Liping Hao

Predominant Contribution of Syntrophic Acetate Oxidation to Thermophilic Methane Formation at High Acetate Concentrations

To quantify the contribution of syntrophic acetate oxidation to thermophilic anaerobic methanogenesis under the stressed condition induced by acidification, the methanogenic conversion process of 100 mmol/L acetate was monitored simultaneously by using isotopic tracing and selective inhibition techniques, supplemented with the analysis of unculturable microorganisms. Both quantitative methods demonstrated that, in the presence of aceticlastic and hydrogenotrophic methanogens, a large percentage of methane (up to 89%) was initially derived from CO(2) reduction, indicating the predominant contribution of the syntrophic acetate oxidation pathway to acetate degradation at high acid concentrations. A temporal decrease of the fraction of hydrogenotrophic methanogenesis from more than 60% to less than 40% reflected the gradual prevalence of the aceticlastic methanogenesis pathway along with the reduction of acetate. This apparent discrimination of acetate methanization pathways highlighted the importance of the syntrophic acetate-oxidizing bacteria to initialize methanogenesis from high organic loadings.

Regulating the hydrolysis of organic wastes by micro-aeration and effluent recirculation

In this study, the effects of micro-aeration and liquid recirculation on the hydrolysis of vegetable and flower wastes during two-phase solid-liquid anaerobic digestion were assessed. To accomplish this, we evaluated the hydrolysis of five batches of waste that were treated under the following conditions: anaerobic, insufficient micro-aeration (aeration for 5 min every 24 h), and sufficient micro-aeration (aeration for 5 min every 12, 4 and 1h). Hydrolysis was found to depend on the level of micro-aeration. Specifically, insufficient micro-aeration led to unstable and decreased performance. Conversely, sufficient micro-aeration promoted the hydrolysis of easily biodegradable carbohydrates and proteins, but the microbial activity was later impaired by liquid recirculation using methanogenic effluent. The hydrolysis efficiency under anaerobic conditions was comparable to the efficiency observed under sufficient micro-aeration, while the cumulative TOC of the anaerobic batch was 1.4-2.4 times higher than that of the micro-aerated batches. In addition, liquid recirculation did not have a negative effect on the development of microbial activity under anaerobic conditions, which resulted in the lignocelluloses having a higher hydrolysis efficiency.

Shift of pathways during initiation of thermophilic methanogenesis at different initial pH

To investigate the metabolic pathways during the initiation of methanogenesis from acid crisis, the influence of initial pH (5.0-6.5) on thermophilic methanogenic conversion of 100mmol/L acetate was monitored based on the isotopic signature and selective-inhibition method combined with analysis of the microbial structure. The results showed, lower pH extended the lag phase for methanogenesis which was inhibited at pH5.0 throughout the incubation. At initial pH6.0-6.5, methanogenesis was primarily initiated via acetoclastic methanogenesis (AM), with the fraction of the hydrogenotrophic pathway (f(mc)) accounting for 21-22% of total methane formation. Conversely, at initial pH5.5, the dominant pathway shifted to syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis (SAO-HM), with f(mc) rising to 51% and the abundance of syntrophic acetate-oxidizing bacteria increasing remarkably. Methanogenesis could initiate independently via SAO-HM pathway when AM pathway was inhibited. Acetate-oxidizing syntrophs could function as the initiation center of methanogenesis from low-pH crisis.

Initiating methanogenesis of vegetable waste at low inoculum-to-substrate ratio: importance of spatial separation.

With the goal of starting-up the methanogenesis of easily biodegradable waste with minimum inoculum, the present work evaluated different inoculum-to-substrate ratios (r(I/S)) in completely mixed systems and in the systems with spatial separation of inoculum and waste. It was found difficult to initiate methanogenesis in the completely mixed systems, even at high r(I/S) 1.105 on a volatile solid basis. Fermentation efficiencies were independent of r(I/S). In the spatial-separation systems with a low total r(I/S) 0.053, the ultimate methane yield (35 °C, 1 atm) reached 445 mL/g-VS added for the inoculum-waste initially completely separated system. The yields decreased to 285, 181, and 34 mL/g-VS added, respectively, for partially separated systems with the ratios controlled at 1.105, 0.254, and 0.113 in the inoculum-containing reactors. This demonstrates the importance of setting spatial separation between inoculum and waste when inoculation is employed. An appropriate inoculation method would initiate methanogenesis rapidly even at low inoculum-to-substrate ratios.

