Microbial Conversion of Glycerol to 1,3-Propanediol: Physiological Comparison of a Natural Producer, Clostridium butyricum VPI 3266, and an Engineered Strain, Clostridium acetobutylicum DG1(pSPD5)

Clostridium acetobutylicum is not able to grow on glycerol as the sole carbon source since it cannot reoxidize the excess of NADH generated by glycerol catabolism. Nevertheless, when the pSPD5 plasmid, carrying the NADH-consuming 1,3-propanediol pathway from C. butyricum VPI 3266, was introduced into C. acetobutylicum DG1, growth on glycerol was achieved, and 1,3-propanediol was produced. In order to compare the physiological behavior of the recombinant C. acetobutylicum DG1(pSPD5) strain with that of the natural 1,3-propanediol producer C. butyricum VPI 3266, both strains were grown in chemostat cultures with glycerol as the sole carbon source. The same “global behavior” was observed for both strains: 1,3-propanediol was the main fermentation product, and the qH₂ flux was very low. However, when looking at key intracellular enzyme levels, significant differences were observed. Firstly, the pathway for glycerol oxidation was different: C. butyricum uses a glycerol dehydrogenase and a dihydroxyacetone kinase, while C. acetobutylicum uses a glycerol kinase and a glycerol-3-phosphate dehydrogenase. Secondly, the electron flow is differentially regulated: (i) in C. butyricum VPI 3266, the in vitro hydrogenase activity is 10-fold lower than that in C. acetobutylicum DG1(pSPD5), and (ii) while the ferredoxin-NAD⁺ reductase activity is high and the NADH-ferredoxin reductase activity is low in C. acetobutylicum DG1(pSPD5), the reverse is observed for C. butyricum VPI 3266. Thirdly, lactate dehydrogenase activity is only detected in the C. acetobutylicum DG1(pSPD5) culture, explaining why this microorganism produces lactate.     

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).     

Impact of anaerobiosis strategy and bioreactor geometry on the biochemical response of Clostridium butyricum VPI 1718 during 1,3-propanediol fermentation

The impact of anaerobiosis strategy on 1,3-propanediol production during cultivation of Clostridium butyricum VPI 1718 in different size bioreactors was studied. In batch trials with N2 gas infusion, the fermentation was successfully accomplished, regardless of initial glycerol concentration imposed and bioreactor geometry. However, in the absence of N2 continual sparging, significant variations concerning the biochemical response of the strain were observed. Specifically, at 1-L bioreactor, the absence of N2 infusion at high initial glycerol concentration induced lactate dehydrogenase activity and thus lactic acid synthesis, probably due to partial blockage of phosphoroclastic reaction caused by insufficient self-generated anaerobiosis environment. During fed-batch cultivation with continual N2 sparging, the strain produced ～71 g L(-1) of 1,3-propanediol, whereas under self-generated anaerobiosis, 1,3-propanediol pathway was evidently restricted, as only 30.5 g L(-1) of 1,3-propanediol were finally produced. Apparently, N2 infusion strategy paired with bioreactor geometry can alter the biochemical behavior of the particular strain.     

Regulation of carbon and electron flow in Clostridium butyricum VPI 3266 grown on glucose-glycerol mixtures

The metabolism of Clostridium butyricum was manipulated at pH 6.5 and in phosphate-limited chemostat culture by changing the overall degree of reduction of the substrate using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids (acetate, butyrate, and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized, 1,3-propanediol became the major product, the specific rate of acid formation decreased, and a low level of hydrogen was observed. Glycerol consumption was associated with the induction of (i) a glycerol dehydrogenase and a dihydroxyacetone kinase feeding glycerol into the central metabolism and (ii) an oxygen-sensitive glycerol dehydratase and an NAD-dependent 1,3-propanediol dehydrogenase involved in propanediol formation. The redirection of the electron flow from hydrogen to NADH formation was associated with a sharp decrease in the in vitro hydrogenase activity and the acetyl coenzyme A (CoA)/free CoA ratio that allows the NADH-ferredoxin oxidoreductase bidirectional enzyme to operate so as to reduce NAD in this culture. The decrease in acetate and butyrate formation was not explained by changes in the concentration of phosphotransacylases and acetate and butyrate kinases but by changes in in vivo substrate concentrations, as reflected by the sharp decrease in the acetyl-CoA/free CoA and butyryl-CoA/free CoA ratios and the sharp increase in the ATP/ADP ratio in the culture grown with glucose and glycerol compared with that in the culture grown with glucose alone. As previously reported for Clostridium acetobutylicum (L. Girbal, I. Vasconcelos, and P. Soucaille, J. Bacteriol. 176:6146-6147, 1994), the transmembrane pH of C. butyricum is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased (cultures grown on glucose-glycerol mixture). For both cultures, the stoichiometry of the H(+) ATPase was shown to remain constant and equal to 3 protons exported per molecule of ATP consumed.     

