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.     

Disruption of the acetate kinase (<Emphasis Type="Italic">ack</Emphasis>) gene of <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> results in delayed acetate production

In microorganisms, the enzyme acetate kinase (AK) catalyses the formation of ATP from ADP by de-phosphorylation of acetyl phosphate into acetic acid. A mutant strain of Clostridium acetobutylicum lacking acetate kinase activity is expected to have reduced acetate and acetone production compared to the wild type. In this work, a C. acetobutylicum mutant strain with a selectively disrupted ack gene, encoding AK, was constructed and genetically and physiologically characterized. The ack (-) strain showed a reduction in acetate kinase activity of more than 97% compared to the wild type. The fermentation profiles of the ack (-) and wild-type strain were compared using two different fermentation media, CGM and CM1. The latter contains acetate and has a higher iron and magnesium content than CGM. In general, fermentations by the mutant strain showed a clear shift in the timing of peak acetate production relative to butyrate and had increased acid uptake after the onset of solvent formation. Specifically, in acetate containing CM1 medium, acetate production was reduced by more than 80% compared to the wild type under the same conditions, but both strains produced similar final amounts of solvents. Fermentations in CGM showed similar peak acetate and butyrate levels, but increased acetoin (60%), ethanol (63%) and butanol (16%) production and reduced lactate (-50%) formation by the mutant compared to the wild type. These findings are in agreement with the proposed regulatory function of butyryl phosphate as opposed to acetyl phosphate in the metabolic switch of solventogenic clostridia.     

Fermentation of 1,3-propanediol by a lactate deficient mutant of Klebsiella oxytoca under microaerobic conditions

Klebsiella oxytoca M5al is an excellent 1,3-propanediol (1,3-PD) producer, but too much lactic acid yielded greatly lessened the fermentation efficiency for 1,3-PD. To counteract the disadvantage, four lactate deficient mutants were obtained by knocking out the ldhA gene of lactate dehydrogenase (LDH) of K. oxytoca M5al. The LDH activities of the four mutants were from 3.85 to 6.92% of the parental strain. The fed-batch fermentation of 1,3-PD by mutant LDH3, whose LDH activity is the lowest, was studied. The results showed that higher 1,3-PD concentration, productivity, and molar conversion rate from glycerol to 1,3-PD can be gained than those of the wild type strain and no lactic acid is produced under both anaerobic and microaerobic conditions. Sucrose fed during the fermentation increased the conversion and sucrose added at the beginning increased the productivity. In fed-batch fermentation with sucrose as cosubstrate under microaerobic conditions, the 1,3-PD concentration, conversion, and productivity were improved significantly to 83.56 g l⁻¹, 0.62 mol mol⁻¹, and 1.61 g l⁻¹ h⁻¹, respectively. Furthermore, 60.11 g l⁻¹ 2,3-butanediol was also formed as major byproduct in the broth.     

Clostridium acetobutylicum mutants isolated for resistance to the pyruvate halogen analogs

Pyruvate halogen analogs, 3-fluoropyruvate and 3-bromopyruvate, are toxic towardClostridium acetobutylicum. After mutagenesis with nitrosoguanidine, mutants resistant to these compounds were selected. In a normal batch culture regulated at pH 4.8, mutants quickly initiated the acid-solvent transition, accumulating more acetoin and lactate than the wild type strain. The maximum rate of specific glucose uptake was higher in the mutants than in the wild type: 1.76 h^–1 and 0.9 h^–1 respectively. When the pH was uncontrolled, mutants converted glucose into solvents, essentially butanol, while the wild-type strain ceased its fermentation at the acidogenic stage. Enzymatic investigations revealed that acetate kinase, butyrate kinase, and acetoacetate decarboxylase activities decreased sooner in the mutants than in the parent strain.     

Construction and characterization of pta gene-deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid fermentation

Clostridium tyrobutyricum ATCC 25755 is an acidogenic bacterium, producing butyrate and acetate as its main fermentation products. In order to decrease acetate and increase butyrate production, integrational mutagenesis was used to disrupt the gene associated with the acetate formation pathway in C. tyrobutyricum. A nonreplicative integrational plasmid containing the phosphotransacetylase gene (pta) fragment cloned from C. tyrobutyricum by using degenerate primers and an erythromycin resistance cassette were constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome inactivated the target pta gene and produced the pta-deleted mutant (PTA-Em), which was confirmed by Southern hybridization. SDS-PAGE and two-dimensional protein electrophoresis results indicated that protein expression was changed in the mutant. Enzyme activity assays using the cell lysate showed that the activities of PTA and acetate kinase (AK) in the mutant were reduced by more than 60% for PTA and 80% for AK. The mutant grew more slowly in batch fermentation with glucose as the substrate but produced 15% more butyrate and 14% less acetate as compared to the wild-type strain. Its butyrate productivity was approximately 2-fold higher than the wild-type strain. Moreover, the mutant showed much higher tolerance to butyrate inhibition, and the final butyrate concentration was improved by 68%. However, inactivation of pta gene did not completely eliminate acetate production in the fermentation, suggesting the existence of other enzymes (or pathways) also leading to acetate formation. This is the first-reported genetic engineering study demonstrating the feasibility of using a gene-inactivation technique to manipulate the acetic acid formation pathway in C. tyrobutyricum in order to improve butyric acid production from glucose.     

