Enrichment Of Mixed Communities Of Methanotrophs And Regulation Strategy For Synthesis Of Polyhydroxyalkanoate

Poly-?-hydroxybutyrate(PHB)has good biodegradability,biocompatibility,and similar mechanical properties to conventional petroleum-based plastics,an ideal substitute for conventional-based plastics.Methane-to-PHB production not only produces biodegradable polymers but also reduces production costs and greenhouse gas emissions.Most researches on the synthesis of PHB by methanotrophic bacteria use pure bacteria,which requires a strict aseptic operation.Few studies on the stable synthesis of PHB by mixed culture and non-sterile operations can reduce the cost of PHB synthesis.This article explored the enrichment of the mixed community and the regulation strategy of PHB synthesis.Methane-oxidizing communities were selectively enriched and acclimated from activated sludge under different conditions.The community structures were analyzed by high-throughput sequencing and combined with the ability of PHB synthesis to select an efficient PHB-producing mixed community and optimize the conditions of PHB synthesis.The results showed that with N₂ as a nitrogen source when the methane-oxygen ratio was 1:1,3:2,and 2:3,PHB accumulation capacity of communities was similar(?20%),and their dominant methane-oxidizing bacteria were Methylococcus,Methyloparacoccus,and Methylophilus,respectively.The PHB synthesis ability of mixed bacteria acclimated with N₂ as the sole nitrogen source was higher than mixed bacteria acclimated with NO₃^-and NH₄⁺as a nitrogen source.A highly efficient methane-oxidizing community was enriched under the condition of N₂ as a nitrogen source and the ratio of methane to oxygen at 2:3.The microbial communities under different conditions are mainly composed of heterotrophs;other dominant heterotrophic bacteria in the mixed communities may use by-products of methanotrophs as a usable carbon source to grow and produce PHB.In the process of PHB synthesis by the mixed community,the optimum methane-oxygen ratio is 2:1,Cu²⁺concentration is 20?mol/L,the temperature is 30?,p H is 7.Salinity could inhibit the PHB accumulation capacity of communities and the enzyme activity of cells.The PHB accumulation capacity of communities was improved when communities were cultivating by nitrate,and the PHB accumulation capacity was slightly inhibited when methane-oxidizing communities were growing by NH₄⁺.Long-term cyclic NO₃^--N₂ cultivation regime was improved the ability of PHB synthesis and the ability of PHB synthesis in long-term cyclic NH₄⁺-N₂ cultivation regime was similar to community in continuous N₂ cultivation.The enhancement experiment of PHB synthesis of mixed community was carried out by adding co-substrate and methanol,and the effects of feast-famine ratio,co-substrate and nitrate on PHB synthesis of the methane-oxidizing community during continuous operation were studied.The results showed that the optimal co-substrates for enhancing PHB synthesis were 0.2 mmol/L sodium citrate and 0.1 mmol/L acetic acids;when sodium citrate,propionic acid,sodium lactate were fed,the methanotrophs communities produced 3-HV and 3-HB copolymers;the optimal methanol concentration was 0.2%(v/v).In continuous operation,the optimal feast-famine ratio for PHB synthesis is 1:3,and 0.1 mmol/L acetic acid had a positive effect on the enhancement experiment of PHB synthesis.The PHB synthesis ability of methane-oxidizing communities briefly cultivated by nitrate was still stable.The PHB synthesis capacity of the community cultured with NO₃^-is slightly higher than community cultured continuously with nitrogen.