Physiologic Mechanisms Involved in Accumulation of 3-Hydroxypropionaldehyde during Fermentation of Glycerol by Enterobacter agglomerans

When grown in 700 mM glycerol within the pH range 6.0 to 7.5, anaerobic pH-regulated cultures of Enterobacter agglomerans exhibited an extracellular accumulation of 3-hydroxypropionaldehyde (3-HPA). This phenomenon, which causes fermentation cessation, occurred earlier when pH was low. In contrast, substrate consumption was complete at pH 8. Levels of glycerol-catabolizing enzymes, i.e., glycerol dehydrogenase and dihydroxyacetone kinase for the oxidative route and glycerol dehydratase and 1,3-propanediol dehydrogenase for the reductive route, as well as the nucleotide pools were determined periodically in the pH 7- and pH 8-regulated cultures. A NAD/NADH ratio of 1.7 was correlated with the beginning of the production of the inhibitory metabolite. Further accumulation was dependent on the ratio of glycerol dehydratase activity to 1,3-propanediol dehydrogenase activity. For a ratio higher than 1, 3-HPA was produced until fermentation ceased, which occurred for the pH 7-regulated culture. At pH 8, a value below 1 was noticed and 3-HPA accumulation was transient, while the NAD/NADH ratio decreased. The low rate of glycerol dissimilation following the appearance of 3-HPA in the culture medium was attributed to the strong inhibitory effect exerted by 3-HPA on glycerol dehydrogenase activity.     

Root colonization by symplasmata-forming Enterobacter agglomerans

Abstract Enterobacter agglomerans strains are able to form cell aggregates called symplasmata when grown in a liquid medium. The nitrogen-fixing E. agglomerans strain NO30, isolated from the rhizosphere soil of rice, was inoculated onto roots of axenically grown wheat and rice seedlings and could colonize the roots of both plants. The ability of NO30 cells to colonize the plant roots seemed comparable in the host and non-host plants, as far as colony forming units (cfu) measurements were concerned. Nevertheless, electron microscopy (SEM, TEM) revealed that, in the case of rice, the normal host plant for NO30, the colonization was characterized by the formation of symplasmata, whereas only individual cells were found on wheat roots. Symplasmata formation seems to be specific for colonization of the host plant, rice. This finding also means that colonization of the host plant may be largely underestimated when measured by conventional techniques. Symplasmata formed in liquid medium or on the roots of rice were stained using Thiery's and Swift's technique, and the presence of polysaccharides and proteins was revealed in the extracellular matrix as well as in fibrils anchoring symplasmata to other symplasmata or to plant cells.     

Glycerol production by microbial fermentation: a review

Microbial production of glycerol has been known for 150 years, and glycerol was produced commercially during World War I. Glycerol production by microbial synthesis subsequently declined since it was unable to compete with chemical synthesis from petrochemical feedstocks due to the low glycerol yields and the difficulty with extraction and purification of glycerol from broth. As the cost of propylene has increased and its availability has decreased especially in developing countries and as glycerol has become an attractive feedstock for production of various chemicals, glycerol production by fermentation has become more attractive as an alternative route. Substantial overproduction of glycerol by yeast from monosaccharides can be obtained by: (1) forming a complex between acetaldehyde and bisulfite ions thereby retarding ethanol production and restoring the redox balance through glycerol synthesis; (2) growing yeast cultures at pH values near 7 or above; or (3) using osmotolerant yeasts. In recent years, significant improvements have been made in the glycerol production using osmotolerant yeasts on a commercial scale in China. The most outstanding achievements include: (1) isolation of novel osmotolerant yeast strains producing up to 130 g/L glycerol with yields up to 63% and the productivities up to 32 g/(L day); (2) glycerol yields, productivities and concentrations in broth up to 58%, 30 g/(L day) and 110-120 g/L, respectively, in an optimized aerobic fermentation process have been attained on a commercial scale; and (3) a carrier distillation technique with a glycerol distillation efficiency greater than 90% has been developed. As glycerol metabolism has become better understood in yeasts, opportunities will arise to construct novel glycerol overproducing microorganisms by metabolic engineering.     

Enterobacter agglomerans: the clinically important plant pathogen.

During a 5-month period Enterobacter agglomerans, now described as a member of the phytopathogenic genus Erwinia, was isolated from 13 patients in a general hospital; in 1 patient it was isolated from two sites. In six instances the organism was the sole pathogen isolated, in two instances it may have contributed to infection and in the remaining instances it was probably a transient saprophyte. The strains showed some variation in biochemical reactions but were similar in colonial morphology and were consistently sensitive to several antibiotics. Although this organism is prevalent in the general environment and usually relatively benign, it does have a potential for nosocomial infection.     

