Toxicity of long chain fatty acids towards acetate conversion by Methanosaeta concilii and Methanosarcina mazei

Summary Long‐chain fatty acids (LCFA) can inhibit methane production by methanogenic archaea. The effect of oleate and palmitate on pure cultures of Methanosaeta concilii and Methanosarcina mazei was assessed by comparing methane production rates from acetate before and after LCFA addition. For both methanogens, a sharp decrease in methane production (> 50%) was observed at 0.5 mmol L−1 oleate, and no methane was formed at concentrations higher than 2 mmol L−1 oleate. Palmitate was less inhibitory than oleate, and M. concilii was more tolerant to palmitate than M. mazei, with 2 mmol L−1 palmitate causing 11% and 64% methanogenic inhibition respectively. This study indicates that M. concilii and M. mazei tolerate LCFA concentrations similar to those previously described for hydrogenotrophic methanogens. In particular, the robustness of M. concilii might contribute to the observed prevalence of Methanosaeta species in anaerobic bioreactors used to treat LCFA‐rich wastewater.


Introduction
Long-chain fatty acids (LCFA) are released during lipid hydrolysis, and hold the majority of the energy potential of these biomolecules . LCFA are degraded by anaerobic bacteria through b-oxidation to form acetate and hydrogen, which are then used by acetoclastic and hydrogenotrophic methanogens to produce methane. In anaerobic bioreactors, approximately 70% of the methane produced from LCFA results from acetoclastic activity, whereas about 30% derives from hydrogenotrophic activity . Low methane production has been reported during continuous bioreactor operation with LCFA, which was associated with inhibition and toxicity of these compounds towards the methanogenic communities (Chen et al., 2014;Dereli et al., 2014).
Studies on the toxic effect of LCFA on the acetoclastic and hydrogenotrophic activities of anaerobic sludge indicate that acetoclastic methanogens are the most sensitive to LCFA Palatsi et al., 2010). Nevertheless, results obtained in our research group showed a significant increase in the relative abundance of methanogens in anaerobic sludge exposed to continuous feeding of oleate (C 18:1 ) and palmitate (C 16:0 ) followed by batch incubation (Sousa et al., 2007). Endurance of acetoclastic methanogens in a continuous bioreactor treating LCFA-rich effluent, at organic loading rates up to 21 kg m À3 day À1 , has also been reported . Additionally, activity of acetoclastic methanogens in sludge incubated with LCFA in batch assays has been shown, as more than 80% of the proteins assigned to the archaeal community were from Methanosaeta concilii (Salvador, 2013). The prevalence of acetoclastic methanogens belonging to Methanosaeta and Methanosarcina genera in LCFA-degrading environments has been reported in other studies (Shigematsu et al., 2006;Palatsi et al., 2010;Baserba et al., 2012;Ma et al., 2015), suggesting some controversy in the reported sensitivity of acetoclastic methanogens to LCFA.
In lipid-containing wastewaters, oleate (C 18:1 ) is generally the most abundant LCFA, and palmitate (C 16:0 ) tends to accumulate in anaerobic bioreactors treating these effluents (Pereira et al., 2002;Dereli et al., 2014). The effect of LCFA on anaerobic sludge has been studied before (Sousa et al., 2007;Palatsi et al., 2010;Silva et al., 2014), and a few studies report the sensitivity of pure cultures of hydrogenotrophic methanogens Zhou et al., 2013). Information on the sensitivity of pure cultures of acetoclastic methanogens is lacking. Methanosaeta concilii and Methanosarcina mazei were the ones commonly found in mesophilic anaerobic bioreactors treating LCFA-based wastewaters (Table S1). In this work, the effect of saturated (C 16:0 , palmitate) and unsaturated (C 18:1 , oleate) LCFA on the acetoclastic methanogenesis of pure cultures of M. concilii and M. mazei was investigated.

