Fed-batch approach to production of 2,3-butanediol by Klebsiella pneumoniae grown on high substrate concentrations.

The bioconversion of sugars present in wood hemicellulose to 2,3-butanediol by Klebsiella pneumoniae grown on high sugar concentrations was investigated. When K. pneumoniae was grown under finite air conditions in the presence of added acetic acid, 50 g of D-glucose and D-xylose per liter could be converted to 25 and 27 g of butanediol per liter, respectively. The efficiency of bioconversion decreased with increasing sugar substrate concentrations (up to 200 g/liter). Butanediol production at low sugar substrate concentrations was less efficient when the organism was grown under aerobic conditions; however, final butanediol values were higher for cultures grown on an initial sugar concentration of 150 g/liter, particularly when the inoculum was first acclimatized to high sugar levels. When a double fed-batch approach (daily additions of sugars together with yeast extract) was used under aerobic conditions, up to 88 and 113 g of combined butanediol and acetyl methyl carbinol per liter could be obtained from the utilization of 190 g of D-xylose and 226 g of D-glucose per liter, respectively.     

Production of 2,3-butanediol by Klebsiella pneumoniae grown on acid hydrolyzed wood hemicellulose

Summary Previously steam explosion had been used to enhance the enzymatic hydrolysis of lignocellulosic substrates to glucose. The conditions for pretreating aspen wood chips were optimized so that highest amounts of undegraded hemicellulose could be obtained after washing the steam exploded chips. The hemicellulose rich water soluble fractions showing highest pentosan yields were then acid hydrolysed to their composite sugars. Approximately 65–75% of the total reducing sugars detected in the wood hydrolysates were in the form of monosaccharides with D-xylose being the major component. Klebsiella pneumoniae was grown in media containing these wood hydrolysates as the substrate and 2,3-butanediol yields of 0.4–0.5 g per g of monosaccharide utilised were obtained.     

Optimized production of 2,3-butanediol by a lactate dehydrogenase-deficient mutant of Klebsiella pneumoniae

To obtain high-yield production of 2,3-butanediol (2,3-BD) from glucose, we optimized the culture conditions for a lactate dehydrogenase-deficient mutant (ΔldhA) of Klebsiella pneumoniae using response surface methodology. 2,3-BD production was successfully improved by optimizing pH (5.6), aeration (3.50 vvm) and concentration of corn steep liquor (45.0 mL/L) as a nitrogen source, resulting in a maximum level of 2,3-BD production of 148.8 g/L and productivity of 2.48 g/L/h. 2,3-BD was also obtained with high concentration (76.24 g/L) and productivity (2.31 g/L/h) from the K. pneumoniae mutant strain using sugarcane molasses as a carbon source.     

Fermentation of glycerol to 1,3-propanediol and 2,3-butanediol by Klebsiella pneumoniae

Klebsiella pneumoniae was shown to convert glycerol to 1,3-propanediol, 2,3-butanediol and ethanol under conditions of uncontrolled pH. Formation of 2,3-butanediol starts with some hours' delay and is accompanied by a reuse of the acetate that was formed in the first period. The fermentation was demonstrated in the type strain of K. pneumoniae, but growth was better with the more acid-tolerant strain GT1, which was isolated from nature. In continuous cultures in which the pH was lowered stepwise from 7.3 to 5.4, 2,3-butanediol formation started at pH 6.6 and reached a maximum yield at pH 5.5, whereas formation of acetate and ethanol declined in this pH range. 2,3-Butanediol and acetoin were also found among the products in chemostat cultures grown at pH 7 under conditions of glycerol excess but only with low yields. At any of the pH values tested, excess glycerol in the culture enhanced the butanediol yield. Both effects are seen as a consequence of product inhibition, the undissociated acid being a stronger trigger than the less toxic diols and acid anions. The possibilities for using the fermentation type described to produce 1,3-propanediol and 2,3-butanediol almost without by-products are discussed. Received: 4 February 1998 / Received revision: 30 March 1998 / Accepted: 13 April 1998     

Enhanced 2,3-butanediol production in recombinant Klebsiella pneumoniae via overexpression of synthesis-related genes