Glycerol fermentation of 1,3-propanediol by Clostridium butyricum. Measurement of product inhibition by use of a pH-auxostat

Summary The fermentation of glycerol to 1,3-propanediol, acetate, and butyrate by Clostridium butyricum was studied with respect to growth inhibition by the accumulating products. The clostridia were grown in a pH-auxostat culture at low cell density and product concentration and near maximum growth rate. The products were then added individually to the medium in increasing concentrations and the resulting depression of growth rate was used as a quantitative estimate of product inhibition. Under these conditions growth was totally inhibited at concentrations of 60 g/l for 1,3-propanediol, 27 g/l for acetic acid and 19 g/l for butyric acid at pH 6.5. Appreciable inhibition by glycerol was found only above a concentration of 80 g/l. In a pH-auxostat without added products but with high cell density as well as in batch cultures the product proportions were different. The 1,3-propanediol concentration may approach the value of complete inhibition while the concentrations of acetic and butyric acids remained below these values by at least one order of magnitude. It was therefore concluded that 1,3-propanediol is the first range inhibitor in this fermentation.     

Conversion of d-psicose to allitol by Enterobacter agglomerans strain 221e

Enterobacter agglomerans strain 221e transformed d-psicose to allitol at a faster rate in the presence of d-glucose in the reaction mixture when the entrance of oxygen was restricted. Cells grown on glycerol were found to be suitable for the transformation reaction. The transformation rates were about 97.0, 95.0 and 62.5%, respectively, when 0.5, 1.0 and 2.0% substrates were used. No consumption of substrate or product was observed in any case. In flask and jar fermentor reactions, the cells of strain 221e showed similar transformation activities. Cells stored at −20°C for 10 d showed almost same transformation activity as intact cells.     

产1,3-丙二醇基因工程菌发酵条件的研究

利用途径工程的方法,将来源于克雷伯氏菌(Klebsiella pneumoniae)的甘油脱水酶基因dhaB和1,3-丙二醇氧化还原酶基因dhaT构建成多顺反子重组质粒pSE-dhaB-dhaT并在大肠杆菌JM 109中进行表达,在大肠杆菌中构建一条新的产1,3-丙二醇代谢途径。研究表明,重组菌株JM 109/pSE-dhaB-dhaT在微好氧条件下,尝试用廉价的乳糖为诱导物、维生素B12为辅酶,可以将甘油转化为1,3-丙二醇,产量达15.34 g/L,甘油转化率为35.7%,对低成本生产1,3-丙二醇作了有益的探索。     

Glycerol fermentation by (open) mixed cultures: a chemostat study

Glycerol is an important byproduct of bioethanol and biodiesel production processes. This study aims to evaluate its potential application in mixed culture fermentation processes to produce bulk chemicals. Two chemostat reactors were operated in parallel, one fed with glycerol and the other with glucose. Both reactors operated at a pH of 8 and a dilution rate of 0.1 h(-1). Glycerol was mainly converted into ethanol and formate. When operated under substrate limiting conditions, 60% of the substrate carbon was converted into ethanol and formate in a 1:1 ratio. This product spectrum showed sensitivity to the substrate concentration, which partly shifted towards 1,3-propanediol and acetate in a 2:1 ratio at increasing substrate concentrations. Glucose fermentation mainly generated acetate, ethanol and butyrate. At higher substrate concentrations, acetate and ethanol were the dominant products. Co-fermentations of glucose-glycerol were performed with both mixed cultures, previously cultivated on glucose and on glycerol. The product spectrum of the two experiments was very similar: the main products were ethanol and butyrate (38% and 34% of the COD converted, respectively). The product spectrum obtained for glucose and glycerol fermentation could be explained based on the general metabolic pathways found for fermentative microorganisms and on the metabolic constraints: maximization of the ATP production rate and balancing the reducing equivalents involved.     

Sensitivity to pH, product inhibition, and inhibition by NAD+ of 1,3-propanediol dehydrogenase purified from Enterobacter agglomerans CNCM 1210

Because of its key role in the metabolism of glycerol during fermentation, 1,3-propanediol dehydrogenase (EC 1.1.1.202) of Enterobacter agglomerans CNCM 1210 was purified to homogeneity and studied with respect to its sensitivity to pH and to nucleotide and 1,3-propanediol concentrations. Enzyme activity was optimal at pH 7.8. The enzyme was competitively inhibited by NAD⁺ (K_i of 0.29 mM), and 1,3-propanediol exerted a strong inhibitory effect according to a mixed-type inhibition with a K_i of 13.7 mM and an a-factor of 9.0. It is proposed that these dehydrogenase properties be extended to the dehydrogenases of Citrobacter freundii and Klebsiella pneumoniae, which exhibited numerous similar physical properties. Received: 4 December 1996 / Accepted: 24 March 1997     

