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1.
The production of 1,3-propanediol (1,3-PD) was investigated with Klebsiella pneumoniae DSM 4799 using raw glycerol without purification obtained from a biodiesel production process. Fed-batch cultures with suspended cells revealed that 1,3-PD production was more effective when utilizing raw glycerol than pure glycerol (productivity after 47 h of fermentation, 0.84 g?L?1?h?1 versus 1.51 g?L?1?h?1 with pure and raw glycerol, respectively). In addition, more than 80 g/L of 1,3-PD was produced using raw glycerol; this is the highest 1,3-PD concentration reported thus far for K. pneumoniae using raw glycerol. Repeated fed-batch fermentation with cell immobilization in a fixed-bed reactor was performed to enhance 1,3-PD production. Production of 1,3-PD increased with the cycle number (1.06 g?L?1?h?1 versus 1.61 g?L?1?h?1 at the first and fourth cycle, respectively) due to successful cell immobilization. During 46 cycles of fed-batch fermentation taking place over 1,460 h, a stable and reproducible 1,3-PD production performance was observed with both pure and raw glycerol. Based on our results, repeated fed batch with immobilized cells is an efficient fermentor configuration, and raw glycerol can be utilized to produce 1,3-PD without inhibitory effects caused by accumulated impurities.  相似文献   

2.
1,3-Propanediol (1,3-PD) is an intermediate in chemical and polymer synthesis. We have previously expressed the genes of a biochemical pathway responsible for 1,3-PD production, thedha regulon ofKlebsiella pneumoniae, inEscherichia coli. An analysis of the maximum theoretical yield of 1,3-PD from glycerol indicates that the yield can be improved by the cofermentation of sugars, provided that kinetic constraints are overcome. The yield of 1,3-PD from glycerol was improved from 0.46 mol/mol with glycerol alone to 0.63 mol/mol with glucose cofermentation and 0.55 mol/mol with xylose cofermentation. The engineeredE. coli also provides a model system for the study of metabolic pathway engineering.  相似文献   

3.
In the present work, mutant strains of Klebsiella pneumoniae with deletions of the als gene encoding acetolactate synthase involved in synthesis of 2,3-butanediol, the ldhA gene encoding lactate dehydrogenase required for lactate synthesis, or both genes, were prepared. Production of 1,3-propanediol (1,3-PD) from glycerol was enhanced in the ldhA mutant strain (ΔldhA), but lower in Δals or Δals ΔldhA mutant strains compared to the parent strain, concomitant with a reduction in the glycerol consumption rate, indicating that deletion of ldhA alone was useful to improve 1,3-PD production. Fed-batch fermentation analysis revealed that, in the ΔldhA mutant strain, 1,3-PD production was higher at low pH than at neutral pH; the reverse was true for the parent strain. Further optimization of culture conditions, by variation of aeration and glycerol feed rates, dramatically improved the production of 1,3-PD by the mutant strain. The maximum level attained was 102.7 g l−1 of 1,3-PD from glycerol.  相似文献   

4.
Novel and selective microbial amperometric biosensors that use Gluconobacter oxydans cells to monitor the bacterial bioconversion of glycerol (Gly) to 1,3-propanediol (1,3-PD) are described. Two different mediators, ferricyanide and flexible polyvinylimidazole osmium functionalized polymer (Os-polymer), were employed to prepare two different microbial biosensors, both of which gave high detection performance. The good operational stabilities of both types of biosensor were underlined by the ability to detect 1,3-PD throughout 140 h of continuous operation. Both microbial biosensor systems showed excellent selectivity for 1,3-PD in the presence of a high excess of glycerol [selectivity ratios (1,3-PD/Gly) of 118 or 245 for the ferricyanide and Os-polymer systems, respectively]. Further, the robustness of each microbial biosensor was highlighted by the high reliability of 1,3-PD detection achieved (average RSD of standards <2%, and well below 4% for samples). The biosensor implementing the Os-polymer mediator exhibited high selectivity towards 1,3-PD detection and allowed moderate sample throughput (up to 12 h−1) when integrated into a flow system. This system was used to monitor the concentration of 1,3-PD during a real bioprocess. Results from biosensor assays of 1,3-PD in bioprocess samples taken throughout the fermentation were in a very good agreement with results obtained from reference HPLC assays (R 2 = 0.999).  相似文献   

