Anaerobic Fermentation Of Glycerol Research Articles

Construction of an energy-conserving glycerol utilization pathways for improving anaerobic succinate production in Escherichia coli

Metabolic engineering of microorganisms for production of succinate from glycerol remains challenging. It was thought that energy supply is a severe problem for anaerobic fermentation of glycerol to produce succinate. In this study, an energy-conserving glycerol utilization pathway was recruited to improve anaerobic succinate production in Escherichia coli. ATP dependent dihydroxyacetone kinase (DhaK) from Klebsiella was used to replace the native phosphoenolpyruvate (PEP) dependent dihydroxyacetone kinase (DhaKLM) of E. coli so that 2 NADH, instead of 1 NADH and 1 menaquinol, can be produced from glycerol to PEP. Besides consumption of 1 NADH and 1 menaquinol for converting PEP to succinate, NADH dehydrogenase I transfers electrons from 1 NADH to 1 menaquinone and pumps 4 protons. Proton-motive force are generated as the additional energy to support cell growth and succinate export. After using this energy-conserving glycerol utilization pathway, succinate titer, productivity and intercellular ATP content increased 282%, 63% and 338% respectively. The best strain YY-GS004 produced 483 mM succinate in 96 h with a yield of 0.92 mol mol-1 glycerol under anaerobic conditions. The specific succinate productivity was 0.47 g g-1 (DCW) h-1, which was the highest one up to date for succinate production from glycerol. This study demonstrated that the energy-conserving glycerol utilization pathway GldA-DhaK can solve the energy problem during anaerobic fermentation of glycerol to produce succinate.

Production of medium-chain carboxylic acids by anaerobic fermentation of glycerol using a bioaugmented open culture

Biological production of carboxylic acids during glycerol fermentation via the chain elongation process was investigated. Glycerol is a by-product of the biodiesel production process and a convenient feedstock alternative for the carboxylate platform. In batch experiments, glycerol was used as substrate, whereas sludge (granular and flocculent) and ruminal liquid served as inocula. To improve the production of carboxylates, bioaugmentation with Clostridium acetobutylicum ATCC 824 was performed. Maximal production of n-caproic and n-caprylic acids was achieved with the use of the granular sludge. Bioaugmentation with C. acetobutylicum enhanced production of n-caproic acid in the presence of 100 mM ethanol. However, increasing ethanol concentration to 200 mM had no further enhancing effect on n-caproic production with or without bioaugmentation. The enhancement of n-caproic acid production was probably due to the increased production of n-butyric acid by C. acetobutylicum, because n-butyric acid is an important intermediate in the chain elongation process when ethanol is used as an electron donor.

A new method to bio-preserve sea bass fillets

In this work a bio-preservation technique was applied to sea bass fillets in order to preserve their quality. The preservation consisted in the application of two kinds of active coatings on the product surface differing in the fermentation time of alginate solution by L. reuteri plus glycerol (24 and 48 h). This technological strategy was chosen because it has been demonstrated that L. reuteri produces the reuterin as an intermediate metabolite during the anaerobic fermentation of glycerol. To assess the antimicrobial effects of sodium alginate with L. reuteri and glycerol, both in vitro and in vivo tests were carried out. The active films, in particular at 48 h fermentation, showed a good antibacterial activity, confirmed also by the major reuterin concentration. To prove the effectiveness of the treatments, microbial and sensory attributes were monitored by in vivo test on fish fillets. Results highlighted that the two active sodium alginate coatings showed a good antibacterial activity. In sea bass fillets stored at 4 °C, proliferation of main spoilage microorganisms was delayed with a consequent preservation of sensory attributes. In particular, it was found that improving the fermentation time (48 h) a better microbiological and sensory quality was achieved.