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.     

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.     

1,3-Propanediol production by Escherichia coli expressing genes from the Klebsiella pneumoniae dha regulon.

The dha regulon in Klebsiella pneumoniae enables the organism to grow anaerobically on glycerol and produce 1,3-propanediol (1,3-PD). Escherichia coli, which does not have a dha system, is unable to grow anaerobically on glycerol without an exogenous electron acceptor and does not produce 1,3-PD. A genomic library of K. pneumoniae ATCC 25955 constructed in E. coli AG1 was enriched for the ability to grow anaerobically on glycerol and dihydroxyacetone and was screened for the production of 1,3-PD. The cosmid pTC1 (42.5 kb total with an 18.2-kb major insert) was isolated from a 1,3-PD-producing strain of E. coli and found to possess enzymatic activities associated with four genes of the dha regulon: glycerol dehydratase (dhaB), 1,3-PD oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK). All four activities were inducible by the presence of glycerol. When E. coli AG1/pTC1 was grown on complex medium plus glycerol, the yield of 1,3-PD from glycerol was 0.46 mol/mol. The major fermentation by-products were formate, acetate, and D-lactate. 1,3-PD is an intermediate in organic synthesis and polymer production. The 1,3-PD fermentation provides a useful model system for studying the interaction of a biochemical pathway in a foreign host and for developing strategies for metabolic pathway engineering.     

1,3-Propanediol production by Escherichia coli expressing genes from the Klebsiella pneumoniae dha regulon.

The dha regulon in Klebsiella pneumoniae enables the organism to grow anaerobically on glycerol and produce 1,3-propanediol (1,3-PD). Escherichia coli, which does not have a dha system, is unable to grow anaerobically on glycerol without an exogenous electron acceptor and does not produce 1,3-PD. A genomic library of K. pneumoniae ATCC 25955 constructed in E. coli AG1 was enriched for the ability to grow anaerobically on glycerol and dihydroxyacetone and was screened for the production of 1,3-PD. The cosmid pTC1 (42.5 kb total with an 18.2-kb major insert) was isolated from a 1,3-PD-producing strain of E. coli and found to possess enzymatic activities associated with four genes of the dha regulon: glycerol dehydratase (dhaB), 1,3-PD oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK). All four activities were inducible by the presence of glycerol. When E. coli AG1/pTC1 was grown on complex medium plus glycerol, the yield of 1,3-PD from glycerol was 0.46 mol/mol. The major fermentation by-products were formate, acetate, and D-lactate. 1,3-PD is an intermediate in organic synthesis and polymer production. The 1,3-PD fermentation provides a useful model system for studying the interaction of a biochemical pathway in a foreign host and for developing strategies for metabolic pathway engineering.     

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.     

Microbial production of 1,3-propanediol

1,3-Propanediol (1,3-PD) production by fermentation of glycerol was described in 1881 but little attention was paid to this microbial route for over a century. Glycerol conversion to 1,3-PD can be carried out by Clostridia as well as Enterobacteriaceae. The main intermediate of the oxidative pathway is pyruvate, the further utilization of which produces CO₂, H₂, acetate, butyrate, ethanol, butanol and 2,3-butanediol. In addition, lactate and succinate are generated. The yield of 1,3-PD per glycerol is determined by the availability of NADH₂, which is mainly affected by the product distribution (of the oxidative pathway) and depends first of all on the microorganism used but also on the process conditions (type of fermentation, substrate excess, various inhibitions). In the past decade, research to produce 1,3-PD microbially was considerably expanded as the diol can be used for various polycondensates. In particular, polyesters with useful properties can be manufactured. A prerequisite for making a “green” polyester is a more cost-effective production of 1,3-PD, which, in practical terms, can only be achieved by using an alternative substrate, such as glucose instead of glycerol. Therefore, great efforts are now being made to combine the pathway from glucose to glycerol successfully with the bacterial route from glycerol to 1,3-PD. Thus, 1,3-PD may become the first bulk chemical produced by a genetically engineered microorganism. Received: 12 January 1999 / Received revision: 9 March 1999 / Accepted: 14 March 1999     

