1,3-丙二醇发酵液后提取技术研究进展

1,3-丙二醇是一种重要的化工原料,以甘油或葡萄糖为原料发酵法制备1,3-丙二醇具有原料可再生、反应条件温和等优点,是近年来国内外的研究热点。由微生物发酵获得的1,3-丙二醇发酵液是含多种强极性的醇及盐类的稀溶液,这使得采用传统的分离方法难以经济、有效地的将1,3-丙二醇从发酵液中纯化出来,后提取过程成为发酵法工业化生产1,3-丙二醇的瓶颈。1,3-丙二醇后提取过程主要包括微生物菌体等高分子物质的去除,盐的去除、回收,有机物的纯化和水的去除。以下对应用于以上分离过程的技术的研究进展进行讨论,提出在该领域应该重视的发展方向。     

基于甘油的1,3-丙二醇生物合成的代谢局限及其改造策略

粗甘油是生物柴油生产中的主要副产物,一些微生物可将甘油转化为重要化工原料1,3-丙二醇(1,3-PD),而利用这些微生物野生菌株生物合成1,3-PD会存在一些局限性,如底物抑制、产物抑制等。文中从1,3-丙二醇的甘油生物转化途径与这些局限性出发,总结了生物合成中存在的问题,并针对这些问题提出了一些基于基因敲除或基因过表达等基因工程技术的改造方法,综述了利用基因工程菌生物转化甘油生成1,3-丙二醇的最新研究进展。     

补料分批发酵过程中两阶段发酵生产1,3-丙二醇

在补料分批发酵过程中提高比生长速率不仅减少乙醇、甲酸的生成,而且提高1,3-丙二醇的得率和比生产速率.发酵后期甘油的浓度在15～26 g/L时有利于提高1,3-丙二醇的生产.采取在发酵前期控制菌体较高比生长速率和发酵后期控制适宜甘油浓度相结合的策略,有效地提高了1,3-丙二醇的生产,降低副产物乳酸和乙醇的生成.     

一步法产1,3-丙二醇酿酒酵母基因工程菌的构建

1,3-丙二醇(1,3-PD)是一种重要的化工原料,发酵法生产1,3-PD是一条新颖且具有潜在竞争力的生产途径。本研究在前期工作的基础上,将分别来源于大肠杆菌和肺炎克雷伯氏菌的基因片段yqhD和dhaB串联表达,构建重组表达载体pYX212-zeocin-pGAP-yqhD-pGAP-dhaB;并得到重组酿酒酵母(Saccharomyces cerevisiae)W303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB。该重组菌和对照S.cerevisiae分别以葡萄糖为底物摇瓶发酵72h后,重组酿酒酵母发酵液中1,3-PD含量约为1.5g/L;而对照菌株不产1,3-PD。以上结果表明本研究在国内首次成功构建了直接以葡萄糖为底物发酵生产1,3-PD的酿酒酵母基因工程菌。为进一步将dhaB、yqhD基因导入其他以葡萄糖为底物高产甘油的酵母宿主中表达,获得以葡萄糖为底物一步法发酵高产1,3-丙二醇工程菌打下了坚实的基础。     

Cloning and Sequence Analysis of the dhaT Gene of the 1,3-Propanediol Regulon from Klebsiella Pneumoniae

1,3-Propanediol oxidoreductase encoded by dhaT gene, a gene of 1,3-propanediol regulon, is important in converting glycerol to 1,3-propanediol in Klebsiella pneumoniae. DhaT gene was amplified from the genome of K. pneumoniae, sequenced and its amino acid sequence deduced. A predicted secondary structure and 3D-structural model was constructed by homology modelling. Based on these results, we infer that 1,3-propanediol oxidoreductase belongs to NAD(P)-dependent alcohol dehydrogenase group III of iron-activated dehydrogenases.     

Klebsiella pneumoniae 1,3-propanediol:NAD+ oxidoreductase.

