【作者】 王晓丽；

【导师】 修志龙；

【作者基本信息】 大连理工大学 ， 生物化工， 2024， 博士

【摘要】 1,3-丙二醇是一种重要的平台化合物,广泛应用于纺织、化妆品和制药等领域,其最大的市场潜力是作为新型材料聚对苯二甲酸丙二醇酯(PTT)的合成单体。微生物转化粗甘油生产1,3-丙二醇因原料可再生、环境友好、反应条件温和等优势越来越受到产业界关注。目前1,3-丙二醇的主流发酵工艺为批式流加发酵,自动化水平有待提高,发酵成本亦需进一步降低,这样才能加快生物基1,3-丙二醇的工业化进程。为此,本研究筛选了一株高产1,3-丙二醇的丁酸梭菌(Clostridium butyricum),重点开发了适用于工业应用的多种新发酵工艺,并研究了有机氮源中调控1,3-丙二醇生产的关键维生素,并利用多组学阐释其调控机制。主要研究内容和结果如下:(1)建立了序列接种自动流加底物的批式流加发酵新工艺。筛选出一株高产1,3-丙二醇的C.butyricum DL07,批式流加发酵1,3-丙二醇的浓度最高可达104.78 g/L。建立了序列接种发酵新工艺,采用上一批次发酵的指数生长期(10 h)的发酵液作为种子,接种至下一批次发酵,实现循环接种,省略了工业上种子逐级放大的过程。该工艺可以稳定运行至少8个批次,1,3-丙二醇生产水平稳定,平均1,3-丙二醇浓度和转化率分别为84.62g/L和0.521 g/g甘油,该工艺的整体1,3-丙二醇生产强度达6.77g/(L·h)。此外,确定了甘油消耗与碱液消耗的线性关系,开发了甘油自动流加控制系统,实现了 1,3-丙二醇的自动化半连续生产。(2)开展了多级连续发酵动力学及过程优化研究。在三级连续发酵中,第一、二、三级发酵分别负责培养种子、积累1,3-丙二醇、降低残余甘油。在不同的初始甘油浓度和补料速率下,第三级发酵的最高1,3-丙二醇浓度为80.05 g/L。基于Monod方程和Pirt模型建立细胞生长、底物消耗和产物形成动力学。进而利用该模型以1,3-丙二醇生产强度为优化目标,获得第一级发酵条件为初始甘油浓度和稀释速率分别为92 g/L和0.341 h-1,此时1,3-丙二醇的生产强度达到最大11.26 g/(L·h)。进一步通过实验验证了该优化结果的可靠性。最后,基于该模型优化了二级连续发酵,预测两发酵罐体积比V2/V1>11.9,1,3-丙二醇浓度可超过80 g/L。(3)开发了原位固定CO2联产1,3-丙二醇、微米碳酸钙和生物氢气的新工艺。利用Ca(OH)2作为发酵pH调节剂兼CO2捕获剂,批式流加发酵进行16 h,1,3-丙二醇浓度为88.60g/L,生产强度高达5.54g/(L·h)。发酵尾气中的CO2低至1.8%(v/v),H2/CO2的比值是NaOH组的149倍。发酵液中可溶性盐和蛋白浓度分别降低了53.6%和44.1%,有助于1,3-丙二醇分离。同时,发酵液中产生了300 nm～20 μm的CaCO3方解石颗粒。该工艺在确保高1,3-丙二醇浓度的前体下,将生产强度提高了95.1%,实现了原位CO2固定,同时获得了高纯度的生物氢气和微米CaCO3。(4)筛选和优化了有机氮源中的关键元素。通过考察不同的有机氮源对1,3-丙二醇产出的影响,发现玉米浆干粉可以代替酵母浸粉。进而利用PB、单因素及CCD实验筛选并优化有机氮源中调控1,3-丙二醇生产的两种关键维生素——生物素和硫胺素,其中生物素是该菌的必需维生素,硫胺素则显著影响1,3-丙二醇浓度,优化的生物素和硫胺素添加量分别为32 μg/L和0.28 mg/L。在优化条件下,1,3-丙二醇浓度高达90.84 g/L。两种维生素的原料成本较低,将发酵辅料有机氮源的成本降低了 99.8%。(5)运用多组学分析探究了生物素和硫胺素的调控机制。硫胺素焦磷酸(TPP)作为硫胺素在生物体内的活性物质,是丙酮酸铁氧还蛋白氧化还原酶(PFOR)的辅因子。蛋白质组学分析表明,添加硫胺素时,甘油到乙酰-CoA代谢途径涉及的所有蛋白表达量均升高,包括PFOR,而乳酸脱氢酶相关基因和蛋白表达量显著降低,此结果支持硫胺素主要影响PFOR表达,调控丙酮酸到乙酰-CoA途径。若硫胺素不足,丙酮酸代谢为乳酸的途径将加强。此外,基因组和转录组分析表明该菌没有完整的生物素合成途径,缺少bioI、bioW和bioF基因,不能合成生物素,但其具备生物素转运蛋白相关基因(BioY、EcfT、EcfA1、EcfA2),可从环境中摄取生物素。尽管该菌含完整的硫胺素合成途径,但thiH、thiG和tenI基因转录水平非常低,相应蛋白未被检出,提示三个基因的低表达可能是该菌硫胺素合成不足的原因。综上,本文开发了适用于工业生产1,3-丙二醇的新发酵技术,并显著降低了 1,3-丙二醇生产的辅料成本,阐释了生物素和硫胺素调控甘油代谢的机制,为推动1,3-丙二醇生产向连续化、自动化、绿色化、低成本发展提供技术支撑。更多还原

【Abstract】 1,3-Propanediol(1,3-PDO)is an important platform chemical with wide application in the fields of textile,cosmetic,and pharmaceutical.Importantly,it is a monomer for the synthesis of a novel polymer,polytrimethylene terephthalate(PTT),which has a large market demand.Bioconversion of crude glycerol to 1,3-PDO has attracted great attention from industry due to renewable substrate,environmentally friendly process,mild reaction conditions.At present,fed-batch fermentation is the most popular process for 1,3-PDO production.Realizing automatic operation and further reducing the fermentation cost could accelerate the industrialization process of bio-based 1,3-PDO production.In this study,a strain,Clostridium butyricum DL07,was screened for producing high concentration of 1,3-PDO.Then various new fermentation processes suitable for industrial