5.
The fermentative production of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae under different fed-batch strategies was investigated. pH-stat fed-batch strategies proved to be not effective for economical 1,3-PD production for the existence of relatively high concentration of byproducts and residual glycerol at the end of the fermentation. However, in the pH-stat fed-batch strategy, an important phenomenon was observed that the yields of two main byproducts, 2,3-butanediol and lactic acid, were closely related to pH value. The dominant byproduct was 2,3-butanediol at a pH value of 5.0 to 6.5 but changed to be lactic acid at a pH value of 7.1 to 8.0. Based on the analysis of the phenomenon, a self-protection mechanism in K. pneumoniae, namely that the growing K. pneumoniae cells switch the metabolic pathways responding to environmental pH changes, was proposed. Thus a kind of feeding strategy was further applied during which the pH value was fluctuated between 6.3 and 7.3 periodically by feeding glycerol–ammonia mixture and sulphuric acid to make the metabolic pathways of 2,3-butanediol and lactic acid sub-active under the periodical low or high pH stress. At last, efficient 1,3-PD production was fulfilled under this fed-batch strategy, and the best results were achieved leading to 70 g/l 1,3-PD with a yield of 0.70 mol/mol glycerol and productivity of 0.97 g/l/h, while the two main byproducts and residual glycerol were under low concentrations.  相似文献   

6.
During the fermentative production of 1, 3-propanediol (1,3-PD), the multiple product inhibitions cannot be negligible to accurately describe the kinetics of fermentation process. A kinetic model for fermentative production of 1,3-PD by Klebsiella pneumoniae HR526 with glycerol as carbon source under aerobic condition was proposed. The inhibitions of multiple products including 1,3-PD, 2, 3-butanediol (2,3-BD), acetate, and succinate were considered in the model. It was found that 1,3-PD, 2,3-BD, and acetate showed strong inhibitions to cell growth depending on their concentrations. The kinetic model was relatively accurate to predict the experimental data of batch, fed-batch, and continuous fermentations. The model thus can serve as a tool for further controlling and optimizing the fermentation process.  相似文献   

7.
The microbial production of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae XJPD-Li under different aeration strategies were investigated. In batch fermentation, the results showed that the final concentration of 1,3-PD and yield on glycerol were 13.44 g/l and 0.73 mol/mol under the anaerobic condition (N2, 0.4 vvm), 11.55 g/l and 0.62 mol/mol without aeration, and 8.73 g/l and 0.47 mol/mol under the aerobic condition (air, 0.4 vvm), respectively. Under the aerobic condition, the yield of 1,3-PD on glycerol was the lowest, while the biomass (optical density at 650 nm) was the highest among these three conditions. In the fed-batch culture, the final concentration and the yield of 1,3-PD was 60.82 g/l and 0.61 mol/mol under the anaerobic condition (N2, 0.4 vvm), 56.43 g/l and 0.53 mol/mol without aeration, and 65.26 g/l and 0.56 mol/mol under the aerobic condition. All these three conditions had good productivities of 1,3-PD, which were 3.35 g/l·h under the anaerobic condition (N2, 0.4 vvm), 3.13 g/l·h without aeration, and 3.16 g/l·h under the aerobic condition within the initial 12 h.  相似文献   