Self-adaption of methane-producing communities to pH disturbance at different acetate concentrations by shifting pathways and population interaction.

To investigate the competition among acetate-utilizing microorganisms at different acetate levels, bioconversion processes of 50, 100, 150 and 200 mM acetate in the presence and absence of methanogenic inhibitor CH3F were monitored in thermophilic methanogenic system. The successive response of methane-producing community during the deteriorative and recovery phases caused by pH disturbance was analyzed. High acetate concentration (>50mM) inhibited the activity of acetoclastic methanogenesis (AM). The increasing pH (>7.5) enhanced this inhibition. The syntrophic acetate oxidizing (SAO) bacteria and hydrogenotrophic methanogens including Methanomicrobiales and Methanobacteirales were more tolerant to the stress from high acetate concentration and high pH. Resumption from alkali condition to normal pH stimulated the growth of acetate oxidizing syntrophs. The reaction rate of SAO-HM was lower than that of AM. These results point to the possibility to regenerate the deteriorated anaerobic digesters by addition of acclimatized inocula rich in acetate-oxidizing syntrophs.

Stable isotope probing of acetate fed anaerobic batch incubations shows a partial resistance of acetoclastic methanogenesis catalyzed by Methanosarcina to sudden increase of ammonia level.

Ammonia inhibition represents a major operational issue for anaerobic digestion. In order to refine our understanding of the terminal catabolic steps in thermophilic anaerobic digestion under ammonia stress, we studied batch thermophilic acetate fed experiments at low (0.26 g L(-1)) and high (7.00 g L(-1)) Total Ammonia Nitrogen concentrations (TAN). Although methane production started immediately for all incubations and resulted in methane yields close to stoichiometric expectations, a 62-72% decrease of methanogenic rate was observed throughout the incubation at 7.00 g L(-1) of TAN compared to 0.26 g L(-1). Stable Isotope Probing analysis of active microbial communities in (13)C-acetate fed experiments coupled to automated ribosomal intergenic spacer analysis and 16S rDNA pyrotag sequencing confirmed that microbial communities were similar for both TAN conditions. At both TAN levels, the (13)C-labeled bacterial community was mainly affiliated to Clostridia-relatives, with OPB54 bacteria being the most abundant sequence in the heavy DNA 16S rDNA pyrotag library. Sequences closely related to Methanosarcina thermophila were also abundantly retrieved in the heavy DNA fractions, showing that this methanogen was still actively assimilating labeled carbon from acetate at free ammonia nitrogen concentrations up to 916 mg L(-1). Stable isotopic signature analysis of biogas, measured in unlabeled acetate fed experiments that were conducted in parallel, confirmed that acetoclastic methanogenic pathway was dominant at both ammonia concentrations. Our work demonstrates that, besides the syntrophic acetate oxidation pathway, acetoclastic methanogenesis catalyzed by Methanosarcina can also play a major role in methane production at high ammonia levels.

Impact of recycled effluent on the hydrolysis during anaerobic digestion of vegetable and flower waste

Two trials were established to investigate the effect of recycled effluent on hydrolysis during anaerobic co-digestion of vegetable and flower waste. Trial I evaluated the effect by regulating the flow rate of recycled effluent, while Trial II regulated the ratio of hydrolytic effluent to methanogenic effluent, which were recycled to hydrolysis reactor. Results showed that the recirculation of methanogenic effluent could enhance the buffer capability and operation stability of hydrolysis reactor. Higher recycled flow rate was favourable for microbial anabolism and further promoted hydrolysis. After 9 days of hydrolysis, the cumulative SCOD in the hydrolytic effluent reached 334, 407, 413, 581 mg/g at recycled flow rates of 0.1, 0.5, 1.0, 2.0 m3/(m3 x d), respectively. It was feasible to recycling a mixture of hydrolytic and methanogenic effluent to the hydrolysis reactor. This research showed that partially introducing hydrolytic effluent into the recycled liquid could enhance hydrolysis, while excessive recirculation of hydrolytic effluent will inhibit the hydrolysis. The flow ratio 1:3 of hydrolytic to methanogenic effluent was found to provide the highest hydrolysis efficiency and degradation rate of lignocelluloses-type biomass, among four ratios of 0:1, 1:3, 1:1 and 3:1. Under this regime, after 9 days of hydrolysis, the cumulative TOC and TN in the hydrolytic effluent reached 162 mg/g and 15 mg/g, the removal efficiency of TS, VS, C and cellulose in the solid phase were 60.66%, 62.88%, 58.35% and 49.12%, respectively. The flow ratio affected fermentation pathways, i.e. lower ratio favoured propionic acid fermentation and the generation of lactic acid while higher ratio promoted butyric acid fermentation.

