3-羟丙醛对Klebsiella pneumoniae发酵产 1,3-丙二醇的影响及其调控

中间产物3-羟丙醛在发酵液中的积累对Klebsiella pneumoniae细胞生长及1,3-丙二醇的合成有显著的抑制作用,而调节发酵的起始甘油浓度及控制发酵pH值可调控发酵液中3-羟丙醛的积累.当起始甘油质量浓度分别为20、30、50、70g/L的批式发酵中,发酵液中3-羟丙醛的积累的高峰分别为4.31、6.87、11.48及13.49mmol/L,当起始甘油质量浓度大于50g/L时,3-羟丙醛在到达积累高峰后不能被菌体有效转化,在发酵后期维持较高浓度,抑制了细胞生长及1,3-丙二醇的合成,发酵不能继续进行.控制发酵pH值为7.75～8.0可促进发酵液堆积的3-羟丙醛被迅速转化.在流加发酵中起始甘油质量浓度采用30g/L,发酵pH值控制为7.75条件下,发酵32 h,1,3-丙二醇质量浓度可达37.16g/L,1,3-丙二醇的生产强度和质量得率分别达到1.16g/(L·h)和52.66%.     

有氧条件下Klebsiella pneumoniae发酵生产1,3-丙二醇的研究

探讨了有氧条件下利用Klebsiella pneumoniae发酵生产1,3-丙二醇的可能性,研究了通气量、初始底物浓度、pH和3-羟基丙醛等因素对发酵过程的影响.当空气通量为0.25vvm时,1,3-丙二醇的得率和生产强度与厌氧时相近.有氧条件下,当初始甘油质量浓度大于50g/L时,发酵中后期出现3-羟基丙醛的长期积累,甘油不能消耗完全.控制pH为7.75～8.00可以促进3-羟基丙醛的转化使甘油完全消耗.     

基于生长代谢耦联的1,3-丙二醇发酵过程底物流加控制策略

研究了克雷伯氏杆菌转化甘油生产1,3-丙二醇的底物流加控制策略,其特征是批式流加发酵过程与细胞生长和代谢相耦联. 通过细胞生长动力学分析,建立了对数生长期和稳定期底物消耗与生物量和碱液消耗量之间的函数关系. 2次在线反馈补料发酵实验结果表明,甘油浓度控制在20±2 g/L时,1,3-丙二醇的终浓度分别达80.3和78.8 g/L以上,与手动反馈补料相比,1,3-丙二醇浓度分别提高了25.0和23.5 g/L.     

若干因素对发酵法生产1,3-丙二醇的调控作用

利用一株新筛选的克雷伯氏肺炎杆菌(Klebsiella pneumoniae ZJU 5205)对发酵法生产1,3-丙二醇进行了研究,考察了 pH、溶氧及添加辅助底物葡萄糖对发酵过程的调控作用。实验结果表明,相比于未调控 pH 发酵,恒定 pH 值发酵得到的菌体浓度明显增高,甘油消耗加快,1,3-丙二醇的最终浓度以及甘油到丙二醇的转化率(Y_(P/S))明显提高,分别达16.39g/L和0.51mol/mol;厌氧发酵时的菌体浓度和甘油消耗速率均高于微氧发酵,但1,3-丙二醇最终浓度和转化率低于微氧发酵;添加葡萄糖为辅助底物能够有效促进菌体生长,但1,3-丙二醇最终浓度和转化率却低于以甘油为单一底物时的发酵结果。     

有机酸对产丙二醇菌生长和生产的影响

为提高克雷伯氏肺炎杆菌厌氧发酵生产1,3-丙二醇的能力,在发酵过程中添加有机酸(OA),考察其对菌体生长、1,3-丙二醇及其他副产物合成的影响。实验表明:添加OA可促进菌体生长,加入质量浓度为0.2~0.5g/L的OA,菌体生长的菌液在590 nm处吸光度(OD)值比参照值高出30%;加入过量的OA(>1.0 g/L),则菌体生长受到抑制,OD值比参照值低37%;添加OA可促进单位细胞合成1,3-丙二醇,加入质量浓度为0.2~0.5g/L的OA,单位菌体的1,3-丙二醇质量浓度比参照高3.8%~17.5%;添加OA能提高甘油转化为1,3-丙二醇的转化速率,加入0.2~0.5 g/L的OA,甘油转化为1,3-丙二醇的最大转化速率达0.81 h-1。在15 L罐上批式发酵48 h,1,3-丙二醇最终质量浓度达42.9 g/L,甘油转化为1,3-丙二醇的平均转化率达64%。     