Glycerol and mixture of carbon sources conversion to hydrogen by Clostridium beijerinckii DSM791 and effects of various heavy metals on hydrogenase activity

Hydrogen is a carbon-neutral energy feedstock which is produced during fermentation of various carbon sources. The genomes of clostridia encode mainly [Fe-Fe]-hydrogenases. Clostridium beijerinckii DSM791 performed anaerobic fermentation of glycerol in batch culture at pH 7.5 and pH 5.5 and produced H2. At pH 7.5, the glycerol consumption rate was 3.7 g/g cell mass/h, which was higher than that at pH 5.5. H2 production reached 5 mmol/h/g cell mass at pH 7.5. The specific hydrogenase activity was ∼1.4 fold higher if cells were grown on glycerol compared to cells grown on glucose. Single (Fe2+, Fe3+, Ni2+) or mixed supply of metals (Fe2+ and Ni2+) increased the specific hydrogenase activity by ∼50%. These results suggest that C. beijerinckii DSM791 could be used as a potential H2 producer. It may help to further enhance H2 production using different industrial or agricultural wastes where glycerol and other carbon sources are present.

Microbial removal of carboxylic acids from 1,3-propanediol in glycerol anaerobic digestion effluent by PHAs-producing consortium

Anaerobic fermentation of glycerol to 1,3-propanediol (1,3-PDO) is conceived as an economic feasible pathway to handle with increasing crude glycerol from biodiesel industry. However, glycerol anaerobic digestion effluent (ADE) consists of carboxylic acids and 1,3-PDO, imposing difficulties for separation. The objective of this study was, therefore, to investigate microbial removal of carboxylic acids from 1,3-PDO in glycerol ADE by polyhydroxyalkanoates (PHAs) producing consortium. Growth tests on carbon sources showed Corynebacterium hydrocarbooxydans had preference for butyrate while Bacillus megaterium for acetate and glycerol. Consequently, their consortium had a higher cell density and a faster substrate utilization rate than single strain grown in glycerol ADE. Acidic pH at 6.0 and 5.2 strongly inhibited cell growth and activity, while C:N ratio (w/w) at 8:1 could balance nitrogen demand for cell growth and PHA synthesis. Kinetic study further revealed over 80% of fed 1,3-PDO was preserved after depletion of carboxylic acids. Correspondingly, total organic carbon (TOC) contribution from 1,3-PDO rose from initial 55.8% to 84%. Produced PHAs comprised 3-hydroxybutyrate (3-HB) units. The results showed this study as the first attempt to provide a win-win solution to remove carboxylic acids from 1,3-PDO in glycerol ADE and converted them into PHAs as a secondary value-added product.

Impact of membrane-associated hydrogenases on the FOF1-ATPase in Escherichia coli during glycerol and mixed carbon fermentation: ATPase activity and its inhibition by N,N′-dicyclohexylcarbodiimide in the mutants lacking hydrogenases

Escherichia coli is able to ferment glycerol and to produce molecular hydrogen (H2) by four membrane-associated hydrogenases (Hyd) changing activity in response to different conditions. In this study, overall ATPase activity of glycerol alone and mixed carbon sources (glucose and glycerol) fermented E. coli wild type and different Hyd mutants and its inhibition by N,N′-dicyclohexylcarbodiimide (DCCD) were first investigated. ATPase activity was higher in glycerol fermented wild type cells at pH 7.5 compared to pH 6.5 and pH 5.5; DCCD inhibited markedly ATPase activity at pH 7.5. The ATPase activity at pH 7.5, compared with wild type, was lower in selC and less in hypF single mutants, suppressed in hyaB hybC selC triple mutant. Moreover, total ATPase activity of mixed carbon fermented wild type cells was maximal at pH 7.5 and lowered at pH 5.5. The ATPase activities of hypF and hyaB hybC selC mutants were higher at pH 5.5, compared with wild type; DCCD inhibited markedly ATPase activity of hypF mutant. These results demonstrate that in E. coli during glycerol fermentation the membrane proton-translocating FOF1-ATPase has major input in overall ATPase activity and alkaline pH is more optimal for the FOF1-ATPase operation. Hyd-1 and Hyd-2 are required for the FOF1-ATPase activity upon anaerobic fermentation of glycerol. The impact of Hyd-1 and Hyd-2 on the FOF1-ATPase is more obvious during mixed carbon fermentation at slightly acidic pH.