Construction and characterization of ack deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid and hydrogen production

Clostridium tyrobutyricum produces butyrate, acetate, H(2), and CO(2) as its main fermentation products from glucose and xylose. To improve butyric acid and hydrogen production, integrational mutagenesis was used to create a metabolically engineered mutant with inactivated ack gene, encoding acetate kinase (AK) associated with the acetate formation pathway. A non-replicative plasmid containing the acetate kinase gene (ack) fragment was constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome should inactivate the target ack gene and produce ack-deleted mutant, PAK-Em. Enzyme activity assays showed that the AK activity in PAK-Em decreased by approximately 50%; meanwhile, phosphotransacetylase (PTA) and hydrogenase activities each increased by approximately 40%. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the expression of protein with approximately 32 kDa molecular mass was reduced significantly in the mutant. Compared to the wild type, the mutant grew more slowly at pH 6.0 and 37 degrees C, with a lower specific growth rate of 0.14 h(-1) (vs 0.21 h(-1) for the wild type), likely due to the partially impaired PTA-AK pathway. However, the mutant produced 23.5% more butyrate (0.42 vs 0.34 g/g glucose) at a higher final concentration of 41.7 g/L (vs 19.98 g/L) as a result of its higher butyrate tolerance as indicated in the growth kinetics study using various intial concentrations of butyrate in the media. The mutant also produced 50% more hydrogen (0.024 g/g) from glucose than the wild type. Immobilized-cell fermentation of PAK-Em in a fibrous-bed bioreactor (FBB) further increased the final butyric acid concentration (50.1 g/L) and the butyrate yield (0.45 g/g glucose). Furthermore, in the FBB fermentation at pH 5.0 with xylose as the substrate, only butyric acid was produced by the mutant, whereas the wild type produced large amounts of acetate (0.43 g/g xylose) and lactate (0.61 g/g xylose) and little butyrate (0.05 g/g xylose), indicating a dramatic metabolic pathway shift caused by the ack deletion in the mutant.     

Metabolic engineering of the non-sporulating, non-solventogenic Clostridium acetobutylicum strain M5 to produce butanol without acetone demonstrate the robustness of the acid-formation pathways and the importance of the electron balance

The primary alcohol/aldehyde dehydrogenase (coded by the aad gene) is responsible for butanol formation in Clostridium acetobutylicum. We complemented the non-sporulating, non-solvent-producing C. acetobutylicum M5 strain (which has lost the pSOL1 megaplasmid containing aad and the acetone-formation genes) with aad expressed from the phosphotransbutyrylase promoter and restored butanol production to wild type levels. Because no acetone was produced, no acids (acetate or butyrate) were re-assimilated leading to high butyrate but especially acetate levels. To counter acetate production, we examined thiolase overexpression in order reduce the acetyl-CoA pool and enhance the butyryl-CoA pool. We combined thiolase overexpression with aad overexpression aiming to also enhance butanol formation. While limiting the formation of acetate and ethanol, the butanol titers were not improved. We also generated acetate kinase (AK) and butyrate kinase (BK) knockout (KO) mutants of M5 using a modified protocol to increase the antibiotic-resistance gene expression. These strains exhibited greater than 60% reduction in acetate or butyrate formation, respectively. We complemented the AKKO M5 strain with aad overexpression, but could not successfully transform the BKKO M5 strain. The AKKO M5 strain overexpressing aad produced less acetate, but also less butanol compared to the M5 aad overexpression strain. These data suggest that loss of the pSOL1 megaplasmid renders cells resistant to changes in the two acid-formation pathways, and especially so for butyrate formation. We argue that the difficulty in generating high butanol producers without acetone and acid production is hindered by the inability to control the electron flow, which appears to be affected by unknown pSOL1 genes.     