8.
A novel expression system of Klebsiella pneumoniae was developed in order to improve 1,3-propanediol (1,3-PD) production using a K. pneumoniae–Escherichia coli shuttle vector pET28a consisting of the kanamycin-resistance gene promoter Pkan. The recombinant plasmid pETPkan-cat carrying the chloramphenicol acetyltransferase gene cat as selectable marker was constructed to test the availability of the promoter Pkan in K. pneumoniae. The results showed that the chloramphenicol acetyltransferase was apparently expressed in K. pneumoniae, and the recombinant strain had a high-level resistance to chloramphenicol, suggesting that the promoter Pkan was efficient in K. pneumoniae. Then, the expression system was applied to the expression of 1,3-PD oxidoreductase in K. pneumoniae. The enzyme was over-expressed, and the recombinant K. pneumoniae showed a nearly 3.0-fold decrease in peak level of the intermediary metabolite 3-hydroxypropionaldehyde and an increase of 16.5% in yield of 1,3-PD with respect to the wild-type strain. From these results, the first reported expression system has paved the way for improvement of 1,3-PD production and will be available and efficient for other heterologous gene expression in K. pneumoniae.  相似文献   

9.
The metabolism of residual glycerol from biodiesel synthesis by Klebsiella pneumoniae BLh-1 was investigated in this study. Batch and fed-batch cultivations were performed in bioreactors under anaerobic and oxygen limitation conditions. Results of batch cultivations showed that the main product was 1,3-propanediol (1,3-PD) in both conditions, although the higher yields and productivities (0.46 mol mol?1 glycerol and 1.22 g?L?1?h?1, respectively) were obtained under anaerobic condition. Large amounts of ethanol were also produced under batch anaerobic condition, peaking at 12.30 g?L?1. Batch cultivations under oxygen limitation were characterized by faster growth kinetics, with higher biomass production but lower conversions of glycerol into 1,3-PD, with yields and productivities of 0.33 mol mol?1 glycerol and 0.99 g?L?1?h?1, respectively. The fed-batch cultivations were carried out in order to investigate the effects of feeding of raw glycerol on cells. Fed-batch under anaerobiosis showed that 1,3-PD and ethanol concentrations increased with the feeding rate, with maximal productions of 26.12 and 19.2 g?L?1, respectively. The oxygen limitation conditions diverted the bacterium metabolism to an elevated lactic acid formation, reaching 59 g?L?1 in higher feeding rates of glycerol, but lowering the production of ethanol.  相似文献   

10.
The microbial production of 1,3-propanediol (1,3-PD) from raw glycerol, a byproduct of biodiesel production, is economically and environmentally advantageous. Although direct use of raw glycerol without any pretreatment is desirable, previous studies have reported that this could cause inhibition of microbial growth. In this study, we investigated the effects of raw glycerol type, different microorganisms, and pretreatment of raw glycerol on the production of 1,3-PD. Raw glycerol from waste vegetable-oil-based biodiesel production generally caused more inhibition of 1,3-PD production and microbial growth compared to raw glycerol from soybean-oil-based biodiesel production. In addition, two raw glycerol types produced from two biodiesel manufacturers using waste vegetable oil exhibited different 1,3-PD production behavior, partially due to different amounts of methanol included in the raw glycerol from the two biodiesel manufacturers. Klebsiella strains were generally resistant to all types of raw glycerol while the growth of Clostridium strains was variably inhibited depending on the type of raw glycerol. The 1,3-PD production of the Clostridium strains using acid-pretreated raw glycerol was significantly enhanced compared to that with raw glycerol, demonstrating the feasibility of using raw glycerol for 1,3-PD production by various microorganisms.  相似文献   

11.
1,3-Propanediol (1,3-PD) biosynthesis plays a key role in NADH consumption to regulate the intracellular reducing equivalent balance of Klebsiella pneumoniae. This study aimed to increase reducing equivalent for enhancing 1,3-PD production through cofermentation of glycerol and xylose. Adding xylose as cosubstrate resulted in more reducing equivalent generation and higher cell growth. In batch fermentation under microaerobic condition, the 1,3-PD concentration, conversion from glycerol, and biomass (OD(600)) relative to cofermentation were increased significantly by 9.1%, 20%, and 15.8%, respectively. The reducing equivalent (NADH) was increased by 1-3 mg/g (cell dry weight) compared with that from glycerol alone. Furthermore, 2,3-butannediol was also doubly produced as major byproduct. In fed-batch fermentation with xylose as cosubstrate, the final 1,3-PD concentration, conversion from glycerol, and productivity were improved evidently from 60.78 to 67.21 g/l, 0.52 to 0.63 mol/mol, and 1.64 to 1.82 g/l/h, respectively.  相似文献   