Short communication: Combined antimicrobial activity of reuterin and diacetyl against foodborne pathogens

Reuterin (β-hydroxypropionialdehyde) is a broad-spectrum antimicrobial substance produced by some strains of Lactobacillus reuteri during anaerobic fermentation of glycerol. Some of these strains are able to survive and produce reuterin in cheese and yogurt when added as adjuncts to the starter. Similarly, in fermented dairy foods, other inhibitory compounds such as lactic acid and diacetyl are produced during fermentation. In this work, we studied the combined effect of reuterin and diacetyl under different pH conditions against Escherichia coli O157:H7, Salmonella Enteritidis, and Listeria monocytogenes. Results from agar spot assays showed that the antimicrobial activity of reuterin-producing strains against the gram-negative bacteria tested was enhanced as the concentration of diacetyl increased to 50mg/kg, and was higher under acidic conditions (pH 5.0) for the 3 pathogenic strains. The combination of reuterin and diacetyl had an additive effect against L. monocytogenes only at diacetyl concentrations of 50mg/kg and pH 5.0. In addition, growth kinetics studies showed that the combination of 1 activity unit (AU)/mL of reuterin with 100mg/kg diacetyl increased the lag time of the 3 pathogens. In milk, synergistic antimicrobial activity was observed with the combination of 1AU/mL reuterin and 50 or 100mg/kg of diacetyl on the gram-negative strains tested, and with 1AU/mL reuterin and 100mg/kg of diacetyl on L. monocytogenes. The greatest inhibition of the 3 pathogens was achieved in acidified milk at pH 5.0 with reuterin (1AU/mL) and diacetyl (100mg/kg). Based on these results, the combination of reuterin and diacetyl in acidified dairy products could be a promising strategy to control food pathogens in these products.

Accelerated glycerol fermentation in Escherichia coli using methanogenic formate consumption

Escherichia coli can ferment glycerol anaerobically only under very defined restrictive conditions. Hence, it was the aim of this study to overcome this limitation via a co-cultivation approach. Anaerobic glycerol fermentation by a pure E. coli culture was compared to a co-culture that also contained the formate-oxidizing methanogen Methanobacterium formicicum. Co-cultivation of the two strains led to a more than 11-fold increased glycerol consumption. Furthermore, it supported a constantly neutral pH and a shift from ethanol to succinate production. Moreover, M. formicicum was analyzed for its ability to grow on different standard media and a surprising versatility could be demonstrated.

Optimizing the production of hydrogen and 1,3-propanediol in anaerobic fermentation of biodiesel glycerol

The conversion of glycerol in biodiesel waste streams to valuable products (e.g. hydrogen and 1,3-propanediol (1,3-PD)) was studied through batch-mode anaerobic fermentation with organic soil as inoculum. The production of hydrogen in headspace and 1,3-PD in liquid phase was examined at different hydrogen retention times (HyRTs), which were controlled by gas-collection intervals (GCIs) and initial gas-collection time points (IGCTs). Two purification stages of biodiesel glycerol (P2 and P3) were tested at three concentrations (3, 5 and 7 g/L). Longer HyRT (longer GCI and longer IGCT) led to lower hydrogen yield but higher 1,3-PD yield. The P3 glycerol at the concentration of 7 g/L had the highest 1,3-PD yield (0.65 mol/mol glycerolconsumed) at the GCI/IGCT of 20 h/65 h and the highest hydrogen yield (0.75 mol/mol glycerolconsumed) at the GCI/IGCT of 2.5 h/20 h), respectively. A mixed-order kinetic model was developed to simulate the effects of GCI/IGCT on the production of hydrogen and 1,3-PD. The results showed that the production of hydrogen and 1,3-PD can be optimized by adjusting HyRT in anaerobic fermentation of glycerol.