2,3-Butanediol (2,3-BD) is a major metabolite produced by Klebsiella pneumoniae KCTC2242, which is a important chemical with wide applications. Three genes important for 2,3-BD biosynthesis acetolactate decarboxylase (budA), acetolactate synthase (budB), and alcohol dehydrogenase (budC) were identified in K. pneumoniae genomic DNA. With the goal of enhancing 2,3-BD production, these genes were cloned into pUC18K expression vectors containing the lacZ promoter and the kanamycin resistance gene to generate plasmids pSB1-7. The plasmids were then introduced into K. pneumoniae using electroporation. All strains were incubated in flask experiments and 2,3-BD production was increased by 60% in recombinant bacteria harboring pSB04 (budA and budB genes), compared with the parental strain K. pneumoniae KCTC2242. The maximum 2,3-BD production level achieved through fedbatch fermentation with K. pneumoniae SGJSB04 was 101.53 g/l over 40 h with a productivity of 2.54 g/l.h. These results suggest that overexpression of 2,3-BD synthesisrelated genes can enhance 2,3-BD production in K. pneumoniae by fermentation.     

Mechanism of 2,3-butanediol stereoisomer formation in Klebsiella pneumoniae

Klebsiella pneumoniae is known to produce meso-2,3-butanediol and 2S,3S-butanediol, whereas 2R,3R-butanediol was detected in the culture broth of K. pneumoniae CGMCC 1.6366. The ratio of 2R,3R-butanediol to all isomers obtained using glycerol as the carbon source was higher than that obtained using glucose as the carbon source. Therefore, enzymes involved in glycerol metabolism are likely related to 2R,3R-butanediol formation. In vitro reactions show that glycerol dehydrogenase catalyzes the stereospecific conversion of R-acetoin to 2R,3R-butanediol and S-acetoin to meso-2,3-butanediol. Butanediol dehydrogenase exhibits high (S)-enantioselectivity in ketone reduction. Genes encoding glycerol dehydrogenase, α-acetolactate decarboxylase, and butanediol dehydrogenase were individually disrupted in K. pneumoniae CGMCC 1.6366, and the 2,3-butanediol synthesis characteristics of these mutants were investigated. K. pneumoniae ΔdhaD lost the ability to synthesize 2R,3R-butanediol. K. pneumoniae ΔbudA showed reduced 2R,3R-butanediol synthesis. However, K. pneumoniae ΔbudC produced a high level of 2R,3R-butanediol, and R-acetoin was accumulated in the broth. The metabolic characteristics of these mutants and in vitro experiment results demonstrated the mechanism of the 2,3-butanediol stereoisomer synthesis pathway. Glycerol dehydrogenase, encoded by dhaD, exhibited 2R,3R-butanediol dehydrogenase activity and was responsible for 2R,3R-butanediol synthesis from R-acetoin. This enzyme also contributed to meso-2,3-butanediol synthesis from S-acetoin. Butanediol dehydrogenase, encoded by budC, was the only enzyme that catalyzed the conversion of diacetyl to S-acetoin and further to 2S,3S-butanediol.     

Effect of succinic acid on 2,3-butanediol production by Klebsiella oxytoca

Summary The effect of succinic acid on the growth of Klebsiella oxytoca and its production of 2,3-butanediol was studied. Increasing succinic acid from 0 g/L to 30 g/L increased the final butanediol concentration. The maximum butanediol productivity occurred at an initial succinic acid concentration of approximately 10 g/L.     

Bioconversion of inulin to 2,3-butanediol by a newly isolated Klebsiella pneumoniae producing inulinase

Inulin could be converted to bio-based chemicals by an inulinase producer without external inulinase, and the production of 2,3-butanediol was less than 50 g/L. In this work, a novel inulinase producer of Klebsiella pneumoniae H3 was isolated, and inulinase catalytic properties as well as 2,3-butanediol fermentation were investigated. The enzyme was an intracellular inulinase with an optimal pH of 6 ∼ 7 and a temperature of 30 °C. The use of inulin by H3 was dependent on the degree of polymerization (DP), and the average DP of inulin in fermentation broth increased from 2.82 to 8.08 in 24-h culture of batch fermentation. Acidic pretreatment was developed to increase inulin utilization by adjusting medium pH to 3.0 prior to sterilization. In batch fermentation with optimized medium and fermentation conditions, the concentration of target product (2,3-butanediol and acetoin) was 80.4 g/L with a productivity of 2.23 g/(L⋅h), and a yield of 0.426 g/g inulin.     