12.
改善细胞通透性促进1,3-丙二醇生物合成   总被引:1,自引:0,他引:1  
吴敏  杨天锦  苗茂栋  倪进波 《化学学报》2009,67(18):2133-2138
克雷伯杆菌发酵生产1,3-丙二醇(1,3-PD)的过程中, 通过加入表面活性剂可改善细胞通透性, 以减少产物和副产物对细胞生长与代谢的抑制作用, 从而促进细菌生长和1,3-PD产出. 对比研究了吐温-80 (Tween-80)、曲拉通X-100 (Triton X-100)、壬基酚聚氧乙烯醚-10 (OP-10)和十六烷基三甲基溴化铵(CTAB)等对发酵中甘油脱氢酶(GDH)、1,3-丙二醇氧化还原酶(PDOR)和甘油脱水酶(GDHt)等3种关键酶活的影响. 实验表明OP-10能较好改善细胞通透性, 胞内释放核酸浓度随添加OP-10量的增加有明显提高. 低浓度的OP-10对GDH, PDOR活性及细胞生长有较好的促进作用; 发酵8~12 h时添加1.0 g&#8226;L-1的OP-10可使1,3-PD浓度和摩尔转化率有较大提高. 结合透射电镜发现非离子表面活性剂OP-10损伤膜结构, 致细胞通透性改变, 有利于充分发挥细胞内酶的催化活性, 对细菌生长和1,3-丙二醇的合成有较大促进作用.  相似文献   

13.
A high-performance liquid chromatographic (HPLC) method with refractive-index detection has been developed for simultaneous analysis of glycerol, dihydroxyacetone (DHA), 3-hydroxypropionaldehyde (3-HPA), and 1,3-propanediol (1,3-PD), four key substances in the metabolic pathway for production of 1,3-PD from glycerol by microorganism fermentation. The compounds were separated on a 300 mm × 7.8 mm ion-exclusion column with a 65:35 (v/v) mixture of deionized water and acetonitrile, containing 0.0005 M H2SO4, as mobile phase. The flow rate was 0.5 mL min?1. Under these conditions the retention times of 3-HPA, DHA, glycerol, and 1,3-PD were 6.87, 14.63, 16.37, and 18.50 min, respectively. Relative standard deviations and average recoveries were between 0.42 and 0.63% and between 96.7 and 103.1%, respectively; detection limits were between 0.017 and 0.038 g L?1. The method enabled separation of these compounds.  相似文献   

14.
This study investigated the biological conversion of crude glycerol generated from a commercial biodiesel production plant as a by-product to 1,3-propanediol (1,3-PD). Statistical analysis was employed to derive a statistical model for the individual and interactive effects of glycerol, (NH4)2SO4, trace elements, pH, and cultivation time on the four objectives: 1,3-PD concentration, yield, selectivity, and productivity. Optimum conditions for each objective with its maximum value were predicted by statistical optimization, and experiments under the optimum conditions verified the predictions. In addition, by systematic analysis of the values of four objectives, optimum conditions for 1,3-PD concentration (49.8 g/L initial glycerol, 4.0 g/L of (NH4)2SO4, 2.0 mL/L of trace element, pH 7.5, and 11.2 h of cultivation time) were determined to be the global optimum culture conditions for 1,3-PD production. Under these conditions, we could achieve high 1,3-PD yield (47.4%), 1,3-PD selectivity (88.8%), and 1,3-PD productivity (2.1/g/L/h) as well as high 1,3-PD concentration (23.6 g/L).  相似文献   

