1. INTRODUCTION
4.3. Results and discussions
4.3.5. Possible sulfide oxidation mechanism
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represent positive and negative correlations, respectively. Edges with solid and dotted lines represent significant (p < 0.05) and marginally significant (0.05 < p < 0.10) correlations, respectively. H2S, H2S content in biogas; Mag, magnetite dose; MC, methane content; MY, methane yield; S0, extracellular S0 concentration.
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To address the possibility of electric syntrophy between ASOBs and electrotrophic methanogens in the presence of magnetite, batch tests where methanogenesis was intentionally blocked by BES addition was performed. The profile of extracellular S0 formation during 28 days of batch experiment under different experimental conditions (i.e., C, M, and MB runs) was monitored (Fig. 4-14). Methane production was not observed in the MB condition, which confirms that the BES addition effectively inhibited the methanogenesis pathway. Interestingly, the residual concentration of extracellular S0 gradually decreased from 30.3 to 4.8 mg/L and from 25.3 to 0.0 mg/L in C and MB runs, repsectively, whereas up to 62.0 mg/L of extracellular S0 formed in M.
Furthermore, the cessation of S0 formation in C and MB was accompanied by the accumulation of H2S content in biogas, which was up to 3,000 ppmv in both C and MB runs. By contrast, H2S content in biogas kept below 100 ppmv in the M run. These results further confirmed that magnetite triggered the anaerobic sulfide oxidation to S0, and it also clearly demonstrated that methanogenesis was of the primary reduction reaction that coupled with anaerobic sulfide oxidation to S0 in the presence of magnetite. In other words, electrotrophic methanogens appears to be a promising candidate for electro-syntrophic partners of ASOBs under magnetite-added conditions.
Fig. 4-14. Profile of extracellular S0 formation during 28 days of batch tests under either magnetite addition or non-magnetite additions, and with methanogenesis being either inhibited or uninhibited by BES. Each experimental condition was quadruplicate. C; control (without BES and magnetite),
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M; magnetite-added condition (with magnetite and without BES), and MB; methanogenesis- inhibited condition (with both magnetite and BES).
The high relative abundance of Methanotrichaceae at both the DNA and RNA levels under magnetite-added conditions (Fig. 4-8) may also support the above observations. Methanotrichaceae species, previously considered to be obligate acetotrophs, are capable of DIET and of electrotrophic reduction of CO2 to CH4 (Rotaru et al., 2014b). It should be noted here that the apparent increase in methane production rate or yield as a result of CO2 reduction to CH4 by the electrons from sulfide oxidation was not observed possibly because the produced amount of H2S was marginal compared with that of methane (Tables 4-3 and 4-4). However, the correlation analysis results showed that the magnetite dose was correlated positively with the methane content but was negatively correlated with H2S content (Fig. 4-13), suggesting the enhancement of methanogenesis, although marginal, by magnetite-assisted DIET. The observation that S0 formation was hindered when methanogenesis was intentionally suppressed in the presence of magnetite crucially supported that magnetite may establish the syntrophic association between anaerobic sulfide oxidation and methanogenesis. In support of this view, the microorganisms in the magnetite-added reactors aggregated to each other in a compact manner, with fine irregular-shaped structures of magnetite nanoparticles (Fig. 4-15).
Such structures indicate that the magnetite particles aid the direct electric syntrophy between microbial cells and their electo-syntrophic metabolism.
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Figure 4-15. SEM images of the digestate samples of RM1 and RM2 under magnetite-added conditions (20 mM Fe).
Recently, a few studies have reported on the influence of conductive materials on the fate of sulfur in AD processes. Li et al. (2017) found that methane production in an upflow anaerobic sludge blanket (UASB) reactors used to treat sulfate-containing wastewater was enhanced by the addition of stainless steel (26 g/L working volume), along with a slight decrease in sulfate removal and ultimately in sulfide production. Although the authors suggested that stainless steel likely drove the electron flow towards methanogenesis by promoting DIET between Geobacter and methanogens, this mechanism was not unambiguously supported by experimental evidences. This study did not monitor the H2S content in the gas phase and could not describe the effect of the potential shifts on the flux of sulfide in the reactor. Another study using UASB reactors has reported that both methanogenic and sulfidogenic activities were enhanced by the addition of magnetite (Jin et al., 2019). The authors observed an increase in the conductivity of sludge and a decrease in the content of conductive c-type cytochromes when magnetite (20 mM Fe) was added, and they proposed that iron-reducing SRB and methanogens formed an electro-syntrophic association via DIET in the presence of magnetite. However, in the proposed syntrophy model, the electrons utilized for CO2 reduction (via DIET or Fe2+/Fe3+ electron shuttle) as well as for sulfate reduction should be derived from the oxidation of organic compounds by the iron-reducing SRBs; this process appears not advantageous to the SRB, and no other potential syntrophy related to sulfur metabolism was considered. A recent study on the treatment of sulfate-containing wastewater using anaerobic sequencing batch reactors found that magnetite enhanced methanogenesis (Liu et al., 2019).
Although the sulfidogenic activity was reported to be somewhat reduced by the addition of magnetite as the level of dissolved sulfide in the mixed liquor decreased, the production of H2S gas was not measured. Based on the metagenomic analysis results, the authors suggested that magnetite promoted electro-syntrophic interactions via DIET and mitigated the inhibition of methanogenesis by sulfate. The different observations described above may be attributable to the differences in reactor operating conditions, which can affect microbial interactions and metabolism, including electron exchange between electroactive microorganisms (Barua & Dhar, 2017; Feng et al., 2017).
It should be noted that none of the previous studies that has investigated the effects of conductive materials on AD has discussed the possible anaerobic oxidation of sulfide to S0 via DIET, making the present study unique and significant. Unlike the initial research hypothesis that
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the magnetite may lead the preferential electron flow from VFA oxidation towards methanogenesis rather than sulfate reduction, via DIET, the syntrophic reactions between anaerobic sulfide oxidation to S0 and electrotrophic methanogenesis was observed. Although further research is required to elucidate the underlying mechanism, the present study proposes a novel electric syntrophy via DIET between ASOBs and electrotrophic methanogens, and this syntrophy couples the oxidation of sulfide to S0 with the reduction of CO2 to CH4. Our observations reveal a new route of anaerobic sulfur metabolism and suggest the possibility to control H2S production in situ by promoting DIET in sulfur-rich methanogenic environments. Additionally, an interesting feature of magnetite-assisted in situ sulfie control is accumulation of extracellular S0. Given that the extracellular S0 is tightly bound to the microbial cells, which are aggregated with magnetite particle (Fig. 4-15), it is suggested that sulfur recovery from the S0-rich sludge, for example, through magnetic separation and physical pretreatment is likely possible.