Fermentation of glucose and xylose in ruminal strains of Butyrivibrio fibrisolvens

Metabolism of glucose and xylose and parameters of growth were investigated in strains of Butyrivibrio fibrisolvens ATCC 19171 and CE 51. In the strain ATCC 19171, the composition of fermentation end-products was the same in cultures supplied with glucose and xylose. The strain CE 51 produced more volatile fatty acids and less lactate from xylose than from glucose. Cells of this strain grown on xylose possessed phosphoketolase activity (EC 4.1.2.9). In both strains the production of cell dry matter and growth rate were higher in cultures supplied with glucose. In xylose-grown cultures butyrivibrios tended to convert more substrate carbon into metabolites and less into cellular material than in cultures grown on glucose.     

Biochemical characteristics and fermentation of glucose and starch by rabbit cæcal strains ofBifidobacterium globosum

Two strains ofBifidobacterium globosum were isolated from cæcal contents of rabbits in a search for potential probiotics. Both strains fermented glucose, galactose, pentoses, maltose, raffinose and starch. Common coccidiostats (monensin, salinomycin) and antimicrobial growth promotors (avoparcin, bacitracin, nitrovin, virginiamycin) supplied at 10 mg/L inhibited their growth in cultures with glucose. Fermentation parameters of bifidobacteria on glucose and starch. When growing on starch, the two strains of bifidobacteria produced 1 mol lactate per 5.6 and 5.7 mol acetate, respectively. Corresponding values during growth on glucose were 17.3 and 8.4 mol of acetate per mol of lactate. Starch-grown cells accumulated more saccharides than cells grown on glucose (1.48vs. 0.41 and 3.12vs. 1.18 mmol glucose units per 1 g of dry matter, respectively).     

Regulation of the lipopolysaccharide-specific sialyltransferase activity of gonococci by the growth state of the bacteria, but not by carbon source, catabolite repression or oxygen supply

The enzyme sialyltransferase (STase) of Neisseria gonorrhoeae is a major pathogenicitiy determinant. Using a refined method for assaying the STase activity, the Km for CMP-NANA was shown to be 14 +/– 2 M, higher than that reported previously. Rates of sialylation by Nonidet extracts, prepared under conditions that optimise solubilisation of the membrane-bound enzyme, were 6 to 20 nmol of NANA transferred from CMP-¹⁴C-NANA onto isolated lipopolysaccharide/min./mg of extracted protein, far higher than the previously reported rates of less than 1 nmol of NANA transferred/min./mg of extracted protein. Gonococci grew more slowly with lactate or pyruvate than with glucose as the carbon source. Although growth with a mixture of limiting concentrations of both glucose and lactate was biphasic, diauxic growth was also found in the control culture supplied with glucose alone. The growth rate in the presence of lactate alone was slower than with glucose. The growth rate increased slightly relative to the glucose culture when both substrates were available; lactate was consumed more rapidly than glucose. Higher STase activities were found in bacteria harvested in the exponential than in the stationary phase of aerobic growth: the activity in aerated cultures was higher than those of oxygen-limited or anaerobic cultures. Similar STase activities were found in bacteria that had been grown with glucose, lactate or pyruvate as the carbon and energy source. Sialyltransferase synthesis is essentially constitutive: it is not regulated by glucose repression or by induction by lactate or anaerobiosis.     

