Fermentation of glycerol to 1,3-propanediol by Klebsiella and Citrobacter strains

Summary Glycerol-fermenting anaerobes were enriched with glycerol at low and high concentrations in order to obtain strains that produce 1,3-propanediol. Six isolates were selected for more detailed characterization; four of them were identified as Citrobacter freundii, one as Klebsiella oxytoca and one as K. pneumoniae. The Citrobacter strains formed 1.3-propanediol and acetate and almost no by-products, while the Klebsiella strains produced varying amounts of ethanol in addition and accordingly less 1,3-propanediol. Enterobacterial strains of the genera Enterobacter, Klebsiella, and Citrobacter from culture collections showed similar product patterns except for one group which formed limited amounts of ethanol, but no propanediol. Seven strains were grown in pH-controlled batch cultures to determine the parameters necessary to evaluate their capacity for 1,3-propanediol production. K. pneumoniae DSM 2026 exhibited the highest final concentration (61 g/l) and the best productivity (1.7 g/l h) whereas C. freundii Zu and K2 achieved only 35 g/l and 1.4 g/l h, respectively. The Citrobacter strains on the other hand gave somewhat better yields which were very close to the theoretical optimum of 65 mol %. Offprint requests to: H. Biebl     

弗氏柠檬酸菌1,3-丙二醇合成的代谢工程研究

【目的】研究弗氏柠檬酸菌(Citrobacter freundii) 1,3-丙二醇合成的代谢过程。【方法】构建甘油脱氢酶基因GSR-lacZ、1,3-丙二醇氧化还原酶基因PDO-lacZ和甘油脱水酶基因GL-lacZ等报告基因。在此基础上,构建3个相应的转座子突变文库。【结果】筛选到6株突变子,其相应关键酶表达水平提高1?11倍,1,3-丙二醇产量提高幅度为3%?50%。对转座子插入位点分析显示,5株突变子插入位点均为β-内酰胺酶(CKO_02592)编码基因,1株突变子插入位点为二氢硫辛酰胺基转移酶(CKO_02433)编码基因。进一步分析发现,β-内酰胺酶基因突变显著提高甘油脱水酶和甘油脱氢酶的表达水平,而1,3-丙二醇氧化还原酶表达水平没有变化;二氢硫辛酰胺基转移酶基因突变显著提高1,3-丙二醇氧化还原酶表达水平,其他两种关键酶基因表达水平不变。【结论】β-内酰胺酶和二氢硫辛酰胺基转移酶基因能够分别影响1,3-丙二醇合成代谢途径关键酶的表达,为构建工程菌株打下基础。     

Fermentation of glycerol to 1,3-propanediol: use of cosubstrates

Three fermentable substances, glucose, 1,2-ethanediol and 1,2-propanediol were checked as cosubstrates for the fermentation of glycerol by Clostridium butyricum and Citrobacter freundii with the aim of achieving a complete conversion of glycerol to 1,3-propanediol. Glucose was fermented by C. butyricum mainly to acetate, CO₂ and reducing equivalents in the presence of glycerol and contributed markedly to the 1,3-propanediol yield. However, because of relatively slow growth on glucose, complete conversion was not achieved. If the two glycols were used as cosubstrates for glycerol fermentation, the 1,3-propanediol yield did not increase but dimished considerably, as they were converted to more reduced products, i.e. alcohols instead of acids. From 1,2-propanediol 2-propanol was formed in addition to 1-propanol. The ratio of the propanols was dependent on the culture conditions.     

