Production of 1,3-Propanediol from Glycerol by Clostridium acetobutylicum and Other Clostridium Species

Glycerol was fermented with the production of 1,3-propanediol as the major fermentation product by four strains of Clostridium acetobutylicum, six of C. butylicum, two of C. beijerinckii, one of C. kainantoi, and three of C. butyricum. 1,3-Propanediol was identified by its retention times in gas chromatography and high-pressure liquid chromatography and by its mass spectrum. During growth of C. butylicum B593 in a chemostat culture at pH 6.5, 61% of the glycerol fermented was converted to 1,3-propanediol. When the pH was decreased to 4.9, growth and 1,3-propanediol production were substantially reduced.     

Isolation and Characterization of Clostridium butyricum DSM 5431 Mutants with Increased Resistance to 1,3-Propanediol and Altered Production of Acids

Clostridium butyricum mutants were isolated from the parent strain DSM 5431 after mutagenesis with N-methyl-N(prm1)-nitro-N-nitrosoguanidine and two selection procedures: osmotic pressure and the proton suicide method. Isolated mutants were more resistant to glycerol and to 1,3-propanediol (1,3-PD) than was the wild type, and they produced more biomass. In batch culture on 62 g of glycerol per liter, the wild type produced more acetic acid than butyrate, with an acetate/butyrate ratio of 5.0, whereas the mutants produced almost the same quantities of both acids or more butyrate than acetate with acetate/butyrate ratios from 0.6 to 1.1. The total acid formation was higher in the wild-type strain. Results of analysis of key metabolic enzymatic activities were in accordance with the pattern of fermentation product formation: either the butyrate kinase activity increased or the acetate kinase activity decreased in cell extracts of the mutants. A decreased level of the hydrogenase and NADH-ferredoxin activities concomitant with an increase in ferredoxin-NAD(sup+) reductase activities supports the conclusion that the maximum percentage of NADH available and used for the formation of 1,3-PD was higher for the mutants (97 to 100%) than for the wild type (70%). In fed-batch culture, at the end of the fermentation (72 h for the wild-type strain and 80 to 85 h for the mutants), 44% more glycerol was consumed and 50% more 1,3-PD was produced by the mutants than by the wild-type strain.     

1,3-Propanediol oxidoreductase production with Klebsiella pneumoniae DSM2026

We report a Klebsiella pneumoniae DSM2026 fermentation procedure for the efficient production of a key enzyme of 1,3-propanediol formation: 1,3-propanediol oxidoreductase (E.C. 1.1.1.202). The fermentation process is composed of an aerobic batch phase on glucose and glycerol and an anaerobic phase on glycerol. The role of the aerobic phase is to produce sufficiently high cell mass (12.9–14.6 g/l dry weight) and to activate the aerobic branch of the Klebsiella glycerol pathway, whereas in the anaerobic phase there is a rapid initiation of 1,3-propanediol oxidoreductase formation. A fast change from an aerobic to an anaerobic environment led to a redox imbalance, which resulted in the abrupt activation of the anaerobic branch of glycerol utilization, with the occurrence of a high 1,3-propanediol-oxidoreductase activity. A mathematical model with substrate inhibition showed that the adequate glycerol concentration for enzyme production was 14–16 g/l. The combination of the optimal substrate concentration together with the subsequent use of glucose and glycerol resulted in 90.6 ± 11.6 U enzyme activity referred to 1 l of fermentation broth and 10.3 ± 0.9 U/(1 h) productivity.     

Genome Sequence of Klebsiella pneumoniae LZ, a Potential Platform Strain for 1,3-Propanediol Production

Klebsiella pneumoniae LZ is a bacterium isolated from soil which can produce 1,3-propanediol from glycerol. Here we present a 5,431,750-bp assembly of its genome sequence. We annotated 9 coding sequences (CDSs) responsible for glycerol fermentation to 1,3-propanediol, 19 CDSs encoding glycerol utilization, and 134 CDSs related to its virulence and defense.     

