Acetate turnover and methanogenic pathways in Amazonian lake sediments

Abstract

Let me know in case you have any questions.Best Tina

Response
Dear Tina, Thank you for the hint to the references.
In fact, Nüsslein et al. 2001 has been cited in the Introduction.We did not include the reference to Beulig et al. 2018, since the work is dealing with a seabed sediment rather than a lake sediment.Nevertheless, both references are now also included in the Discussion (see marked ms).However, we did not include the Beulig et al. reference in the ISME journal, since this work is mainly dealing with anaerobic methane oxidation, and the data on acetate oxidation are not thus obvious in this paper.

Ralf Introduction
Acetate is an important intermediate in the anoxic degradation of organic matter and is produced by fermentation processes and chemolithotrophic homoacetogenesis.The contribution of these two processes to acetate production is difficult to determine, but seems to be quite different for different environments (Fu et al., 2018;Hädrich et al., 2012;Heuer et al., 2010;Lokshina et al., 2019;Ye et al., 2014).The degradation of acetate requires a suitable oxidant such as oxygen, nitrate, ferric iron or sulfate.If such oxidants are not or no longer available, such as in many freshwater environments (e.g., paddy fields, lake sediments, peat), acetate sometimes accumulates until suitable electron acceptors become again available.
Temporal accumulation and subsequent oxidative consumption has for example been observed in peatlands during increase and decrease, respectively, of the water table (Duddleston et al., 2002).However, it is generally assumed that acetate degradation in the absence of inorganic electron acceptors is accomplished by aceticlastic methanogenesis (Zinder, 1993).If aceticlastic methanogenesis is operative, the methyl group of the acetate is converted to CH4.
If methanogenesis is the exclusive final step in the anaerobic degradation of organic matter, polysaccharides (one of the most important compounds from primary production) will be dismutated to equal amounts of CH4 and CO2.Furthermore, acetate usually accounts for more than two third of total methane production, especially if polysaccharides are the predominant degradable organic matter (Conrad, 1999).However, CO2 has often been found to be the main product in many anoxic environments despite the absence of inorganic electron acceptors (O2, nitrate, ferric iron, sulfate) (Keller et al., 2009;Yavitt and Seidmann-Zager, 2006).Such results have been explained by the assumption that organic substances (e.g.humic acids) may also serve as electron acceptors (Gao et al., 2019;Keller et al., 2009;Klüpfel et al., 2014).Organic electron acceptors also allow the oxidation of acetate (Coates et al., 1998;Lovley et al., 1996).The role of organic electron acceptors during anaerobic degradation of organic matter is potentially important, but still not well known (Corbett et al., 2013) There are also many reports that methane production in lake sediments is dominated by hydrogenotrophic rather than aceticlastic methanogenesis (Conrad, 1999;Conrad et al., 2011;Ji et al., 2016).Such observations were explained (1) by incomplete degradation of organic matter producing predominantly H2 and CO2 without concomitant acetate production (Conrad et al., 2010;Hodgkins et al., 2014;Liu et al., 2017), (2) by acetate oxidation coupled to the reduction of organic substances (see above), or (3) by syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis (Lee and Zinder, 1988;Vavilin et al., 2017).If acetate oxidation is operative, the methyl group of the acetate is converted to CO2.However, if acetate oxidation is syntrophic, it does not require a chemical compound (other than H + ) as electron acceptors, since it is the hydrogenotrophic methanogenesis that eventually accepts the electrons released during acetate oxidation.
Syntrophic acetate oxidation can replace aceticlastic methanogenesis and thus, has been found when aceticlastic methanogenic archaea were not present in the microbial community of lake sediment (Nüsslein et al., 2001).This may also happen in other anoxic environments when conditions are not suitable for aceticlastic methanogens, e.g., at elevated temperatures (Conrad et al., 2009;Liu and Conrad, 2010;Liu et al., 2018), in the presence of high concentrations of ammonium (Müller et al., 2016;Schnürer et al., 1999;Zhang et al., 2014), or phosphate (Conrad et al., 2000).However, syntrophic acetate oxidation has also been found in lake sediments that contained populations of putatively aceticlastic methanogens (Vavilin et al., 2017).It is presently unkown under which conditions syntrophic acetate oxidizers can successfully compete with aceticlastic methanogens and co-occur with acetate oxidation that is coupled to the reduction of organic substances.
As a further step in understanding the ecology of acetate oxidizers (syntrophic or nonsyntrophic ones) versus aceticlastic methanogens, we attempted to document their coexistence by studying lake sediments, which had been reported containing 16S rRNA genes of putatively aceticlastic Methanosaetaceae (Methanotrichaceae (Oren, 2014)) (Ji et al., 2016).
We used these sediments and measured the fractions of hydrogenotrophic methanogenesis and of the methyl group of acetate being oxidized to CO2 rather than reduced to CH4, and compared the turnover of acetate to the production rate of CH4.

