Therefore, in The Netherlands, sulfate and nitrate concentrations in the water may control the methane emission from methanogenic environments.The influence of sulfate and nitrate on methanogenesisMethane is produced by methanogenic archaea (methanogenesis) living in syntrophic association with fermentative and acetogenic bacteria. In presence of sulfate and nitrate, sulfate- and nitrate-reducing populations may successfully compete with these methanogenic consortia. In Chapter 4 the sediment was investigated for its potential methanogenic and syntrophic activity and the influence of sulfate and nitrate on these potential activities. Addition of acetate stimulated both methane formation and sulfate reduction, indicating that an active acetate-utilizing population of methanogens and sulfate reducers was present in the sediment. When inorganic electron acceptors were absent, substrates like propionate and butyrate were converted by syntrophic methanogenic consortia. However, addition of sulfate or nitrate resulted in the complete inhibition of these consortia. Our results showed that propionate and butyrate were directly used by the sulfate and nitrate reducers. This indicated that the syntrophic methanogenic consortia could not compete with nitrate and sulfate reducers.Acetate, a key intermediate in the anaerobic degradation of organic matterIn Chapter 5 the importance of methanogenesis and sulfate reduction in a freshwater sediment was investigated by using (non) specific inhibitors. Only the combined inhibition of methanogenesis and sulfate reduction resulted in the accumulation of intermediates (acetate, propionate and valerate). Acetate was the most important compound in the accumulation (93 mole %) and thereby confirming its role as a key intermediate in the terminal step of organic matter mineralization. Furthermore, the inhibition studies showed that about 70-80% of the total carbon flow to CH 4 was through acetate. This clearly demonstrated that acetate was quantitatively the most important substrate for methanogens in the sediment. Addition of chloroform (CHCl 3 ) inhibited methanogens and acetate-utilizing sulfate reducers in the sediment. Pure culture studies showed that CHCl 3 was an inhibitor of growth and product formation by methanogenic archaea, homoacetogenic bacteria, a syntrophic bacterium ( Syntrophobacter fumaroxidans ) and the sulfate-reducing bacterium ( Desulfotomaculum acetoxidans ) operating the acetylCoA-pathway.In the sediment acetate is quantitatively the most important substrate for methanogens (chapter 5). Therefore, the anaerobic conversion of [2- 13C] acetate in the presence of sulfate or nitrate was investigated (Chapter 6). Aceticlastic methanogenesis was the dominant acetate-utilizing process when the sulfate concentration was below 70 m M. At higher sulfate concentrations the formation of 13C-labeled CH 4 decreased significantly, indicating that methanogens and sulfate reducers were competing for the same substrate. When sufficient sulfate (>500 m M) was present the outcome of the competition was in favor of the sulfate reducers. Unexpectedly, nitrate-reducing bacteria hardly competed with methanogens and sulfate reducers for the available acetate. The electron-acceptor/acetate ratio indicated that denitrification was coupled to the oxidation of reduced sulfur compounds or other electron donors rather than to the oxidation of acetate. Furthermore, nitrate reduction seemed to have a direct inhibitory effect on methanogenesis, and an indirect effect as a consequence of the oxidation of reduced sulfur-compounds to sulfate. The inhibition of methanogenesis by nitrate was probably not the result of competition for substrate but was due to the formation of toxic intermediates of the denitrification processes. The fact that acetate-utilizing nitrate reducers were outnumbered by the methanogens and sulfate reducers and hardly competed with these types of microorganisms for the available acetate indicated that acetate-utilizing nitrate reducers played a minor role in the degradation of acetate in the sediment (Chapter 6).Anaerobic acetate-utilizing microorganismsEnumeration of acetate-utilizing anaerobes gave insight into the different groups of microorganisms involved in the acetate metabolism in the sediment. In Chapter 7 the physiological properties of the acetate-utilizing anaerobes obtained by direct serial dilution of freshwater sediment are described. An acetate-utilizing methanogen (culture AMPB-Zg) was enriched and appeared to be closely related to Methanosaeta concilii . The most dominant acetate-utilizing sulfate reducer (strain ASRB-Zg) in the sediment was related to Desulfotomaculum nigrificans and Desulfotomaculum thermosapovorans . This result supported our hypothesis that acetate is a competitive substrate for methanogens and sulfate reducers in the sediment (Chapter 5 and 6). An acetate-utilizing nitrate reducer (strain ANRB-Zg) was isolated which showed to be related to Variovorax paradoxus . In the presence of acetate and nitrate, strain ANRB-Zg was capable of oxidizing reduced sulfur compounds to sulfate. Strain ANRB-Zg may have been involved in the oxidation of reduced sulfur compounds to sulfate in the sediment (Chapter 6). However, at this moment too little information is available to understand the exact role of strain ANRB-Zg in the sulfur and carbon cycle of the sediment. The degradation of acetate in the absence and presence of SO 42-and NO 3-is depicted in Fig. 1. The dominant acetate-utilizing anaerobes and their metabolic interactions are given as well.Figure 1: The influence of sulfate and nitrate on aceticlastic methanogenesis in freshwater sediment. AMPB: aceticlastic methanogen, ASRB: acetate-utilizing sulfate reducer, ANRB: acetate-utilizing nitrate reducer. Thick stripped lines represent competition for acetate between AMPB and ASRB. Thick dotted lines represent inhibition caused by toxic intermediates.Finally, the conversion of acetate by methanogenic and sulfidogenic communities under acetate-limited conditions was studied in Chapter 8. Our results showed that the acetate-utilizing methanogens were able to compete efficiently with the sulfate reducers for the available acetate in an acetate-limited chemostat with sulfate in excess during a long-term experiment (1 year). From the chemostat studies it became clear that the kinetic properties of the acetate-utilizing methanogen and sulfate reducer were almost similar. Unfortunately, exact values for these kinetic properties are still lacking. Therefore predictions based on these parameters about the outcome of the competition of methanogens and sulfate reducers for acetate could not be made. In Chapter 2 a review of the physiological, ecological and biochemical aspects of acetate-utilizing anaerobes and their metabolic interactions are presented.Concluding remarksThe results which are presented in this thesis advanced our knowledge of the effect of sulfate and nitrate on methane formation in sediments which are found in a typical Dutch polder. The sediment is a potential source of methane but it remains unclear if the sediment emits high quantities of methane. It was assumed that the methane emission is in the same order of magnitude (42-225 kg CH 4 ha -1yr -1) as reported for another sediment. The presence of sulfate appeared to be a major factor in controlling the formation of methane. This is due to the competition between acetate-utilizing methanogens and sulfate reducers. Nevertheless, the origin of sulfate and its effect on methane emission on the long-term is not fully understood. The inhibitory effect of nitrate on methanogenesis appears to be the result of the formation of toxic intermediates of the denitrification processes but tangible proof is still lacking at this moment. Also the physiology and ecophysiology of some of the dominant acetate-utilizing anaerobes, and the metabolic interactions among them are not completely resolved. Further investigations of these topics are needed to get a better understanding of the environment as a source of methane and the emission from it. Intriguingly, measurements of CH 4 emissions from grasslands near the location of the sediments have shown that a net methane consumption in the area is possible. This indicates that methane produced in the ditches and originating from other sources may be oxidized again by the grassland soils. To determine a methane budget for Dutch polders the potential sink and/or source capacity of the grasslands should be included to get insight in the contribution to the emission of methane to the atmosphere.</p

