Enhanced Anaerobic Digestion Of Straw Via Co-cultivated Consortia And Its Underlying Microbial Mechanisms

Although anaerobic digestion is an important way of straw resources utilization,hydrolysis is the rate-limiting step of this process.Due to lack of appropriate environment selection pressure,some inocula including biogas slurry and residues,anaerobic sludge,livestock and poultry manure etc.could not form efficient hydrolytic consortia.Thus this study constructed co-cultivatied consortia by inoculating anaerobic sluge into ruminal microbiota.And the enhanced anaerobic digestion of straw was investigated at the batch and semicontinuous mode,respectively.By means of molecular ecology techniques,metal speciation,and Excitation-Emission Matrix and Parallel Factor Analysis(EEM-PARAFAC)etc.,microbial mechanisms were illustrated and the factors of "instability" stage using straw materials were analyzed.The main results are as follows:Under the semi-continuous conditions with different organic loading rates(OLR)of 1.5 g/(L·d),3.5 g/(L·d)and 7 g/(L·d),the methane yields of co-cultivation increased 1.79-,2.07-and 2.26-fold compared with the single-rumen system.The methane content of biogas varied from 32 to 44%,significantly different(p < 0.05)from the control reactor.There was no corresponding accumulation of volatile fatty acids(VFAs)and pH values varied from 6.8 to 7.68 during the whole anaerobic digestion.The cellulolytic activities including CMCase and FPase improved significantly(p < 0.05)compared with the control samples.After anaerobic digestion,Methanobrevibacter became the dominant genus,and acetotrophic Methanosarcina was the second-most abundant genus.Compositions of hydrolytic bacteria were changed in cocultivated system.The proportion of Ruminococcus evidently increased compared with the initial co-inoculum even though Clostridium was still the dominant genus.Bacteroides,Fibrobacter and Acetivibrio from rumen disappeared.Mutual cooperation formed between Methanobrevibacter and Ruminococcus was achieved for high cellulolytic activity and methanogenic efficiency in co-cultivated system.Ruminal microbiota(RM)were co-inoculated with anaerobic sludge(AS)at different volatile solid(VS)ratios including 1:1,1:2,2:1,1:0 and 1:0 to study the digestion of rice straw in the batch experiments.Methane yields in co-cultivated system(including ratio of 1:1,1:2,2:1)increased 3-fold compared with the single-inoculum and the methane yield reached 273.64 mL/g VS at a co-inoculum ratio of 1:1,which equaled to 78.21% of the theoretical yield.Cocultivation significantly improved the xylanase and cellulase activities(p < 0.05),which made the digestion rates varing from 43.95% to 44.64%.And it could have pivotal importance in the efficient degradation of rice straw.Microbial compositions were clearly altered after digestion in the co-cultivated system.Clostridiales members became the main cellulolytic bacteria including polysaccharide-degraders(Proteiniclasticum,Lutispora)and acidogens(Tissierella,Sedimentibacter).Hemicellulose-degraders belonged to members of Lysinibacillus.Hydrolytic bacteria and acetoclastic methanogens(Methanosarcina and Methanosaeta)coexisted in the solid residues.In the liquid phase,syntrophy involving propionate oxidizers(Syntrophobacter or Pelotomaculum)with associated methanogens(Methanosaeta)accelebrated the startup time of anaerobic digestion by reducing propionate accumulation.Digestion performance was studied in a semi-continuous reactor by combination with different OLR and digestate recirculation ratio using straw materials.Methane yield was 217 mL/g VS at the OLR of 4 g/(L·d)and 1:1 of recirculation ratio,which reached 56-58% of the theoretical value.In the stable stage,the dry degradation rates ranged from 79.86% to 88.81%.Hydrolytic bacteria belonged to Clostridiales.Lachnospiraceae and Bacteroidales were main acid-producing bacteria while Spirochaetaceaea and Clostridiales were the mumbers of acidogens.Syntrophic oxidation consortia consisted mainly of Synergistetes,propionate oxidizers(Syntrophobacter and Pelotomaculum),and butyrate oxidizers(Syntrophus and Syntrophomonas).The total methanogens,Methanobacteriales and Methanosarcinales increased to 6.20×108 copies/m L,3.30×108 copies/m L and 1.75×107 copies/mL respectively.At the OLR of 6 g/(L·d),the concentrations of dissolved organic matter(DOM)increased to 13.67 g/L.Bioavailable concentrations of Zn and Fe in biogas residues decreased significantly(p < 0.05),equal to 4.39 mg/kg and 5.54 mg/kg.Bioavailable concentrations of Cu and Ni declined to 0.40 mg/kg and 1.33 mg/kg respectively while the bioavailable concentration of Mo was 0.98 mg/kg.The total methanogens and Methanobacteriales deceased significantly to 8.56×105 copies/m L and 4.14×103 copies/mL(p < 0.05).And then methanogenic activities decreased.Microbial diversity dropped,and especially the hydrolytic bacteria and syntrophic oxidizers decreased.In contrast,the proportion of Bacteroidales increased significantly(p < 0.05).In addition to butyrate cumulation,acetate and propionate concentrations were 2.02 g/L and 6.54 g/L,respectively,which indicated the “rancidity” of the digestion reactor.Effects of high solid content and feedstock to inoculum(F/I)on the digestion performance of rice straw were studied at the batch experiment.The results showed that methane yields were on decline with the increasing of solid content.At the solid content of 20% with F/I of 2 and 6,the lag phase(?)reached to 6.24 d and 9.16 d,respectively.Two fluorescent components were decomposed by PARAFAC model.The dynamic changes of the coenzyme F420 correlated significantly with methane yields(p < 0.01),which could be directly used to characterize the methanogenic activities.During high solid-state digestion using the semi-continuous mode,the highest volume biogas productivity reached to 1.04 L/(L·d).The average methane yield reached to 280.90 mL/g VS with around 54.39% methane content at the OLR of 2.26 g/(L·d),which is up to 80.29% of the theoretical yield of rice straw.At the same stage,the cellulase activities increased obviously in the horizontal reactor,which was indispensable to the degradation of cellulose.Three fluorescent components were decomposed by PARAFAC model.Fluorescence intensities of coenzyme F420 and NADH reflected the dynamic changes of methanogenic activities and anaerobic digestion efficiency,respectively.At the OLR of 2.47 g/(L·d),the average concentration of ammonia nitrogen reached to 1082.63 mg/L,which resulted in the hydrogenotrophic Methanobacteriales decreasing to 1.04×10~6 copies/g TS.In contrast,the acetotrophic Methanosarcinales increased to 9.44×10~6 copies/g TS,which indicated adaptive shifts.At the same time,the methanogenic activities characterized by coenzyme F420 decreased and propionate began to accumulate in the digestion reactor.