氧对Klebsiella pneumoniae产1,3-丙二醇代谢的影响

考察了氧对Klebsiella pneumoniae发酵产1,3-丙二醇(PDO)代谢的影响. 研究结果表明,在厌氧或供氧条件下,K. pneumoniae都能利用甘油产PDO. 起始甘油浓度20 g/L,发酵时间4 h,在充分供氧条件下,PDO产量仅为1.1 mmol/L;但在微量供氧条件下,PDO产量为16 mmol/L,是厌氧发酵时的1.28倍. 在微量供氧条件下,调控PDO合成的关键酶甘油脱氢酶、甘油脱水酶及1,3-丙二醇氧化还原酶的活性分别为7.28, 1.14, 0.52 U/mg,高于厌氧或充分供氧条件下的相应酶活性. 氧对细胞内NADH的合成也有影响,厌氧及微量供氧条件下菌体内NADH含量分别为3.78及3.72 μmol/g (DCW),高于充分供氧发酵时的0.85 μmol/g (DCW).     

微生物转化甘油生产1,3-丙二醇的研究进展

甘油是油裂解和生物柴油生产过程中的主要副产物,生物柴油市场的蓬勃发展将会造成甘油价格的下降。这些甘油-水混合物不用再次处理就可以直接用于微生物发酵。在众多甘油的利用当中,附加值最高的应该是1,3-丙二醇(PDO),利用甘油在厌氧条件下发酵生产PDO与传统工艺相比产物浓度从70～80g/L提高到了100g/L,而且如果用固定化细胞发酵,产率可以从2g/(L·h)提高到30g/(L·h)。综述了微生物转化甘油为1,3-丙二醇的研究进展。     

葡萄糖作辅助碳源对klebsiella pneumoniae发酵生产1,3-丙二醇的影响

在Klebsiella pneumoniae发酵甘油生产1,3 丙二醇的种子培养及发酵实验中,考察了葡萄糖作辅助碳源对菌体生长及1,3 丙二醇生成的影响. 结果表明,在种子培养期,以葡萄糖和甘油为混合碳源可缩短种子培养周期;在批次发酵和流加发酵中,葡萄糖作辅助碳源可使1,3 丙二醇产率及得率明显提高,但不同的葡萄糖加入方式对产率及得率促进的效果不同. 在初始发酵培养基中添加5 g/L葡萄糖、并在4 40 h进行葡萄糖与甘油混合液连续流加的条件下,1,3 丙二醇浓度、产率及得率较单一甘油为底物的流加发酵结果分别提高41.2 , 38.6 和8.3 .     

蔗糖与葡萄糖作辅助碳源对重组Klebsiella pneumoniae发酵生产 1,3-丙二醇的影响

实验研究了重组Klebsiella pneumoniae批式发酵生产1,3-丙二醇过程中辅助碳源蔗糖与葡萄糖对发酵过程的影响,对发酵工艺进行了放大,并对流加策略进行了优化. 结果表明,葡萄糖为发酵生产1,3-丙二醇的辅助碳源优于蔗糖;以重组Klebsiella pneumoniae为菌种,以葡萄糖为辅助碳源,采用指数流加策略,30 L发酵罐中1,3-丙二醇的产量最高达85.2 g/L,产率达0.63 mol/mol,比单纯以甘油为碳源分别提高37.35%和25.00%.     

甘油发酵生产1,3-丙二醇工艺的技术经济性分析

针对甘油生物转化1,3-丙二醇的连续发酵过程,通过发酵和分离过程的模拟与优化,得到不同初始条件下的最大产物浓度、生产强度、1,3-丙二醇得率及整个工艺过程的物耗和能耗,并进行了经济成本核算. 结果表明,提高发酵过程的生产强度未必能带来利润的增长,反而可能导致利润下降;提高1,3-丙二醇的得率有利于增加利润,但得率高于0.68 mol/mol时,利润增长趋缓;敏感性分析显示,甘油成本是影响利润的关键因素,通过联产生物柴油可以降低成本,税后利润提高31%.     