1,3-丙二醇产生菌的诱变育种及培养基优化

以实验室自然筛选的克雷伯氏杆菌(Klebsiella sp.)为出发株,采用紫外诱变及亚硝基胍和超声波协同处理获得一株1,3-丙二醇高产突变株。在摇瓶发酵中,其产1,3-丙二醇产量由17.39 g/L提高到24.11 g/L,提高38.64%。变异株经10次传代培养,发酵能力稳定。对发酵培养基成分进行了优化,优化后1,3-丙二醇产量为30.05g/L,为优化前的1.25倍。     

Exopolysaccharide production on low-grade maple sap by Enterobacter agglomerans grown in small scale bioreactors

Enterobacter agglomerans isolated from a soil sample collected in the vicinity of a sugar house was grown on low-grade maple sap: When cultivated in shake flask, Ent. agglomerans produced exopolysaccharide and α-ketoglutarate. No polysaccharide and no α-ketoglutarate were produced during growth at dissolved oxygen concentrations lower than 1% saturation in a 3.5 1 bioreactor, but succinate, lactate and formate were accumulated. In order to increase polysaccharide production, various agitation systems were adapted to 7 1 bioreactors with working volumes of 41. Highest polysaccharide yield (Y_p/s= 0.70) and biomass yield (Y_x/s= 0.17) were obtained with bioreactors agitated with four-and six-bladed Rushton turbines. Accumulation of mixed organic acids during polysaccharide production indicated that both aerobic and anaerobic conditions prevailed inside the bioreactors. Maple sap concentration higher than 60 g 1⁻¹ (as sucrose equivalent) decreased both growth and exopolysaccharide production.     

Glycerol conversion to 1,3-propanediol by newly isolated clostridia

Summary From pasteurized mud and soil samples glycerol-fermenting clostridia that produced 1,3-propanediol, butyrate and acetate were obtained. The isolates were taxonomically characterized and identified as Clostridium butyricum. The most active strain, SH1 = DSM 5431, was able to convert up to 110 g/l of glycerol to 56 g/l of 1,3-propanediol in 29 h. A few Clostridium strains from culture-collections (3 out of 16 of the C. butyricum group) and some isolates of Kutzner from cheese samples were also able to ferment glycerol, but the final concentration and the productivity of 1,3-propanediol was lower than in strain SH1. Strain SH1 grew well in a pH range between 6.0 and 7.5, with a weak optimum at 6.5, and was stimulated by sparging with N₂. Best overall productivity was obtained in fed-batch culture with a starting concentration of 5% glycerol. In all fermentations the yield of 1,3-propanediol in relation to glycerol was higher than expected from NADH production by acid formation. On the other hand the H₂ production was lower than expected, if per mole of acetyl coenzyme A one mole of H₂ is released. The observations point to a substantial transfer of reducing potential from ferredoxin to NAD, which finally results in increased 1,3-propanediol production.     

N-carbobenzyloxy-D-aspartic acid as a competitive inhibitor of indole-3-acetyl-L-aspartic acid hydrolase of Enterobacter agglomerans

The gene for a specific IAA-asp hydrolase from Enterobacteragglomerans, iaaspH, is a potentially useful tool for modificationofIAA homeostasis in higher plants that use the IAA-asp oxidation pathway forauxin catabolism. In order to optimize the utility of this gene for plantmodification and to increase the success of obtainingiaaspH transformed plants from culture, we haveinvestigated aspects of IAA-asp hydrolase catalysis. The catalyticcharacteristics of the IAA-asp hydrolase from Enterobacteragglomerans was studied using ten compounds that are structuralanalogues of IAA-asp. These compounds were tested as potential IAA-asphydrolasesubstrates as well as for inhibition of IAA-asp hydrolysis. Among them,N-carbobenzyloxy-D-aspartic acid (N-CBZ-D-asp) and N-CBZ-L-asp were found to bethe strongest inhibitors with more than 80% inhibition of IAA-asp hydrolysis.Aspartyl-L-aspartic acid and a asp-ser-asp-pro-arg peptide also showed stronginhibitory activities, reducing rates of IAA-L-asp hydrolysis, when added atequal molar amounts relative to the substrate, by 60% and 65%, respectively.N-CBZ-D-asp was chosen for further kinetic studies and for studies of itstoxicity in relation to seed germination because it was a strong inhibitor,exhibited a very low rate of hydrolysis by the IAA-asp hydrolase and wascommercially available. N-CBZ-D-asp was shown to be a competitive inhibitor forthe Enterobacter agglomerans IAA-asp hydrolase with aK _i value of 1.22 mM. Studies oftomato seed germination showed that N-CBZ-D-asp did not affect the rate of seedgermination at up to 1 mM, but the growth rate of seedlings wassignificantly reduced when the concentration in the medium was 0.5mM and higher. These results indicate that, at suitableconcentrations, N-CBZ-D-asp should be a useful tool for control of low levelexpression of the iaaspH in transgenic plants duringcritical stages of plant regeneration from culture.     