Production of 1,3-propanediol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> VPI 3266 using a synthetic medium and raw glycerol

Growth inhibition of Clostridium butyricum VPI 3266 by raw glycerol, obtained from the biodiesel production process, was evaluated. C. butyricum presents the same tolerance to raw and to commercial glycerol, when both are of similar grade, i.e. above 87% (w/v). A 39% increase of growth inhibition was observed in the presence of 100 g l^–1 of a lower grade raw glycerol (65% w/v). Furthermore, 1,3-propanediol production from two raw glycerol types (65% w/v and 92% w/v), without any prior purification, was observed in batch and continuous cultures, on a synthetic medium. No significant differences were found in C. butyricum fermentation patterns on raw and commercial glycerol as the sole carbon source. In every case, 1,3-propanediol yield was around 0.60 mol/mol glycerol consumed.     

Taxonomy of the glycerol fermenting clostridia and description of Clostridium diolis sp. nov

Six Clostridium strains which ferment glycerol to 1,3-propanediol were tested for their taxonomic and phylogenetic relatedness. All but one were known as C butyricum. By physiological tests, 16S rDNA sequences and fatty acid composition two groups were distinguished. The first comprised the strains VPI 3266, DSM 2478 and DSM 523 (C. "kainantoi") and was consistent with the type strain of C. butyricum in almost all characters. The second group comprising the strains DSM 5430, DSM 5431 and E5 was related to C. beijerinckii. The 16S rDNAs of these strains were almost identical with that of the type strain of C. beijerinckii, DSM 791. The DNA-DNA hybridization value of DSM 5431 and ES with C. beijerinckii DSM 791 was markedly but not decisively lower (67 and 72%, respectively). However, there were significant physiological differences to C. beijerinckii which suggested to describe the strains as a separate species, Clostridium diolis with strain SH1 (= DSM 5431) as the type strain. The new species is distinguished from C. beijerinckii, which requires complex nutrients, by its ability to grow in glucose mineral medium with biotin as the only growth factor and by differences in substrate utilization. "C. kainantoi" Takeda and Matsui was recognized as a later synonym of C. butyricum.     

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.     

Separation and characterization of the 1,3-propanediol and glycerol dehydrogenase activities from Clostridium butyricum E5 wild-type and mutant D

AIMS: Clostridium butyricum E5 wild-type and mutant E5-MD were cultivated in chemostat culture on glycerol in order to compare the properties of two key enzymes of glycerol catabolism, i.e. propanediol and glycerol dehydrogenase. METHODS AND RESULTS: These two enzymes, which belong to the dha regulon, were separated by gel filtration. Both dehydrogenase activities displayed similar properties, such as pH optimum values, specificity towards physiological substrates and dependence on Mn2+. Both strains accumulate glycerol at high levels. CONCLUSION: The mutant D strain contained a propanediol dehydrogenase activity which had a low affinity for its physiological substrate, leading to the conclusion that this strain would seem more resistant to the toxic effect of 3-hydroxypropionaldehyde than the wild-type. SIGNIFICANCE AND IMPACT OF THE STUDY: These properties make Cl. butyricum mutant D strain the best candidate so far to be used as a biotechnological agent for the bioconversion of glycerol to 1,3-propanediol.     

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.     

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.     

Adaptation dynamics of Clostridium butyricum in high 1,3-propanediol content media

Aim of the present study was to evaluate the effect of exogenous additions of 1,3-propanediol (1,3-PDO) on microbial growth and metabolites production of Clostridium butyricum VPI 1718 strain, during crude glycerol fermentation. Preliminary batch cultures in anaerobic Duran bottles revealed that early addition of 1,3-PDO caused growth cessation in rather low quantities (15?g/L), while 1,3-PDO additions during the middle exponential growth phase up to 70?g/L resulted in an almost linear decrease of the specific growth rate (μ), accompanied by reduced glycerol assimilation, with substrate consumption being used mainly for energy of maintenance requirements. During batch trials in a 3-L bioreactor, the strain proved able to withstand more than 70?g/L of both biologically produced and externally added 1,3-PDO, whereas glycerol assimilation and metabolite production were carried on at a lower rate. Adaptation of the strain in high 1,3-PDO concentration environments was validated during its continuous cultivation with pulses of 1,3-PDO in concentrations of 31 and 46?g/L, where no washout phenomena were noticed. As far as C. butyricum cellular lipids were concerned, during batch bioreactor cultivations, 1,3-PDO addition was found to favor the biosynthesis of unsaturated fatty acids. Also, fatty acid composition was studied during continuous cultures, in which additions of 1,3-PDO were performed at steady states. Lipids were globally more saturated compared to batch cultures, while by monitoring of the transitory phases, it was noticed that the gradual diol washout had an evident impact in the alteration of the fatty acid composition, by rendering them more unsaturated.     