A comparative study of solvent-assisted pretreatment of biodiesel derived crude glycerol on growth and 1,3-propanediol production from Citrobacter freundii

The rapidly growing biodiesel industry has created a scenario, where it is both important and challenging to deal with the enormous amount of crude glycerol generated as an inherent by-product. With every 100 gallons of biodiesel produced, 5-10 gallons of the crude glycerol is left behind containing several impurities which makes its disposal difficult. The objective of the present investigation was to evaluate the impact of biodiesel-derived crude glycerol upon microbial growth and production of 1,3-propanediol by Citrobacter freundii. Five different grades of crude glycerol (obtained from biodiesel preparation using jatropha, soybean, sunflower, rice bran and linseed oils) were used. Crude glycerol caused significant inhibition of microbial growth and subsequently 1,3-propanediol production as compared to pure glycerol. Therefore, a process was developed for the treatment of crude glycerol using solvents before fermentation wherein four different non-polar solvents were examined yielding different grades of pretreated glycerol. Subsequently, the potential toxic effects of pretreated glycerol on the growth and 1,3-propanediol production by C. freundii was evaluated. In case of petroleum ether-treated crude glycerol obtained from jatropha & linseed and hexane-treated crude glycerol obtained from rice bran, the yields obtained were comparable to the pure glycerol. Similarly, soybean-derived glycerol gave comparable results after treatment with either hexane or petroleum ether.     

Optimized 1,3-propanediol production from crude glycerol using mixed cultures in batch and continuous reactors

Bioprocess and Biosystems Engineering - The production of 1,3-propanediol from crude glycerol and mixed anaerobic sludge was investigated in batch experiments and continuous reactors. Using a 23...     

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     

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.     

Development of an immobilized cell reactor for the production of 1,3-propanediol by Citrobacter freundii

Citrobacter freundii DSM 30040 immobilized on modified polyurethane carrier particles PUR 90/16 was used for continuous glycerol fermentation in an anaerobic fixed bed reactor with effluent recycle and pH control (fixed bed loop reactor). The fermentor was run with buffered mineral medium under growth conditions resulting in the permanent renewal of active biomass. The effects of glycerol concentration in the feed, dilution rate (D), pH and temperature (T) were investigated to optimize the process. With 400 mm glycerol in the feed, pH 6.9, T = 36°C and D = 0.5 h^–1 the maximum productivity could be determined as 8.2 g/l per hour of 1,3-propanediol.     

Microbial conversion of glycerol to 1,3-propanediol

Abstract: Glycerol produced by cleavage of natural fats can microbially be converted to 1,3-propanediol (PD) by Citrobacter, Klebsiella and Clostridia strains. The fermentation by C. butyricum , product recovery and purification has been investigated in detail up to the 2 m³ scale. Estimation of product costs for a 10,000 t/a plant indicates that the microbial process is obviously more attractive than the chemical route. Presently, 1,3-propanediol has only a low market volume; however, its use for special polycondensates, in particular polyesters, could reduce glycerol surpluses and make plastics a easily biodegradable part of natural cycles.     

Elevated 3-hydroxypropionaldehyde production from glycerol using a Citrobacter freundii mutant

A mutant strain of Citrobacter freundii capable of elevated 3-hydroxypropionaldehyde production from glycerol was isolated using chemical mutagenesis and a screening protocol. The protocol involved screening mutagenized bacterial cells on solid minimal medium containing 5 % (v/v) glycerol. Colonies were picked onto duplicate solid minimal medium plates and one plate was stained with 1 % (w/v) phloroglucinol. Those colonies staining red were further screened and a mutant, HPAO-1, was identified. The mutant strain produced a several-fold higher 3-hydroxypropionaldehyde concentration than did the parent strain when grown on 5 % (v/v) glycerol. The ratio of culture volume to flask volume influenced 3-hydroxypropionaldehyde production by the mutant cells compared to the parent cells. Aldehyde production was highest when the mutant strain was grown on 5 % (v/v) glycerol at a ratio of culture volume to flask volume of 1:3 or 1:12.5.     