Isolation and characterization of acetate-utilizing anaerobes from a freshwater sediment

Acetate-degrading anaerobic microorganisms in freshwater sediment were quantified by the most probable number technique. From the highest dilutions a methanogenic, a sulfate-reducing, and a nitrate-reducing microorganism were isolated with acetate as substrate. The methanogen (culture AMPB-Zg) was non-motile and rod-shaped with blunted ends (0.5-1 mm x 3-4 mm long). Doubling times with acetate at 30-35 degrees C were 5.6-8.1 days. The methanogen grew only on acetate. Analysis of the 16S rRNA sequence showed that AMPB-Zg is closely related to Methanosaeta concilii. The isolated sulfate-reducing bacterium (strain ASRB-Zg) was rod-shaped with pointed ends (0.5-0.7 mm x 1.5-3.5 mm long), weakly motile, spore forming, and gram positive. At the optimum growth temperature of 30 degrees C the doubling times with acetate were 3.9-5.3 days. The bacterium grew on a range of organic acids, such as acetate, butyrate, fumarate, and benzoate, but did not grow autotrophically with H2, CO2, and sulfate. The closest relative of strain ASRB-Zg is Desulfotomaculum acetoxidans. The nitrate-reducing bacterium (strain ANRB-Zg) was rod-shaped (0.5-0.7 mm x 0.7-1 mm long), weakly motile, and gram negative. Optimum growth with acetate occurred at 20-25 degrees C. The bacterium grew on a range of organic substrates, such as acetate, butyrate, lactate, and glucose, and did grow autotrophically with H2, CO2, and oxygen but not with nitrate. In the presence of acetate and nitrate, thiosulfate was oxidized to sulfate. Phylogenetically, the closest relative of strain ANRB-Zg is Variovorax paradoxus

