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Acetate is quantitatively the most important substrate for methane production in a freshwater sediment in The Netherlands. In the presence of alternative electron acceptors the conversion of acetate by methanogens was strongly inhibited. By modelling the results, obtained in experiments with and without 13C-labelled acetate, we could show that the competition for acetate between methanogens and sulfate reducers is the main cause of inhibition of methanogenesis in the sediment. Although nitrate led to a complete inhibition of methanogenesis, acetate-utilising nitrate-reducing bacteria hardly competed with methanogens for the available acetate in the presence of nitrate. Most-probable-number enumerations showed that methanogens (2×108 cells cm−3 sediment) and sulfate reducers (2×108 cells cm−3 sediment) were the dominant acetate-utilising organisms in the sediment, while numbers of acetate-utilising nitrate reducers were very low (5×105 cells cm−3 sediment). However, high numbers of sulfide-oxidising nitrate reducers were detected. Denitrification might result in the formation of toxic products. We speculate that the accumulation of low concentrations of NO (<0.2 mM) may result in an inhibition of methanogenesis.
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Acetate is quantitatively the most important substrate for methanogens in methanogenic environments [1–4]. Up to 70–80% of the methane in freshwater sediments may be derived from acetate; the remainder is formed by reduction of bicarbonate with hydrogen. The consumption of acetate or hydrogen by methanogens is strongly affected by the competition with anaerobic respiring microorganisms. Consequently, one of the factors regulating methane formation in nature is the availability of inorganic electron acceptors such as nitrate, sulfate, sulfur and oxidised metals (FeIII, MnIV) .
Insight into the effect of sulfate on H2-dependent methanogenesis has been obtained in sediment studies and in studies with pure cultures of H2-consuming methanogens and sulfate reducers [5–8]. It was shown in batch cultures that sulfate reducers outcompete methanogens for H2 due to a higher affinity and higher growth yield [5,8]. Unfortunately, less is known about the competition between methanogens and sulfate reducers for acetate. Schönheit et al.  showed that Desulfobacter postgatei has a higher affinity for acetate than Methanosarcina barkeri. This could explain why Desulfobacter species are the main acetate-degrading microorganisms in marine sediments. However, Methanosaeta rather than Methanosarcina species are the dominant acetate-degrading methanogens in various methanogenic environments [3,10]. These methanogens display maximum acetate uptake rates (Vmax), half-saturation constants (Km) and maximum growth rates (μmax) similar to acetate-utilising sulfate reducers isolated from freshwater environments [11,12]. Thus, the effect of sulfate on the fate of acetate is not as clear-cut as its effect on the fate of H2.
Nitrate is also known to suppress methane formation [13–16]. It was speculated that production of the toxic intermediates of denitrification (nitrite, NO, N2O), rather than the competition for acetate between methanogens and denitrifiers, was responsible for the inhibition of methanogenesis [13,17–19]. Remarkably, little is known about the role of acetate as electron donor for nitrate reduction in natural environments. Most nitrate-reducing bacteria have only been tested for their capacity to grow on acetate and nitrate. Information about their growth kinetic properties on acetate is scarce. Quantification and identification of acetate-utilising denitrifiers may give insight into their role in acetate metabolism in anoxic environments.
We analysed the effect of sulfate and nitrate on methane formation in freshwater sediment by using 2-13C-labelled acetate. To obtain insight into the effect of inorganic electron acceptors on potential methanogenesis, we quantified the acetate-utilising methanogens, sulfate reducers and nitrate reducers in the sediment. Furthermore, a mathematical model was used to analyse possible interactions between the different microbial populations in the sediment.
