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 methanogenesis

Methane 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 matter

In 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 microorganisms

Enumeration 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 ₄^2-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 remarks

The 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.