Fermentation and evaluation of Klebsiella pneumoniae and K. oxytoca on the production of 2,3-butanediol

Klebsiella is one of the genera that has shown unbeatable production performance of 2,3-butanediol (2,3-BD), when compared to other microorganisms. In this study, two Klebsiella strains, K. pneumoniae (DSM 2026) and K. oxytoca (ATCC 43863), were selected and evaluated for 2,3-BD production by batch and fed-batch fermentations using glucose as a carbon source. Those strains' morphologies, particularly their capsular structures, were analyzed by scanning electron microscopy (SEM). The maximum titers of 2,3-BD by K. pneumoniae and K. oxytoca during 10 h batch fermentation were 17.6 and 10.9 g L(-1), respectively; in fed-batch cultivation, the strains showed the maximum titers of 50.9 and 34.1 g L(-1), respectively. Although K. pneumoniae showed higher productivity, SEM showed that it secreted large amounts of capsular polysaccharide, increasing pathogenicity and hindering the separation of cells from the fermentation broth during downstream processing.     

Effects of aeration on 2,3-butanediol production from glucose by Klebsiella oxytoca

Higher cell concentrations and greater 2,3-butanediol production were observed in aerobic cultures of Klebsiella oxytoca than with anaerobic cultures. The concentration of butanediol inhibitors such as ethanol and lactic acid are partially suppressed by adequate aeration-agitation. Excessive aeration-agitation leads to the formation of acetoin and acetic acid at the expense of butanediol. With 94.3 g/l of glucose in the media, aerobic batch cultures produced 38.1 g/l butanediol with complete substrate use and a productivity of 0.39 g/l/h.     

Reduction of acetoin to 2,3-butanediol in Klebsiella pneumoniae: a new model

Fermentation of xylose by Klebsiella pneumoniae (ATCC 8724, formerly known as Aerobacter aerogenes) carried out in our laboratory yields 2,3-butanediol as the major product. Experimental data obtained in this work cannot be explained by the model presently in the literature for the formation of 2,3-butanediol isomers from acetoin isomers. A new model is proposed with the existence of two acetoin reductases and an acetoin racemase. The two reductases were separated and their stereospecificity determined. Extension of the model of other microorganisms is discussed.     

Effect of oxygen supply and dilution rate on the production of 2,3-butanediol in continuous bioreactor by Klebsiella pneumoniae

Kinetics of 2,3-butanediol production by Klebsiella pneumoniae (NRRL B199) from glucose have been studied in a continuous bioreactor. The effect of oxygen supply rate and dilution rate on the product output rate and yield of 2,3-butanediol were investigated. For a feed glucose concentration of 100 g l⁻¹, the optimum oxygen transfer rate is between 25.0–35.0 mmol l⁻¹ h⁻¹. Under these conditions, maximum product concentration obtained was 35 g l⁻¹ at a dilution rate of 0.1 h⁻¹ and the maximum product output rate obtained was 4.25 g l⁻¹ h⁻¹. The product yield based on the substrate utilized approached the theoretical value (50%) at low values of oxygen transfer rate but decreased with increasing oxygen transfer rate.     

Production of 2,3-butanediol by Klebsiella oxytoca

Summary High glucose concentrations result in high levels of 2,3-butanediol, improved yield and productivity, and a decrease in cell growth in batch cultures of Klebsiella oxytoca. A maximum of 84.2 g butanediol/l and a yield of 0.5 was obtained with an initial glucose concentration of 262.6g/l. Adding the substrate in two steps in a modified fed-batch operation resulted in 85.5 g butanediol/l, 6.4 g acetoin/l and 3.4 g ethanol/l with a net yield of 0.5. Increasing the cell density to 60g/l resulted in productivities as high as 3.22 g/l.h.     