Fermentation of pectin and glucose, and activity of pectin-degrading enzymes in the rabbit caecal bacterium Bacteroides caccae

AIMS: To compare fermentation pattern in cultures of Bacteroides caccae supplied with pectin and glucose, and identify enzymes involved in metabolism of pectin. METHODS AND RESULTS: A strain KWN isolated from the rabbit caecum was used. Fermentation pattern, changes of viscosity and enzyme reactions products were determined. Cultures grown on pectin produced significantly more acetate and less formate, lactate, fumarate and succinate than cultures grown on glucose. Production of cell dry matter and protein per gram of substrate used was the same in pectin- and glucose-grown cultures. The principal enzymes that participated in the metabolism of pectin were extracellular exopectate hydrolase (EC 3.2.1.67), extracellular endopectate lyase (EC 4.2.2.2) and cell-associated 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EC 4.1.2.14). The latter enzyme is unique to the Entner-Doudoroff pathway. Activities of pectinolytic enzymes in cultures grown on glucose were low. Activity of KDPG aldolase was similar in pectin- and glucose-grown cells. CONCLUSIONS: Metabolites and activities of pectin-degrading enzymes differed in cultures of B. caccae KWN grown on pectin and glucose. Yields of dry matter and protein were the same on both substrates. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on metabolism of pectin in animal strains of Bacteroides is incomplete. This study extends the knowledge on metabolism in bacteria from the rabbit caecum.     

The kinetics of end-product inhibition of l-lactate production from xylose and glucose by Lactococcus lactis IO-1

Summary The relative contributions of lactate inhibition and the generation of sterile (undividing) cells to the low xylose utilisation rate of Lactococcus lactis IO-1 was investigated. The lactate inhibition constant of xylose grown cells was shown to be 9.3 times more than that of glucose grown cells. However, the sterile cell production rate and LDH inactivation rate of the xylose cultures were at least 10 times less than the glucose cultures. Thus, it is suggested that the slower substrate consumption rate in xylose medium is caused mainly by the large inhibition constant for the end product.     

Radiorespirometry evidence for the discrimination between 13C-enriched glucose and unlabelled glucose molecules by Paracoccus denitrificans

Paracoccus denitrificans was grown on either unlabelled glucose, [1-13C]glucose or [6-13C]glucose as the sole carbon source for growth. The cells were then incubated with a range of 14C-glucose substrates to compare the 14CO2-evolution rates between cells grown on the glucose and the 13C-labelled glucose. Cells grown on 13C-glucose had significantly faster rates of 14CO2-evolution than those grown on unlabelled glucose. The % yields of 14CO2, per [1-14C]-, [6-14C]- and [U-14C]glucose supplied were also substantially greater than those measured for cells grown on unlabelled glucose. The data indicated that growth of Paracoccus on 13C-enriched glucose substrates resulted in cells with notably different 14C-glucose oxidation metabolism compared to that observed in cells grown on unlabelled glucose.     

Growth of Rhodopseudomonas capsulata on l- and d-malic acid

d-malate replaced l-malate in supporting both photosynthetic (anaerobic, light) and heterotrophic (aerobic, dark) growth of Rhodopseudomonas capsulata. Growth rates and cell yields were nearly equivalent with both enantiomorphs. Addition of glucose to malate culture media increased the growth rate and doubled the cell yield of heterotrophic cultures, but had little effect on photosynthetic cultures. Aerobically-grown cells showed a higher level of substrate-dependent oxygen uptake with l-malate than with d-malate. This preference for l-malate occured even in cells grown on d-malate. No malic racemase activity was detected in extracts of heterotrophically- or photosynthetically-grown cells.     

Fermentation of carbohydrates and yield of microbial protein in mixed cultures of rabbitcaecal microorganisms