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The pore water profiles showed that sulfate concentrations varied with depth and season (Fig. 1). The observed steep concentration gradients of sulfate indicated that sulfate reduction occurred in the first 0.5 cm of the sediment. Below 5 cm the sulfate concentrations increased again to constant values in the bottom centimetres. This suggests that either sulfate reduction did not occur or that sulfate reduction was in equilibrium with sulfate production by chemolithotrophic denitrifiers (discussed below). Unfortunately, the measurement of sulfate reduction in situ (by tracer method) to confirm that the steep concentration gradients were indeed a result of ongoing sulfate reduction was technically not possible in this study. However, pore water profiles were made every 2 months (3–5 cores per sample date) over a 2-year period and these steep concentration gradients of sulfate were observed especially during the spring, summer and autumn . During the winter months the profiles showed practically continuous concentration gradients. Remarkably, most of the sulfate reduction seems to take place in the first centimetre of the sediment. We were not able to measure the penetration depth of oxygen in the sediment but it was shown to be at a maximum of 2–3 mm in similar freshwater sediment . This suggests that oxygen levels at the sediment/water interface were low enough to allow sulfate reduction to take place. In the summer and spring profiles organic acid concentrations were generally below detection limit (<10 μM) but sporadically in autumn (1994 and 1995) in the first centimetre of the sediment, formate (10 μM), acetate (300 μM) and lactate (25 μM) accumulated. This accumulation of acetate in the sediment was probably due to an increased input of dead organic matter. It justifies the addition of relatively high concentrations of 13C-acetate (120–150 μM) in our sediment incubations.
Our experiments showed that in summer, aceticlastic methanogenesis is the dominant acetate-consuming process in the freshwater sediment from Zegvelderbroek. The quantitative importance of acetate to methanogenesis could not be judged in these experiments, but an inhibition study revealed that about 70–80% of the total carbon flow to CH4 was through acetate . In our experiments, methanogenesis seemed to account for more than 70% of the acetate mineralisation in the sediment when sulfate concentrations were between 50 and 70 μM. The reduction of sulfate at these low concentrations was not stimulated by the presence of acetate. However, addition of sulfate stimulated sulfate reduction, suggesting that the acetate-utilising sulfate reducers are sulfate limited at less than 70 μM. Indeed, sulfate reduction dominated the consumption of acetate (>75%) when sulfate concentrations in the sediment were higher than 500 μM (Fig. 3A,B). Stimulation of both processes by the addition of acetate showed that acetate-utilising methanogens and sulfate reducers were competing directly for the available acetate. This was confirmed by our modelling results (Fig. 3A,B). Previous studies had shown that methanogenesis and sulfate reduction were mainly confined to the first 10 cm of the sediment (Scholten, unpublished). The pore water profiles showed steep concentration gradients of sulfate, indicating that sulfate reduction occurred in the first 0.5 cm of the sediment (Fig. 1). As the sulfate concentration increased again below 10 cm it seemed that sulfate reducers became substrate limited. Thus, the presence of sulfate may be the most important factor controlling the formation of methane in this sediment. However, it is still unclear why such high sulfate concentrations and profiles are observed in sediment from Zegvelderbroek. The influx and oxidation of S-rich organic matter and iron sulfide might explain these relatively high concentrations  and its seasonal dynamics might also explain the parallel displacement of the observed concentration profiles. The observed potential consumption rates for 13C-acetate, sulfate and nitrate were comparable to rates determined in summer 1994 and spring 1995. The potential consumption rates for 13C-acetate, sulfate and nitrate determined in autumn 1995 and winter 1995/1996 were 4–6 times lower, suggesting a seasonal pattern (Scholten, unpublished).
Enumerations showed that the dominant acetate-utilising microorganisms were methanogens (2×108 cells cm−3 sediment) and sulfate reducers (2×108 cells cm−3 sediment). The most abundant acetate-utilising methanogen and sulfate reducer were isolated from the highest dilution step of the MPN series and were characterised physiologically and phylogenetically . On the basis of their partial 16S rRNA sequences it became clear that the methanogen and sulfate reducer were closely related to Methanosaeta concilii and Desulfotomaculum acetoxidans, respectively. Both the methanogen (strain AMPB-Zg) and sulfate reducer (strain ASRB-Zg) displayed Vmax, Km and maximum growth rate (μm) values  similar to and even slightly lower than described for other acetate-utilising methanogens and sulfate reducers [11,12,33]. From the Vmax and Km values for acetate, determined in resting cell suspensions, it became clear that the most abundant acetate-degrading sulfate reducer strain ASRB-Zg had slightly better kinetic properties than the most abundant acetate-degrading methanogen strain-AMPB. This, in combination with the low acetate concentrations compared to the Km values, explains why acetate-utilising sulfate reducers (in the presence of sufficient sulfate) were able to outcompete acetate-utilising methanogens for the available acetate in the sediment incubations. Besides, strain ASRB-Zg is a generalist and it is possible that acetate degradation is not the only activity of the strain in the sediment. The ability to use other substrates besides acetate can give strain ASRB-Zg a competitive advantage over strain AMPB-Zg (a specialist) when sufficient sulfate is present . At low sulfate concentrations versatile acetate-degrading sulfate reducers may prefer other substrates than acetate . Unfortunately, no information is available on how mixed substrate utilisation may affect the competition between strain AMPB-Zg and strain ASRB-Zg.
