Multiple Taxonomy Of A Noval Homoacetogen Blautia Coccoides Strain GA-1 And Its Metabolic Mechanism In Mixotrophism

Homoacetogens are autotrophic anaerobes with CO₂ as sole carbon source for acetate production and biomass formation.These anaerobes can also utilize organic compounds heterotrophically to produce acetate and form biomass.The unique performance in nutrition and metabolism shows them a broad application prospects in resource utilization of waste biomass,organic wastewater,and capture of CO₂ released by burning of fossil fuels.The strain resources and application technologies can also find a great significance in acetic acid chemical industry with a sustainable raw material supply.However,research on the application technology of homoacetogens is only in its infancy with limited results,especially the scarcity of strain resources and the metabolic mechanism that is not well understood by now.It is essential to obtain more novel homoacetogens which have extensive applied prospect.A good deal of insight into the performances of the strains in physiological ecology,and growth and metabolism will be helpful to find and evaluate their potential industrial applications,and lay a sound theoretical basis and provide guidance to the development of practical technologies.To develop strain resource of homoacetogens,three main types of activated sludge were separately collected and enriched with H₂/CO₂?v:v,4:1?as sole carbon source and 2-bromoethanesulfonate as methanogens inhibitor.The three types of activated sludge were sampled from a continuously stirred tank reactor?CSTR?,and the acidogenic and methanogenic phase of a four-compartment anaerobic baffled reactor,respectively.Both reactors were performed for treating high density organic wastewater.Enriched for 240 h,three mixed bacteria communities rich in homoacetogens were obtained from the sampled sludges.The maximum homoacetogenic metabolic capacity came to the enrichment culture from the activated sludge sampled from the CSTR,with a specific H₂/CO₂ autotrophic assimilation of 54.79 mmol/gMLVSS.The high throughput sequencing of bacterial 16 S rRNA revealed that Acetobacterium and Blautia were the dominant bacteria in the enrichment culture with a relative abundance of 23.1% and 6.1%,respectively.Most strains in the two genera can produce acetate by assimilating H₂/CO₂.A few homoacetogen strains were isolated from the enrichment culture of the activated sludge sampled from the CSTR,among which a pure culture with a better homoacetogenic metabolic capacity was identified.The identified bacterium is ellipsoidal or ovoid in shape with the diameter of 0.2⁰.5 ?m,having a polysaccharide capsule,non-spore,G+.Sixteen and fourteen carbon saturated fatty acids and G+C in the cell is about 42.66%,21.20% and 48.40%,respectively.The strain is autotrophy or heterotrophy with H₂/CO₂ or monosaccharoses as the sole carbon source,respectively,with acetic acid as the main product.The bacterium was identified as a novel strain of Blautia coccoides by multiple taxonomy and then named B.coccoides GA-1.With H₂/CO₂?v:v,4:1?as the sole carbon source,the specific acetic acid production of strain GA-1 reached 5.32 g/g-dry cells.It was found that the strain's homoacetogenesis would be observably inhibited by heterotrophic metabolism under mixed nutritional condition,resulting in a remarkable decrease in autotrophic assimilation of H₂/CO₂.The following three potential mechanism were involved in the inhibition of heterotrophic metabolism to homoacetogenesis: 1)competition for CoA between the glycolytic and the Wood-Ljungdahl pathway;2)competition for ATP between cell synthesis and CO₂ assimilation;3)intracellular NADH/NAD+ balance being affected by the low pH due to the accumulation of acetic acid produced by heterotrophic metabolism.Both autotrophic and heterotrophic metabolism could be kept very well in B.coccoides GA-1 under the following condition: initial pH about 7.5,35³7?,salinity less than 2.5 g/L,yeast powder 1 g/L and dicarbonate 1 g/L.With H₂ /CO₂?v:v,4:1?as the sole carbon source,strain GA-1 grew weakly and the autotrophic metabolism would gradually decline with subcultures.Supplement of glucose was found to enhance the growth of strain GA-1,but the strain's autotrophic metabolism would be remarkably inhibited by a high concentration of glucose.Subculturing in 200 mg/L glucose medium with H₂/CO₂ as gas phase,strain GA-1 could grow well and a specific autotrophic acetic acid production of 2.16 g/g-dry cells was maintained in the subcultures.The heterotrophic process of B.coccoides GA-1 could be well simulated by Gompertz model or Logistic model,but Logistic model was better to simulate the autotrophic process of the strain.The results of model analysis showed that the maximum growth rate,substrate degradation rate and acetic acid production rate of the strain in heterotrophic process reached 93.24,370.26 and 79.11 mg/?L·h?,respectively,at 37? and p H 7.3,with 2000 mg/L glucose and 10%?v:v?inoculum in the medium.Meanwhile,the maximum growth rate,H₂/CO₂ assimilation rate and acetic acid production rate in autotrophic process with H₂/CO₂ as the sole carbon source averaged 4.97 mg/?L·h?,0.05 mmol/?L·h?and 2.87 mg/?L·h?,respectively.Test and analysis on feasible application indicated that B.coccoides GA-1 is a potential bacterial resource with a wide variety of application.Its potential in acetic acid production from lignocellulosic biomass,organic wastewater and CO₂ released by burning of fossil fuels were evaluated,and then calculated the prospect of the acetic acid production processes in the future.The present research would give valuable reference and ideas for the subsequent development of homoacetogen resources.