Fermentation pattern and yields of microbial protein were investigated in cultures of the rabbit caecal contents supplied with glucose, xylose, starch, pectin and xylan. Rabbits at the age of 4 weeks (before weaning) and 3 months were slaughtered, their caecal contents added at 1.1% to growth media and incubated anaerobically at 39°C for 18 h. Caecal microorganisms of 4‐week‐old rabbits produced no methane and caproate, less butyrate, but more propionate than microorganisms of 3‐month‐old rabbits. In both groups of rabbits, fermentation of xylose produced significantly more propionate and less butyrate than fermentation of glucose. More propionate and less acetate was formed from starch than from pectin. In caecal cultures from 4‐week‐old rabbits with pectin, the molar percentages of acetate was significantly higher and percentages of other short‐chain fatty acids (SCFA) lower than in cultures with starch or xylan. In cultures from 3‐month‐old rabbits, fermentation of pectin and xylan produced similar SCFA profiles, different from SCFA molar composition in cultures with starch. Average production of microbial protein was 129mg per lg of carbohydrate digested (range 110 to 141mg/g). Protein yields were the same on glucose and xylose, but nonsignificantly higher on starch than on pectin and xylan. It can be concluded that the characteristics of substrate affected fermentation pattern in mixed cultures of rabbit caecal microorganisms. Substrate effects on protein yields were not statistically significant, due to high variation.     

Growth of the thermophilic bacterium Clostridium thermocellum in continuous culture

Clostridium thermocellum is an anaerobic thermophilic bacterium that produces enthanol from cellulosic substrates. When the organism was grown in continuous culture at dilution rates ranging from 0.04 to 0.25 h^-1, growth yields on cellobiose were higher than on glucose, and even higher yields were observed on cellotetraose. However, differences in bacterial yield were much greater at slow growth rates, and it appeared that glucose-grown cells had a fourfold higher (0.41 g substrate/g protein/h) maintenance energy requirement than cellobiose-grown cultures. Cellobiose and glucose were co-utilized in dual substrate continuous culture, and this was in contrast to batch culture experiments which indicated that the organism preferred the disaccharide. These experiments demonstrate that carbohydrate utilization patterns in continuous culture are different from those in batch culture and that submaximal growth rates affect substrate preference and bioenergetic parameters. The mechanisms regulating carbohydrate use may be different in batch versus continuous culture.Published with the approval of the Director of the Kentucky Agricultural Experiment Station as journal article no. 95-07-064.     

A comparison of orbitally‐shaken and stirred‐tank bioreactors: pH modulation and bioreactor type affect CHO cell growth and protein glycosylation

Orbitally shaken bioreactors (OSRs) support the suspension cultivation of animal cells at volumetric scales up to 200 L and are a potential alternative to stirred‐tank bioreactors (STRs) due to their rapid and homogeneous mixing and high oxygen transfer rate. In this study, a Chinese hamster ovary cell line producing a recombinant antibody was cultivated in a 5 L OSR and a 3 L STR, both operated with or without pH control. Effects of bioreactor type and pH control on cell growth and metabolism and on recombinant protein production and glycosylation were determined. In pH‐controlled bioreactors, the glucose consumption and lactate production rates were higher relative to cultures grown in bioreactors without pH control. The cell density and viability were higher in the OSRs than in the STRs, either with or without pH control. Volumetric recombinant antibody yields were not affected by the process conditions, and a glycan analysis of the antibody by mass spectrometry did not reveal major process‐dependent differences in the galactosylation index. The results demonstrated that OSRs are suitable for recombinant protein production from suspension‐adapted animal cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1174–1180, 2016     

L-lactate production from xylose employingLactococcus lactis IO-1

Summary The growth, substrate utilisation and L-lactate production ofLactococcus lactis IO-1 were examined on xylose, and glucose and xylose media. The yield of lactate on xylose was 0.47 g lactate/g xylose at an initial xylose concentration of 51.2 g/l and the _max was 0.72 h^–1. Xylose cultures were more susceptible to lactate inhibition than were glucose cultures but showed similar kinetic behaviour. The organism was capable of complete sugar utilisation when grown on a mixture of 20 g/l xylose and 20 g/l glucose and synthesised 0.66 g lactate/g sugar.     