Pyrrolnitrin Production by an Enterobacter agglomerans Strain with a Broad Spectrum of Antagonistic Activity Towards Fungal and Bacterial Phytopathogens

Strain IC1270 of Enterobacter agglomerans has been previously described as a producer of a complex of chitinolytic enzymes and as an antagonist of many fungal phytopathogens [Chernin et al. (1995) Appl. Env. Microbiol. 61:1720–1726]. Here we show that this strain also produces an antibiotic that was purified by TLC and HPLC and identified by UV, IR, MS, and NMR analyses as pyrrolnitrin [3-chloro-4-(2′-nitro-3′-chlorophenyl)pyrrole]. The purified antibiotic is efficient against many phytopathogenic bacteria and fungi in vitro. This is the first piece of evidence showing that pyrrolnitrin can be produced by bacteria other than Pseudomonas and that one bacterial strain can simultaneously produce chitinolytic enzymes and pyrrolnitrin. The possible role of a combination of chitinases and pyrrolnitrin in antagonism is discussed.     

NonepidemicErwinia herbicola (Enterobacter agglomerans) in blood cultures: Bacteriological analysis of fifteen cases

The finding ofErwinia herbicola (Enterobacter agglomerans) in blood cultures of 15 inpatients, during a 53/4-year period in a nonepidemic context, was analyzed for clinical significance. Mixed cultures were seen in 5 patients; multiple sets were taken in 10 patients. In only 6 patients couldErwinia be significantly correlated with clinical septicemia; in 9, it was of doubtful significance, more likely reflecting transient bacteremia than skin contamination or laboratory handling. These findings contrast to previous reports that stress a preponderance of clinically significant strains ofEnterobacter species andErwinia from blood cultures. The only bacteriological differences between the “significant” and “doubtful” groups involved the time required to detectErwinia.     

Decolorization of Azo Dyes with Enterobacter agglomerans Immobilized in Different Supports by Using Fluidized Bed Bioreactor

Immobilized cells of Enterobacter agglomerans, able to reduce azo dyes enzymatically, were used as a biocatalyst for the decolorization of synthetic medium containing the toxic azo dye methyl red (MR). This bacterial strain exhibits high ability to completely decolorize 100 mg/L of MR after only 6 h of incubation under aerobic conditions. Cells of E. agglomerans were immobilized in calcium alginate, polyacylamide, cooper beech, and vermiculite, and were used for the decolorization of MR from synthetic water by using a fluidized bed bioreactor. The highest specific decolorization rate was obtained when E. agglomerans was entrapped in calcium alginate beads and was of about 3.04 mg MR/g cell/h with a 50% conversion time (t_1/2) of about 1.6 h. Moreover, immobilized cells in calcium alginate continuously decolorized MR even after seven repeated experiments without significant loss of activity, while polyacrylamide-, cooper beech-, and vermiculite-immobilized cells retained only 62, 15, and 13% of their original activity, respectively.     

Utilization of Glycerol as a Hydrogen Acceptor by Lactobacillus reuteri: Purification of 1,3-Propanediol:NAD Oxidoreductase

Lactobacillus reuteri utilizes exogenously added glycerol as a hydrogen acceptor during carbohydrate fermentations, resulting in higher growth rates and cell yields than those obtained during growth on carbohydrates alone. Glycerol is first converted to 3-hydroxypropionaldehyde by a coenzyme B(12)-dependent glycerol dehydratase and then reduced to 1,3-propanediol by an NAD -dependent oxidoreductase. The latter enzyme was purified and determined to have a molecular weight of 180,000; it is predicted to exist as a tetramer of identical 42,000-molecular-weight subunits.     

Water relations of ethanol fermentation by Saccharomyces cerevisiae: Glycerol production under solute stress

The effect of water activity (a_w) upon glycerol production during ethanol fermentation by Saccharomyces cerevisiae was studied in continuous culture using sorbitol to adjust the a_w to 0.971. A reduction of the a_w of the medium resulted in more glycerol being produced. The glycerol yield was 3–4-fold greater at 0.971 a_w than 0.994 a_w. Ethanol yield and specific rate of ethanol production was slightly greater at 0.971 than 0.994 a_w whereas the biomass concentration was lower. Reduced a_w increased the specific rate of glucose uptake and the maintenance requirements of the organism. The results suggest that the effect of a_w on glycerol production can be reduced by controlled feeding of substrate during fermentation.