Optimizing the production of hydrogen and 1,3-propanediol in anaerobic fermentation of biodiesel glycerol

Abstract The conversion of glycerol in biodiesel waste streams to valuable products (e.g. hydrogen and 1,3-propanediol (1,3-PD)) was studied through batch-mode anaerobic fermentation with organic soil as inoculum. The production of hydrogen in headspace and 1,3-PD in liquid phase was examined at different hydrogen retention times (HyRTs), which were controlled by gas-collection intervals (GCIs) and initial gas-collection time points (IGCTs). Two purification stages of biodiesel glycerol (P2 and P3) were tested at three concentrations (3, 5 and 7 g/L). Longer HyRT (longer GCI and longer IGCT) led to lower hydrogen yield but higher 1,3-PD yield. The P3 glycerol at the concentration of 7 g/L had the highest 1,3-PD yield (0.65 mol/mol glycerol consumed ) at the GCI/IGCT of 20 h/65 h and the highest hydrogen yield (0.75 mol/mol glycerol consumed ) at the GCI/IGCT of 2.5 h/20 h), respectively. A mixed-order kinetic model was developed to simulate the effects of GCI/IGCT on the production of hydrogen and 1,3-PD. The results showed that the production of hydrogen and 1,3-PD can be optimized by adjusting HyRT in anaerobic fermentation of glycerol.

Acrolein in Wine: Importance of 3-Hydroxypropionaldehyde and Derivatives in Production and Detection

Certain lactic acid bacteria strains belonging to the genus Lactobacillus have been implicated in the accumulation of 3-hydroxypropionaldehyde (3-HPA) during anaerobic glycerol fermentation. In aqueous solution 3-HPA undergoes reversible dimerization and hydration, resulting in an equilibrium state between different derivatives. Wine quality may be compromised by the presence of 3-HPA due to the potential for spontaneous conversion into acrolein under winemaking conditions. Acrolein is highly toxic and has been implicated in the development of bitterness in wine. Interconversion between 3-HPA derivatives and acrolein is a complex and highly dynamic process driven by hydration and dehydration reactions. Acrolein is furthermore highly reactive and its steady-state concentration in complex systems very low. As a result, analytical detection and quantification in solution is problematic. This paper reviews the biochemical and environmental conditions leading to accumulation of its precursor, 3-HPA. Recent advances in analytical detection are summarized, and the roles played by natural chemical derivatives are highlighted.

Influence of environmental parameters on production of the acrolein precursor 3-hydroxypropionaldehyde by Lactobacillus reuteri DSMZ 20016 and its accumulation by wine lactobacilli

Lactic acid bacteria belonging to the genus Lactobacillus are known to convert glycerol into 3-hydroxypropionaldehyde (3-HPA) during anaerobic glycerol fermentation. Wine quality can be gravely compromised by the accumulation of 3-HPA, due to its spontaneous conversion to acrolein under wine making conditions. Acrolein is not only a dangerous substance for the living cell, but has been implicated in the development of unpleasant bitterness in beverages. This study evaluates the effect of individual environmental parameters on 3-HPA production by Lactobacillus reuteri DSMZ 20016, which only proved possible under conditions that allow accumulation well below the threshold concentration affecting cell viability. 3-HPA production was optimal at pH 6 and in the presence of 300 mM glycerol. Production increased with an increase in cell concentration up to an OD 600 of 50, whereas higher cell concentrations inhibited accumulation. Data presented in this study suggest that 3-HPA plays a role in regulating its own production through quorum sensing. Glycerol dehydratase possessing bacterial strains isolated from South African red wine, L. pentosus and L. brevis, tested positive for 3-HPA accumulation. 3-HPA is normally intracellularly reduced to 1,3-propanediol. This is the first study demonstrating the ability of wine lactobacilli to accumulate 3-HPA in the fermentation media. Recommendations are made on preventing the formation of acrolein and its precursor 3-HPA in wine.

Anaerobic fermentation of glycerol in Paenibacillus macerans: metabolic pathways and environmental determinants.

