Polyhydroxyalkanoates Production From Methane By Methanotrophs And Mixed Communities

Polyhydroxyalkanoates（PHA）is a thermoplastic linear polymers synthesised by various bacteria.PHA is a potential substitute for tradition plastics with the advantage of biodegradability,biocompatibility and mechanical properties similar to petrochemical plastic.It is very important to solve the environmental problems caused by petrochemical plastics.Poly-β-hydroxybutyrate（PHB）is the most abundant PHA homopolymer.However,the expansion of PHA utilization has been restrained due to their high production cost,which is mainly attributed to expensive carbon sources.The development of inexpensive feedstock is one of the main ways to reduce PHA production cost.Methane is the second most abundant greenhouse gas with rich sources.The utilization of methane to produce PHA not only cuts the emissions of greenhouse gases but also greatly reduces PHA production cost.Moreover,methane could be regenerated after PHA-based products being discared and degreaded in anaerobic systems.PHA production cost can be further reduced with mixed cultures as microorganism.Nevertheless,the production of PHA from methane is mainly conducted with pure bacteria and the selection pressure to enrich methane-oxidizing communities with excellent PHA production performance remains unclear.What’s more,factors affecting PHA production by methanotrophs also requires detailed research.Therefore,in this dissertation,the influencing factors and metabolic mechanism of PHB production by methanotrophs were explored with Methylosinus trichosporium OB3b as microorganism.Afterwards,methane-oxidizing communities were enriched from activated sludge under different seletion pressures.The relationship between PHB synthesis capacity and microbial community structure was analyzed.Meanwhile,the effects of cosubstrtate and nitrogen source on the PHA production of communities were studied.The main content and results are as follows:（1）Effects of nitrogen source（NH₄⁺,NO₃^-and N₂）on the growth and PHB accumulation capacity of methanotrphs were explored and the expression of methane monooxygenase（MMO）in different forms was controlled by varying Cu²⁺ concentration.The results showed that the response of methanotrphs to nitrogen source was regulated by MMO.Ammonia-supplied methanotroph grew at higher rate（0.64 day-1）with the expression of soluble MMO（sMMO）,while its growth was inhibited by hydroxylamine and nitrite with the expression of particulate MMO（pMMO）.But ammonia-supplied methanotroph containing pMMO accumulated more PHB（45.2%）.Nitrate-supplied methanotrophs had higher growth rate（0.61 day-1）and PHB content（51.0%）with the expression of pMMO.The growth rate of N₂-fixing bacteria was low（0.22-0.23 day-1）.PHB contents of bacteria expressing sMMO showed no significant change under different nitrogen source conditions.But in the presence of pMMO,the PHB content of methanotroph was greatly decreased after continuously cultivated by N₂（32.1%）.The growth rate and PHB content of methanotrophs continuously cultivated by N₂ with the expression of pMMO were greatly increased by 50%and 26.2%in the cyclic NO₃^--N₂ cultivation regime,indicating that long-term N₂-fixing cultivation was detrimental to the activity of methanotroph expressing pMMO.（2）The effects of methane,oxygen and common biogas sulfur compounds on the PHB production of nitrate-supplied methanotroph were investigated with the expression of pMMO.The results showed that the optimal ratio of methane to oxygen in growth phase and PHB accumulation phase was 1:1 and 2:1 respectively.With fixed methane concentration,the effects of oxygen on the PHB accumulation in the absence of N₂ was different from that in the presence of N₂.In the absence of N₂,PHB accumulation by methanotroph could tolerate high oxygen concentration.In the presence of N₂,with headspace gas replenished only when oxygen was almost depleted,the increase and decrease cycle of PHB content was observed.In the regime of updating headspace gas at the point when the PHB content began to decrease,higher PHB content（55.5%）and higher PHB productivity（12.5 mg/L/h）was obtained at 0.2 atm O2,while PHB accumulation was depressed with oxygen concentration greater than 0.3 atm.PHB production capacity of methanotroph was improved by the addition of H2S,but dimethyl sulfide has a significant inhibitory effect on the activity of methanotroph.（3）Methane-oxidizing communities were selectively enriched from sewage sludge to obtain a mixed culture with high levels of PHB accumulation capacity.Methane and N₂ was used as sole carbon source and sole nitrogen source,while oxygen and Cu²⁺ concentration was varied.The results showed that the PHB accumulation capacity of communities was mainly regulated by Cu²⁺ concentration.All communities enriched with Cu²⁺ could accumulate high content of PHB（43.2-45.9%）,while only small amounts of PHB were accumulated by cultures enriched without Cu²⁺（11.9-17,5%）.Batch assays revealed that communities grown with Cu²⁺ and 0.2 atm O2 synthesized more PHB,which had a wider optimal CH₄:O2 range and produced a high PHB content（48.7%）even though in the presence of N₂.Although methane was added as the sole carbon source,heterotrophs dominated with abundances between 77.2%and 85.6%.All methanotrophs detected belonged to type Ⅱgenera and mainly consisted of Methylocella and Methylosinus.（4）Effects of cosubstrate on the PHA production were investigated in methane-oxidizing communities enriched with Cu²⁺ at 0.2 atm O2.The optimal cosubstrate was propionate and malate and the optimal concentration was 0.4 mmol/L and 0.2 mmol/L respectively,which could increase the PHA content to 65.7%and 72.8%.Structural analysis showed that only when fed propionate as cosubstrate,copolymer was accumulated containing 3-hydroxybutyrate（3-HB）and 3-hydroxyvalerate（3-HV）.In order to improve the growth rate and PHA production capacity of communities continuously cultivated by N₂,the communities was briefly cultivated by nitrate.The results showed that the optimal inoculum quantity was 20%and the biomass concentration and PHA content of communities enriched with Cu²⁺ at 0.2 atm O2 were increased by 68.5%and 11%.Correspondingly,the PHA productivity was increased by 2 times.In long-term cyclic NAO₃^--N₂ cultivation regime,commuties enriched with Cu²⁺ at 0.2 atm O2 could also maintain excellent PHA production capacity.