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,     

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.     

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.     

Carbon and electron flow in Clostridium butyricum grown in chemostat culture on glucose-glycerol mixtures

Summary The metabolism of C. butyricum was manipulated, at neutral pH and in carbon limited chemostat cultures by changing the overall degree of reduction of the substrate, using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids (acetate, butyrate and lactate). When the glycerol (in C moles)/glucose+glycerol (in C moles) ratio was progressively changed from 0 to 1 a corresponding increase of 1,3-propanediol production occured and an immediate and drastic decrease of the specific rate of acetate production was observed while the specific rate of butyrate production only decreased slightly. For glycerol (in C moles)/glucose+glycerol (in C moles) ratios higher than 0.5, the q_{NAD(P)H from Fd} and the CO₂/H₂ molar ratio increased sharply, the first becoming positive and the second higher than 1. This indicates a complete reversion of the electron flow: part of reduced ferredoxin produced by the phosphoroclastic cleavage of pyruvate to acetyl-CoA was diverted from H₂ formation toward NAD(P) reduction by the ferredoxin-NAD(P) reductase(s) in order to produce NAD(P)H. This change in the electron flow was associated to an increase in the specific rate and the yield of 1,3-propanediol production related to glycerol.     

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.     

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。     

Inactivation of <Emphasis Type="Italic">dhaD</Emphasis> and <Emphasis Type="Italic">dhaK</Emphasis> abolishes by-product accumulation during 1,3-propanediol production in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

1,3-Propanediol (1,3-PD) can be used for the industrial synthesis of a variety of compounds, including polyesters, polyethers, and polyurethanes. 1,3-PD is generated from petrochemical and microbial sources. 1,3-Propanediol is a typical product of glycerol fermentation, while acetate, lactate, 2,3-butanediol, and ethanol also accumulate during the process. Substrate and product inhibition limit the final concentration of 1,3-propanediol in the fermentation broth. It is impossible to increase the yield of 1,3-propanediol by using the traditional whole-cell fermentation process. In this study, dhaD and dhaK, the genes for glycerol dehydrogenase and dihydroxyacetone kinase, respectively, were inactivated by homologous recombination in Klebsiella pneumoniae. The dhaD/dhaK double mutant (designated TC100), selected from 5,000 single or double cross homologous recombination mutants, was confirmed as a double cross by using polymerase chain reaction. Analysis of the cell-free supernatant with high-performance liquid chromatography revealed elimination of lactate and 2,3-butanediol, as well as ethanol accumulation in TC100, compared with the wild-type strain. Furthermore, 1,3-propanediol productivity was increased in the TC100 strain expressing glycerol dehydratase and 1,3-PDO dehydrogenase regulated by the arabinose P_BAD promoter. The genetic engineering and medium formulation approaches used here should aid in the separation of 1,3-propanediol from lactate, 2,3-butanediol, and ethanol and lead to increased production of 1,3-propanediol in Klebsiella pneumoniae.     

Identification of the gene encoding NADH-rubredoxin oxidoreductase in Clostridium acetobutylicum.

NADH-rubredoxin oxidoreductase (NROR), a flavoprotein from the obligately anaerobe Clostridium acetobutylicum is encoded by an ORF (nror) of 1140 nucleotides. Whereas primary structure analysis reveals that NROR has amino acid sequence patterns homologous with those involved in FAD and NAD-binding, the enzyme is distantly related to other flavoproteins in the databank. NROR is highly active for reducing clostridial rubredoxin (Rd) especially against C. acetobutylicum Rd with an efficiency (k(cat)/K(m)) of 400,000 mM(-1)s(-1). These results suggest that Rd from C. acetobutylicum, C. pasteurianum, C. butyricum, and C. cellulolyticum can be interchanged with each other. Since C. acetobutylicum is the sole Clostridium strain that possesses such an enzyme, possible functions are discussed with regard to Desulfovibrio gigas and Pyrococcus furiosus, the only two other anaerobic systems for which a similar activity was reported, but no gene isolated.