Direct Regulation of Na+-Dependent myo-Inositol Transport by Sugars in Retinal Pigment Epithelium: Role of Phorbol Ester and Staurosporin

An Na⁺-dependent active process for myo-inositol (MI) uptake, sharing a common carrier system with glucose and sensitive to phlorizin, was previously established in primary cultures of bovine retinal pigment epithelial (RPE) cells (26, 32). The present report further examines the nature of glucose-induced inhibition of MI transport in primary cultures of RPE cells. RPE cells were grown in supplemented Dulbecco's modification of Eagle's medium (DMEM) containing 5 mM D-glucose (basic growth media) or 40 mM D-glucose or its nonmetabolizable analogue, α-methyl-D-glucoside (αMG); 1–5 mM nonradioactive MI, pyruvate, or lactate; or 0.2–20 µM phorbol 12-myristate 13-acetate (TPA) or straurosporin (modified growth media), for up to 4 weeks. The capacity of RPE cells to accumulate ³H-MI (ratios of intracellular transported radioactive MI, [MI]_i, to external free MI concentration, [MI]_i/[MI]₀) decreased by up to 41% or 34% when cells were grown for 10 days or longer with 40 mM D-glucose or 40 mM αMG, respectively, compared to cells grown in basic growth media. The rate of uptake of ³H-MI also was reduced to 63 ± 15% or 48 ± 8% of the control values when cells were fed 1 or 5 mM nonradioactive MI, respectively. In addition, cellular capacity to bind to [³H]phlorizin was reduced to 52 ± 7%, 61 ± 5%, or 38 ± 6% of the controls when RPE cells were fed 40 mM D-glucose, 40 mM αMG, or 5 mM nonradioactive MI, respectively. Growth media containing either pyruvate or lactate, the glucose metabolites, did not suppress the ability of RPE cells to accumulate MI. An 18 ± 8% reduction in [³H]thymidine incorporation into DNA occurred when cells were grown in 40 mM glucose for 12–14 days, compared to cells grown with 5 mM glucose. Chronic treatment (12–14 days) of the cells with phorbol ester, an activator of protein kinase C, caused up to twofold increase in MI uptake, [³H]phlorizin binding, cell number, and DNA synthesis. However, when the rates of MI uptake into cells grown in basic growth media or TPA-treated media were normalized to cell number, no significant difference in MI uptake was found between the treated and untreated cells. Addition of staurosporin, a protein kinase C inhibitor, together with TPA, in the growth media reversed the phorbol-induced increase of MI uptake. In contrast to its chronic effect, a 60-min incubation (acute effect) of cells in the presence of TPA, with or without inclusion of stauropsorin, did not alter the uptake of ³H-MI into RPE cells, regardless of glucose levels in the growth media. These studies indicated that glucose itself, and not glucose metabolites, regulated uptake of MI into primary cultures of RPE cells. In addition, glucose-induced down-regulation of MI uptake was not mediated through the protein kinase C pathway, but the staurosporin-inhibited, TPA-stimulated protein kinase C was partly responsible for growth and proliferation of RPE cells.     

Energy conservation in malolactic fermentation by Lactobacillus plantarum and Lactobacillus sake

A comparably poor growth medium containing 0.1% yeast extract as sole non-defined constituent was developed which allowed good reproducible growth of lactic acid bacteria. Of seven different strains of lactic acid bacteria tested, only Lactobacillus plantarum and Lactobacillus sake were found to catalyze stoichiometric conversion of l-malate to l-lactate and CO₂ concomitant with growth. The specific growth yield of malate fermentation to lactate at pH 5.0 was 2.0 g and 3.7 g per mol with L. plantarum and L. sake, respectively. Growth in batch cultures depended linearly on the malate concentration provided. Malate was decarboxylated nearly exclusively by the cytoplasmically localized malo-lactic enzyme. No other C₄-dicarboxylic acid-decarboxylating enzyme activity could be detected at significant activity in cell-free extracts. In pH-controlled continuous cultures, L. plantarum grew well with glucose as substrate, but not with malate. Addition of lactate to continuous cultures metabolizing glucose or malate decreased cell yields significantly. These results indicate that malo-lactic fermentation by these bacteria can be coupled with energy conservation, and that membrane energetization and ATP synthesis through this metabolic activity are due to malate uptake and/or lactate excretion rather than to an ion-translocating decarboxylase enzyme.     