Paenibacillus macerans is one of the species with the broadest metabolic capabilities in the genus Paenibacillus, able to ferment hexoses, deoxyhexoses, pentoses, cellulose, and hemicellulose. However, little is known about glycerol metabolism in this organism, and some studies have reported that glycerol is not fermented. Despite these reports, we found that several P. macerans strains are capable of anaerobic fermentation of glycerol. One of these strains, P. macerans N234A, grew fermentatively on glycerol at a maximum specific growth rate of 0.40 h(-1) and was chosen for further characterization. The use of [U-13C]glycerol and further analysis of extracellular metabolites and proteinogenic amino acids via nuclear magnetic resonance (NMR) spectroscopy allowed identification of ethanol, formate, acetate, succinate, and 1,2-propanediol (1,2-PDO) as fermentation products and demonstrated that glycerol is incorporated into cellular components. A medium formulation with low concentrations of potassium and phosphate, cultivation at acidic pH, and the use of a CO2-enriched atmosphere stimulated glycerol fermentation and are proposed to be environmental determinants of this process. The pathways involved in glycerol utilization and synthesis of fermentation products were identified using NMR spectroscopy in combination with enzyme assays. Based on these studies, the synthesis of ethanol and 1,2-PDO is proposed to be a metabolic determinant of glycerol fermentation in P. macerans N234A. Conversion of glycerol to ethanol fulfills energy requirements by generating one molecule of ATP per molecule of ethanol synthesized. Conversion of glycerol to 1,2-PDO results in the consumption of reducing equivalents, thus facilitating redox balance. Given the availability, low price, and high degree of reduction of glycerol, the high metabolic rates exhibited by P. macerans N234A are of paramount importance for the production of fuels and chemicals.

Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli

Given its availability, low prices, and high degree of reduction, glycerol has become an ideal feedstock for the production of reduced compounds. The anaerobic fermentation of glycerol by Escherichia coli could be an excellent platform for this purpose but it requires expensive nutrients such as tryptone and yeast extract. In this work, microaerobic conditions were used as a means of eliminating the need for rich nutrients. Availability of low amounts of oxygen enabled redox balance while preserving the ability to synthesize reduced products. A fermentation balance analysis showed approximately 95% recovery of carbon and reducing equivalents. The pathways involved in glycerol dissimilation were identified using different genetic and biochemical approaches. Respiratory (GlpK-GlpD/GlpABC) and fermentative (GldA-DhaKLM) routes mediated the conversion of glycerol to glycolytic intermediates. Although pyruvate formate-lyase (PFL) and pyruvate dehydrogenase contributed to the synthesis of acetyl-CoA from pyruvate, most of the carbon flux proceeded through PFL. The pathways mediating the synthesis of acetate and ethanol were required for the efficient utilization of glycerol. The microaerobic metabolism of glycerol was harnessed by engineering strains for the co-production of ethanol and hydrogen (EH05 [pZSKLMgldA]), and ethanol and formate (EF06 [pZSKLMgldA]). High ethanol yields were achieved by genetic manipulations that reduced the synthesis of by-products succinate, acetate, and lactate. Co-production of hydrogen required the use of acidic pH while formate co-production was facilitated by inactivation of the enzyme formate-hydrogen lyase. High rates of product synthesis were realized by overexpressing glycerol dehydrogenase (GldA) and dihydroxyacetone kinase (DhaKLM). Engineered strains efficiently produced ethanol and hydrogen and ethanol and formate from glycerol in a minimal medium without rich supplements.

A new model for the anaerobic fermentation of glycerol in enteric bacteria: Trunk and auxiliary pathways in Escherichia coli

Anaerobic fermentation of glycerol in the Enterobacteriaceae family has long been considered a unique property of species that synthesize 1,3-propanediol (1,3-PDO). However, we have discovered that Escherichia coli can ferment glycerol in a 1,3-PDO-independent manner. We identified 1,2-propanediol (1,2-PDO) as a fermentation product and established the pathway that mediates its synthesis as well as its role in the metabolism of glycerol. We also showed that the trunk pathway responsible for the conversion of glycerol into glycolytic intermediates is composed of two enzymes: a type II glycerol dehydrogenase (glyDH-II) and a dihydroxyacetone kinase (DHAK), the former of previously unknown physiological role. Based on our findings, we propose a new model for glycerol fermentation in enteric bacteria in which: (i) the production of 1,2-PDO provides a means to consume reducing equivalents generated in the synthesis of cell mass, thus facilitating redox balance, and (ii) the conversion of glycerol to ethanol, through a redox-balanced pathway, fulfills energy requirements by generating ATP via substrate-level phosphorylation. The activity of the formate hydrogen-lyase and F 0F 1-ATPase systems were also found to facilitate the fermentative metabolism of glycerol, and along with the ethanol and 1,2-PDO pathways, were considered auxiliary or enabling. We demonstrated that glycerol fermentation in E. coli was not previously observed due to the use of medium formulations and culture conditions that impair the aforementioned pathways. These include high concentrations of potassium and phosphate, low concentrations of glycerol, alkaline pH, and closed cultivation systems that promote the accumulation of hydrogen gas.