产1,3-丙二醇菌株丁酸梭菌的诱变育种

甘油由丁酸梭菌转化成1,3-丙二醇的研究是厌氧条件下进行。为了获得1,3-丙二醇的高产突变株,以丁酸梭菌为出发菌株进行诱变处理。经过硫酸二乙酯（DES）化学诱变得到2株高产正突变株C.but2031和C.but2046,再经过紫外线和亚硝基胍（NTG）复合诱变得到突变株C.but3037。经过初筛、复筛和传代实验,表明其是稳定的突变株。C.but3037的1,3-丙二醇产量由出发菌株的2.2g/L提高到15.7g/L,提高了6.13倍,     

Complete Genome Sequence of Clostridium clariflavum DSM 19732

Clostridium clariflavum is a Cluster III Clostridium within the family Clostridiaceae isolated from thermophilic anaerobic sludge (Shiratori et al, 2009). This species is of interest because of its similarity to the model cellulolytic organism Clostridium thermocellum and for the ability of environmental isolates to break down cellulose and hemicellulose. Here we describe features of the 4,897,678 bp long genome and its annotation, consisting of 4,131 protein-coding and 98 RNA genes, for the type strain DSM 19732.     

基因组重排筛选高产谷氨酰胺转胺酶菌株

谷氨酰胺转胺酶(TGase)的产量不足的问题一直限制其工业化生产规模,故采用基因组重排的方法,筛选高产谷氨酰胺转胺酶菌株。通过对不同制备条件下原生质体纯度和形成率的考量,获得制备原生质体的最优条件为以6mg/ml的溶菌酶浓度进行酶解,酶解时间2h。再优化融合条件,以2min紫外灭活和40min热灭活结合的方法挑选出融合子。通过两轮基因组重排,经过96孔板发酵高通量筛选和摇瓶发酵复筛验证,获得了一株产酶达7.12U/ml的茂源链霉菌,相比最初选用菌株的平均酶活提高65.5%。发酵结果显示,酶活提高的原因可能是在重组后原酶成熟更快、更彻底,且得到的菌株遗传稳定性良好。证明基因组重排能够有效提高菌株的产酶水平,同时为谷氨酰胺转胺酶产量提高提供理论依据。     

1,3-Propanediol formation with product-tolerant mutants of Clostridium butyricum DSM 5431 in continuous culture: productivity, carbon and electron flow

Glycerol fermentation and product formation of two product-tolerant mutants of Clostridium butyricum DSM 5431 were investigated in continuous culture at increasing glycerol feed concentrations. Under conditions of glycerol excess (above 55 g l⁻¹ at D = 0·15 h⁻¹), the mutants maintained a constant level of glycerol consumption and product formation, whereas the parent strain exhibited a substantial decrease in substrate conversion, 1,3-propanediol and butyrate formation, and an increase in acetate formation. The activities of the glycerol dehydrogenase, the glycerol dehydratase and the 1,3-propanediol dehydrogenase showed only slight changes with glycerol concentrations in the mutants, but dropped markedly at high concentrations in the wild type. Intracellular concentrations of NADH, NAD ⁺ and acetyl-CoA remained at a relatively constant level in the mutants, but increased sharply with the wild type strain. The NADH content was always higher than the NAD ⁺ content in the mutants as well as in the wild type.     

Production of 1,3-Propanediol by Clostridium butyricum VPI 3266 in continuous cultures with high yield and productivity

The effects of dilution rate and substrate feed concentration on continuous glycerol fermentation by Clostridium butyricum VPI 3266, a natural 1,3-propanediol producer, were evaluated in this work. A high and constant 1,3-propanediol yield (around 0.65 mol/mol), close to the theoretical value, was obtained irrespective of substrate feed concentration or dilution rate. Improvement of 1,3-propanediol volumetric productivity was achieved by increasing the dilution rate, at a fixed feed substrate concentration of 30, 60 or 70 g l⁻¹. Higher 1,3-propanediol final concentrations and volumetric productivities were also obtained when glycerol feed concentration was increased from 30 to 60 g l⁻¹, at D=0.05–0.3 h⁻¹, and from 60–70 g l⁻¹, at D=0.05 and 0.1 h⁻¹·30 g l⁻¹ of 1,3-propanediol and the highest reported value of productivity, 10.3 g l⁻¹ h⁻¹, was achieved at D=0.30 h⁻¹ and 60 g l⁻¹ of feed glycerol. A switch to an acetate/butyrate ratio higher than one was observed for 60 g l⁻¹ of feed glycerol and a dilution rate higher than 0.10 h⁻¹; moreover, at D=0.30 h⁻¹ 3-hydroxypropionaldehyde accumulation was observed for the first time in the fermentation broth of C. butyricum.     