The acetate-utilising microorganisms in the sediment were quantified by the MPN method in liquid media. Our MPN counts were similar or 1–2 orders of magnitude higher than the numbers obtained by other investigators in rice field soil, lake and marine sediments for acetate-utilising methanogens and sulfate reducers [41–44]. In these studies the longest incubation period for the MPN counts was 3 months and relatively high temperatures (28–35°C). To obtain true numbers, our incubations needed up to 9 months of incubation after which the results were unaffected by another 3 months of incubation. For example, after 4–5 months of incubation our MPN counts were 2–3 orders of magnitude lower than the final numbers. Bak and Pfennig  already mentioned that prolonged incubation times might positively influence the counting efficiency. A few parameters that can be identified as being responsible for prolonged incubation times are growth rate and incubation temperature. Normally the type of MPN counts applied here results in an underestimation of the number of cells . However, the estimation of the number of acetate-utilising methanogens and sulfate reducers based on consumption rates of acetate coincided with the numbers obtained with the MPN method. The results obtained with the independent approach of the consumption rate method, despite its errors, add credence to the estimates of active in situ populations of acetate-utilising microorganisms obtained by the MPN method.
Nitrate-reducing bacteria hardly competed with methanogens and sulfate reducers for the available acetate in the presence of nitrate (Table 2). Nitrate reducers (5×105 cells cm−3 sediment) were clearly outnumbered by the methanogens and sulfate reducers. Furthermore, the pore water profiles showed that nitrate concentrations were below 1 μM. These factors may explain why acetate-utilising nitrate reducers played a minor role in the degradation of acetate in the sediment. Although acetate was consumed, our model was not able to describe this consumption on the basis of competition between acetate-utilising nitrate-reducing bacteria and other microorganisms (Fig. 3C). The low number of denitrifiers in our MPN counts may be a result of the sediment mixing of the upper 10 cm prior to the enumeration and sediment incubation studies. They may be found in large number in the upper part of the sediment where oxygen and nitrate are present at higher concentrations. In this case the competition with the other functional groups may be on kinetic grounds. However, it seemed that in the presence of nitrate other interactions besides competition for acetate played a role in the sediment.
The calculation of electron recovery indicated that, in addition to acetate, nitrate reducers (Table 4) used other electron donors. We observed that part of the nitrate reduction was coupled to the oxidation of reduced sulfur compounds (formation of sulfate) rather than to the oxidation of acetate (Figs. 2C and 3C). Nitrate reduction coupled to the oxidation of reduced sulfur compounds has been reported in studies of natural environments [47–49]. We isolated an acetate-utilising nitrate reducer (strain ANRB-Zg) that was capable of oxidising thiosulfate to sulfate in the presence of acetate . Furthermore, we observed the oxidation of sulfide to sulfate in MPN incubations (106 cells cm−3 sediment) with medium containing sulfide and NO3− (results not shown). This strongly suggests that sulfur-oxidising nitrate reducers were present in the sediment and are most likely responsible for the sulfate accumulation in the presence of nitrate (Figs. 2C and 3C). Chemolithoautotrophic denitrifiers may compete with heterotrophic (acetate-utilising) denitrifiers in the sediment for the available nitrate and this might have led, in combination with possible mixed substrate utilisation, to the observed incomplete acetate and sulfate balances. The model, although it included chemolithoautotrophic denitrification, was also unable to describe acetate and sulfate dynamics satisfactorily at high nitrate concentrations (Figs. 2C and 3C). Only at those conditions, model simulations were significantly different (i.e. differing more than 2 times the standard error) from experimental results. Our experimental and modelling results suggest that the role of chemolithoautotrophic denitrification and mixed substrate utilisation needs more attention to describe substrate dynamics in the presence of available nitrate.