Isolation and Characterization of Human Intestinal Bacteria Capable of Transforming the Dietary Carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[4,5- b ]Pyridine

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogenic heterocyclic aromatic amine formed in meat products during cooking. Although the formation of hazardous PhIP metabolites by mammalian enzymes has been extensively reported, research on the putative involvement of the human intestinal microbiota in PhIP metabolism remains scarce. In this study, the in vitro conversion of PhIP into its microbial derivate, 7-hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3',2':4,5]imidazo[1,2-a]pyrimidin-5-ium chloride (PhIP-M1), by fecal samples from 18 human volunteers was investigated. High-performance liquid chromatography analysis showed that all human fecal samples transformed PhIP but with efficiencies ranging from 1.8 to 96% after 72 h of incubation. Two PhIP-transforming strains, PhIP-M1-a and PhIP-M1-b, were isolated from human feces and identified by fluorescent amplified fragment length polymorphism and pheS sequence analyses as Enterococcus faecium strains. Some strains from culture collections belonging to the species E. durans, E. avium, E. faecium, and Lactobacillus reuteri were also able to perform this transformation. Yeast extract, special peptone, and meat extract supported PhIP transformation by the enriched E. faecium strains, while tryptone, monomeric sugars, starch, and cellulose did not. Glycerol was identified as a fecal matrix constituent required for PhIP transformation. Abiotic synthesis of PhIP-M1 and quantification of the glycerol metabolite 3-hydroxypropionaldehyde (3-HPA) confirmed that the anaerobic fermentation of glycerol via 3-HPA is the critical bacterial transformation process responsible for the formation of PhIP-M1. Whether it is a detoxification is still a matter of debate, since PhIP-M1 has been shown to be cytotoxic toward Caco-2 cells but is not mutagenic in the Ames assay.

Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry

Although biofuels such as biodiesel and bioethanol represent a secure, renewable and environmentally safe alternative to fossil fuels, their economic viability is a major concern. The implementation of biorefineries that co-produce higher value products along with biofuels has been proposed as a solution to this problem. The biorefinery model would be especially advantageous if the conversion of byproducts or waste streams generated during biofuel production were considered. Glycerol-rich streams generated in large amounts by the biofuels industry, especially during the production of biodiesel, present an excellent opportunity to establish biorefineries. Once considered a valuable 'co-product', crude glycerol is rapidly becoming a 'waste product' with a disposal cost attributed to it. Given the highly reduced nature of carbon in glycerol and the cost advantage of anaerobic processes, fermentative metabolism of glycerol is of special interest. This review covers the anaerobic fermentation of glycerol in microbes and the harnessing of this metabolic process to convert abundant and low-priced glycerol streams into higher value products, thus creating a path to viability for the biofuels industry. Special attention is given to products whose synthesis from glycerol would be advantageous when compared with their production from common sugars.

Improving 1,3-propanediol production from glycerol in a metabolically engineered Escherichia coli by reducing accumulation of sn-glycerol-3-phosphate.

High levels of glycerol significantly inhibit cell growth and 1,3-propanediol (1,3-PD) production in anaerobic glycerol fermentation by genetically engineered Escherichia coli (E. coli) strains expressing genes from the Klebsiella pneumoniae dha (K.pneumoniae) regulon. We have previously demonstrated that 1,3-PD production by the engineered E. coli can be improved by reducing the accumulation of methylglyoxal. This study focuses on investigation of another lesser-known metabolite in the pathways related to 1,3-PD production-glycerol-3-phosphate (G3P). When grown anaerobically on glycerol in the absence of an exogenous acceptor, the engineered E. coli strains have intracellular G3P levels that are significantly higher than those in K. pneumoniae, a natural 1,3-PD producer. Furthermore, in the engineered E. coli strains, the G3P levels increase with increasing glycerol concentrations, whereas, in K. pneumoniae, the concentrations of G3P remain relatively constant. Addition of fumarate, which can stimulate activity of anaerobic G3P dehydrogenase, into the fermentation medium led to a greater than 30-fold increase in the specific activity of anaerobic G3P dehydrogenase and a significant decrease in concentrations of intracellular G3P and resulted in better cell growth and an improved production of 1,3-PD. This indicates that the low activity of G3P dehydrogenase in the absence of an exogenous electron acceptor is one of the reasons for G3P accumulation. In addition, spent media from E.coli Lin61, a glycerol kinase (responsible for conversion of glycerol to G3P) mutant, contains greatly decreased concentrations of G3P and shows improved production of 1,3-PD (by 2.5-fold), when compared to media from its parent strain E. coli K10. This further suggests that G3P accumulation is one of the reasons for the inhibition of 1,3-PD production during anaerobic fermentation.

