Enhanced exopolysaccharide production by metabolic engineering of Streptococcus thermophilus

It is possible that the low levels of production of exopolysaccharides (EPSs) by lactic acid bacteria could be improved by altering the levels of enzymes in the central metabolism that influence the production of precursor nucleotide sugars. To test this hypothesis, we identified and cloned the galU gene, which codes for UDP glucose pyrophosphorylase (GalU) in Streptococcus thermophilus LY03. Homologous overexpression of the gene led to a 10-fold increase in GalU activity but did not have any effect on the EPS yield when lactose was the carbon source. However, when galU was overexpressed in combination with pgmA, which encodes phosphoglucomutase (PGM), the EPS yield increased from 0.17 to 0.31 g/mol of carbon from lactose. A galactose-fermenting LY03 mutant (Gal(+)) with increased activities of the Leloir enzymes was also found to have a higher EPS yield (0.24 g/mol of carbon) than the parent strain. The EPS yield was further improved to 0.27 g/mol of carbon by overexpressing galU in this strain. However, the highest EPS yield, 0.36 g/mol of carbon, was obtained when pgmA was knocked out in the Gal(+) strain. Measurements of the levels of intracellular metabolites in the cultures revealed that the Gal(+) strains had considerably higher glucose 1-phosphate levels than the other strains, and the strain lacking PGM activity had threefold-higher levels of glucose 1-phosphate than the other Gal(+) strains. These results show that it is possible to increase EPS production by altering the levels of enzymes in the central carbohydrate metabolism.     

Enhanced Exopolysaccharide Production by Metabolic Engineering of Streptococcus thermophilus

It is possible that the low levels of production of exopolysaccharides (EPSs) by lactic acid bacteria could be improved by altering the levels of enzymes in the central metabolism that influence the production of precursor nucleotide sugars. To test this hypothesis, we identified and cloned the galU gene, which codes for UDP glucose pyrophosphorylase (GalU) in Streptococcus thermophilus LY03. Homologous overexpression of the gene led to a 10-fold increase in GalU activity but did not have any effect on the EPS yield when lactose was the carbon source. However, when galU was overexpressed in combination with pgmA, which encodes phosphoglucomutase (PGM), the EPS yield increased from 0.17 to 0.31 g/mol of carbon from lactose. A galactose-fermenting LY03 mutant (Gal⁺) with increased activities of the Leloir enzymes was also found to have a higher EPS yield (0.24 g/mol of carbon) than the parent strain. The EPS yield was further improved to 0.27 g/mol of carbon by overexpressing galU in this strain. However, the highest EPS yield, 0.36 g/mol of carbon, was obtained when pgmA was knocked out in the Gal⁺ strain. Measurements of the levels of intracellular metabolites in the cultures revealed that the Gal⁺ strains had considerably higher glucose 1-phosphate levels than the other strains, and the strain lacking PGM activity had threefold-higher levels of glucose 1-phosphate than the other Gal⁺ strains. These results show that it is possible to increase EPS production by altering the levels of enzymes in the central carbohydrate metabolism.     

Lactic acid production from corn stover using mixed cultures of Lactobacillus rhamnosus and Lactobacillus brevis

Mixed cultures of Lactobacillus rhamnosus and Lactobacillus brevis was studied for improving utilization of both cellulose- and hemicellulose-derived sugars from corn stover for lactic acid production. During simultaneous saccharification and fermentation (SSF) of NaOH-treated corn stover by the mixed cultures, a lactic acid yield of 0.70 g/g was obtained, which was about 18.6% and 29.6% higher than that by single cultures of L. rhamnosus and L. brevis, respectively. Our results indicated that lactic acid yield from NaOH-pretreated corn stover by mixed cultures of L. rhamnosus and L. brevis was comparable to that from pure sugar mixtures (0.73 g/g of glucose/xylose mixture at 3:1 w/w).     

Exopolysaccharide production by Streptococcus thermophilus SY: production and preliminary characterization of the polymer

AIMS: To evaluate the effect of yeast extract (YE) concentration, temperature and pH on growth and exopolysaccharide (EPS) production in a whey-based medium by Streptococcus thermophilus SY and to characterize the partially purified EPS. METHODS AND RESULTS: Factorial experiments and empirical model building were used to optimize fermentation conditions and the chemical composition, average molecular weight (MW) and rheological properties of aqueous dispersions of the EPS were determined. Exopolysaccharide production was growth associated and was higher (152 mg l(-1)) at pH 6.4 and 36 degrees C with 4 g l(-1) YE. High performance size exclusion chromatography of the partially purified EPS showed two peaks, with a weight average MW of 2 x 10(6) and 5 x 10(4), respectively. The EPS was a heteropolysaccharide, with a glucose : galactose : rhamnose ratio of 2 : 4.5 : 1. Its water dispersions had a pseudoplastic behaviour and showed a higher viscosity of xanthan solutions. SIGNIFICANCE AND IMPACT OF THE STUDY: The fermentation conditions and some properties of an EPS produced by Strep. thermophilus, a dairy starter organism, were described.     

