产酸克氏杆菌1,3-丙二醇发酵液中2,3-丁二醇的定性和定量分析

产酸克氏杆菌(Klebsiella oxytoca)在厌氧和微好氧条件下均可以发酵甘油产生1,3-丙二醇。相对于厌氧发酵液,在微好氧发酵液的高效液相色谱图中出现两个未知物峰。对微好氧发酵液进行离心,脱盐和蒸馏处理,以两未知物质为主要成分的馏分通过气相色谱-质谱联机分析显示两种未知物质均为2,3-丁二醇,通过气相色谱和高效液相色谱分析确定两未知物质分别为meso-2,3-丁二醇和L-( )-2,3-丁二醇。建立了用HPLC法对meso-2,3-丁二醇和L-( )-2,3-丁二醇进行外标定量的测定方法,在标准品浓度100~1200mg/L范围内,meso-2,3-丁二醇线性方程为y=7×10-6x,r=0.9991;L-( )-2,3-丁二醇线性方程为y=7×10-6x,r=0.9998。在发酵液样品中meso-2,3-丁二醇的回收率为99.67%;相对标准偏差RSD为0.35%,L-( )-2,3-丁二醇的回收率为99.89%;相对标准偏差RSD为0.09%。     

Enhanced Reducing Equivalent Generation for 1,3-Propanediol Production Through Cofermentation of Glycerol and Xylose by Klebsiella pneumoniae

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.     

Microbial Fed-batch Production of 1,3-Propanediol Using Raw Glycerol with Suspended and Immobilized Klebsiella pneumoniae

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.     

Effect of Biodiesel-derived Raw Glycerol on 1,3-Propanediol Production by Different Microorganisms

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.     

Simultaneous HPLC Determination of Four Key Metabolites in the Metabolic Pathway for Production of 1,3-Propanediol from Glycerol

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.     

Comparative Evaluation of Pumice Stone as an Alternative Immobilization Material for 1,3-Propanediol Production from Waste Glycerol by Immobilized Klebsiella pneumoniae

In this study, pumice stone (PS), which is a vastly available material in Turkey, was evaluated as an alternative immobilization material in comparison to other commercially available immobilization materials such as glass beads and polyurethane foam. All immobilized bioreactors resulted in much better 1,3-propanediol production from waste glycerol in comparison to the suspended cell culture bioreactor. It was also demonstrated that the locally available PS material is as good as the commercially available immobilization material. The maximum volumetric productivity (8.5?g?L^?1?h^?1) was obtained by the PS material, which is 220?% higher than the suspended cell system. Furthermore, the immobilized bioreactor system was much more robust against cell washout even at very low hydraulic retention time values.     

生物制造2,3-丁二醇:回顾与展望

2,3-丁二醇是生物制造产品体系中一种重要的精细化工原料和潜在平台化合物,广泛应用于材料、医药、食品及航空航天等领域。利用生物质可再生资源为原料生产2,3-丁二醇符合当前发展低碳经济的国家需求。本文回顾了生物制造2,3-丁二醇的研究历史,分析了微生物合成2,3-丁二醇的代谢机理,总结了提高生物制造2,3-丁二醇经济性的有效途径,包括廉价原料的替代、菌株选育与遗传改造和发酵过程控制等,并对2,3-丁二醇的各种下游分离过程进行了对比分析;指出今后研究重点应着眼于努力提高生物质的利用效率,同时实现高效的2,3-丁二醇生物转化两方面,并在此基础上开发2,3-丁二醇的系列高值衍生物,以进一步拓展其应用领域。     

Radiation-Induced Dehydration and Destruction of 1,2-Propanediol, 1,2-Butanediol,and 2,3-Butanediol in Deaerated Aqueous Solutions

High Energy Chemistry - The γ-radiation-induced transformations of α-diols in deaerated aqueous solutions at pH 7 have been studied. The composition and radiation-chemical yields of the...     

介孔氧化钨担载Pt催化剂上甘油氢解制备1,3-丙二醇

采用蒸发诱导自组装法制备了介孔氧化钨(m-WO3),担载Pt后应用于催化甘油氢解制1,3-丙二醇.结果表明,与商业氧化钨(c-WO3)相比,m-WO3具有高比表面积和易还原的优点,从而使得Pt纳米粒子高度分散于其上.在180℃和5.5 MPa H2下反应12h,Pt/m-WO3催化剂上甘油转化率和1,3-丙二醇的选择性分别为18.0％和39.2％,明显高于Pt/c-WO3催化剂.     

Production of 1,3-propanediol by Klebsiella pneumoniae

1,3-Propanediol (1,3-PD) has numerous applications from polymers to cosmetics, foods, lubricants, and medicines. Recently, there are strong industrial interests in a new kind of polyester, polytrimethylene terephthalate, with 1,3-PD as a monomer. This new polyester shows significant promise for use in carpeting and textiles. In this article we introduce a mild aerobic fermentation process using a strain screened from Klebsiella pneumoniae ATCC 25955, which is insensitive to oxygen, to produce 1,3-PD. We also describe a two-step fermentation process starting with glucose that was converted into glycerol with a glycerol-producing yeast, followed by K. pneumoniae that converts glycerol into 1,3-PD without intermediate isolation and purification of glycerol.     

生物法制备2,3-丁二醇的最新进展

2,3-丁二醇及其衍生物作为重要的液体燃料和化工原料,具有广阔的工业应用前景。高效、经济的2,3-丁二醇生物制备方法,对我国低碳经济和循环经济的建设具有重要的促进作用。针对近三年间生物法制备2,3-丁二醇领域的最新研究成果,本文综述了当前国内外学者在该领域研究的热点,即关键基因和酶的鉴定、新菌种的开发和代谢工程改造、同步糖化和共培养等发酵条件的优化、耦合工艺等分离纯化技术改进等。使用非致病的高产单一2,3-丁二醇手性异构体的代谢工程菌株,作为细胞炼制工厂,利用廉价的非粮原料作底物,采用经济、简单、环保的分离纯化方式,是2,3-丁二醇产业化发展的可靠保障。     

Optimization of Culture Conditions for 1,3-Propanediol Production from Glycerol Using a Mutant Strain of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

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.     