application were developed.Some key vitamins included in organic nitrogen sources were found to significantly regulate 1,3-PDO production and their regulatory mechanisms were studied based on multi-omics data.The main results were obtained as follows:(1)Sequential fed-batch fermentation with an automatic substrate feeding system.A strain named C.butyricum DL07 was obtained for high-level titer of 1,3-PDO.The maximum 1,3PDO concentration of 104.78 g/L was achieved by fed-batch fermentation.A novel sequential fed-batch fermentation was developed,in which the next fermenter was inoculated with cells growing at exponential phase(10 h)in the prior fermenter.In this way,fermentations were performed with repeated inoculation without seed amplification.The fermentation could steadily run at least 8 batches with the stable 1,3-PDO production.The average concentration of 1,3-PDO was 84.62 g/L with the average yield of 0.521 g/g glycerol,and the overall productivity reached 6.77 g/(L·h).Additionally,the glycerol consumption had a good linear relationship with alkali consumption.Based on this formula,an automatic glycerol feeding system was developed to achieve semi-continuous and automatic production of 1,3-PDO.(2)Kinetics and process optimization of multi-stage continuous fermentation.In threestage continuous fermentation,the first,second,and third stages of fermentation were responsible for seed culture,1,3-PDO accumulation,and the consumption of residual glycerol,respectively.Under different initial glycerol concentrations and feeding rates,the highest 1,3PDO concentration of 80.05 g/L was obtained in the third stage fermentation.Based on Monod and Pirt models,kinetic models were established to describe the relationships among the product production,substrate consumption,and cell growth.This model was then used to optimize the fermentation condition for the highest 1,3-PDO productivity as feeding glycerol and dilution rate of 92 g/L and 0.341 h-1,respectively.The predicted highest 1,3-PDO was 11.26 g/(L·h).The reliability of the predicted results was experimentally verified.Finally,the kinetics-based two-stage continuous fermentation was optimized to achieve a target 1,3-PDO of 80 g/L.The optimized minimum volume ratio of two fermenters(V2/V1)was 11.9.(3)In situ carbon dioxide capture to co-produce 1,3-PDO,micro-calcium carbonate and biohydrogen.Based on Ca(OH)2 as the pH regulator and CO2 capture agent,the maximum concentration of 1,3-PDO reached 88.60 g/L with an productivity of 5.54 g/(L·h)in fed-batch fermentation.Furthermore,the CO2 proportion in exhaust gas was only 1.8%(v/v).Compared with NaOH group,the ratio of H2 to CO2 in exhaust gas showed a remarkable 149-fold increase in the Ca(OH)2 group.Soluble salts and proteins of broths decreased by 53.6%and 44.1%,respectively,which would contribute to the separation of 1,3-PDO.Meanwhile,calcite CaCO3 with sizes in the range of 300 nm to 20 μm were formed in the fermentation.1,3-PDO productivity increased by 95.1%with high 1,3-PDO concentration,and achieved in situ CO2 fixation to produce micro-calcium carbonate and collect high-purity biohydrogen.