15.
NADH is a coenzyme which plays a central role in cellular growth and metabolism. It is an intracellular fluorophore which fluoresces at 460 nm when cells are irradiated by 340 nm wavelength of light. The application of NADH+H+ fluorescence measurement for characterization of biomass and its metabolic activity during batch fermentation of 1,3-propanediol (1,3-PD) using Clostridium diolis was investigated in this study. A linear correlation between net fluorescence and biomass concentration was observed during both the initial and final phases of 1,3-PD fermentation. This could be used as an on-line indicator of biomass concentration inside the bioreactor thereby eliminating the need for sampling and off-line analysis for establishing biomass concentration during these phases. Also a sharp decline in the NADH+H+ fluorescence value was obtained towards the end of fermentation which could be a significant on-line, in situ signal of substrate depletion in the bioreactor and therefore possible fresh nutrient feed for enhanced production of 1,3-PD by repetitive and/or various fed-batch cultivation(s). This is the first report on the use of NADH + H+ fluorescence measurement technique for 1,3-PD fermentation.  相似文献   

16.
Glycerol metabolism is a typical biological oxidoreductive reaction. 1,3-Propanediol (1,3-PD) is the final product of the reductive branch, while acetate, succinate, lactate, 2,3-butanediol (2,3-BD), and ethanol were produced in the oxidative branch. 2,3-BD, which has similar properties of high boiling point and water solubility with 1,3-PD, not only contests the carbon flow and NADH with 1,3-PD but also serves as an obstacle for obtaining high purity 1,3-PD in downstream processes. In this study, a 2,3-BD pathway-deficient mutant of Klebsiella oxytoca ZG36 was constructed by knocking out the budA gene of the wild-type strain M5al. The results of fed-batch fermentation by ZG36 indicated that the glycerol flux and the distribution of metabolites were altered in the K. oxytoca when the 2,3-BD pathway was blocked. No 2,3-BD was produced, and the activity of α-acetolactate decarboxylase (α-ALDC) can not be detected in the fermentation processes. The indexes of the 1,3-PD titer, the conversion from glycerol to 1,3-PD, and the productivity per cell dry weight (CDW) increased by 42%, 62%, and 46%, respectively, compared with the M5al, and the yield of the byproducts also increased obviously. The assay of the enzyme activities in the oxidative branch and the reductive branch of the glycerol metabolism, as well as the intracellular redox state, exposited the results logically.  相似文献   

17.
Repeated batch cultivation (empty-and-fill protocol) using obligate anaerobe Clostridium diolis was attempted in the present study to improve the production of 1,3-propanediol (1,3-PD). In repeated batch operation, 20?% (v/v) culture broth was removed from the bioreactor and supplemented with an equal volume of fresh nutrient medium when the residual glycerol concentration in the bioreactor decreased below 15?g/L. Four cycles of culture broth withdrawal and subsequent replacement resulted in achieving a 1,3-PD concentration of 67.8?g/L with a productivity of 1.04?g/L/h at the end of 65?h. This represented a 1,3-PD concentration and productivity enhancement by 2.6-fold and 1.5-fold, respectively, as compared to batch 1,3-PD fermentation. This is the first report on the use of repeated batch mode of bioreactor operation for enhanced 1,3-PD production.  相似文献   

18.
19.
Supplementation with acetate under low levels was used as a novel approach to control the morphological development of Klebsiella pneumoniae aimed to improve 1,3-propanediol (1,3-PD) production. A full range of morphological types formed from rod shape to oval shape even round shape in response to different concentrations of acetate. The cell growth and 1,3-PD productions in the shake flasks with 0.5 g/L acetate addition were improved by 9.4 and 28.37 %, respectively, as compared to the control, while the cell became shorter and began to lose its original shape. The cell membrane penetration by acetate was investigated by the biomimetic vesicles, while higher concentration of acetate led to more moderate colorimetric transitions. Moreover, the percentage composition of unsaturated fatty acid (UFA) was increased as well as the increased concentrations of acetate, whereas higher UFA percentage, higher fluidity of bacterial cell membrane.  相似文献   