弗氏柠檬酸菌甘油脱水酶基因在大肠杆菌中的克隆和表达

以弗氏柠檬酸菌（Citrobacter freundii）基因组DNA为模板,通过PCR得到甘油脱水酶（glycerol dehydratase）基因dhaB、dhaC、dhaE,克隆到表达载体pSE380上,得到重组质粒pSn-dhaBCE。将此重组质粒转化到E．coli JM109中,重组菌株SDS-PAGE结果显示有明显的61kD、22kD、16kD三条特异性蛋白条带出现。重组菌株经诱导表达,酶活力为11．59U／mL。     

Efficient production of 1,3-propanediol from fermentation of crude glycerol with mixed cultures in a simple medium

The objective of this study was to examine the applicability of mixed cultures for 1,3-propanediol (1,3-PDO) production from crude glycerol. Three different sources of mixed cultures were tested, where the mixed culture from a municipal wastewater treatment plant showed the best results. 1,3-PDO can be produced as the main product in this mixed culture with typical organic acids like acetic and butyric acids as by-products. The yield was in the range of 0.56–0.76 mol 1,3-PDO per mol glycerol consumed depending on the glycerol concentration. A final product concentration as high as 70 g/L was obtained in fed-batch cultivation with a productivity of 2.6 g/L h. 1,3-PDO can be kept in the culture several days after termination of the fermentation without being degraded. Degradation tests showed that 1,3-PDO is degraded much slower than other compounds in the fermentation broth. In comparison to 1,3-PDO production in typical pure cultures, the process developed in this work with a mixed culture achieved the same levels of product titer, yield and productivity, but has the decisive advantage of operation under complete non-sterile conditions. Moreover, a defined fermentation medium without yeast extract can be used and nitrogen gassing can be omitted during cultivation, leading to a strong reduction of investment and production costs.     

Purification of 1,3-propanediol dehydrogenase from Citrobacter freundii and cloning, sequencing, and overexpression of the corresponding gene in Escherichia coli.

1,3-Propanediol dehydrogenase (EC 1.1.1.202) was purified to homogeneity from Citrobacter freundii grown anaerobically on glycerol in continuous culture. The enzyme is an octamer of a polypeptide of 43,400 Da. When tested as a dehydrogenase, the enzyme was most active with substrates containing two primary alcohol groups separated by one or two carbon atoms. In the physiological direction, 3-hydroxypropionaldehyde was the preferred substrate. The apparent Km values of the enzyme for 3-hydroxypropionaldehyde and NADH were 140 and 33 microM, respectively. The enzyme was inhibited by chelators of divalent cations but could be reactivated by the addition of Fe2+. The dhaT gene, encoding the 1,3-propanediol dehydrogenase, was cloned, and its nucleotide sequence (1,164 bp) was determined. The deduced dhaT gene product (387 amino acids, 41,324 Da) showed a high level of similarity to a novel family (type III) of alcohol dehydrogenases. The dhaT gene was overexpressed in Escherichia coli 274-fold by using the T7 RNA polymerase/promoter system.     

Expression of 1,3-propanediol oxidoreductase and its isoenzyme in Klebsiella pneumoniae for bioconversion of glycerol into 1,3-propanediol

In the Klebsiella pneumoniae reduction pathway for 1,3-propanediol (1,3-PD) synthesis, glycerol is first dehydrated to 3-hydroxypropionaldehyde (3-HPA) and then reduced to 1,3-PD with NADH consumption. Rapid conversion of 3-HPA to 1,3-PD is one of the ways to improve the yield of 1,3-PD from glycerol and to avoid 3-HPA accumulation, which depends on enzyme activity of the reaction and the amount of reducing equivalents available from the oxidative pathway of glycerol. In the present study, the yqhD gene, encoding 3-propanediol oxidoreductase isoenzyme from Escherichia coli and the dhaT gene, encoding 3-propanediol oxidoreductase from K. pneumoniae were expressed individually and co-expressed in K. pneumoniae using the double tac promoter expression plasmid pEtac-dhaT-tac-yqhD. The three resultant recombinant strains (K. pneumoniae/pEtac-yqhD, K. pneumoniae/pEtac-dhaT, and K. pneumoniae/pEtac-dhaT-tac-yqhD) were used for fermentation studies. Experimental results showed that the peak values for 3-HPA production in broth of the three recombinant strains were less than 25% of that of the parent strain. Expression of dhaT reduced formation of by-products (ethanol and lactic acid) and increased molar yield of 1,3-PD slightly, while expression of yqhD did not enhance molar yield of 1,3-PD, but increased ethanol concentration in broth as NADPH participation in transforming 3-HPA to 1,3-PD allowed more cellular NADH to be used to produce ethanol. Co-expression of both genes therefore decreased by-products and increased the molar yield of 1,3-PD by 11.8%, by catalyzing 3-HPA conversion to 1,3-propanediol using two cofactors (NADH and NADPH). These results have important implications for further studies involving use of YqhD and DhaT for bioconversion of glycerol into 1,3-PD.     