Determination of kinetic parameters of 1,3-propanediol fermentation by Clostridium diolis using statistically optimized medium

1,3-propanediol (1,3-PD) is a chemical compound of immense importance primarily used as a raw material for fiber and textile industry. It can be produced by the fermentation of glycerol available abundantly as a by-product from the biodiesel plant. The present study was aimed at determination of key kinetic parameters of 1,3-PD fermentation by Clostridium diolis. Initial experiments on microbial growth inhibition were followed by optimization of nutrient medium recipe by statistical means. Batch kinetic data from studies in bioreactor using optimum concentration of variables obtained from statistical medium design was used for estimation of kinetic parameters of 1,3-PD production. Direct use of raw glycerol from biodiesel plant without any pre-treatment for 1,3-PD production using this strain investigated for the first time in this work gave results comparable to commercial glycerol. The parameter values obtained in this study would be used to develop a mathematical model for 1,3-PD to be used as a guide for designing various reactor operating strategies for further improving 1,3-PD production. An outline of protocol for model development has been discussed in the present work.     

混合菌发酵1,3-丙二醇的初步研究

将表达酿酒酵母3-磷酸甘油脱氢酶基因(GPD1)和3-磷酸甘油酯酶基因(HOR2)的质粒PSE-gpd1-hor2转化到甘油激酶基因(glpK)和甘油脱氢酶基因(gldA)双缺失的大肠杆菌JM109C中,构建产甘油的工程菌JM109C/PSE-gpd1-hor2.接种JM109C/pSE-gpd1-hor2和Klebsiella在含1%葡萄糖的摇瓶发酵培养基中37℃发酵56 h,1,3-丙二醇的最高产量为1.28 g/L,葡萄糖摩尔转化率为37.5%;在30 L发酵罐中发酵68 h,1,3-丙二醇的最高产量为24.09 g/L,葡萄糖摩尔转化率为38.0%;5 g/L的乙酸、乳酸,10 g/L的乙醇分别使1,3-丙二醇的产量降低了91.41%、54.68%和51.56%.     

Production of 1,3-propanediol by Clostridium butyricum DSM 5431 and product tolerant mutants in fedbatch culture: Feeding strategy for glycerol and ammonium

Summary A fedbatch strategy was developed coupling the feeding of the two inhibitory substrates glycerol and ammonium to alkali consumption. A continuous, automated substrate addition was achieved responding directly to the needs of the culture. Thus substrate concentrations were kept on a constant low, but non limiting level. The feeding was applied for the cultivation of Clostridium butyricum DSM 5431 and mutants with increased product tolerance. Compared to fedbatch cultivations with intermittent feeding cultivation times were considerably shortened.     

Glycerol dehydratase activity: the limiting step for 1,3-propanediol production by Clostridium butyricum DSM 5431

S. ABBAD-ANDALOUSSI, E. GUEDON, E. SPIESSER AND H. PETITDEMANGE. 1996. Glycerol catabolism by Clostridium butyricum DSM 5431 into acetate, butyrate and 1,3-propanediol (1,3-PD) was studied in chemostat culture. The fact that the intracellular concentrations of NADH (18–22 μUmol g^-1dry cell mass) were extremely high suggested that the dehydratase activity was the rate limiting step in 1,3-PD formation. This limitation was proved by the addition of propionaldehyde, another substrate of propanediol dehydrogenase, into the culture medium. This resulted in an increase in (i) glycerol utilization, (ii) biomass formation and (iii) product biosynthesis.     

微生物菌种选育中的基因组改组技术及其应用进展

该文论述了基因组改组技术的产生和原理、方法和特点,以及该技术的应用、意义及其发展前景.基因组改组技术是首先对微生物菌株进行诱变,筛选出正向突变的菌株,然后通过原生质体"递推式融合"使这些正向突变的若干个菌株进行基因组重组,从中筛选出符合育种要求的重组子,从而在短时间内获得性状得到大幅度提高的菌株.     

