Microbial production of 1,3-propanediol by Klebsiella pneumoniae using crude glycerol from biodiesel preparations

1,3-Propanediol (1,3-PD) was produced by Klebsiella pneumoniae using crude glycerol obtained from biodiesel production. The 1,3-PD concentration of 51.3 g/l⁻¹ on crude glycerol from alkali-catalyzed methanolysis of soybean oil was comparable to that of 53 g/l⁻¹ on crude glycerol derived from a lipase-catalyzed process. The productivities of 1.7 g l⁻¹ h⁻¹ on crude glycerol were comparable to that of 2 g l⁻¹ h⁻¹ on pure glycerol. It could be concluded that the crude glycerol could be directly converted to 1,3-PD without any prior purification.     

丁酸梭杆菌发酵甘油制备1,3-丙二醇的研究

本研究采用丁酸梭芽孢杆菌(Clostridium butyricum)从甘油发酵制备1,3—丙二醇。该发酵需厌氧培养,发酵培养基为：甘油6％,葡萄糖1％,玉米浆2％,(NH4)2SO40．2％。发酵温度为34℃,pH为6．5～7。在最佳条件下,发酵50h可产1,3—丙二醇40．7g／L,甘油摩尔转化率达68％。     

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...     

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.     

Radiation effects on microcrystalline cellulose in 1-butyl-3-methylimidazolium chloride ionic liquid

The radiation processing of cellulose in ionic liquids (ILs) demands a comprehensive knowledge of radiation effects on cellulose in ILs. Herein, gamma radiation-induced degradation kinetics of microcrystalline cellulose (MCC) in 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) was studied by viscometry. The intrinsic viscosity of MCC in [Bmim]Cl decreased slightly with increasing dose; while chemical structure and crystalline state of cellulose has no obvious change up to 300kGy. The radiation degradation rate constant (k) of MCC in [Bmim]Cl was 2.60×10(-7)/kGy, lower than that of solid cellulose, but higher than that in N-methylmorpholine-N-oxide (NMMO) solvent. Furthermore, k value decreased to 1.12×10(-7)/kGy in dimethyl sulfoxide (DMSO)/[Bmim]Cl system due to the free radicals scavenging of DMSO. The radicals generated during irradiation play main role in the radiation degradation of MCC in [Bmim]Cl. This work provides a new way to control the average molecular weight of cellulose by radiation-induced degradation of cellulose in ILs.     

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.     

Pathway and kinetic analysis of 1,3-propanediol production from glycerol fermentation by Clostridium butyricum

Stoichiometric analysis is applied to continuous glycerol fermentation by Clostridium butyricum to calculate theoretical maximum yields and to predict preferred pathways under different conditions. The upper limits of product concentration and productivity as a function of dilution rate in continuous culture is also predicted from product inhibition kinetic. The theoretical maximum propanediol yield (0.72 mol/mol glycerol) which is calculated for a culture without hydrogen and butyric acid formation agrees well with the experimental maximum value (around 0.71 mol/mol). Comparisons of experimental results (product concentration and productivity) with theoretical calculations and those of the glycerol fermentation by Klebsiella pneumoniae reveal that the production of 1,3-propanediol by C. butyricum is far below the optimum performance available with the present strain. One of the reasons is the relatively high formation of butyric acid under the culture conditions so far applied. The distribution of reducing equivalents to propanediol and hydrogen is also suboptimal. The utilization of the reducing power from pyruvate oxidation for propanediol production is about 60–70% of the theoretical maximum under the present experimental conditions.     

Metabolic engineering for the microbial production of 1,3-propanediol

Improvements in the biological production of 1,3-propanediol, a key component of an emerging polymer business, have been realized. Utilizing genes from natural strains that produce 1,3-propanediol from glycerol, metabolic engineering has enabled the development of a recombinant strain that utilizes the lower cost feedstock D-glucose. This accomplishment bodes well for future metabolic engineering efforts and, ultimately, for increased societal benefit obtained through the production of chemicals from renewable resources.     