Kinetic study of the conversion of different substrates to lactic acid using Lactobacillus bulgaricus

Lactic acid fermentation includes several reactions in association with the microorganism growth. A kinetic study was performed of the conversion of multiple substrates to lactic acid using Lactobacillus bulgaricus. Batch experiments were performed to study the effect of different substrates (lactose, glucose, and galactose) on the overall bioreaction rate. During the first hours of fermentation, glucose and galactose accumulated in the medium and the rate of hydrolysis of lactose to glucose and galactose was faster than the convesion of these substrates. Once the microorganism built the necessary enzymes for the substrate conversion to lactic acid, the conversion rate was higher for glucose than for galactose. The inoculum preparation was performed in such a way that healthy young cells were obtained. By using this inoculum, shorter fermentation times with very little lag phase were observed. The consumption patterns of the different substrates converted to lactic acid were studied to determine which substrate controls the overall reaction for lactic acid production. A mathematical model (unstructured Monod type) was developed to describe microorganism growth and lactic acid production. A good fit with a simple equation was obtained. It was found experimentally that the approximate ratio of cell to substrate was 1 to 10, the growth yield coefficient (Y(XS)) was 0.10 g cell/g substrate, the product yield (Y(PS)) was 0.90 g lactic acid/g substrate, and the alpha parameter in the Luedeking-Piret equation was 9. The Monod kinetic parameters were obtained. The saturation constant (K(S)) was 3.36 g/L, and the specific growth rate (microm ) was 1.14 l/h.     

Engineering of Saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose

For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as l-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of l-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of l-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the l-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h(-1) g [dry weight](-1)) and ethanol production (0.29 g h(-1) g [dry weight](-1)) and a high ethanol yield (0.43 g g(-1)) during anaerobic growth on l-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.     

Optimization of fermentation conditions and rheological properties of exopolysaccharide produced by deep-sea bacterium Zunongwangia profunda SM-A87

Zunongwangia profunda SM-A87 isolated from deep-sea sediment can secrete large quantity of exopolysaccharide (EPS). Response surface methodology was applied to optimize the culture conditions for EPS production. Single-factor experiment showed that lactose was the best carbon source. Based on the Plackett–Burman design, lactose, peptone and temperature were selected as significant variables, which were further optimized by the steepest ascent (descent) method and central composite design. The optimal culture conditions for EPS production and broth viscosity were determined as 32.21 g/L lactose, 8.87 g/L peptone and an incubation temperature of 9.8°C. Under these conditions, the maximum EPS yield and broth viscosity were 8.90 g/L and 6551 mPa•s, respectively, which is the first report of such high yield of EPS from a marine bacterium. The aqueous solution of the EPS displayed high viscosity, interesting shearing thinning property and great tolerance to high temperature, a wide range of pH, and high salinity.     

Multiple and reversible hydrogenases for hydrogen production by Escherichia coli: dependence on fermentation substrate, pH and the F(0)F(1)-ATPase

Molecular hydrogen (H(2)) can be produced via hydrogenases during mixed-acid fermentation by bacteria. Escherichia coli possesses multiple (four) hydrogenases. Hydrogenase 3 (Hyd-3) and probably 4 (Hyd-4) with formate dehydrogenase H (Fdh-H) form two different H(2)-evolving formate hydrogen lyase (FHL) pathways during glucose fermentation. For both FHL forms, the hycB gene coding small subunit of Hyd-3 is required. Formation and activity of FHL also depends on the external pH ([pH](out)) and the presence of formate. FHL is related with the F(0)F(1)-ATPase by supplying reducing equivalents and depending on proton-motive force. Two other hydrogenases, 1 (Hyd-1) and 2 (Hyd-2), are H(2)-oxidizing enzymes during glucose fermentation at neutral and low [pH](out). They operate in a reverse, H(2)-producing mode during glycerol fermentation at neutral [pH](out). Hyd-1 and Hyd-2 activity depends on F(0)F(1). Moreover, Hyd-3 can also work in a reverse mode. Therefore, the operation direction and activity of all Hyd enzymes might determine H(2) production; some metabolic cross-talk between Hyd enzymes is proposed. Manipulating of different Hyd enzymes activity is an effective way to enhance H(2) production by bacteria in biotechnology. Moreover, a novel approach would be the use of glycerol as feedstock in fermentation processes leading to H(2) production, reduced fuels and other chemicals with higher yields than those obtained by common sugars.     