Fermentative utilization of glycerol, a more reduced carbohydrate than aldoses and ketoses, requires the disposal of the two extra hydrogen atoms. This is accomplished by sacrificing an equal quantity of glycerol via an auxiliary pathway initiated by glycerol dehydratase. The product, 3-hydroxypropionaldehyde, is then reduced by 1,3-propanediol NAD+:oxidoreductase (1,3-propanediol dehydrogenase; EC 1.1.1.202), resulting in the regeneration of NAD+ from NADH. The pathway for the assimilation of glycerol is initiated by an NAD-linked dehydrogenase. In Klebsiella pneumoniae the two pathways are encoded by the dha regulon which is inducible only anaerobically. In this study 1,3-propanediol:NAD+ oxidoreductase was purified from cells grown anaerobically on glycerol. The enzyme was immunochemically distinct from the NAD-linked glycerol dehydrogenase and was an octamer or hexamer of a polypeptide of 45,000 +/- 3,000 daltons. When tested as a dehydrogenase, only 1,3-propanediol served as a substrate; no activity was detected with ethanol, 1-propanol, 1,2-propanediol, glycerol, or 1,4-butanediol. The enzyme was inhibited by chelators of divalent cations. An enzyme preparation inhibited by alpha,alpha'-dipyridyl was reactivated by the addition of Fe2+ or Mn2+ after removal of the chelator by gel filtration. As for glycerol dehydrogenase, 1,3-propanediol oxidoreductase is apparently inactivated by oxidation during aerobic metabolism, under which condition the enzyme becomes superfluous.     

An improved screening method for microorganisms able to convert crude glycerol to 1,3-propanediol and to tolerate high product concentrations

A new screening method was developed and established to find high-performance bacteria for the conversion of crude glycerol to 1,3-propanediol. Three soil samples from palm oil-rich habitats were investigated using crude glycerol of a German biodiesel plant. Nine promising 1,3-propanediol producers could be found. Because of a special pH buffer system, a fast evaluation on microscale and high 1,3-propanediol concentrations up to 40 g L⁻¹ could be achieved. Three strains demonstrated very high product tolerance and were identified as Clostridium butyricum. Two strains, AKR91b and AKR102a, grew and produced 1,3-propanediol in the presence of 60 g L⁻¹ initial 1,3-propanediol, the strain AKR92a even in the presence of 77 g L⁻¹ 1,3-propanediol. The strains AKR91b and AKR102a tolerated up to 150 g L⁻¹ crude glycerol and produced 80% of the 1,3-propanediol attained from pure glycerol of the same concentration. Further criteria for the choice of a production strain were the pathogenicity (risk class), ability to grow on low-cost media, e.g., with less yeast extract, and robustness, e.g., process stability after several bioconversions. Overall, the strain C. butyricum AKR102a was chosen for further process optimization and scale-up due to its high productivity and high final concentration in a pH-regulated bioreactor.     

甘油歧化为1,3-丙二醇的代谢及关键酶研究进展

微生物发酵生产1,3-丙二醇因对环境友好而成为研究热点。通过对发酵菌种、代谢途径、调节子和关键酶的分析,阐述了微生物转化甘油为1,3-丙二醇的分子机理。尤其对还原途径的限速酶-甘油脱水酶的分子结构及再激活因子进行了详细分析,为菌种的遗传改造提供了理论依据。     

甘油歧化为1,3-丙二醇的代谢及关键酶研究进展

微生物发酵生产1,3-丙二醇因对环境友好而成为研究热点。通过对发酵菌种、代谢途径、调节子和关键酶的分析,阐述了微生物转化甘油为1,3-丙二醇的分子机理。尤其对还原途径的限速酶-甘油脱水酶的分子结构及再激活因子进行了详细分析,为菌种的遗传改造提供了理论依据。     

Glycerol fermentation by a new 1,3-propanediol-producing microorganism:Enterobacter agglomerans