(4)Screening and optimization of key elements from organic nitrogen sources.Firstly,1,3-PDO production levels were estimated with different organic nitrogen sources.Dried corn steep liquor could replace yeast extract for 1,3-PDO production.Two vitamins,biotin and thiamine,were identified to significantly regulate 1,3-PDO production by PB design,singlefactor,and CCD experiments.Biotin is an essential vitamin for the strain,and thiamine significantly affects 1,3-PDO concentration.The optimized addition amounts of biotin and thiamine were 32 μg/L and 0.28 mg/L,respectively,resulting in a 1,3-PDO concentration as high as 90.84 g/L.This fermentation condition greatly reduces the raw materials cost of 1,3PDO production.The raw material cost of the two vitamins is lower,reducing the raw cost of organic nitrogen by 99.8%.(5)The regulatory mechanisms of biotin and thiamine were proposed based on multiomics analysis.Thiamine pyrophosphate(TPP),the active form of thiamine,is a co-factor of pyruvate:ferredoxin oxidoreductase(PFOR).Proteomic analysis showed that when thiamine was added into fermentation,the expression levels of all proteins involved in metabolic pathway form glycerol to acetyl-CoA,including PFOR,increased,while the expression levels of genes and proteins related lactate dehydrogenase decreased significantly.The results support that thiamine mainly affect PFOR expression,regulating the pathway from pyruvate to acetyl-CoA.If thiamine is insufficient in fermentation,the pathway involved in pyruvate to lactate would be strengthened.In addition,according to genomic and transcriptomics data,C.butyricum DL07 has incomplete biotin synthesis pathway due to the absence of bioI,bio W,and bioF genes,which leads to the failure to synthesize biotin.Fortunately,it has biotin transporter proteins encoded by BioY、EcfT、EcfA1、EcfA2 genes,which can help cells uptake biotin from the environment.Although a complete thiamine synthesis pathway was found in the strain,thiH,thiG,and tenI genes have very low expression levels and the corresponding proteins were not detected.These results suggest that low expression levels of these three genes are the possible reason for insufficient thiamine synthesis in C.butyricum DL07.In summary,this study had developed some novel fermentation processes,which have potential for industrial production of 1,3-PDO.The raw material cost of 1,3-PDO production was reduced significantly.Moreover,the regulatory mechanisms of biotin and thiamine on glycerol metabolism were explored.These processes promote 1,3-PDO production towards continuous,automatic,green,and low-cost operation,and has great reference value for industrial application.更多还原