Production of a Novel Extracellular Polysaccharide by Lactobacillus sake 0-1 and Characterization of the Polysaccharide

A novel exopolysaccharide (EPS) produced by Lactobacillus sake 0-1 (CBS 532.92) has been isolated and characterized. When the strain was grown on glucose, the produced EPS contained glucose and rhamnose in a molar ratio of 3:2 and the average molecular mass distribution (M(infm)) was determined at 6 x 10(sup6) Da. At a concentration of 1%, the 0-1 EPS had better viscosifying properties than xanthan gum when measured over a range of shear rates from 0 to 300 s(sup-1), while shear-thinning properties were comparable. Rheological data and anion-exchange chromatography suggested the presence of a negatively charged group in the EPS. Physiological parameters for optimal production of EPS were determined in batch fermentation experiments. Maximum EPS production was 1.40 g (middot) liter(sup-1), which was obtained when L. sake 0-1 had been grown anaerobically at 20(deg)C and pH 5.8. When cultured at lower temperatures, the EPS production per gram of biomass increased from 600 mg at 20(deg)C to 700 mg at 10(deg)C but the growth rate in the exponential phase decreased from 0.16 to 0.03 g (middot) liter(sup-1) (middot) h(sup-1). EPS production started in the early growth phase and stopped when the culture reached the stationary phase. Growing the 0-1 strain on different energy sources such as glucose, galactose, mannose, fructose, lactose, and sucrose did not alter the composition of the EPS produced.     

Growth and exopolysaccharide production during free and immobilized cell chemostat culture of Lactobacillus rhamnosus RW-9595M

AIMS: Biomass and exopolysaccharide (EPS) production were studied during chemostat cultures in whey permeate medium with Lactobacillus rhamnosus RW-9595M-free cells and cells immobilized on solid porous supports (ImmobaSil). METHODS AND RESULTS: A continuous culture with free cells was conducted for 9 days at dilution rates (D) between 0.3 and 0.8 h(-1) in yeast extract (YE)/mineral supplemented whey permeate. Maximum EPS production (1808 mg l(-1)) and volumetric productivity (542.6 mg l(-1) h(-1)) were obtained for a low D of 0.3 h(-1). A continuous fermentation in a two-stage bioreactor system, composed of a first stage with immobilized cells and a second stage inoculated with free cells produced in the first reactor, was carried out for 32 days. The influence of YE concentration, temperature and dilution rate, and their interactions on biomass, EPS and lactic acid production was investigated. A statistically significant model was found only for lactic acid production. Marked cell morphological and physiological changes led to the formation of very large cell-containing aggregates and a low mean soluble EPS production (138 mg l(-1)). Aggregate volumetric productivity of the two-stage system varied between 5.7 and 49.5 g l(-1) h(-1) for different fermentation conditions and times. Aggregates contained a very high biomass concentration, estimated at 74% of aggregate dry weight by nitrogen analysis and 4.3 x 10(12) CFU g(-1) by a DNA extraction method and a high nonsoluble polysaccharide content (14.2%). At age 24 days, insoluble EPS concentration and volumetric productivity were 1250 mg l(-1) and 2240 mg l(-1) h(-1) respectively. The physiological changes were shown to be reversible when cells were incubated during three successive batch cultures. CONCLUSIONS: EPS production and volumetric productivity during continuous free-cell chemostat cultures with L. rhamnosus RW-9595M are among the highest values reported for lactobacilli in literature. Immobilization and continuous culture resulted in low soluble EPS production and large morphological and physiological changes of L. rhamnosus RW-9595M, with formation of macroscopical aggregates mainly composed of biomass and nonsoluble EPS. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on continuous EPS production by immobilized LAB. Immobilization and culture time-induced cell aggregation and could be used to produce new synbiotic products with very high viable cell and EPS concentrations.     

Change from homo- to heterolactic fermentation by Streptococcus lactis resulting from glucose limitation in anaerobic chemostat cultures.