 According to their ability to synthesize 1,3-propanediol from glycerol, two species were isolated from the anoxic mud of a distillery waste-water digestor: Clostridium butyricum and Enterobacter agglomerans. The latter, a facultatively anaerobic gram-negative bacterium, is described for the first time as a microorganism producing 1,3-propanediol from glycerol. The products of glycerol conversion by E. agglomerans were identified using nuclear magnetic resonance. A 20-g/l glycerol solution was fermented mainly to 1,3-propanediol (0.51 mol/mol) and acetate (0.18 mol/mol). Ethanol, formate, lactate and succinate were formed as by-products. Gas production was very low; 1,3-propanediol production perfectly balanced the oxido-reduction state of the microorganism. Acetate was the predominant metabolite generating energy for growth. High-glycerol-concentration fermentations (71 g/l and 100 g/l) resulted in an increase of the 1,3-propanediol yield (0.61 mol/mol) at the expense of lactate and ethanol production. Specific rates of glycerol consumption and 1, 3-propanediol and acetate production increased whereas the growth rate decreased. The decrease in ATP yield was linearly correlated with the specific rate of 1,3-propanediol production. Incomplete glycerol consumption (about 40 g/l) was systematically observed when high glycerol concentrations were used. The unbalanced oxido-reduction state, the low carbon recovery and the detection of an unknown compound by HPLC observed in these cases indicate the formation of another metabolite, which is possibly an inhibitory factor. Received: 17 November 1994 / Accepted: 15 December 1994     

1,3-Propanediol:NAD+ oxidoreductases of Lactobacillus brevis and Lactobacillus buchneri.

In the cofermentation of glycerol with a sugar by Lactobacillus brevis and Lactobacillus buchneri, a 1,3-propanediol:NAD+ oxidoreductase provides an additional method of NADH disposal. The enzyme has been purified from both L. brevis B22 and L. buchneri B190 and found to have properties very similar to those reported for the enzyme from Klebsiella pneumoniae. The enzymes required Mn2+ and are probably octamers with a molecular mass of 350 kDa. Although not absolutely specific for 1,3-propanediol when tested as dehydrogenases, the enzymes have less than 10% activity with glycerol, ethanol, and 1,2-propanediol. These properties contrast sharply with those of a protein isolated from another Lactobacillus species (L. reuteri) that ferments glycerol with glucose and previously designated a 1,3-propanediol dehydrogenase.     

1,3-Propanediol:NAD+ oxidoreductases of Lactobacillus brevis and Lactobacillus buchneri.

In the cofermentation of glycerol with a sugar by Lactobacillus brevis and Lactobacillus buchneri, a 1,3-propanediol:NAD+ oxidoreductase provides an additional method of NADH disposal. The enzyme has been purified from both L. brevis B22 and L. buchneri B190 and found to have properties very similar to those reported for the enzyme from Klebsiella pneumoniae. The enzymes required Mn2+ and are probably octamers with a molecular mass of 350 kDa. Although not absolutely specific for 1,3-propanediol when tested as dehydrogenases, the enzymes have less than 10% activity with glycerol, ethanol, and 1,2-propanediol. These properties contrast sharply with those of a protein isolated from another Lactobacillus species (L. reuteri) that ferments glycerol with glucose and previously designated a 1,3-propanediol dehydrogenase.     

3-Hydroxypropionaldehyde guided glycerol feeding strategy in aerobic 1,3-propanediol production by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

3-Hydroxypropionaldehyde (3-HPA) is a toxic intermediary metabolite in the biological route of 1,3-propanediol biosynthesis from glycerol. 3-HPA accumulated in culture medium would arouse an irreversible cessation of the fermentation process. The role of substrate (glycerol) on 3-HPA accumulation in aerobic fermentation was investigated in this paper. 1,3-Propanediol oxidoreductase and glycerol dehydratase, two key enzyme catalyzing reactions of 3-HPA formation and consumption, were sensitive to high concentration of 3-HPA. When the concentration of 3-HPA increased to a higher level in medium (ac 10 mmol/L), the activity of 1,3-propanediol oxidoreductase in cell decreased correspondingly, which led to decrease of the 3-HPA conversion rate, then the 3-HPA concentration increasing was accelerated furthermore. 3-HPA accumulation in culture medium was triggered by this positive feedback mechanism. In the cell exponential growth phase, the reaction catalyzed by 1,3-propanediol oxidoreductase was the rate limiting step in 1,3-propanediol production. The level of 3-HPA in culture medium could be controlled by the substrate (glycerol) concentration, and lower level of glycerol could avoid 3-HPA accumulating to a high, lethal concentration. In fed batch fermentation, under the condition of initial glycerol concentration 30 g/L, and keeping glycerol concentration lower than 7–8 g/L in cell exponential growth phase, 3-HPA accumulation could not be incurred. Based on this result, a glycerol feeding strategy was set up in fed batch fermentation. Under the optimized condition, 50.1 g/L of 1,3-propanediol was produced in 24 h, and 73.1 g/L of final 1,3-propanediol concentration was obtained in 54 h.     