W掺杂多级孔SiO2纳米球负载Pt用于催化甘油氢解制1,3-丙二醇

合成了原位W掺杂的多级孔SiO₂纳米球材料(W-HPSN), 系统考察了W-HPSN合成过程中短链醇类共溶剂(甲醇、乙醇、正丙醇)的加入对Pt/W-HPSN催化剂甘油氢解制1,3-丙二醇(1,3-PDO)性能的影响. 与仅以水为溶剂合成的材料制备的Pt/W-HPSN-H₂O催化剂相比, 加入醇类共溶剂后, 催化剂的比表面积均有不同程度的增大, 并在除1.4 nm的微孔和>2 nm的介孔以外, 在1.7 nm处出现了新的微孔结构. 在甘油氢解反应中, 加入醇类共溶剂合成的材料制备的催化剂的催化性能也更高. 在最佳的以甲醇作为共溶剂合成的Pt/W-HPSN-Me催化剂上, 甘油转化率和1,3-PDO选择性分别为88.8%和56.3%, 而Pt/W-HPSN-H₂O催化剂上二者分别为64.1%和40.7%. 根据表征结果, 推测更小的Pt粒径、更多原位产生的Brønsted酸位, 有利于提高Pt/W-HPSN催化剂的催化性能. 通过对W-HPSN-Me的组成进行优化, 发现当W/Si物质的量比为1/320时, Pt/W-HPSN-Me催化剂在423 K、氢气压力4 MPa、反应时间仅为12 h的反应条件下, 甘油转化率和1,3-PDO选择性进一步提高至98.7%和58.8%, 1,3-PDO得率可达58.0%, 展示了HPSN材料作为甘油选择氢解制1,3-PDO催化剂载体的良好应用前景.     

Statistical Optimization of 1,3-Propanediol (1,3-PD) Production from Crude Glycerol by Considering Four Objectives: 1,3-PD Concentration,Yield, Selectivity,and Productivity

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).     

生物柴油及1,3-丙二醇联产工艺产业化进展

生物柴油作为可再生的清洁能源,已在美国、欧盟等多个国家和地区推行使用。在生物柴油的生产过程中,最高可得到约10%的副产物甘油,副产物甘油的去向将成为生物柴油大规模产业化发展所面临的严峻问题。1,3-丙二醇是一种重要的化工原料,作为合成新型聚酯PTT的原料,1,3-丙二醇已引起人们的广泛关注。以生物柴油副产物甘油为原料耦合生产1,3-丙二醇,不仅解决了生物柴油副产物甘油的出路问题,同时降低了1,3-丙二醇的生产成本。本文详细介绍了生物柴油及1,3-丙二醇生产技术及联产工艺的研究进展,并对其应用前景进行了展望。     

生物柴油及1,3-丙二醇联产工艺产业化进展

生物柴油作为可再生的清洁能源,已在美国、欧盟等多个国家和地区推行使用.在生物柴油的生产过程中,最高可得到约10%的副产物甘油,副产物甘油的去向将成为生物柴油大规模产业化发展所面临的严峻问题.1,3-丙二醇是一种重要的化工原料,作为合成新型聚酯PTT的原料,1,3-丙二醇已引起人们的广泛关注.以生物柴油副产物甘油为原料耦合生产1,3-丙二醇,不仅解决了生物柴油副产物甘油的出路问题,同时降低了1,3-丙二醇的生产成本.本文详细介绍了生物柴油及1,3-丙二醇生产技术及联产工艺的研究进展,并对其应用前景进行了展望.     

Metabolism of methylated arsenic compounds by arsenic-resistant bacteria (Klebsiella oxytoca and Xanthomonas sp.)

Two bacteria exhibiting resistance to toxic arsenic were isolated. These had been contaminated with arsenic in a Chlorella sp. culture medium containing arsenic. The two bacteria were identified as Klebsiella oxytoca and Xanthomonas sp., and grew well in a peptone medium at neutral pH at 30°C, reaching the stationary phase in ca 100h and 70h, respectively. The growth of the bacteria was not affected by arsenic(V) concentrations in the medium as high as 1000mg dm^?3. The bacteria bioaccumulated arsenic, a part of the arsenic being methylated. The bioaccumulation exhibited its peak around the turing point from the log phase to the stationary phase. The relative content of methylated arsenic in the excrement was greater than that in the bacterial cells. Adaptation treatment of inorganic arsenic caused an increase in the bioaccumulation of inorganic arsenic by K. oxytoca. Such a situation was not observed in the case of Xanthomonas sp. The bacteria also bioaccumulated methylated arsenic compounds, and demethylation of these species was observed. When the bacteria were killed by ethanol, arsenic was not taken up by the cells.     

高效液相色谱和质谱法对产酸克雷伯氏菌SG-11生物合成吲哚-3-乙酸代谢途径的研究

产酸克雷伯氏菌 (Klebsiellaoxytoca)SG 1 1是从水稻根面分离的植物根际促生细菌 ,具有生物固氮能力。利用HPLC和GC MS对该菌的代谢产物进行了定性定量分析 ,结果表明该菌能产生较高浓度的吲哚 3 乙酸(IAA) ;对其代谢途径的研究结果证明 ,该菌以吲哚 3 丙酮酸代谢途径合成IAA。