Accumulation of Methylglyoxal in Anaerobically Grown Escherichia coli and Its Detoxification by Expression of the Pseudomonas putida Glyoxalase I Gene

Anaerobic glycerol fermentation by Escherichia coli strains expressing genes from the Klebsiella pneumoniae dha regulon showed that cell growth and 1,3-propanediol (1,3-PD) production are significantly inhibited when 5g/L or higher of glycerol is initially present. One reason for this inhibition may be methylglyoxal (MG) accumulation. Assays of both intracellular and extracellular MG levels indicated an accumulation of MG in anaerobic glycerol fermentation of transgenic E. coli. Pseudomonas putida glyoxalase I was expressed in the transgenic E. coli to enhance MG detoxification. The activity of glyoxalase I in the transgenic E. coli with the P. putida glyoxalase I under anaerobic conditions was 12-fold higher than that in the control cells. Compared to the control cells, the transgenic cells with the P. putida glyoxalase I displayed a reduction of 35–43% in intracellular MG and a decrease of 30% in extracellular MG. These decreases were statistically significant (P>94). Furthermore, the expression of the P. putida glyoxalase I in the transgenic E. coli markedly improved cell growth and resulted in a 50% increase in 1,3-PD production.

The Effect of Reuterin on Listeria monocytogenes and Escherichia coliO157:H7 in Milk and Cottage Cheese

A broad-spectrum reuterin produced during anaerobic fermentation of glycerol by Lactobacillus reuteri strain 12002 was found to be inhibitory and bactericidal for Listeria monocytogenes and Escherichia coli O157:H7. Lyophilized reuterin was prepared by a two-step fermentation process. A batch fermentation in a 15-liter fermentor was applied to produce a maximum biomass of L. reuteri using a modified MRS broth at pH 4.3. Further, harvested cells were used to ferment glycerol (250 mM) under anaerobic conditions. The sensitivity to reuterin of 10 strains of Listeria spp., including 6 strains of L. monocytogenes, and 6 strains of E. coli, including one enterotoxigenic E. coli strain and two enterohemorrhagic E. coli strains, was estimated. Strains of L. monocytogenes were more resistant to reuterin than E. coli strains. In cottage cheese, pH 5.4, L. monocytogenes increased by 0.4 log while E. coli O157:H7 decreased by 0.5 log in 21 days at 7°C; addition of reuterin (50 to 250 units per g) to the cottage cheese reduced the viability of both organisms. The inactivation rate was more pronounced (P ≤ 0.05) with E. coli O157:H7 than L. monocytogenes and it was dependent on reuterin concentration. The rate of E. coli O157:H7 population reduction reached to 2, 3, and 6 log cycles by day 7 for reuterin concentrations of 50, 100, and 150 units per g of creamed cottage cheese, respectively. While, 100, 150, and 250 units of reuterin per g caused reductions in L. monocytogenes counts by 2, 5, and greater than 5 log cycles, respectively. In UHT skim milk with 150 units of reuterin per ml, stored at 7°C, the decline in the numbers of L. monocytogenes cells was higher than that in cottage cheese. Milk fat in the range of 0.5 to 3% did not affect the reuterin activity (P ≤ 0.05). Addition of 3% salt enhanced the lethal effect of reuterin and diminished the initial population of L. monocytogenes by 4.5 log cycles in three days at 7°C.