Stoichiometric analysis and experimental investigation of glycerol–glucose co-fermentation in Klebsiella pneumoniae under microaerobic conditions

The ratio between two substrates is an important parameter in microbial co-fermentation, such as 1,3-propanediol production from glycerol by Klebsiella pneumoniae using glucose as the cosubstrate. In this study, the glycerol–glucose cometabolism by K. pneumoniae is stoichiometrically analyzed according to energy (ATP), reducing equivalent (NADH₂) and product balances. The theoretical analysis reveals that the yield of 1,3-propanediol to glycerol under microaerobic conditions depends not only on the ratio of glucose to glycerol initially added, but also on the molar fraction of reducing equivalent oxidized completely by molecular oxygen in tricarboxylic acid (TCA) cycle (δ) and the molar fraction of TCA cycle in acetyl-CoA metabolism (γ). The maximum ratio of 0.32 mol glucose per mol glycerol is needed to convert glycerol completely to 1,3-propanediol under anaerobic conditions if glycerol neither enters oxidation pathways nor forms biomass. The ratio can be reduced under microaerobic conditions. The experimental results of batch cultures demonstrate that the biomass concentration and yield of 1,3-propanediol on glycerol could be enhanced by using glucose as a co-substrate. The theoretical analysis reveals the relationship between yield of 1,3-propanediol to glycerol, ratio of glucose to glycerol and respiratory quotient (RQ). These results are helpful for the experimental design and control.     

Effect of glucose on glycerol metabolism by Clostridium butyricum DSM 5431

The levels of 1,3-propanediol dehydrogenase and of the glycerol dehydrogenase in Clostridium butyricum grown on glucose–glycerol mixtures were similar to those found in extracts of cells grown on glycerol alone, which can explain the simultaneous glucose–glycerol consumption. On glycerol, 43% of glycerol was oxidized to organic acids to obtain energy for growth and 57% to produce 1,3-propanediol. With glucose–glycerol mixtures, glucose catabolism was used by the cells to produce energy through the acetate–butyrate production and NADH, whereas glycerol was used chiefly in the utilization of the reducing power since 92–93% of the glycerol flow was converted through the 1,3-propanediol pathway. The apparent K _ms for the glycerol dehydrogenase was 16-fold higher for the glycerol than that for the glyceraldehyde in the case of the glyceraldehyde-3-phosphate dehydrogenase and fourfold higher for the NAD⁺, providing an explanation for the shift of the glycerol flow toward 1,3-propanediol when cells were grown on glucose–glycerol mixtures.     

Glycerol and propanediols degradation by Desulfovibrio alcoholovorans in pure culture in the presence of sulfate, or in syntrophic association with Methanospirillum hungatei

Abstract In a mineral medium containing sulfate as terminal electron acceptor, the sulfate-reducing bacterium Desulfovibrio alcoholovorans oxidized stoichiometrically 1 mol glycerol to 1 mol acetate and 1 mol 1,3-propanediol to 1 mol acetate with the concomitant reduction of 0.75 and 1 mol sulfate, respectively; 1 mol 1,2-propanediol was degraded to 0.8 mol acetate and 0.1 mol proprionate, with the reduction of approximately 1 mol sulfate. The maximum specific growth rates (μ_max in h⁻¹) were 0.22, 0.086 and 0.09 with glycerol, 1,3-propanediol and 1,2-propanediol, respectively. The growth yields were 12.7 g, 11.1 g and 7.2 g dry weight/mol 1,3-propanediol, glycerol and 1,2-propanediol degraded, respectively. The growth yields and maximum specific growth rates of the H₂-transferring associations were also calculated. In the absense of sulfate, all these reduced substrates were degraded to acids and methane when D. alcoholovorans was cocultured with Methanospirillum hungatei . Changes in the metabolic pathway were observed in the degradation of 1,2- and 1,3-propanediol. The metabolic efficiency of D. alcoholovorans to degrade glycerol, 1.2- and 1,3-propanediol is discussed.     