Improved 1,3-propanediol production with hemicellulosic hydrolysates (corn straw) as cosubstrate: Impact of degradation products on Klebsiella pneumoniae growth and 1,3-propanediol fermentation

To improve 1,3-propanediol (1,3-PD) production by an economic and efficient approach, hemicellulosic hydrolysates (HH) used as cosubstrate resulted in more biomass and higher reducing power for 1,3-PD production. The effects of primary degradation products such as individual sugars (xylose, glucose, mannose, arabinose and galactose) and major inhibitors (furfural, acetate and formate) on the Klebsiella pneumoiae growth and 1,3-PD production were investigated in this study. Xylose and mannose could efficiently promote the 1,3-PD production and cell growth. Furfural (0.28 g/l) and sodium acetate (1.46 g/l) in low concentration were not inhibitory to Klebsiella pneumoniae, rather they have stimulatory effect on the growth and 1,3-PD biosynthesis, especially the acetate. In fed-batch fermentation with HH as cosubstrate, the final 1,3-PD production, conversion from glycerol and productivity were 71.58 g/l, 0.65 mol/mol and 1.93 g/l/h, respectively, which were 17.8%, 25.0% and 17.7% higher than that from glycerol alone.     

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.     

Improved 1,3-propanediol production by engineering the 2,3-butanediol and formic acid pathways in integrative recombinant Klebsiella pneumoniae

In the biotechnological process, insufficient cofactor NADH and multiple by-products restrain the final titer of 1,3-propanediol (1,3-PD). In this study, 1,3-PD production was improved by engineering the 2,3-butanediol (2,3-BD) and formic acid pathways in integrative recombinant Klebsiella pneumoniae. The formation of 2,3-BD is catalysed by acetoin reductase (AR). An inactivation mutation of the AR in K. pneumoniae CF was generated by insertion of a formate dehydrogenase gene. Inactivation of AR and expression of formate dehydrogenase reduced 2,3-BD formation and improved 1,3-PD production. Fermentation results revealed that intracellular metabolic flux was redistributed pronouncedly. The yield of 1,3-PD reached 0.74 mol/mol glycerol in flask fermentation, which is higher than the theoretical yield. In 5 L fed-batch fermentation, the final titer and 1,3-PD yield of the K. pneumoniae CF strain reached 72.2 g/L and 0.569 mol/mol, respectively, which were 15.9% and 21.7% higher than those of the wild-type strain. The titers of 2,3-BD and formic acid decreased by 52.2% and 73.4%, respectively. By decreasing the concentration of all nonvolatile by-products and by increasing the availability of NADH, this study demonstrates an important strategy in the metabolic engineering of 1,3-PD production by integrative recombinant hosts.     

Physiologic mechanisms of sequential products synthesis in 1,3-propanediol fed-batch fermentation by Klebsiella pneumoniae

The glycerol fed-batch fermentation by Klebsiella pneumoniae CGMCC 1.6366 exhibited the sequential synthesis of products, including acetate, 1,3-propanediol (1,3-PD), 2,3-butanediol, ethanol, succinate, and lactate. The dominant flux distribution was shifted from acetate formation to 1,3-PD formation in early- exponential growth phase and then to lactate synthesis in late-exponential growth phase. The underlying physiological mechanism of the above observations has been investigated via the related enzymes, nucleotide, and intermediary metabolites analysis. The carbon flow shift is dictated by the intrinsic physiological state and enzymatic activity regulation. Especially, the internal redox state could serve as a rate-controlling factor for 1,3-PD production. The q(1,3-PD) formation was the combined outcomes of regulations of glycerol dehydratase activity and internal redox balancing. The q(ethanol)/q(acetate) ratios demonstrated the flexible adaptation mechanism of K. pneumoniae preferring ATP generation in early-exponential growth phase. A low PEP to pyruvate ratio corresponded LDH activity increase, leading to lactate accumulation in stationary phase.     

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.     

1,3-Propanediol production and tolerance of a halophilic fermentative bacterium, Halanaerobium saccharolyticum subsp. saccharolyticum

1,3-Propanediol (1,3-PD) is widely used in polymer industry in production of polyethers, polyesters and polyurethanes. In this article, a study on 1,3-PD production and tolerance of Halanaerobium saccharolyticum subsp. saccharolyticum is presented. 1,3-PD production was optimized for temperature, vitamin B(12) and acetate concentration. The highest 1,3-PD concentrations and yields (0.6 mol/mol glycerol) were obtained at vitamin B?? concentration 64 μg/l and an inverse correlation between 1,3-PD and hydrogen production was observed with varying vitamin B?? concentrations. In the studied temperature range and initial acetate concentrations up to 10 g/l, no significant variations were observed in 1,3-PD production. High initial acetate (29-58 g/l) was observed to cause slight decrease in 1,3-PD concentrations produced but no effects on 1,3-PD yields (mol/mol glycerol). Initial 1,3-PD concentrations inhibited the growth of H. saccharolyticum subsp. saccharolyticum. When initial 1,3-PD concentration was raised from 1g/l to 57 g/l, a decrease of 12% to 75%, respectively, in the highest optical density was observed.