Commensalistic Interaction Between Lactobacillus acidophilus and Propionibacterium shermanii

Propionibacterium shermanii and Lactobacillus acidophilus were grown in batch mixed culture in a 5-liter fermenter under controlled conditions of pH 5.8 and 35°C on a semisynthetic medium with glucose as an energy source. Cellular efficiencies and fermentation balances were developed for this pair and compared with P. shermanii grown in pure culture on glucose, lactate, and a mixture of these substrates and with L. acidophilus grown on glucose. P. shermanii had ATP yield coefficient values of 17 for each substrate alone but had an average value of 30 for substrate mixtures. Growth rates were similar for P. shermanii on glucose or lactate but higher cell yields were observed for glucose. P. shermanii used both lactate and glucose in mixed substrate until lactate was exhausted, and growth rates slowed thereafter. L. acidophilus had a similar ATP yield coefficient of 15 but produced lower cell yields than did P. shermanii on glucose. Mixed culture of both microorganisms on glucose resulted in much faster and nearly equal growth rates for both and no lactate accumulation in the medium. Acetic acid production rates per generation were lower in mixed culture, suggesting use by the growing culture. The cause of the synergistic effect was not determined but may be due to the rapid production and removal of lactate or CO₂ enhancement in mixed culture.     

Effect of oxygen on the metabolism of Zymomonas mobilis

The specific growth rate of the ethanol producing bacterium Zymomonas mobilis was 25–40% lower in the presence of oxygen than under anaerobic conditions, provided the cultures were supplied with a low substrate concentration (20 g glucose/l). However, the molar growth yield of these cultures was not influenced by oxygen. With washed cell suspensions, an oxygen consumption could be initiated by the addition of either glucose, fructose, or ethanol. Cell extracts catalyzed the oxidation of NADH with oxygen at a molar ratio of 2:1. Further experiments showed that this NADH oxidase is located in the cell membrane. The specific oxygen consumption rates of cell suspensions correlated with the intracellular NADH oxidizing activities; both levels decreased with increasing concentrations of the fermentation end-product ethanol. The addition of 5 mM NaCN completely inhibited both the intracellular oxygen reduction and also the oxygen consumption of whole cells. Both catalase and superoxide dismutase were present even in anaerobically grown cells. Aeration seemed to have little effect on the level of catalase, but the superoxide dismutase activity was 5-fold higher in cells grown aerobically. Under aerobic conditions considerable amounts of acetaldehyde and acetic acid were formed in addition to the normal fermentation products, ethanol and carbon dioxide.Dedicated to Professor Dr. H. G. Schlegel on the occasion of his 60th birthday     

Influence of carbon source on production of a heat stable protease from Thermomonospora fusca YX

Summary Thermomonospora fusca YX produced a very active heat stable protease when incubated in media containing cellulose as the substrate. Cultures grown on Solka-floc generated the highest amount of protease whereas the protease was produced at significantly lower levels when T. fusca YX was grown on cellobiose or glucose. Negligible growth or protease production was observed when protein was used as a carbon source. The production of the protease did not appear to be constitutive. While rapid growth was observed on either cellobiose or glucose, protease levels were at least two to fourfold lower than for the T. fusca YX cultures grown on Solka-floc wich generated 33% less cell mass. Protease production was four times lower in cultures which employed casein hydrolysate (tryptone) or xylan as carbon sources than for cellulose.     