Production of 1,3-propanediol by Klebsiella pneumoniae from glycerol broth

Broth containing 152 g glycerol l⁻¹ from Candida krusei culture was converted to 1,3-propanediol by Klebsiella pneumoniae. Residual glucose in the broth promoted growth of K. pneumoniae while acetate was inhibitory. After desalination treatment of glycerol broth by electrodialysis, the acetate in the broth was removed. A fed-batch culture with electrodialytically pretreated broth as␣substrate was developed giving 53 g 1,3- propanediol l⁻¹ with a yield of 0.41 g g⁻¹ glycerol and a productivity of 0.94 g l⁻¹ h⁻¹.     

Fermentative production of 1,3-propanediol from glycerol by Clostridium butyricum up to a scale of 2m3

Summary The conversion of glycerol to 1,3-propanediol (PD) by Clostridium butyricum DSM 5431 was studied in anaerobic culture. Growth and product formation were optimal at pH = 7.0 and T = 35° C, while aeration rate and stirrer speed were found to have no significant influence. As increasing amounts of initial glycerol led to inhibition of growth, cultivations were done in fed-batch operation. Comparative cultivations were carried out in an air-lift (ALR) and a stirred-tank reactor (STR) having equal working volumes (V _L = 30 l) and no difference in product formation was found. The process was scaled up to reactor sizes of 1.2 m³ (ALR) and 2.0 m³ (STR). The same results were obtained irrespective of reactor volume as well as reactor type (STR/ALR). PD concentrations of approximately 50–58 g·l^–1 and overall productivities of 2.3–2.9 g·l^–1 ·h^–1 could be reached. Offprint requests to: W.-D. Deckwer     

Simultaneous production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol by a recombinant strain of Klebsiella pneumoniae

In this study, an aldehyde dehydrogenase (ALDH) was over-expressed in Klebsiella pneumoniae for simultaneous production of 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO). Various genes encoding ALDH were cloned and expressed in K. pneumoniae, and expression of Escherichia colialdH resulted in the highest 3-HP titer in anaerobic cultures in shake flasks. Anaerobic fed-batch culture of this recombinant strain was further performed in a 5-L reactor. The 3-HP concentration and yield reached 24.4 g/L and 0.18 mol/mol glycerol, respectively, and at the same time 1,3-PDO achieved 49.3 g/L with a yield of 0.43 mol/mol in 24 h. The overall yield of 3-HP plus 1,3-PDO was 0.61 mol/mol. Over-expression of the E. coli AldH also reduced the yields of by-products except for lactate. This study demonstrated the possibility of simultaneous production of 3-HP and 1,3-PDO by K. pneumoniae under anaerobic conditions without supply of vitamin B₁₂.     

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.     

High production of 1,3-propanediol from industrial glycerol by a newly isolated Clostridium butyricum strain

Batch and continuous cultures of a newly isolated Clostridium butyricum strain were carried out on industrial glycerol, the major by-product of the bio-diesel production process. For both types of cultures, the conversion yield obtained was around 0.55 g of 1,3-propanediol formed per 1 g of glycerol consumed whereas the highest 1,3-propanediol concentration, achieved during the single-stage continuous cultures was 35-48 g l-1. Moreover, the strain presented a strong tolerance at the inhibitory effect of the 1,3-propanediol, even at high concentrations of this substance at the chemostat (e.g. 80 g l-1). 1,3-Propanediol was associated with cell growth whereas acetate and butyrate seemed non growth-associated products. At low and medium dilution rates (until 0.1 h-1), butyrate production was favoured, whereas at higher rates acetate production increased. The maximum 1,3-propanediol volumetric productivity obtained was 5.5 g l-1 h-1. A two-stage continuous fermentation was also carried out. The first stage presented high 1,3-propanediol volumetric productivity, whereas the second stage (with a lower dilution rate) served to further increase the final product concentration. High 1,3-propanediol concentrations were achieved (41-46 g l-1), with a maximum volumetric productivity of 3.4 g l-1 h-1. A cell concentration decrease was reported between the second and the first fermentor.     