Table 4. Consumed 13C-acetate, sulfate and nitrate and produced 13C-methane and sulfate (μmol l−1 h−1) obtained from incubations of freshwater sediment samples for 6 h (September 1995) or 7 h (April 1996) at 17°C
|Incubation||Consumed||Produced 13C-CH4||Electron recovery (%)|
| ||13C-acetate||SO42−||NO3−|| ||ma||srb||nrc||total|
|Acetate||131 (18)||20 (17)||0 (0)||82 (12)||72||5||0||77|
|Unsupplemented control||0 (0)||14 (4)||0 (0)||0 (0)|| || || || |
|Acetate+SO42−||124 (23)||83 (18)||0 (0)||43 (8)||40||23||0||62|
|Acetate omitted control||0 (0)||55 (13)||0 (0)||0 (0)|| || || || |
|Acetate+NO3−||100 (27)||0 (0)||180 (12)||1 (5)||1||1||12||13|
|Acetate omitted control||0 (0)||0 (0)||163 (34)||0 (0)|| || || || |
|Acetate+SO42−||126 (12)||144 (48)||0 (0)||34 (1)||30||94||0||124|
|Acetate omitted control||0 (0)||26 (28)||0 (0)||0 (0)|| || || || |
|Acetate+NO3−||65 (6)||99 (78)||215 (25)||0 (0)||0||60||5||65|
|Acetate omitted control||0 (0)||60 (47)||210 (27)||0 (0)|| || || || |
In addition to substrate competition, some processes may be inhibited by the accumulation of products. Possible inhibiting products are sulfide, nitrite, NO and N2O [13,15,16]. However, within the sediments sulfide concentrations (0.2–0.5 mM) were always below the concentrations inhibitory of sulfate reduction and methanogenesis (>1.5 mM) [50,51]. Thus this inhibition will be relatively unimportant in the freshwater sediments investigated. However, this might not have been the case for products of nitrate reduction. Brunet and Garcia-Gil  mentioned that the initial concentration of free sulfide determines the type of nitrate reduction. At very low concentrations of free sulfide (<50 μM) nitrate was reduced to N2 whereas at high sulfide (>0.3 mM) incomplete reduction to nitrite, NO and N2O took place [52,53]. We were not able to detect the formation of nitrite, NO and N2O in our experiments (detection limits: 1 μM for nitrite and 0.5 mM for NO and N2O, respectively) but sulfide concentrations in the incubations were high enough to affect the type of nitrate reduction. Furthermore, methane production was inhibited in our incubations with nitrate. This inhibition could not be explained by competition for acetate (as acetate concentrations were not limiting (Fig. 3C). Earlier studies have shown that nitrite, NO and N2O inhibited methanogenesis [13,15,16]. Klüber and Conrad  showed that NO at concentrations as low as 1–2 μM inhibited acetate-dependent methanogenesis completely. If we take into account that the sulfide concentrations were between 0.1 and 0.5 mM in the incubations, inhibition of methanogenesis in the incubations with nitrate might be explained by the accumulation of low concentrations of NO (<0.2 mM) that were below the detection limit. Therefore, we suggest that inhibition of methanogenesis by NO has to be incorporated in the model in order to describe the influence of nitrate on methanogenesis correctly.
In conclusion, the combination of experiments and modelling proved to be very helpful to understand the fate of acetate under different redox conditions in freshwater sediment. This combination revealed that the competition for acetate between methanogens and sulfate reducers is the main cause of inhibition of methanogenesis in the sediment studied. Nitrate-reducing bacteria hardly competed with methanogens and sulfate reducers for the available acetate. The low number of nitrate reducers may explain why acetate-utilising nitrate reducers played a minor role in the degradation of acetate in the sediment. Furthermore, a significant part of the nitrate consumption was coupled to electron donors other than acetate, most likely reduced sulfur compounds. To fully understand these processes more work has to be done on the organisms involved in the conversion of alternative electron donors in the presence of nitrate.