紫外诱变选育高耐受1,3-丙二醇的克雷伯氏杆菌

1,3-丙二醇具有许多优良的化学性质,可以作为中间体合成多种有经济价值的工业用化合物,是一种潜在的重要化工平台产品。文章以产1,3-丙二醇的肺炎克雷伯氏菌为出发菌株,通过紫外诱变筛选得到耐受1,3-丙二醇130 g/L的突变株。该突变株以甘油为底物,发酵48 h,发酵液中1,3-丙二醇的质量浓度达到96.57 g/L。     

Solvent Extraction of Biologically Derived 1,3-Propanediol with Ethyl Acetate and Ethanol Cosolvent

This study examined the use of ethyl acetate and its mixture with ethanol as cosolvent for the extraction of biologically derived 1,3-propanediol (1,3-PDO) from a fermentional process. Experimental results on extraction of the fermentation model mixture revealed that ethyl acetate was a suitable solvent, having the distribution coefficient of 1,3-PDO of 0.22 at 303.15 K. The temperature (303.15 to 323.15 K) was found not to have a significant effect on the distribution coefficient. On the other hand, the addition of glycerol into the feed aqueous stream (at the concentrations of 4, 8, 12 g/L) was found to increase the distribution coefficient of 1,3-PDOs, however, the compound selectivity decreased. When ethanol was used as a cosolvent at the volume ratio of ethyl acetate to ethanol of 90:10, the distribution coefficient increased from 0.22 to 0.31 at 303.15 K. This decreased the number of theoretical stages (NTS) required to achieve 90% recovery of 1,3-PDO from the aqueous phase from 3 to 2 stages at the solvent to feed (S/F) ratio of 9. In addition, the extraction results with actual fermentation broth at 303.15 K indicated that the use of ethanol cosolvent could improve the distribution coefficient of 1,3 PDO from 0.14 to 0.20.     

外源添加ATP对K.pneumoniae菌体生长和产物合成的影响

基于K.pneumoniae生成1,3 丙二醇过程中甘油脱水酶催化失活与复活的特性,在发酵培养基中添加不同质量浓度的三磷酸腺苷(ATP),考察ATP对菌体生长、1,3 丙二醇(1,3 PD)、其他副产物合成的影响。实验结果表明:添加ATP抑制菌体生长,加入质量浓度为0 4g/LATP,OD值比参照低53%;添加ATP促进单位细胞合成1,3 丙二醇,加入质量浓度为0 4g/LATP,1,3 丙二醇质量浓度/OD值比参照高90%;添加ATP能提高甘油到1,3 丙二醇转化率,加入0 8g/LATP,甘油到1,3 PD的最大转化率达到0 76mol/mol。     

1,3-丙二醇发酵过程中底物抑制及其对策的研究

研究了底物甘油浓度对Klebsiellapneumoniae发酵生产 1 ,3 丙二醇的影响 ,并通过实验确定了最佳的初始甘油浓度和甘油开始对产物生成产生抑制作用的浓度。针对底物抑制现象 ,提出了菌种驯化和流加甘油发酵两种解决途径。结果表明 :采用原始菌株发酵 ,适宜的甘油初始浓度为 642 .4mmol/L ,此时 1 ,3 丙二醇浓度和转化率分别可达 2 60mmol/L和 0 .492mol/mol;在较高甘油初始浓度 (789mmol/L)的情况下 ,经驯化培养获得的耐底物抑制菌株 ,可将最终 1 ,3 丙二醇浓度和转化率分别提高到 30 0 .9mmol/L和 0 .530mol/mol;采用流加甘油发酵工艺 ,1 ,3 丙二醇浓度和转化率分别可提高到 397.7mmol/L和 0 .62 5mol/mol。     

Adsorption of 1,3-propanediol from synthetic mixture using polymeric resin as adsorbents