Optimization of medium compositions favoring butanol and 1,3-propanediol production from glycerol by Clostridium pasteurianum

Medium compositions favoring butanol and 1,3-propanediol (1,3-PDO) production from glycerol by Clostridium pasteurianum DSM525 were investigated using statistical experimental designs. Medium components affecting butanol and 1,3-PDO production were screened using a fractional factorial experimental design. Among the six tested variables (phosphate buffer, MnSO4·H2O, MgSO4·7H2O, FeSO4·7H2O, (NH4)2SO4, and yeast extract), FeSO4·7H2O, (NH4)2SO4, and yeast extract were found to be significant variables for further optimization of medium using a Box-Behnken design. Optimal butanol (0.98 g/L/h) and 1,3-PDO (1.19 g/L/h) productivities were predicted by the corresponding quadratic model for each product and the models were validated experimentally under optimized conditions. The optimal medium composition for butanol production was significantly different from that for 1,3-PDO production (0.06 vs. 0 g/L for FeSO4·7H2O, 7.35 vs. 0 g/L for (NH4)2SO4, and 5.08 vs. 8.0 g/L for yeast extract), suggesting that the product formation from glycerol by C. pasteurianum DSM525 can be controlled by changing medium compositions.     

Metabolic engineering of Clostridium acetobutylicum for the industrial production of 1,3-propanediol from glycerol

Clostridium butyricum is to our knowledge the best natural 1,3-propanediol producer from glycerol and the only microorganism identified so far to use a coenzyme B12-independent glycerol dehydratase. However, to develop an economical process of 1,3-propanediol production, it would be necessary to improve the strain by a metabolic engineering approach. Unfortunately, no genetic tools are currently available for C. butyricum and all our efforts to develop them have been so far unsuccessful. To obtain a better "vitamin B12-free" biological process, we developed a metabolic engineering strategy with Clostridium acetobutylicum. The 1,3-propanediol pathway from C. butyricum was introduced on a plasmid in several mutants of C. acetobutylicum altered in product formation. The DG1(pSPD5) recombinant strain was the most efficient strain and was further characterized from a physiological and biotechnological point of view. Chemostat cultures of this strain grown on glucose alone produced only acids (acetate, butyrate and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized in chemostat culture, 1,3-propanediol became the major product, the specific rate of acid formation decreased and a very low level of hydrogen was observed. In a fed-batch culture, the DG1(pSPD5) strain was able to produce 1,3-propanediol at a higher concentration (1104 mM) and productivity than the natural producer C. butyricum VPI 3266. Furthermore, this strain was also successfully used for very long term continuous production of 1,3-propanediol at high volumetric productivity (3 g L-1 h-1) and titer (788 mM).     

The effects of cell recycling on the production of 1,3-propanediol by Klebsiella pneumoniae

The effects of both biomass age and cell recycling on the 1,3-propanediol (1,3-PDO) production by Klebsiella pneumoniae were investigated in a membrane-supported bioreactor using hollow-fiber ultrafiltration membrane module in two separate experiments. It was determined that older cells have a negative effect on 1,3-PDO production. The concentrations of by-products, such as acetic acid and ethanol, increased in cultures with older cells, whereas the concentrations of succinic acid, lactic acid and 2,3-butanediol decreased. The effect of cell recycling was comparatively studied at a cell recycling ratio of 100 %. The results showed that cell recycling had also negative effects on 1,3-PDO fermentation. It was hypothesized that both cell recycling and biomass age caused metabolic shifts to undesired by-products which then inhibited the 1,3-PDO production. On the other hand, the use of hollow-fiber ultrafiltration membrane module was found to be very effective in terms of removal of cells from the fermentation broth.     

Study of two-stage processes for the microbial production of 1,3-propanediol from glucose

The microbial production of 1,3-propanediol (1,3-PD) from glucose was studied in a two-stage fermentation process on a laboratory scale. In the first stage, glucose was converted to glycerol either by the osmotolerant yeast Pichia farinosa or by a recombinant Escherichia coli strain. In the second stage, glycerol in the broth from the first stage was converted to 1,3-PD by Klebsiella pneumoniae. The culture broth from P. farinosa was shown to contain toxic metabolites that strongly impair the growth of K. pneumoniae and the formation of 1,3-PD. Recombinant E. coli is more suitable than P. farinosa for producing glycerol in the first stage. The fermentation pattern from glycerol can be significantly altered by the presence of acetate, leading to a significant reduction of PD yield in the second stage. However, in the recombinant E. coli culture acetate formation can be prevented by fed-batch cultivation under limiting glucose supply, resulting in an effective production of 1,3-PD in the second stage with a productivity of 2.0 g l(-1) h(-1) and a high yield (0.53 g/g) close to that of glycerol fermentation in a synthetic medium. The overall 1,3-PD yield from glucose in the two stage-process with E. coli and K. pneumoniae reached 0.17 g/g.