Fermentation strategies for 1,3-propanediol production from glycerol using a genetically engineered Klebsiella pneumoniae strain to eliminate by-product formation

We generated a genetically engineered Klebsiella pneumoniae strain (AK-VOT) to eliminate by-product formation during production of 1,3-propanediol (1,3-PD) from glycerol. In the present study, the glycerol-metabolizing properties of the recombinant strain were examined during fermentation in a 5 L bioreactor. As expected, by-product formation was completely absent (except for acetate) when the AK-VOT strain fermented glycerol. However, 1,3-PD productivity was severely reduced owing to a delay in cell growth attributable to a low rate of glycerol consumption. This problem was solved by establishing a two-stage process separating cell growth from 1,3-PD production. In addition, nutrient co-supplementation, especially with starch, significantly increased 1,3-PD production from glycerol during fed-batch fermentation by AK-VOT in the absence of by-product formation.     

Physiological predisposition of various Clostridium species to synthetize 1,3-propanediol from glycerol

1,3-Propanediol (1,3-PD) production by fermentation of glycerol was first described in 1881 but little attention was paid to this biosynthesis for over a century. An increasing interest in microbial 1,3-PD production is observed since late 1980s. The high growth rate of the biofuel market and the perspective of glycerol becoming abundant attract even more attention to this valuable chemical.Glycerol conversion to 1,3-PD is known to occur in Clostridia, Enterobacteriaceae and Lactobacillaceae. Some clostridial species are among the best 1,3-PD producers.This work is a review of the current state of research on Clostridium spp. strains that ferment glycerol to 1,3-PD. It focuses on the metabolic pathways and factors that influence the production of this diol. The effects of different environmental stresses on the process of 1,3-PD synthesis are also covered. Moreover, various genetic engineering methods utilized in order to improve the capabilities of bacteria used in this process are presented.     

Conversion of glycerol to 1,3-propanediol by a newly isolated thermophilic strain

Of 60 different thermophilic enrichment cultures, 16 converted glycerol anaerobically to 1,3-propanediol. Two PD-forming strains were further enriched, isolated, and characterised. For the most active strain, AT1, the optimal cultivation parameters for pH and temperature were determined as 5.8 to 6.0 and 58°C, respectively. In batch-fermentations with AT1, 6.4 g propanediol per litre was formed with a productivity of 0.17 g l^–1 h^–1.     

Identification and utilization of a 1,3-propanediol oxidoreductase isoenzyme for production of 1,3-propanediol from glycerol in Klebsiella pneumoniae

In a previous study, we showed that 1,3-propanediol (1,3-PD) was still produced from glycerol by the Klebsiella pneumoniae mutant strain defective in 1,3-PD oxidoreductase (DhaT), although the production level was lower compared to the parent strain. As a potential candidate for another putative 1,3-PD oxidoreductase, we identified and characterized a homolog of Escherichia coli yqhD (88% homology in amino acid sequence), which encodes an alcohol dehydrogenase and is well known to replace the function of DhaT in E. coli. Introduction of multiple copies of the yqhD homolog restored 1,3-PD production in the mutant K. pneumoniae strain defective in DhaT. In addition, by-product formation was still eliminated in the recombinant strain due to the elimination of the glycerol oxidative pathway. An increase in NADP-dependent 1,3-PD oxidoreductase activity was observed in the recombinant strain harboring multiple copies of the yqhD homolog. The level of 1,3-PD production during batch fermentation in the recombinant strain was comparable to that of the parent strain; further engineering can generate an industrial strain producing 1,3-propanediol.