Direct inhibition of methanogenesis by ferric iron

Observed inhibition of methanogenesis under Fe(III)-reducing conditions is usually explained by competition of methanogens and Fe(III)-reducing bacteria for the common Substrates acetate and hydrogen. However, substrate competition alone cannot explain the strong inhibition of methanogenesis during Fe(III)-reduction. We demonstrate direct inhibition of methanogenesis by amorphous Fe(OH)(3) at concentrations between 0 and 10 mM in experiments with pure cultures of inethanogens. The sensitivity toward Fe(III) was higher for Methanospirillum hungatei and Methanosarcina barkeri grown with H-2/CO2 than for Methanosaeta concilii and Methanosarcina barkeri grown with acetate. Cultures of Methanosarcina barkeri grown with H-2/CO2 and methanol demonstrated a capacity for Fe(III) reduction, which suggests that Fe(III)-reduction by methanogens may also contribute to Fe(III) inhibition of methanogenesis. Our results have important implications for kinetic modelling of microbial redox processes in anoxic soils and sediments. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies

Global transcriptomics analysis of the Desulfovibrio vulgaris change from syntrophic growth with Methanosarcina barkeri to sulfidogenic metabolism

Desulfovibrio vulgaris is a metabolically flexible micro-organism. It can use sulfate as an electron acceptor to catabolize a variety of substrates, or in the absence of sulfate can utilize organic acids and alcohols by forming a syntrophic association with a hydrogen-scavenging partner to relieve inhibition by hydrogen. These alternative metabolic types increase the chance of survival for D. vulgaris in environments where one of the potential external electron acceptors becomes depleted. In this work, whole-genome D. vulgaris microarrays were used to determine relative transcript levels as D. vulgaris shifted its metabolism from syntrophic in a lactate-oxidizing dual-culture with Methanosarcina barkeri to a sulfidogenic metabolism. Syntrophic dual-cultures were grown in two independent chemostats and perturbation was introduced after six volume changes with the addition of sulfate. The results showed that 132 genes were differentially expressed in D. vulgaris 2 h after addition of sulfate. Functional analyses suggested that genes involved in cell envelope and energy metabolism were the most regulated when comparing syntrophic and sulfidogenic metabolism. Upregulation was observed for genes encoding ATPase and the membrane-integrated energy-conserving hydrogenase (Ech) when cells shifted to a sulfidogenic metabolism. A five-gene cluster encoding several lipoproteins and membrane-bound proteins was downregulated when cells were shifted to a sulfidogenic metabolism. Interestingly, this gene cluster has orthologues found only in another syntrophic bacterium, Syntrophobacter fumaroxidans, and four recently sequenced Desulfovibrio strains. This study also identified several novel c-type cytochrome-encoding genes, which may be involved in the sulfidogenic metabolis

Spatial and temporal variability of particle flux at the NW European continental margin

A synopsis of results from two sediment trap moorings deployed at the mid- and outer slope (water depths 1450 and 3660 m, respectively) of the Goban Spur (N.E. Atlantic Margin) is presented. Fluxes increase with trap deployment depth; below 1000 m resuspended and advected material contributes increasingly to bulk flux. Fluxes of dry weight, POC and diatoms in the traps 400 m above bottom (mab) are smaller than those recorded at the sediment surface due to lateral fluxes in the benthic nepheloid layer. These near-bottom fluxes are larger at shallower water depths. Pa-231/Th-230 ratios in sedimenting material suggest that boundary scavenging is not significant at the Goban Spur. Fluxes of Pb-210 in the intermediate and deep traps are comparable to the Pb-210 supply rate at this site. At the outer slope, sediment Pb-210 fluxes are similar to those measured in the traps 400 mab; at the mid- slope they are a factor of 2 higher, once again indicating large near-bottom lateral particle input. Based on POC- normalised biomarkers in sedimenting material, we followed changes in the quality of sedimenting material with differing trap depth and on seasonal and event-related time scales. In spring fresh, diatom-dominated sedimentation occurs, with progressive degradation of POC with time (to winter) and depth (from 600 to 3220 m). Deeper traps are distinguished on the basis of opal and aluminium fluxes that are dominant in lateral input. A storm event during late September 1993 was clearly reflected in the delta N-15 isotope ratio of sedimenting material, with a time lag of 2-3 weeks. Diatom and opal fluxes were elevated in this storm-related signal, and its biomarker composition in the 600-m trap was similar to that during spring. An estimate made of upward nitrate flux (new production) at the shelf break and at the outer slope indicated a 2-fold higher new (export) production at the shelf break. Particulate organic carbon export from the shelf break to below the depth of maximal seasonal mixing ranges between 3 and 9% of primary production. [KEYWORDS: Isotopic composition; north-atlantic; goban spur; organic-matter; open-ocean; sediments; nitrogen; carbon; phytoplankton; 20-degrees-w]