Results and discussion
Methanosaeta concilii (DSM 3671 T ) and M. mazei (DSM 2053 T ) were grown on sodium acetate as substrate for methane production, which was quantified over time before and after LCFA addition (Figs S1 and S2). Differences in methane production rate before and after LCFA addition were used to determine the methanogenic inhibition at oleate or palmitate concentrations of 0.5, 1, 2 and 4 mmol L À1 (see Fig. 1 as example).
Slope ratio (S ratio ) was calculated for each incubation condition according to equation S ratio = SlopeA/SlopeB, where SlopeB and slopeA represent the cumulative methane production slopes (mmol L À1 day À1 ) before and after the headspace flushing, second acetate addition and LCFA (oleate or palmitate) addition. Methane production rate of M. concilii was affected by oleate, as shown by the sharp decrease in the S ratio with 0.5 mmol L À1 oleate, compared with the S ratio obtained in the control (Table 1). This corresponds to 67% methanogenic inhibition and thus the IC 50 of oleate was below 0.5 mmol L À1 . No methane was produced, nor was acetate consumed, after the addition of 2 and 4 mmol L À1 oleate to M. concilii cultures (Table 1 and Table S2), which suggests complete methanogenic inhibition. Palmitate also affected methane production of M. concilii, although not as much as oleate, i.e. palmitate concentrations up to 2 mmol L À1 resulted in a maximum inhibition of 11%, whereas in the presence of 4 mmol L À1 methanogenesis was inhibited by 94% (Table 1).
Similar results were obtained with M. mazei in the presence of oleate, which caused 64% inhibition at 0.5 mmol L À1 and complete inhibition at 2 and 4 mmol L À1 (Table 1 and Table S2). However, although Methanosarcina spp. are reported as highly tolerant to others toxicants, such as ammonia and salts (De Vrieze et al., 2012;Hao et al., 2015), M. mazei was more vulnerable to palmitate than M. concilii. Palmitate concentrations of 2 mmol L À1 caused a 64% decrease of methane production by M. mazei, while for the same concentration the methanogenic inhibition of M. concilii was only 11% (Table 1).
The predominance of Methanosaeta spp. in anaerobic reactors containing high concentrations of palmitate (Shigematsu et al., 2006;Salvador et al., 2013)   Cumulative methane production from acetate by M. mazei before and after the addition of 0.5 mmol L À1 of oleate: (A) cumulative methane content measured in the bottles headspace; (B) cumulative methane production mathematically adjusted. Dashed lines represent the methane production rate (mmol L À1 day À1 ) before (SlopeB) and after (SlopeA) LCFA addition. Arrow points the moment of headspace flushing and second acetate addition (↑) and LCFA addition (↓). M. mazei (DSM 2053 T ) was acquired from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Germany) and was grown under strict anaerobic conditions as described by Stams et al. (1993) with sodium acetate as substrate at a final concentration of 20 mmol L À1 . Methane production was quantified over time until the mid-exponential phase was achieved. At this point, the methane accumulated in the headspace was quantified by gas chromatography, and after was removed under sterile conditions by flushing with 80% N 2 and 20% CO 2 gas mixture. To avoid substrate limitation during the exposure to LCFA, 10 mmol L À1 of acetate was added at the moment of LCFA addition. Assays were performed in duplicate and bottles were incubated in the dark at 37°C, without agitation. was found to persist and dominate over Methanosarcina in unstable anaerobic bioreactors with acetate concentrations up to 44 mmol L À1 (Chen and He, 2015). The prevalence of these acetoclastic microorganisms in LCFA-degrading environments might also be influenced by their different sensitivity to these compounds. A comparison between IC 50 values obtained for oleate and palmitate towards acetoclastic methanogens in this study, and towards hydrogenotrophic methanogens  is presented in Table 2. Our results show that M. concilii and M. mazei are similarly affected by the presence of oleate as the hydrogenotroph Methanospirillum hungatei, and M. concilii seems to be even more tolerant to the presence of palmitate than M. hungatei. Previous studies on the toxicity of LCFA towards anaerobic sludge highlighted the higher sensitivity of acetoclasts compared with hydrogenotrophs. Since LCFA can absorb to the cells at variable amounts, its toxicity might be explained by a physical inhibition phenomenon rather than by direct metabolic inhibition (Pereira et al., 2005). Mass transfer limitations exerted by LCFA are likely more pronounced for acetate than for hydrogen transport, since hydrogen is a smaller molecule (Pereira et al., 2005).