20.
杨曼  赵晓晨  任煜京  王佳  雷念  王爱琴  张涛 《催化学报》2018,39(6):1027-1037
随着生物柴油产业的快速发展, 甘油作为副产物大量过剩, 通过甘油氢解制备具有高附加值的丙二醇既符合原子经济的原则, 又具有重要的学术研究意义和应用价值. 其中选择氢解制备1,3-丙二醇, 因其产物在新型聚酯材料合成中的重要应用而备受关注, 被认为是最具工业应用潜力的甘油转化工艺之一. 但是由于反应空间位阻效应和热力学上的限制, 甘油氢解制备1,3-丙二醇仍存在很大的挑战. 目前, 可高选择性制得1,3-丙二醇的催化剂体系主要是铱-铼催化剂和铂-钨催化剂两类; 且反应通常需要较苛刻的压力. 在我们的前期工作中, 单原子/准单原子Pt催化剂(Pt/WOx)成功突破了这一压力壁垒, 实现了在较低(1 MPa)氢气压力下优异的甘油选择氢解制1,3-丙二醇活性; 但是该催化剂的耐压性能较差, 是长期反应后失活的一个主要原因, 且随着氢气压力的进一步升高, Pt/WOx催化剂活性大幅下降. 研究发现, 在高压氢气下, 单原子/准单原子催化剂Pt/WOx中的WOx易被过度还原, 导致催化剂失活, 大大限制了该系列单原子催化剂在涉氢催化反应中的应用. 因此, 通过载体改性设计制备具有良好氢气稳定性的载体, 有可能拓宽催化反应的氢压窗口.一般来讲, 可以通过掺杂来改变载体的电子结构、表面化学性质等, 进而改变催化剂活性、选择性和稳定性. 为了提高单原子/准单原子催化剂(Pt/WOx)在高压氢气中的稳定性, 本文采用原位掺杂法制备了一系列具有不同掺杂量、结构、及物化性质的Nb-WOx载体, 并通过物理吸附, XRD, SEM, HRTEM, Raman, XPS对其进行表征. 结果发现Nb掺杂的介孔WOx仍保持较高的比表面积(~136 m2/g); 且当Nb掺杂量增加至2 wt%时, WOx由一维针状堆积转变为二维片状堆积;至5 wt%时,变为三维球状堆积. 以所得的Nb-WOx为载体, 我们制备了一系列Pt高度分散的Pt/Nb-WOx催化剂, 并考察了其在甘油选择性氢解制备1,3-丙二醇反应中的催化性能. 结果表明, 当H2压力由1 MPa升到5 MPa后, Pt/WOx催化剂上1,3-丙二醇的收率由13.9%急剧下降到8.3%; 但随着Nb掺杂量的增加(>2wt%), 1,3-丙二醇的收率随压力升高并没有明显变化, 说明该催化剂在高压氢气下的过度还原被有效抑制, 反应的最佳氢气压力由1 MPa拓宽到5 MPa. 其中, Nb的最佳掺杂量为2 wt%, 在Pt/2%Nb-WOx催化剂上1 MPa甘油氢解转化率为40.0%, 1,3-丙二醇收率11.9%; 当氢气压力为5 MPa时, 甘油氢解转化率为40.3%, 1,3-丙二醇收率11.1%. 值得一提的是, 虽然Nb的掺杂使WOx的表面酸量增加, 并且表现出了更优的酸催化纤维二糖水解活性, 但是在甘油氢解制备1,3-丙二醇的反应中Pt/Nb-WOx催化剂的性能并没有得到提升反而略有下降, 进一步证实了催化剂本身的表面酸性位并不是催化甘油选择氢解的活性中心, 真正的活性中心很可能是氢气辅助下原位产生的B酸.对WOx载体进行Nb的掺杂改性, 显著提高了单原子/准单原子Pt/WOx催化剂在高压氢气反应条件下的稳定性, 进而在更宽压力范围内实现了甘油氢解制1,3-丙二醇的高效催化转化. 同时, 载体的改性也拓宽了该系列催化剂在其他涉氢反应中的应用范围, 尤其是需要较高氢气压力的反应.  相似文献   

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