Materials and Methods
The sediment samples were obtained from floodplain lakes in the Amazon region and have already been used for a study on structure and function of methanogenic microbial communities (Ji et al., 2016).In particular, these sediment have been assayed for the percentage of hydrogenotrophic methanogenesis and for the percentage contribution of putatively aceticlastic methanogens to the total archaeal community (Ji et al., 2016).Here, we used six of these sediments for incubation experiments with radioactive tracers.These are the same sediment samples as those listed in our previous publication (Ji et al., 2016).The identity of the lake sediments and the percentage content of putatively aceticlastic methanogens is summarized in Table 1.The experiments were carried out at the same time as those in our previous publication (Ji et al., 2016) and were basically using the same incubation techniques.However, the experimental approach to determine the fractions of hydrogenotrophic methanogenesis (fH2) were different.In our previous experiment, values of fH2 were determined from the δ 13 C of CH4 in the presence (δ 13 CCH4-mc) and absence (δ 13 CCH4) of methyl fluoride, an inhibitor of aceticlastic methanogenesis, and from the δ 13 C of the methyl group of acetate (δ 13 Cac-methyl): fH2 = (δ 13 CCH4δ 13 Cac-methyl)/(δ 13 CCH4-mcδ 13 Cac-methyl) (1).
The CH4 production rates and fH2 values from this experiment are shown in Fig. 1 for comparison.
In the present experiment, however, values of fH2 were determined by addition of NaH 14 CO3 and measurement of the specific radioactivities in CH4 and CO2.Briefly, about 10-15 ml of each replicate (n =3) were filled into 27-ml sterile tubes, flushed with N2, closed with butyl rubber stoppers, and incubated at 25ºC.After preincubation for 12 days (in order to deplete eventually present inorganic oxidants), 0.5 ml of a solution of carrier-free NaH 14 CO3 (about 1 µCi; 50 Ci mol -1 ) was added, the tubes flushed again with N2, and incubation was continued at 25ºC for about 100 days.Partial pressures of CH4 and CO2 as well as their contents of 14 C were measured at different time points after mixing the slurries by heavy manual shaking.The gas partial pressures were measured by gas chromatography with a flame ionization detector (Ji et al., 2016), the radioactivities were analyzed with a radiodetector (RAGA) (Conrad et al., 1989).The data were used to calculate the fractions of hydrogenotrophic methanogenesis (fH2) from the specific radioactivites of gaseous CH4 (SRCH4) and CO2 (SRCO2): For determination of acetate turnover, the same conditions were used, except that preincubation was for 25 days, 0.5 ml of a solution of carrier-free Na[2-14 C]acetate (about 2 µCi; 50 Ci mol -1 ), equivalent to about 20 nmol acetate, was added, and incubation was continued for about 8 h.During this time gas samples were repeatedly taken and the radioactivities in CH4 and CO2 were analyzed in a gas chromatograph with a radiodetector (RAGA) (Conrad et al., 1989).In the end, the sediment samples were acidified with 1 ml of 1M H2SO4 to liberate CO2 from carbonates, and the radioactivities in CH4 and CO2 were analyzed again.The data were used to calculate the, the acetate turnover rate constants (kac) and the respiratory index (RI) values from the radioactivities of gaseous CH4 and CO2 as described by Schütz et al. ( 1989).The RI is defined as: RI = 14 CO2/( 14 CO2 + 14 CH4) (3).
The acetate concentration (ac) was analyzed in the sediments at the end of the incubation using high pressure liquid chromatography.The acetate concentrations are summarized in Table 1.The rates of acetate-dependent CH4 production (Pac) were calculated from the acetate turnover rates and the RI:

Results
Six different lake sediments from Amazonia were incubated in the presence of H 14 CO3.
Methane production started without lag phase indicating that the inorganic electron acceptors, which were present in the original sediment (Ji et al., 2016) had been depleted during the anaerobic preincubation and did not suppress methanogenesis.The CH4 production rates were compared to those obtained in our previous experiments without addition of H 14 CO3 (Ji et al., 2016).Although the rates of CH4 production were different in the two different incubations, the orders of magnitude were similar for the different lake sediments (Fig. 1A).The incubations in the presence of H 14 CO3 were used to follow the specific radioactivities of CH4 (Fig. 2A) and CO2 (Fig. 2B) over the incubation time.The specific radioactivities of CH4 changed only little but were slightly different for the different lake sediments.The specific radioactivities of CO2 decreased with time as expected due to the production of nonradioactive CO2.Both specific radioactivities were used to calculate the fractions of hydrogenotrophic methanogenesis (fH2), which increased with incubation time and eventually reached a plateau.The values of fH2 averaged between 30 and 60 d of incubation are summarized in Fig. 1B.Only the incubations of sediment "Grande" did not reach a plateau but still increased after 260 d of incubation due to the continuously decreasing specific radioactivities of CO2 (data not shown).Averaging these values over the 4 data points between 160 and 260 d resulted in fH2 of about 60% (Fig. 1B).The thus determined values of fH2 were comparable to those determined in the absence of H 14 CO3 using values of δ 13 C, which have already been published (Ji et al. 2016) (Fig. 1B).
The same sediments were used to determine the turnover of [2-14 C]acetate by measuring the increase of radioactive CH4 (Fig. 3A) and CO2 (Fig. 3B).These data were used to determine the rate constants of acetate turnover (Fig. 3C), which ranged between 0.02 and 1.7 h -1 .The respiratory indices (RI) were generally larger than 0.2 except those of the sediments Tapari and Verde, which were smaller than 0.2 (Fig. 4B).The RI values and the acetate turnover rate constants were used to calculate the rates of CH4 production from acetate in comparison to the rates of total CH4 production (Fig. 4A).Interestingly, acetate-dependent CH4 production was always larger than total CH4 production, except in those sediments exhibiting a RI <0.2.