In situ visualization of the change in lignocellulose biodegradability during extended anaerobic bacterial degradation

To investigate the variation in biodegradability of lignocellulose under anaerobic degradation, extended anaerobic bacterial degradation experiments with four stages were conducted for both untreated and dilute-acid pretreated rice straw. In situ biodegradability visualization of the interior and exterior lignocellulosic biomass surfaces was carried out by fluorescent protein GFP-CBM3 labeling with or without bovine serum albumin blocking and observation with confocal laser scanning microscopy. The amount of fermentation products and the degradation rate of cellulose and hemicellulose decreased significantly along the extended experiments, suggesting a decrease of biodegradability under anaerobic degradation, although 50% (untreated) and 63% (pretreated) of the cellulose left at the end of the experimental period. In contrast, the quantity of cellulose-GFP-CBM3 points on plant cell walls increased slightly and the spatial distribution obviously changed, suggesting an increase in accessible cellulose and revealing that most of the cellulose exposed on the surface could not be hydrolyzed effectively under anaerobic degradation. From the combined results of SEM, AFM and FTIR, the increased accessibility and decreased biodegradability were attributed to the cell wall deconstruction by hydrolysis and the reaggregation, redistribution and redeposition of lignin and non-productive cellulose with increasing crystallinity. Furthermore, the initially conducted dilute-acid pretreatment of raw biomass did not enhance the successive biodegradability of residual biomass, even though the accessibility of pretreated biomass was higher than that of untreated biomass. Hence, the biodegradability cannot be immediately predicated by accessibility; rather, it mainly depends on the crystallinity of cellulose combined with enzymes, as well as the spatial structure of lignocellulose.

Quantification of the inhibitory effect of methyl fluoride on methanogenesis in mesophilic anaerobic granular systems

The inhibitory effect of CH(3)F on methanogenesis in mesophilic anaerobic granules was tested at different concentrations (0-10% v/v, in the gas phase) and verified by the stable carbon isotopic signatures of CH(4) and CO(2). The results showed that the inhibitory effect increased with the initial CH(3)F concentration up to 5%. The CH(3)F concentration causing 50% metabolic inhibition was 0.32%. Complete inhibition of acetoclastic methanogenesis with a 91% reduction in total methanogenic activity was achieved when 5% CH(3)F was initially added to the headspace, which resulted in 870 μM dissolved CH(3)F in the liquid. It was much higher than that applied in other natural anoxic non-granular systems, indicating that the layered granular structure influenced the inhibitory effect. The obvious increase in hydrogen content indicated that high concentrations of CH(3)F (≥5%) suppressed hydrogenotrophic methanogenesis as well. The stable inhibition lasted for at least 6d as the CH(3)F concentration decreased slowly with incubation time. These results suggested that CH(3)F could be used for investigating methanogenic processes in anaerobic granular systems after the CH(3)F concentration and incubation time for specific inhibition of acetoclastic methanogenesis were carefully determined. In the present system, CH(3)F concentration of 5% was suggested to be optimal.

Response of anaerobes to methyl fluoride, 2-bromoethanesulfonate and hydrogen during acetate degradation

To use the selective inhibition method for quantitative analysis of acetate metabolism in methanogenic systems, the responses of microbial communities and metabolic activities, which were involved in anaerobic degradation of acetate, to the addition of methyl fluoride (CH3F), 2-bromoethanesulfonate (BES) and hydrogen were investigated in a thermophilic batch experiment. Both the methanogenic inhibitors, i.e., CH3F and BES, showed their effectiveness on inhibiting CH4 production, whereas acetate metabolism other than acetoclastic methanogenesis was stimulated by BES, as reflected by the fluctuated acetate concentration. Syntrophic acetate oxidation was thermodynamically blocked by hydrogen (H2), while H2-utilizing reactions as hydrogenotrophic methanogenesis and homoacetogenesis were correspondingly promoted. Results of PCR-DGGE fingerprinting showed that, CH3F did not influence the microbial populations significantly. However, the BES and hydrogen notably altered the bacterial community structures and increased the diversity. BES gradually changed the methanogenic community structure by affecting the existence of different populations to different levels, whilst H2 greatly changed the abundance of different methanogenic populations, and induced growth of new species.