Community Structures And Diversity Of Bacteria And Archaea In Low Temperature Biogas Systems

Biogas technology is a popular strategy to avoid environmental pollution.It can provide energy on a local scale,and as such contribute to the development of sustainable agriculture.A variety of organic wastes can be used to efficiently produce biogas by natural fermentation,with nutrient-rich bioslurry as the secondary product.However,the success of biogas fermentors is limited in areas at higher altitudes with colder climates: it is estimated that more than 75 % of China household biogas digesters are functioning sub-optimally due to low temperatures.For this situation to be improved,more research is needed.The performance of a biogas system is highly dependent on the microorganisms responsible for the fermentation process.Preliminary research was carried out to identify key microorganisms that are responsible for fermentation at low temperatures in a biogas system(the ―Gray Box‖),to reveal the community composition and to analyse their functions.The results indicated data gaps in our knowledge of the key factors influencing the fermentation process at low temperatures,such as the succession of population shifts,i.e.,the dynamics in time of changing dominant communities;the relationship between these communities and environmental factors(both biotic and abiotic);and the interrelationships between the biological communities.These factors will need to be studied in future research.Inocula were prepared for biogas fermentation at three different temperatures,15 ℃(for system A),9 ℃(for system B)and 4 ℃(for system C).These inocula were applied in batch fermentation experiments A,B and C,respectively,using pig manure as the substrate.Fermentation was performed at an external temperature of 9 ℃.During the process,the microorganisms responsible for fermentation were characterized by 16 S r DNA amplicon sequencing.A statistical correlation analysis was performed to provide insights how biogas production at low temperatures can be improved by targeted inoculums of specific microorganisms.Results:(1)Performance of Systems A,B and C was analysed with regard to the following parameters.The fermentation was studied for 120 d(System A)or 160 d(Systems B and C).The total biogas output was measured as 68,650 m L for System A,61,750 m L for system B and 19,150 m L for System C.Methane(CH4)production was measured as 31,191 m L,20,906 m L and 3,031 m L,respectively.The total biogas output of System A was 1.1 times that of System B and 3.6 times that of System C,while the CH4 output of System A was 1.5 times higher than that of System B and 10.3 times that of System C.(2)The abundance of microbial communities was determined based on bacterial and archaeal OTU averages.For System A there were 1,215 bacterial and 787 archaeal OTUs detected.For System B these numbers were 968 and 758,and for System C 732 and 720,respectively.During the fermentation,the bacterial and archaeal OTU averages of System A were thus 1.3 and 1.0 times those of System B and 1.7 and 1.1 times those of System C,respectively.(3)The community structure of microorganisms was analysed for dominant phyla and genera.In all three systems,the dominant bacterial phyla were determined as Firmicutes,Bacteroidetes,Proteobacteria and Synergistetes,and archaeal were dominant for Euryarchaeota,MCG,Thaumarchaeota and Woesearchaeota.At the genus level,dominant hydrolytic bacterial genera were Clostridium,Streptococcus and Vibrio.Dominant fermentative acid-producing bacteria were determined as Terrisporobacter,Turicibacter and members of the vadin BC27_wastewater-sludge_group.Moreover,dominant hydrogen-producing acetogens were identified such as Syntrophomonas.Finally,dominant methanogens such as Methanobacterium and Methanocorpusculum were detected.The most abundant bacteria were Pseudomonas,Clostridium,and Terrisporobacter while the dominant archaea were Methanosphaera for System C.(4)The dynamics of the communities were also studied.In System A,the relative abundance of Clostridium peaked at 60 d and again at 90~100d.The relative abundance of Streptococcus peaked at day 20 and Vibrio was highest after 10 d of fermentation.Turicibacter primarily peaked at days 20 and 40.Methanobacterium peaked at 20 d,50 d and 70 d.In System B,the relative abundance of Clostridium peaked after 100 d of fermentation and a second peak appeared at 140~150d.Streptococcus peaked relatively early,<30 d of fermentation.The relative abundance of Terrisporobacter first peaked <60 d,while it reached maximum levels at 110 d and 140 d.The relative abundance of Methanocorpusculum peaked three times,after 30 d,50 d and 150 d since fermentation,respectively.Conclusions:(1)Biogas production performed at an external temperature of 9 ℃ was highest using an inoculum prepared at 15 ℃(System A).Fermentation with that inoculum was significantly better than results obtained with inoculums prepared at 9 ℃ or 4 ℃.Although all three systems tested here produced biogas,only optimal results were obtained with the proper acclimatized inoculum.(2)In the biogas system performing at low temperatures,the dominant hydrolytic bacteria included cellulolytic or hemi-cellulolytic,proteolytic and lipolytic bacteria.The dominant fermentative acid-producing bacteria were mostly responsible for degradation of monosaccharides and amino acids.Hydrogenotrophic methanogens were the dominant methanogens.In combination,these produced a steady fermentation food chain,ultimately reducing carbon dioxide to generate CH4.Nevertheless,a low temperature can negatively affect this process,so that hydrolysis and fermentation,hydrogenesis and acetogenesis,and methanogenesis are severely inhibited but denitrifying bacteria thrive.When these become the dominant bacterial population,the undesired production of inert N2 will increase.(3)In the test systems applied here,the water-insoluble macromolecular organic matrices cellulose,hemicellulose,protein and fat were sequentially hydrolysed in the order of fat,then protein,then hemicellulose,then cellulose.Thus,the hydrolysis of fat and protein occurred mainly at initial fermentation stages.This was followed by hydrolysis of hemicellulose during the intermediate fermentation stage,and at a final stage hydrolysis of cellulose mainly occurred.Moreover,metabolism of monosaccharides by fermentative acid-producing bacteria primarily peaked at the initial and intermediate fermentation stages;in addition,hydrogenotrophic methanogens were mostly active primarily at the initial and intermediate fermentation stages.(4)This study has shown that acclimatized inoculums may be used to improve the efficiency of biogas fermentation at low temperatures,so that the process becomes cold-tolerant.The optimal temperature for such acclimatization is 15 ℃.This provides a practical and effective method to improve the efficiency of a biogas system performing at low temperatures.It is particularly suitable to improve the efficiency of biogas systems located at high latitude and plateau regions.