The aim of this work was to separate 1,3-PDO from a synthetic mixture using polymeric resins, Amberlite XAD-7 and XAD-16 resins. The equilibrium adsorption of 1,3-PDO onto two polymeric resins were investigated in binary and tertiary systems. Experimental results of binary component adsorption equilibrium indicated that the adsorption capacity (q) of 1,3-PDO at 160 g/L onto XAD-7 and XAD-16 was 835.96 and 584.61 mg 1,3- PDO/g dry resin, respectively. The adsorption isotherms were closely predicted by the Langmuir-Freundlich model among the two isotherm model tested. The value of n of 1,3-PDO adsorbed on XAD-7 are much higher than those on XAD-16. This result suggested that XAD-7 resin has a higher affinity for the 1,3-PDO adsorption than XAD-16 resin. Moreover, the value of adsorption capacity of 1,3-PDO in the binary and tertiary component were compared at the same conditions. In the tertiary system, although the selectivity of 1,3-PDO from XAD-7 was approximately six times higher than XAD-16, the adsorption capacity of 1,3-PDO at 160 g/L onto XAD-16 was higher than XAD-7. Interestingly, the reusability of XAD-7 and XAD-16 resins in the three cycle times shows a slight loss of adsorption capacity. Furthermore, the investigation about desorption by an ethanol/water mixture at 50% (V/V) indicated that the desorption yield of 1,3-PDO from XAD-7 was lower than XAD-16 resin for both the binary and tertiary component. This was due to the more favorable adsorption characteristics of XAD-7 resin than XAD-16 resin.     

微生物发酵法中试生产1,3-丙二醇

以甘油为原料,利用克雷伯氏菌进行了发酵罐容积规模分别为1 m3和20 m3的发酵法1,3-丙二醇生产的中试研究。试验结果表明:1m3和20m3的发酵罐最终发酵液中1,3-丙二醇平均质量浓度分别达到了74.6 g/L和63.9 g/L,1,3-丙二醇相对于甘油的得率分别为61.2%和49.9%。采用发酵液醇沉预处理、再精馏的专利技术可分离得到纯度大于99%的1,3-丙二醇产品,分离收率大于85%,产品质量达到了聚对苯二甲酸丙二酯(PTT)聚合反应的要求。成本分析表明,生物法生产1,3-丙二醇技术是经济可行的。     

Adaptive evolution of Clostridium butyricum and scale-Up for high-Concentration 1,3-propanediol production

In order to obtain the excellent 1,3-propanediol (1,3-PDO) producer from wild-type Clostridium butyricum, adaptive evolution was carried out to select the strain for fast growth. The most significant change was that fermentation time decreased from 36 h to 20 h after adaptive evolution. Thus, it led to the corresponding volumetric productivity of 1,3-PDO increasing from 0.97 to 2.14 (g/L·h⁻¹) which increased by 114%. Adaptive evolution was also applied to butyric acid tolerant strain selected based on the fast-growing one through a simple equipment. 1,3-PDO concentration increased from 40.28 to 66.23 g/L through fed-batch fermentation by butyric acid tolerant strain compared with the fast-growing one. In addition, the endpoint strain was successfully and steadily used in the 50-L scale fermentation. Thus, adaptive evolution is an excellent strategy which can help us select the fast-growing strain and reduce the negative effect from substrate and metabolite inhibition. © 2018 American Institute of Chemical Engineers AIChE J, 65: 32–39, 2019     

Production of 3-hydroxypropionic acid by recombinant <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> based on aeration and ORP controlled strategy

A biosynthetic pathway for the production of 3-hydroxypropionic acid (3-HP) from glycerol was established in recombinant Klebsiella pneumoniae by introducing the aldehyde dehydrogenase gene from Escherichia coli. The activity of aldehyde dehydrogenase, which oxidized 3-hydroxypropionaldehyde (3-HPA) to 3-HP, was detected and 3-HP was produced by the recombinant strains. Three different oxygen supply strategies, associated with measuring the oxidoreduction potential (ORP) during the fermentation under these conditions, were adopted for higher production of 3-HP by the recombinant cells. About 0.8 g/l 3-HP and more 1,3-propanediol production by the recombinant Klebsiella pneumoniae were obtained under completely aerobic conditions. Under micro-aerobic conditions, 3-HP production could be increased to 2.2 g/l and 1,3-propanediol production was almost the same as in the original strain. Under the anaerobic conditions, 1,3-propanediol was the main product and about 1.3 g/l 3-HP was produced. Finally, 3-HP production of the recombinant strain was increased to 2.8 g/l under micro-aerobic condition with a further two-stage ORP controlled strategy.