Discussion
The RI value quantifies the fraction of the methyl group of acetate that is oxidized to CO2 rather than reduced to CH4.Since some oxidation of acetate methyl is also happening in pure cultures of aceticlastic methanogens (Weimer and Zeikus, 1978), and since a RI of around 0.2 has often been found in environments where acetate turnover was dominated by aceticlastic methanogenesis (Phelps and Zeikus, 1984;Rothfuss and Conrad, 1993;Winfrey and Zeikus, 1979), an RI value of 0.2 may in practice be used as the threshold for the change of methanogenic to oxidative acetate turnover.Based on this criterion, i.e.RI < 0.2, the lake sediments of Tapari and Verde behaved as when acetate turnover was exclusively caused by aceticlastic methanogenesis.The percentage of acetate-dependent CH4 production was fairly consistent with the fraction of hydrogenotrophic methanogenesis, which made up the remainder of total CH4 production.In conclusion, the acetate turnover and CH4 production in these lake sediments behaved as expected as when aceticlastic methanogenesis was the sole process of acetate consumption (reaction 1 in Fig. 5).
However, the sediments of Jua and in particular those of Jupinda, Cataldo, and Grande exhibited RI values >0.2, showing that a substantial fraction of the acetate-methyl was oxidized to CO2.Hence, acetate was not exclusively consumed by aceticlastic methanogenesis, but it was oxidized, for example by syntrophic acetate oxidation producing H2 and CO2.Similarly, RI values > 0.2 have been observed in the sediment of Lake Kinneret in Israel and interpreted as syntrophic acetate oxidation (Nüsslein et al., 2001).Also in the methanogenic zone of an anoxic seabed in the Baltic Sea, acetate has been shown to be degraded syntrophically (Beulig et al., 2018).The H2 and CO2 from acetate oxidation may subsequently be used as methanogenic substrates, thus supporting CH4 production (reactions 2 and 3 in Fig. 5).Such support would be consistent with the relatively high fractions (fH2) of hydrogenotrophic methanogenesis observed in the sediments of Lakes Juas, Jupinda, Cataldo and Grande.However, it would not explain why acetate turnover rates were higher than necessary for supporting the observed rates of total CH4 production.A possible conclusion is that acetate was converted to CO2 without concomitant production of H2.Possibly, electrons from acetate were transferred to organic electron acceptors (reaction 4 in Fig. 5), such as suggested in the literature (Coates et al., 1998;Lovley et al., 1996).Alternatively, acetate may have first been converted to H2 plus CO2 followed by the oxidation of H2 with organic electron acceptors (reactions 2 and 5 in Fig. 5) rather than syntrophic formation of CH4 from H2 plus CO2 (reactions 2 and 3 in Fig. 5).In conclusion, these lake sediments behaved as when acetate consumption was accomplished not only by acetate-dependent methanogenesis, but also by oxidative consumption.
Our conclusions are mainly based on radiotracer measurements, which may be biased.For example, acetate turnover rate constants are calculated from acetate concentrations and turnover rate constants.Acetate concentrations were only measured at the end of incubation and thus, may not have been representative for the entire incubation time.Furthermore, acetate in the sediment may occur in several pools with different turnover (Christensen and Blackburn, 1982).Therefore, acetate turnover rates and acetate-dependent CH4 production rates may be overestimated, if the actual acetate turnover depends on a pool size that is smaller than that analyzed.Overestimation may also result from too low RI values, such as when carbonate-bound radioactivity is neglected.However, such bias was avoided by acidification prior to determination of the RI.Finally, a potential bias may arise from the fact that the rates of CH4 production and the acetate turnover rates were measured in two different sets of incubation, with different incubation times.While CH4 production (and fH2) was measured over tens of days (Fig. 2), acetate turnover was determined within 8 h (Fig. 3).
Nevertheless, the data in the lake sediments of Tapari and Verde resulted in CH4 production and acetate turnover consistent with the operation of aceticlastic methanogenesis, which is the canonical acetate consumption pathway for methanogenic sediments.Therefore, we are confident that our results obtained from the sediments of Jua, Jupinda, Cataldo and Grande were also in a realistic range.
The determination of fractions of hydrogenotrophic methanogenesis (fH2) depends on the specific radioactivity of the dissolved CO2 pool that is involved in CH4 production.However, it is the pool of gaseous CO2 that is analyzed in the assay, assuming that its specific radioactivity is identical to that of the active dissolved pool.Since non-radioactive CO2 is permanently produced from oxidation of organic matter, there may be disequilibrium.
Nevertheless, determinations of fH2 using radioactive bicarbonate exhibited the same tendencies as those based on δ 13 C values, and thus are probably quite reliable.Furthermore, the fH2 values were fairly consistent with the fractions of acetate-dependent methanogenesis determined from the turnover of radioactive acetate.
Despite these reservations, our results collectively demonstrated that acetate turnover in tropical lake sediments did not necessarily follow a canonical pattern with aceticlastic methanogenesis as sole or predominant process of acetate turnover, despite the fact that all these sediments contained populations of putative aceticlastic methanogenic archaea.Acetate consumption in Methanosaeta species is known to have a relatively high affinity and a low threshold for acetate (Jetten et al., 1992).Therefore, the question arises why oxidative processes, including syntrophic acetate oxidation, could successfully compete with aceticlastic methanogenesis.The data in the absence of radioactive bicarbonate are the same as published in Ji et al. (2016), when fH2 was determined from values of δ 13 C; mean ±SE.

Figure captions Fig. 1 :
Figure captionsFig.1:Methane production in sediments of different Amazonian lakes: (A) rates of CH4 production, and (B) fractions of hydrogenotrophic methanogenesis, both determined in the absence and the presence of radioactive bicarbonate.

Fig. 4 :
Fig. 4: (A) Rates of total and acetate-derived CH4 production in sediments of different Amazonian lakes and (B) respiratory indices (RI) of the turned over [2-14 C]acetate; mean ±SE.