2,3-丁二醇代谢途径关键酶基因敲除对克雷伯氏菌发酵产1,3-丙二醇的影响

2,3-丁二醇是克雷伯氏菌发酵产1,3-丙二醇的主要副产物,为减少2,3-丁二醇的产生,利用Red重组技术对克雷伯氏菌2,3-丁二醇合成途径关键酶基因budC和budA进行了敲除。突变株发酵性能实验结果表明,所获得的两株突变株生长性能受到不同程度的影响;budC基因的缺失使菌株1,3-丙二醇产量提高了10%,2,3-丁二醇降低为原来的70%,而budA基因缺失则使菌株无2,3-丁二醇和1,3-丙二醇的产生,但乳酸、琥珀酸、乙醇和乙酸的产量较出发菌株都有明显增长。通过进一步对budC基因缺失菌株主要产物分析,推测在该菌中存在2,3-丁二醇回补途径,这一结果为低副产物克雷伯氏菌的改造提供了新依据。     

Analysis of 2,3-butanediol production with Klebsiella pneumoniae using sugar mixture under different oxygen supply conditions

以筛选的肺炎克雷伯氏菌(Klebsiella pneumoniae UV-86)为对象,考察供氧条件分别对菌体生长、葡萄糖和木糖双底物利用和产物合成的影响.研究发现生物量随氧供应量增加而增加.不同供氧条件对菌体消耗葡萄糖过程的影响较小,而代谢木糖的能力随氧供应量的增大而增强.微氧条件下2,3-丁二醇的生物合成能力最强,2,3-丁二醇产量在1.5 vvm下达到最高为30.1 g/L,是好氧时的2.5倍,最大体积产率为0.485 g/(L·h).不同条件下两底物产物分布有所区别,木糖代谢中乙酸生产增强.因此根据不同阶段代谢特点选择适合的供氧策略可以提高过程产量和产率.     

氧供应量对Klebsiella pneumoniae利用混合糖生物合成2,3-丁二醇过程的影响分析

以筛选的肺炎克雷伯氏茵（Klebsiella pneumoniaeUV-86）为对象,考察供氧条件分别对茵体生长、葡萄糖和木糖双底物利用和产物合成的影响。研究发现生物量随氧供应量增加而增加。不同供氧条件对茵体消耗葡萄糖过程的影响较小,而代谢木糖的能力随氧供应量的增大而增强。微氧条件下2,3-丁二醇的生物合成能力最强,2,3-丁二醇产量在1．5wm下达到最高为30．1g／L,是好氧时的2．5倍,最大体积产率为0．485g／（L·h）。不同条件下两底物产物分布有所区别,木糖代谢中乙酸生产增强。因此根据不同阶段代谢特点选择适合的供氧策略可以提高过程产量和产率。     

产酸克雷伯氏杆菌发酵产2，3-丁二醇的培养基优化

采用不同设计方法相结合的策略对耐高糖产酸克雷伯氏杆菌（Klebsiella oxytoca）ME—UD-3-4发酵产2,3-丁二醇的培养基进行优化。首先在单因素实验的基础上采用Plackett—Burrnan设计法对影响ME—UD-3-4发酵产2,3-丁二醇的相关因素进行研究,筛选到3种有显著效应的因素（P〈0．05）：葡萄糖、玉米浆和MgSO4·7H2O。然后利用响应曲面法（Response Surface Methodology,RSM）对这3种因素的最佳水平范围进一步探讨;对得到的回归模型进行分析,得最佳条件（g／L）：葡萄糖220、玉米浆19和MgSO4·7H2O 0．4;在最佳条件下,发酵80h,2,3-丁二醇产量从原来的57．3 g／L提高到86．1 g／L,生产强度由0．72g／（L·h）提高到1．08g/（L·h）。     

Fed-batch with biomass recycle and batch production of 2,3-butanediol from glucose by Bacillus polymyxa

The production of 2,3-butanediol from glucose by Bacillus polymyxa in batch was sensitive to both protein concentration and aeration rate. Two fed-batch experiments which were resupplied from a reservoir containing urea as the sole source of nitrogen, and incorporated total biomass recycle resulted in yields of 65.84 mM and 69.66 mM of 2,3-butanediol per 100 mM of glucose utilized. No massive sporulation events were detected. In addition, fed-batch with recycle yielded more butanediol and less acetate than any batch run.     

The effect of wood-derived inhibitors on 2, 3-butanediol production by Klebsiella pneumoniae

Four potential inhibitors, hydroxymethylfurfural, furfural, syringaldehyde and vanillin, were assayed over a range of concentrations and times to simulate the amounts that might be expected to accumulate when the hydrolyzed water-soluble fraction from steam-treated aspenwood was added in a fed-batch mode to K. pneumoniae. Combinations of the individual materials resulted in a cumulative inhibitory effect. Prolonged incubation could alleviate some of the inhibition.