Differences in cell envelopes composition might also influence the sensitivity of microorganisms. For example, the cell wall of Methanosarcina contains methanochondroitin and the one of Methanosaeta contains a sheath surrounding the S-layer and the cytoplasmic membranes. The sheath might have a protective effect since a. Differences in methane production rate before and after LCFA addition were expressed as a slope ratio (S ratio ) that was calculated for each condition using the cumulative methane production slope, according to the equation (S ratio = SlopeA/SlopeB). For control assays, in which no LCFA was added, S ratio were equally calculated and SlopeA determined after the headspace flushing and second acetate addition (Figs S1 and S2). b. The inhibitory effect of the different LCFA concentrations on methane production was expressed in percentage, by comparing the S ratio obtained from the LCFA supplemented assays (S ratio_L ) with the slopes ratio obtained from the control assays (S ratio_C ), according to equation (Inhibition = ((S ratio_C À S ratio_L )/S ratio_C )*100). c. Average AE standard deviation of duplicate assays. it is reported to be resistant to detergents (Claus and K€ onig, 2010). Although the studies with mixed communities degrading LCFA are important, information about the sensitivity of individual species growing in pure cultures show the unequivocal metabolic behaviour of each tested species in the presence of LCFA.
The tolerance to LCFA can be higher when methanogens are growing in complex microbial communities than in pure cultures, due to a structural protection provided by aggregation of different microbial species. IC 50 values between 0.1 and 1 mmol L À1 and approximately 3 mmol L À1 were reported for suspended and granular sludge respectively (Table S3). These values are, however, close to the ones obtained in this study for acetoclastic methanogens, and by Sousa et al. (2013) for pure cultures of hydrogenotrophic methanogens, and are indicative of LCFA concentrations that might cause operational problems due to direct inhibition of methanogens. These studies, all together, allow to discriminate between mass transport-related physical inhibition and metabolic inhibition, which impacts practical applications of anaerobic processes for treatment of LCFA-containing wastewater.
Because in continuous bioreactors oleate is for a large part converted to palmitate (Pereira et al., 2002), the potential toxicity of palmitate is, in this context, most relevant. Palmitate concentrations between 1 and 2 mmol L À1 can be tolerated by methanogenic communities, allowing to feed higher oleate concentrations than the IC 50 for oleate (<0.5 mmol L À1 ). The higher IC 50 exhibited by the acetoclastic M. concilii and the hydrogenotroph Methanobacterium formicicum (Table 2), particularly for palmitate, may explain why these species are commonly found in bioreactors and point to their importance in the conversion of LCFA to methane in anaerobic wastewater treatment systems.

Conclusions
In this work, two acetoclastic methanogens revealed different tolerance to LCFA. Methanosaeta concilii demonstrated a tolerance to palmitate similar to hydrogenotrophic methanogens, which generally are considered to be more resistant. These results are relevant in the context of lipid-rich wastewater treatment, where the presence and prevalence of Methanosaeta species could be a good indicator of the system potential to efficiently convert LCFA to methane. Fig. S1. Cumulative methane production from acetate consumption by Methanosaeta concilii during exposure to oleate or palmitate. Fig. S2. Cumulative methane production from acetate consumption by Methanosarcina mazei during exposure to oleate or palmitate. Table S1. Main acetoclastic methanogens detected in anaerobic sludges from LCFA-fed reactors. Table S2. Acetate concentration (mmol L À1 ) determined before LCFA was added (Ac init ) and at the end of the assays (Ac end ). Table S3. Inhibition of acetoclastic methanogenic activity by LCFA, in several sludges exposed to different wastewater compositions.