Lactic streptococci, classically regarded as homolactic fermenters of glucose and lactose, became heterolactic when grown with limiting carbohydrate concentrations in a chemostat. At high dilution rates (D) with excess glucose present, about 95% of the fermented sugar was converted to l-lactate. However, as D was lowered and glucose became limiting, five of the six strains tested changed to a heterolactic fermentation such that at D = 0.1 h(-1) as little as 1% of the glucose was converted to l-lactate. The products formed after this phenotypic change in fermentation pattern were formate, acetate, and ethanol. The level of lactate dehydrogenase, which is dependent upon ketohexose diphosphate for activity, decreased as fermentation became heterolactic with Streptococcus lactis ML(3). Transfer of heterolactic cells from the chemostat to buffer containing glucose resulted in the nongrowing cells converting nearly 80% of the glucose to l-lactate, indicating that fine control of enzyme activity is an important factor in the fermentation change. These nongrowing cells metabolizing glucose had elevated (ca. twofold) intracellular fructose 1,6-diphosphate concentrations ([FDP](in)) compared with those in the glucose-limited heterolactic cells in the chemostat. [FDP](in) was monitored during the change in fermentation pattern observed in the chemostat when glucose became limiting. Cells converting 95 and 1% of the glucose to l-lactate contained 25 and 10 mM [FDP](in), respectively. It is suggested that factors involved in the change to heterolactic fermentation include both [FDP](in) and the level of lactate dehydrogenase.     

Alterations in glycohydrolase activities in streptozotocin-diabetic Chinese hamsters (Cricetulus griseus).

1. Six weeks after the injection of streptozotocin at 125 mg/kg i.p. in the AV line nondiabetic Chinese hamsters, the animals showed hyperglycemia, increased kidney, pancreas and stomach weights and stomach glucagon contents and depletion of insulin and glucagon in the pancreas. 2. Plasma beta-D-galactosidase and N-acetyl-beta-D-glucosaminidase were elevated; whereas alpha-D-glucosidase was decreased and alpha-D-galactosidase remained unchanged in the plasma. 3. In the kidney, streptozotocin-diabetes led to depression of alpha-D-mannosidase, beta-D-fucosidase and N-acetyl-beta-D-glucosaminidase activities in both 12,000 g supernatant and precipitate fractions, decreases in alpha-D-glucosidase in the supernatant only and no change in alpha-L-fucosidase, alpha-D-galactosidase, beta-D-galactosidase and beta-D-glucuronidase. 4. In the liver, significant increases in N-acetyl-beta-D-glucosaminidase, alpha-D-galactosidase, beta-D-galactosidase, beta-D-fucosidase, beta-D-glucosidase and alpha-D-mannosidase were found in either the supernatant or the precipitate fraction of the diabetic animals. The data indicate diabetes-dependent tissue-specific changes in glycohydrolases in the Chinese hamster.     

Production of green biodegradable plastics of poly(3-hydroxybutyrate) from renewable resources of agricultural residues

This work describes potential opportunities for utilization of agro-industrial residues to produce green biodegradable plastics of poly(3-hydroxybutyrate) (PHB). Wheat straws were examined with good efficacy of carbon substrates using Cupriavidus necator. Production was examined in separate hydrolysis and fermentation (SHF) in the presence and absence of WS hydrolysis enzymes, and in simultaneous saccharification and fermentation (SSF) with enzymes. Results showed that production of PHB in SSF was more efficient in terms of viable cell count, cell dry weight, and PHB production and yield compared to those of SHF and glucose-control cultures. While glucose control experiment produced 4.6 g/L PHB; SSF produced 10.0 g/L compared to 7.1 g/L in SHF when utilizing enzymes during WS hydrolysis. Results showed that most of sugars produced during the hydrolysis were consumed in SHF (~98 %) compared to 89.2 % in SSF. Results also demonstrated that a combination of glucose and xylose can compensate for the excess carbon required for enhancing PHB production by C. necator. However, higher concentration of sugars at the beginning of fermentation in SHF can lead to cell inhibition and consequently catabolite repressions. Accordingly, results demonstrated that the gradual release of sugars in SSF enhanced PHB production. Moreover, the presence of sugars other than glucose and xylose can eliminate PHB degradation in medium of low carbon substrate concentrations in SSF.     