pH对克雷伯氏菌1,3-丙二醇合成关键酶酶活及发酵特性的影响

在5 L发酵罐进行甘油脉冲流加发酵,分析了不同pH值对克雷伯氏肺炎杆菌发酵特性的影响,pH 6.5为菌体最佳生长条件,克雷伯氏肺炎杆菌合成1,3-丙二醇的产量最高。在1,3-丙二醇合成速率较大的对数中前期,进行甘油脉冲流加发酵,提高甘油浓度促进甘油脱水酶、1,3-丙二醇氧化还原酶和甘油脱氢酶活性。不同pH值的脉冲试验表明,甘油脱水酶,2,3-丁二醇脱氢酶比酶活随着pH值的升高而升高,1,3-丙二醇氧化还原酶,乳酸脱氢酶比酶活在pH6.5最高,因此偏酸性的发酵条件和对数期维持一定的甘油浓度能够促进1,3-丙二醇的合成。     

Fermentation of raw glycerol to 1,3-propanediol by new strains ofClostridium butyricum

Industrial glycerol obtained through the transesterification process using rapeseed oil did not support growth of several strains ofClostridium butyricum obtained from bacterial culture collections. Ten new strains ofC. butyricum were obtained from mud samples from a river, a stagnant pond, and a dry canal. These new isolates fermented the commercial glycerol and produced 1,3-propanediol as a major fermentation product with concomitant production of acetic and butyric acids. Four of the ten isolates were able to grow on industrial glycerol obtained from rapeseed oil. One strain,C. butyricum E5, was very resistant to high levels of glycerol and 1,3-propanediol. Using fed-batch fermentation, 109 g L^–1 of industrial glycerol were converted into 58 g of 1,3-propanediol, 2.2 g of acetate and 6.1 g of butyrate per liter.     

Microbial production of 1,3-propanediol: Recent developments and emerging opportunities

1,3-Propanediol, a valuable bifunctional molecule, can be produced from renewable resources using microorganisms. It has several promising properties for many synthetic reactions, particularly for polymer and cosmetic industries. By virtue of being a natural product, relevant biochemical pathways can be harnessed into fermentation processes to produce 1,3-propanediol. Various strategies for the microbial production of 1,3-propanediol are reviewed and compared in this article with their promises and constraints. Furthermore, genetic and metabolic engineering could significantly improve product yields and overcome the limitations of fermentation technology. Present review gives an overview on 1,3-propanediol production by wild and recombinant strains. It also attempts to encompass the various issues concerned in utilization of crude glycerol for 1,3-propanediol production, with particular emphasis laid on biodiesel industries. This review also summarizes the present state of strategies studied for the downstream processing and purification of biologically produced 1,3-propanediol. The future prospect of 1,3-propanediol and its potential as a major bulk chemical are discussed under the light of the current research.     

Overflow metabolism during anaerobic growth of Klebsiella aerogenes NCTC 418 on glycerol and dihydroxyacetone in chemostat culture

Klebsiella aerogenes NCTC 418 was grown anaerobically in chemostat culture with glycerol as source of carbon and energy. Glycerol-limited cultures did not ferment the carbon source with maximal efficiency but produced considerable amounts of 1,3-propanediol. The fraction of glycerol converted to this product depended on the growth rate and on the limitation: faster growing cells produced relatively more of this compound. Under glycerol excess conditions the energetic efficiency of fermentation was decreased due to the high 1,3-propanediol excretion rate. Evidence is presented that 1,3-propanediol accumulation exerts a profound effect on the cells' metabolic behaviour.When steady state glycerol-limited cultures were instantaneously relieved of the growth limitation a vastly enhanced glycerol uptake rate was observed, accompanied by a shift in the fermentation pattern towards 1,3-propanediol and acetate. This observation was consistent with the extremely high glycerol dehydrogenase activity that was measured in vitro. Some mechanisms that could be responsible for the energy dissipation during this response are discussed.