【作者】 张婷婷；

【导师】 周集体；

【作者基本信息】 大连理工大学 ， 环境工程， 2017， 博士

【摘要】 聚羟基烷酸酯（PHA）是一类由微生物合成的热塑性线性高分子聚合物,因其具有良好生物可降解性、生物相容性和与石化塑料相似的机械性能,是一种非常具有前景的石化塑料的替代品,对于解决石化塑料引起的环境问题有很重要的意义,其中,聚-β-羟基丁酸酯（PHB）是存在最广泛的一种PHA均聚物。然而,PHA的生产成本较高,阻碍了 PHA的广泛应用。PHA的生产成本主要来自底物成本,廉价底物的开发是降低PHA生产成本的重要途径之一。甲烷是全球第二大温室气体,具有丰富的来源,若以甲烷为底物合成PHA,不仅能实现PHA生产成本的降低,同时能够减少温室气体的排放,而且废弃的PHA产品在厌氧条件下可以被微生物分解转化为甲烷,重新进入PHA合成过程。目前,利用甲烷合成PHA的研究多以纯菌为菌种,以混合菌群代替纯菌可进一步降低PHA生产成本,但对于非无菌条件下具有稳定的高PHA合成能力的甲烷氧化混合菌群的驯化条件尚不清楚,而且甲烷氧化菌PHA合成特性的调控因素也尚未探明。针对这些问题,本论文,首先以MethylosinustrichosporiumOB3b为菌种,探究了甲烷氧化菌PHB合成特性的调控因素和代谢机理,然后以活性污泥为菌种,在不同环境压力下,进行甲烷氧化混合菌群的驯化,解析了混合菌群PHB合成能力与微生物群落结构的关系,探索了共底物和氮源调控混合菌群PHA合成能力的方法。主要研究内容和结果如下:（1）分别以NH₄⁺、NO₃^-和N₂为氮源,通过改变Cu²⁺浓度控制不同形式甲烷单加氧酶（MMO）的表达,探究了氮源对表达不同MMO甲烷氧化菌的生长和PHB合成特性的影响。结果显示,甲烷氧化菌对氮源的响应受MMO的调控。以NH₄⁺为氮源的条件下,溶解性MMO（sMMO）表达时,甲烷氧化菌的生长速率较高（0.64day-1）,而颗粒性MMO（pMMO）表达时,甲烷氧化菌的生长受羟氨和亚硝酸盐抑制,但具有较高的PHB含量（45.2%）。以N03-为氮源时,表达pMMO的甲烷氧化菌具有较高的生长速率（0.61 day-1）和PHB含量（51.0%）。以N₂为氮源的条件下,甲烷氧化菌的生长速率较低（0.22-0.23day-1）,sMMO表达时的PHB含量,与以NH₄⁺和NO₃^-为氮源时相比,无明显变化,但pMMO表达时的PHB含量明显下降,仅为32.1%。构建NO₃^--N₂循环培养模式,与持续N₂培养相比,固氮生长表达pMMO的甲烷氧化菌的生长速率和PHB含量分别提高了 50%和26.2%,证明长期的N₂培养会抑制表达pMMO甲烷氧化菌的活性。（2）探究了 CH₄和O₂及生物气常见含硫化合物对N03-培养表达pMMO的甲烷氧化菌PHB合成特性的影响,发现生长段和PHB合成段最适甲烷氧气比分别为1:1和2:1。在甲烷浓度固定的条件下,不存在N₂时氧气浓度对甲烷氧化菌PHB合成的影响规律与存在N₂时不同。不存在N₂时,甲烷氧化菌的PHB合成对氧气具有较高的耐受能力。而存在N₂时,若仅在氧气耗尽时更换顶空气体,PHB含量先上升后下降,若在PHB开始降解时更换顶空气体,PHB含量和PHB生产效率在0.2 atm O2时较高,分别为55.5%、和12.5 mg/L/h,但氧气浓度高于0.3 atm时,PHB合成受到抑制。研究表明生物气中的H2S可以促进甲烷氧化菌的PHB合成,但二甲基硫醚对甲烷氧化菌的生长和PHB合成有明显的抑制作用。（3）以甲烷和氮气为唯一碳源和氮源,在不同氧气和Cu²⁺浓度条件下,从剩余活性污泥中富集驯化具有高PHB合成能力的混合菌群,结果显示甲烷氧化混合菌群的PHB合成特性主要受Cu²⁺浓度的调控。含Cu²⁺培养基中驯化的混合菌群具有较高的PHB合成能力,PHB含量为43.2-45.9%,而不含Cu²⁺培养基中驯化的混合菌群的PHB含量较低,仅为11.9-17.5%。进一步实验结果表明,含Cu²⁺培养基中0.2 atm O2条件下驯化的混合菌群具有最佳的PHB合成能力,其不仅具有较宽的最适甲烷氧气比范围,而且存在N₂时,PHB含量可增至48.7%。虽然甲烷为唯一的底物,但所有混合菌群都主要由异养菌构成,异养菌丰度为77.2-85.6%,混合菌群中的甲烷氧化菌由四种II型菌构成,主要为 Methylocella 和 Methylosinus。（4）考察共底物对含Cu²⁺培养基中0.2 atm O₂条件下驯化的混合菌群PHA合成的影响,发现最适共底物为丙酸和苹果酸,其最适浓度分别为0.4mmol/L和0.2mmol/L,可使PHA含量分别提高至65.7%和72.8%,而且通过结构分析发现,以丙酸为共底物时,PHA为3-羟基丁酸（3-HB）和3-羟基戊酸（3-HV）的共聚物,而添加其它共底物时,仅有PHB生成。为提高持续固氮生长的混合菌群的生长速率和PHA合成能力,对混合菌群进行短暂NO₃^-培养,结果表明接种量为20%时,含Cu²⁺培养基中0.2atmO2条件下驯化的混合菌群的PHA合成能力最佳,其生物量浓度和PHA含量分别提高了 68.5%和11%,相应的PHA生产效率提高了约2倍,而且在长期NO₃^--N₂循环培养模式中,其依然可以保持稳定的高PHA合成能力,PHA含量与持续氮气培养相当。更多还原

【Abstract】 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.更多还原