Production of 1,3-propanediol by Clostridium butyricum in continuous culture with cell recycling

The continuous fermentation of 1,3-propanediol from glycerol by Clostridium butyricum was subjected to cell recycling by filtration using hollow-fibre modules made from polysulphone. The performance of the culture system was checked at a retention ratio (dilution rate/bleed rate) of 5, dilution rates between 0.2 h⁻¹ and 1.0 h⁻¹ and glycerol input concentrations of 32 g l⁻¹ and 56 g l⁻¹. The near-to-optimum propanediol concentration of 26.5 g l⁻¹ (for 56 g l⁻¹ glycerol) was maintained up to a dilution rate of 0.5 h⁻¹ and then decreased while the propanediol productivity was highest at 0.7 h⁻¹. The productivity could be increased by a factor of four in comparison to the continuous culture without cell recycling. By application of the model of Zeng and Deckwer [(1995) Biotechnol Prog 11: 71–79] for cultures under substrate excess, it was shown that the limitations resulted exclusively from product inhibition and detrimental influences from the cell recycling system, such as shear stress, were not involved. Received: 20 October 1997 / Received revision: 12 December 1997 / Accepted: 14 December 1997     

1,3-Propanediol production in a two-step process fermentation from renewable feedstock

In this work, the production of 1,3-propanediol from glucose and molasses was studied in a two-step process using two recombinant microorganisms. The first step of the process is the conversion of glucose or other sugar into glycerol by the metabolic engineered Saccharomyces cerevisiae strain HC42 adapted to high (>200 g l⁻¹) glucose concentrations. The second step, carried out in the same bioreactor, was performed by the engineered strain Clostridium acetobutylicum DG1 (pSPD5) that converts glycerol to 1,3-propanediol. This two-step strategy led to a flexible process, resulting in a 1,3-propanediol production and yield that depended on the initial sugar concentration. Below 56.2 g l⁻¹ of sugar concentration, cultivation on molasses or glucose showed no significant differences. However, at higher molasses concentrations, glycerol initially produced by yeast could not be totally converted into 1,3-propanediol by C. acetobutylicum and a lower 1,3-propanediol overall yield was observed. In our hand, the best results were obtained with an initial glucose concentration of 103 g l⁻¹, leading to a final 1,3-propanediol concentration of 25.5 g l⁻¹, a productivity of 0.16 g l⁻¹ h⁻¹ and 1,3-propanediol yields of 0.56 g g⁻¹ glycerol and 0.24 g g⁻¹ sugar, which is the highest value reported for a two-step process. For an initial sugar concentration (from molasses) of 56.2 g l⁻¹, 27.4 g l⁻¹ of glycerol were produced, leading to 14.6 g l⁻¹ of 1.3-propanediol and similar values of productivity, 0.15 g l⁻¹ h⁻¹, and overall yield, 0.26 g g⁻¹ sugar.     

Genetic engineering of Clostridium thermocellum DSM1313 for enhanced ethanol production

Background

The twin problem of shortage in fossil fuel and increase in environmental pollution can be partly addressed by blending of ethanol with transport fuel. Increasing the ethanol production for this purpose without affecting the food security of the countries would require the use of cellulosic plant materials as substrate. Clostridium thermocellum is an anaerobic thermophilic bacterium with cellulolytic property and the ability to produce ethanol. But its application as biocatalyst for ethanol production is limited because pyruvate ferredoxin oxidoreductase, which diverts pyruvate to ethanol production pathway, has low affinity to the substrate. Therefore, the present study was undertaken to genetically modify C. thermocellum for enhancing its ethanol production capacity by transferring pyruvate carboxylase (pdc) and alcohol dehydrogenase (adh) genes of the homoethanol pathway from Zymomonas mobilis.

Results

The pdc and adh genes from Z. mobilis were cloned in pNW33N, and transformed to Clostridium thermocellum DSM 1313 by electroporation to generate recombinant CTH-pdc, CTH-adh and CTH-pdc-adh strains that carried heterologous pdc, adh, and both genes, respectively. The plasmids were stably maintained in the recombinant strains. Though both pdc and adh were functional in C. thermocellum, the presence of adh severely limited the growth of the recombinant strains, irrespective of the presence or absence of the pdc gene. The recombinant CTH-pdc strain showed two-fold increase in pyruvate carboxylase activity and ethanol production when compared with the wild type strain.

Conclusions

Pyruvate decarboxylase gene of the homoethanol pathway from Z mobilis was functional in recombinant C. thermocellum strain and enhanced its ability to produced ethanol. Strain improvement and bioprocess optimizations may further increase the ethanol production from this recombinant strain.