Chemical inhibitors of hexokinase-2 enzyme reduce lactate accumulation,alter glycosylation processing,and produce altered glycoforms in CHO cell cultures

Chinese hamster ovary (CHO) cells, predominant hosts for recombinant biotherapeutics production, generate lactate as a major glycolysis by-product. High lactate levels adversely impact cell growth and productivity. The goal of this study was to reduce lactate in CHO cell cultures by adding chemical inhibitors to hexokinase-2 (HK2), the enzyme catalyzing the conversion of glucose to glucose 6-phosphate, and examine their impact on lactate accumulation, cell growth, protein titers, and N-glycosylation. Five inhibitors of HK2 enzyme at different concentrations were evaluated, of which 2-deoxy- d -glucose (2DG) and 5-thio- d -glucose (5TG) successfully reduced lactate accumulation with only limited impacts on CHO cell growth. Individual 2DG and 5TG supplementation led to a 35%–45% decrease in peak lactate, while their combined supplementation resulted in a 60% decrease in peak lactate. Inhibitor supplementation led to at least 50% decrease in moles of lactate produced per mol of glucose consumed. Recombinant EPO-Fc titers peaked earlier relative to the end of culture duration in supplemented cultures leading to at least 11% and as high as 32% increase in final EPO-Fc titers. Asparagine, pyruvate, and serine consumption rates also increased in the exponential growth phase in 2DG and 5TG treated cultures, thus, rewiring central carbon metabolism due to low glycolytic fluxes. N-glycan analysis of EPO-Fc revealed an increase in high mannose glycans from 5% in control cultures to 25% and 37% in 2DG and 5TG-supplemented cultures, respectively. Inhibitor supplementation also led to a decrease in bi-, tri-, and tetra-antennary structures and up to 50% lower EPO-Fc sialylation. Interestingly, addition of 2DG led to the incorporation of 2-deoxy-hexose (2DH) on EPO-Fc N-glycans and addition of 5TG resulted in the first-ever observed N-glycan incorporation of 5-thio-hexose (5TH). Six percent to 23% of N-glycans included 5TH moieties, most likely 5-thio-mannose and/or 5-thio-galactose and/or possibly 5-thio-N-acetylglucosamine, and 14%–33% of N-glycans included 2DH moieties, most likely 2-deoxy-mannose and/or 2-deoxy-galactose, for cultures treated with different concentrations of 5TG and 2DG, respectively. Our study is the first to evaluate the impact of these glucose analogs on CHO cell growth, protein production, cell metabolism, N-glycosylation processing, and formation of alternative glycoforms.     

Ruminal Fermentation and Sugar Concentrations

The maximal amounts of growth of Selenomonas ruminantium were examined in the media containing various amounts of glucose. The yields of cells per unit weight of glucose are linear functions to glucose concentrations in the ranges between zero to 0.005% and 0.005 to 0.7%, Cell yields per glucose are greater in the former range, indicating greater a-mounts of energy are available per glucose at lower concentrations. Growth responses in lactate media containing various amounts of glucose showed that the preincubation with larger amounts of glucose is inhibitory for the following growth and metabolism of lactate. The organism produces predominantly lactate in the glucose medium. However, volatile fatty acid productions increase when the initial concentrations of glucose become low. Isotopic studies showed that the lactate utilization yielding volatile fatty acids is inhibited by the preceding metabolism of high concentrations of glucose. These results were discussed with regard to normal and abnormal fermentations in the rumen.     

Growth of Rhodopseudomonas capsulata on l- and d-malic acid

d-malate replaced l-malate in supporting both photosynthetic (anaerobic, light) and heterotrophic (aerobic, dark) growth of Rhodopseudomonas capsulata. Growth rates and cell yields were nearly equivalent with both enantiomorphs. Addition of glucose to malate culture media increased the growth rate and doubled the cell yield of heterotrophic cultures, but had little effect on photosynthetic cultures. Aerobically-grown cells showed a higher level of substrate-dependent oxygen uptake with l-malate than with d-malate. This preference for l-malate occured even in cells grown on d-malate. No malic racemase activity was detected in extracts of heterotrophically- or photosynthetically-grown cells.