Production of exopolysaccharides by submerged mycelial culture of a mushroom Tremella fuciformis

The optimization of submerged culture conditions for mycelial growth and exopolysaccharide (EPS) production in an edible mushroom Tremella fuciformis was studied in shake flasks and bioreactors. The temperature of 28 degrees C and pH 8 in the beginning of fermentation in agitated flasks was the most efficient condition to obtain maximum mycelial biomass and EPS. The optimal medium constituents were as follows (gL(-1)): glucose 20, tryptone 2, KH(2)PO(4) 0.46, K(2)HPO(4) 1 and MgSO(4).7H(2)O 0.5. The fungus was cultivated under various agitation and aeration conditions in a 5L stirred-tank bioreactor. The maximum cell mass and EPS production were obtained at a relatively high agitation speed of 200 rpm and at an aeration rate of 2 vvm. The flow behavior of the fermentation broth was Newtonian and the maximum apparent viscosity (35 cP) was observed at a highly aerated condition (2 vvm). The EPS productivity in an airlift reactor was higher than that in the stirred-tank reactor. The morphological study revealed that the fungus grows in mainly three different yeast-like forms: ovoid, elongated, and double yeast forms. The high population of the elongated yeast has a very close relationship to high EPS production. The EPS were protein-bound polysaccharides consisted of mainly mannose, xylose, and fucose. The molecular weights of EPS were determined to be (1.3-1.5)x10(6).     

Membrane anchoring and surface distribution of glycohydrolases of human erythrocyte membranes

The membrane anchoring of the following glycohydrolases of human erythrocyte plasma membranes was investigated: alpha- and beta-D-glucosidase, alpha- and beta-D-galactosidase, beta-D-glucuronidase, N-acetyl-beta-D-glucosaminidase, alpha-D-mannosidase, and alpha-L-fucosidase. Optimized fluorimetric methods for the assay of these enzymes were set up. Treatment of the ghost preparation with 1.0 mol/l (optimal concentration) NaCl caused release ranging from 4.2% of alpha-D-glucosidase to 70% of beta-D-galactosidase; treatment with 0.4% (optimal concentration) Triton X-100 liberated 5.1% of beta-D-galactosidase to 89% of alpha-D-glucosidase; treatment with 1.75% (optimal concentration) octylglucoside yielded solubilization from 6.3% of beta-D-galactosidase to 85% of alpha-D-glucosidase. Treatment with phosphoinositide-specific phospholipase C caused no liberation of any of the studied glycohydrolases. These results are consistent with the notion that the above glycohydrolases are differently anchored or associated with the erythrocyte plasma membrane, and provide the methodological basis for inspecting the occurrence of these enzymes in different membrane microdomains.     

Butanol production from corn fiber xylan using Clostridium acetobutylicum

Acetone, butanol, and ethanol (ABE) were produced from corn fiber arabinoxylan (CFAX) and CFAX sugars (glucose, xylose, galactose, and arabinose) using Clostridium acetobutylicum P260. In mixed sugar (glucose, xylose, galactose, and arabinose) fermentation, the culture preferred glucose and arabinose over galactose and xylose. Under the experimental conditions, CFAX (60 g/L) was not fermented until either 5 g/L xylose or glucose plus xylanase enzyme were added to support initial growth and fermentation. In this system, C. acetobutylicum produced 9.60 g/L ABE from CFAX and xylose. This experiment resulted in a yield and productivity of 0.41 and 0.20 g/L x h, respectively. In the integrated hydrolysis, fermentation, and recovery process, 60 g/L CFAX and 5 g/L xylose produced 24.67 g/L ABE and resulted in a higher yield (0.44) and a higher productivity (0.47 g/L x h). CFAX was hydrolyzed by xylan-hydrolyzing enzymes, and ABE were recovered by gas stripping. This investigation demonstrated that integration of hydrolysis of CFAX, fermentation to ABE, and recovery of ABE in a single system is an economically attractive process. It is suggested that the culture be further developed to hydrolyze CFAX and utilize all xylan sugars simultaneously. This would further increase productivity of the reactor.     

Carbon Source Requirements for Exopolysaccharide Production by Lactobacillus casei CG11 and Partial Structure Analysis of the Polymer

Exopolysaccharide production by Lactobacillus casei CG11 was studied in basal minimum medium containing various carbon sources (galactose, glucose, lactose, sucrose, maltose, melibiose) at concentrations of 2, 5, 10, and 20 g/liter. L. casei CG11 produced exopolysaccharides in basal minimum medium containing each of the sugars tested; lactose and galactose were the poorest carbon sources, and glucose was by far the most efficient carbon source. Sugar concentrations had a marked effect on polymer yield. Plasmid-cured Muc^- derivatives grew better in the presence of glucose and attained slightly higher populations than the wild-type strain. The values obtained with lactose were considerably lower for both growth and exopolysaccharide yield. The level of specific polymer production per cell obtained with glucose was distinctively lower for Muc^- derivatives than for the Muc⁺ strain. The polymer produced by L. casei CG11 in the presence of glucose was different from that formed in the presence of lactose. The polysaccharide produced by L. casei CG11 in basal minimum medium containing 20 g of glucose per liter had an intrinsic viscosity of 1.13 dl/g. It was rich in glucose (76%), which was present mostly as 2- or 3-linked residues along with some 2,3 doubly substituted glucose units, and in rhamnose (21%), which was present as 2-linked or terminal rhamnose; traces of mannose and galactose were also present.     

