| Biogas technology,which is based on the decomposition and stabilization of organic materials from various sources via anaerobic digestion（AD）,can effectively alleviate the environmental pollution and provide clean energy in the form of biogas;thus,it plays a positive role in energy structure optimization and energy security enhancements,which is helpful for“carbon neutrality.”However,the frequent occurrence of process instability decreased the economic feasibility of the existing commercial biogas plants,limiting their further sustainable development.To guarantee an AD reactor’s stable and efficient operation,it is of great significance to understand the characteristics of process instability in AD reactors,diagnose process stability in a timely and accurate manner,and explore the mechanism of process instability in detail.In this context,controlled instability simulation batch and semi-continuous experiments were conducted in laboratory-scale reactors treating energy crops.Based on the techniques,including physical-chemical analysis,thermodynamic calculation,high throughput sequencing,metagenomics,and metaproteomics,the response characteristics of various state indicators and thermodynamic parameters in the process destabilization of AD were investigated,and the metabolic deterrence mechanism of process instability was systematically revealed.The results are listed as follows.（1）Batch experiments show that the continuous increase of organic loading in the form of substrate-to-inoculum ratio（S/I）will aggravate the acidification phenomenon caused by excessive accumulation of Volatile Fatty Acid（VFA）,thereby resulting in the adverse effects on methane generation kinetics,including reduced methane yield,decreased methanogenic activity,and prolonged lag phase,or even worse,the failure of AD.The coupling microbial analysis and thermodynamic calculations showed that the change in the Gibbs free energy（△G）for the degradation of the key VFAs correlated with process stability and increased progressively with the increasing S/I.The△G of propionate and acetate degradation gradually increased as the S/I ratio increased,and positive values were observed when S/I reached 30,corresponding to the irreversible acidification of the AD reactor,indicating the ability of the△G for VFA to diagnose process stability.（2）Based on the results of the batch experiments,the semi-continuous experiments further introduced disturbances to AD reactors treating energy crops by gradually increasing the Organic Loading Rate（OLR）and reducing the Hydraulic Retention Time（HRT）to induce process instability.The physicochemical analysis showed that,during the long-term experiment of 400 days,although a decline in methane yield was observed several times,self-recovery was also observed several times with the continuous operation of the AD process.Eventually,the tolerable OLR gradually increased,and the HRT steadily shortened.The full-load stable operating OLR is 18 g VS m-3·d-1,and HRT is 4 d.However,suppose physicochemical indicators were used to diagnose process stability,both in terms of the magnitude of abrupt changes and thresholds.In that case,all types of state indicators provide early warning information several times at conditions well below full load operating conditions.Thus,process management based on the above diagnostic mode will prevent the AD reactor from achieving the optimum operational efficiency and therefore working in sub-optimal conditions.Microbial analysis showed that ecological parameters could rebound during the destabilization induction process,indicating the ability of anaerobic microorganisms to self-adapt to environmental perturbations,which is consistent with AD reactor performance deteriorating and then recovering itself over the continuous operation process.However,due to the difficulties such as sampling and pretreatment,there are limitations to the commercial application of ecological indicators for diagnosing process stability.Thermodynamic analysis showed that the?G of propionate and valerate degradation did not indicate an imbalance in the AD reactors in the presence of repeated reversible acidification but only increased in response to progressively more significant levels of stressed conditions,eventually changing abruptly at an extreme operating condition of OLR is 20 g VS m-3·d-1and HRT is 4 d,which is 2-3 days ahead of the sharp decreased in methane yield.Thus,using?G of VFA degradation to identify and diagnose process stability can avoid the excessive warning phenomenon caused by reversible acidification but still retains the early warning and responsive early warning functions.So,the?G of VFA degradation has the potential to be used as an ideal state indicator for identifying process stability and providing early warning.（3）Metagenomic and metaproteomic analysis showed that the accumulated butyrate and valerate during the semi-continuous reactors were degraded via theβ-oxidation pathway dominated by Syntrophomonas.In contrast,the propionate is degraded mainly by Pelotomaculum,Desulfotomaculum,and Syntrophobacter through Methyl-Malonyl-Co A（MMC）pathway.During the repeated reversible acidification induced by process disturbance,although a slight decrease in the relative abundance of the active members of the above dominant genus was observed,the dominant Syntrophomonas and Pelotomaculum restored the normal metabolic performance to degrade those short-chain fatty acids by acetyl-Co A acyltransferase（Acca）and acetate kinase（ack A）and phosphate acetyltransferase（Pta）mediated metabolic pathways.The conversion of acetate to methane is dominated by Syntrophic Acetate Oxidation Bacteria（SAOB）,Methanosarcina,Methanoculleus,and Methanobacterium.The relative abundance of SAOB and methanogens,however,was not observed to decrease significantly during reversible acidification,but rather to increase,so repeated abrupt changes in VFA during these phases with early warning information do not imply the actual failure of the AD reactors.As the AD reactors further ran into extreme operating conditions(OLR is 20 g VS m-3·d-1and HRT is 4 d),the differentially expressed proteins（DEPs）of the dominant genus responsible for the degradation of the accumulated VFAs were gradually dominated by down-regulated proteins.Based on the performance of DEPs,the key to process instability of the AD system treating energy crops was the degradation of acetate and propionate.The metabolism of acetate and propionate is progressively inhibited with increasing stressed conditions,and the ability to regulate the production and conversion of these two is subsequently reduced.The accumulated acetate and propionate inhibited the degradation of butyrate and valerate through feedback inhibition.As the acid-base equilibrium of the AD system was disrupted entirely,the DEPs of theβ-oxidation pathway associated with the degradation of valerate and butyrate were also significantly down-regulated.VFAs continued to accumulate and resulted in complete irreversible acidification.Comparing the dynamics of DEPs during the stabilization and destabilization of acetate and propionate under high stress operating conditions,the key to targeted regulation was finally identified as the activities of methylmalonyl-Co A epimerase（Mce）,methylmalonyl-Co A mutase（Mcm）and succinyl-Co A synthetase（Scs）in the MMC pathway,and ack A,pta and methyl-coenzyme M reductase（mcr）activities in the conversion process of acetate to methane.In this study,based on the traditional physicochemical analysis,an innovative coupling of microbial analysis and thermodynamic calculations was made to improve the calculation ofΔG for VFA degradation at the boundary of the AD system from the perspective of macroscopic biochemical reaction;the thermodynamic characteristics of process instability in AD reactors were investigated;the potential ofΔG for critical VFA degradation as an ideal state indicator for identifying process stability and providing early warning information was discussed;the key regulatory factors that control the overall performance of AD reactors are identified at the level of active microorganisms and functional proteins.The above results can provide a reference value for the improvement of online process monitoring and diagnosis and provide a theoretical basis for developing targeted regulation countermeasures,which is of great application and significance to ensure the stable and efficient operation of commercial AD reactors. |
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