Influence of transport energization on the growth yield of Escherichia coli.

The growth yields of Escherichia coli on glucose, lactose, galactose, maltose, maltotriose, and maltohexaose were estimated under anaerobic conditions in the absence of electron acceptors. The yields on these substrates exhibited significant differences when measured in carbon-limited chemostats at similar growth rates and compared in terms of grams (dry weight) of cells produced per mole of hexose utilized. Maltohexaose was the most efficiently utilized substrate, and galactose was the least efficiently utilized under these conditions. All these sugars were known to be metabolized to glucose 6-phosphate and produced the same pattern of fermentation products. The differences in growth yields were ascribed to differences in energy costs for transport and phosphorylation of these sugars. A formalized treatment of these factors in determining growth yields was established and used to obtain values for the cost of transport and hence the energy-coupling stoichiometries for the transport of substrates via proton symport and binding-protein-dependent mechanisms in vivo. By this approach, the proton-lactose stoichiometry was found to be 1.1 to 1.8 H+ per lactose, equivalent to approximately 0.5 ATP used per lactose transported. The cost of transporting maltose via a binding-protein-dependent mechanism was considerably higher, being over 1 to 1.2 ATP per maltose or maltodextrin transported. The formalized treatment also permitted estimation of the net ATP yield from the metabolism of these sugars; it was calculated that the growth yield data were consistent with the production of 2.8 to 3.2 ATP in the metabolism of glucose 6-phosphate to fermentation products.     

Factors affecting exocellular polysaccharide production by Lactobacillus delbrueckii subsp. bulgaricus grown in a chemically defined medium

We developed a chemically defined medium (CDM) containing lactose or glucose as the carbon source that supports growth and exopolysaccharide (EPS) production of two strains of Lactobacillus delbrueckii subsp. bulgaricus. The factors found to affect EPS production in this medium were oxygen, pH, temperature, and medium constituents, such as orotic acid and the carbon source. EPS production was greatest during the stationary phase. Composition analysis of EPS isolated at different growth phases and produced under different fermentation conditions (varying carbon source or pH) revealed that the component sugars were the same. The EPS from strain L. delbrueckii subsp. bulgaricus CNRZ 1187 contained galactose and glucose, and that of strain L. delbrueckii subsp. bulgaricus CNRZ 416 contained galactose, glucose, and rhamnose. However, the relative proportions of the individual monosaccharides differed, suggesting that repeating unit structures can vary according to specific medium alterations. Under pH-controlled fermentation conditions, L. delbrueckii subsp. bulgaricus strains produced as much EPS in the CDM as in milk. Furthermore, the relative proportions of individual monosaccharides of EPS produced in pH-controlled CDM or in milk were very similar. The CDM we developed may be a useful model and an alternative to milk in studies of EPS production.     

Low-cost propionate salt as road deicer: evaluation of cheese whey and other media constituents

Propionate and acetate salts are environmentally friendly, effective road deicer substitutes for widely used sodium chloride. A low-cost medium, using raw cheese whey and hydrolyzed whey permeate/whey permeate powder as substrates, and corn-steep liquor as a nutrient supplement, was studied for lactic acid production, replacing synthetic lactose and other high-cost nutrients. A non-sterile stage-I fermentation process for improved lactate productivity using an inexpensive commercial medium was performed at a 20-L fermenter level. A lactate yield of 0.98 g/g lactose and a productivity of 1.1 g/L/h was obtained with complete lactose utilization. When synthetic lactate and glucose were used as substrates in propionate and acetate fermentation, a total acid yield of 0.55 g/g glucose and lactate consumed and a batch productivity of 0.22 g/L/h was obtained. A stage-II fermentation process to produce propionate and acetate salts from cheese whey-derived lactate (stage-I fermentation broth) resulted in 1.6%( w/v) propionate after a total of 161 h (stages I and II).