Kinetics of product formation in batch and continuous culture of Clostridium barkeri

Summary The main fermentation end products in batch culture (unlimited glucose supply) of Clostridium barkeri were butyrate and lactate. The specific rate of butyrate production was linearly proportional to the growth rate while the specific rate of lactate production increased at low growth rates. In a glucose limited chemostat culture butyrate production was partly growth associated while acetate and lactate production was growth associated. Lactate was, however, only produced at high dilution rates. By varying the glucose concentration in the inflowing medium it was shown that lactate production was stimulated by a high feeding rate of the carbon source. These results are discussed in view of the fructose-1,6-diphosphate dependent lactate dehydrogenase activity in many other organisms.     

Production of propionate by fed-batch fermentation of Propionibacterium acidipropionici using mixed feed of lactate and glucose

Propionibacterium acidipropionici was grown in a fed-batch culture, fed with glucose or lactate, or mixtures of lactate and glucose. Lactate and glucose were always simultaneously consumed. As co-substrate, glucose modified the propionate:acetate molar ratio (P/A) and increased the fraction of carbon used for biomass production. A P/A of 7.63 was obtained with a lactate:glucose molar ratio of 4; a P/A value of 1.34 was obtained with lactate alone and 1.85 with glucose alone. The fraction of carbon recovered in biomass was 0.09 for glucose, 0.12 for lactate, and 0.21 for a lactate:glucose molar ratio of 4.     

Alternative schemes of butyrate production in Butyrivibrio fibrisolvens and their relationship to acetate utilization, lactate production, and phylogeny

Butyrivibrio fibrisolvens strains D1 and A38 produced little lactate, but strain 49 converted as much as 75% of its glucose to lactate. Strain 49 had tenfold more lactate dehydrogenase activity than strains D1 or A38, this activity was stimulated by fructose 1,6-bisphosphate, and had a pH optimum of 6.25. A role for fructose 1,6-bisphosphate or pH regulation of lactate production in strain 49 was, however, contradicted by the observations that very low concentrations (< 0.2 mM) of fructose 1,6-bisphosphate gave maximal activity, and continuous cultures did not produce additional lactate when the pH was decreased. The lactate production of strain 49 was clearly inhibited by the presence of acetate in the growth medium. When strain 49 was supplemented with as little as 5 mM acetate, lactate production decreased dramatically, and most of the glucose was converted to butyrate. Strain 49 did not possess butyrate kinase activity, but it had a butyryl-CoA/acetate CoA transferase that converted butyryl-CoA directly to butyrate, using acetate as an acceptor. The transferase had a low affinity for acetate (K _m of 5 mM), and this characteristic explained the acetate stimulation of growth and butyrate formation. Strains D1 and A38 had butyrate kinase but not butyryl-CoA/acetate CoA transferase, and it appeared that this difference could explain the lack of acetate stimulation and lactate production. Based on these results, it is unlikely that B. fibrisolvens would ever contribute significantly to the pool of ruminal lactate. Since relatives of strain 49 (strains Nor37, PI-7, VV1, and OB156, based on 16S rRNA sequence analysis) all had the same method of butyrate production, it appeared that butyryl-CoA/acetate CoA transferase might be a phylogenetic characteristic. We obtained a culture of strain B835 (NCDO 2398) that produced large amounts of lactate and had butyryl-CoA/acetate CoA transferase activity, but this strain had previously been grouped with strains A38 and D1 based on 16S rRNA sequence analysis. Our strain B835 had a 16S rRNA sequence unique from the one currently deposited in GenBank, and had high sequence similarity with strains 49 and Nor37 rather than with strains A38 or D1. Received: 3 December 1998 / Accepted: 18 February 1999     

Acetate accumulation and regulation by process parameters control in Chinese hamster ovary cell culture

Chinese hamster ovary (CHO) cells represent a group of predominantly used mammalian hosts for producing recombinant therapeutic proteins. Known for their rapid proliferation rates, CHO cells undergo aerobic glycolysis that is characterized by fast glucose consumption, that ultimately gives rise to a group of small-molecule organic acids. However, only the function of lactate has been extensively studied in CHO cell culture. In this study, we observed the accumulation of acetate from the late exponential phase to harvest day, potentially contributing to the pH decline in late culture stage regardless of lactate consumption. In addition, we evaluated the acidification of the fresh media and the cell culture suspension, and the data revealed that acetate presented a lower acidification capacity compared to lactate and exhibited limited inhibitory effect on cells with less than 20 mM supplemented in the media. This study also explored the ways to control acetate accumulation in CHO cell culture by manipulating the process parameters such as temperature, glucose, and pH control. The positive correlation between the specific glucose consumption rate and acetate generation rate provides evidence of the endogenous acetate generation from overflow metabolism. Reducing these parameters (temperature, glucose consumption) and HCl-controlled low pH ultimately suppress acetate build-up. In addition, the specific acetate generation rate and relevant glucose consumption rate are found to be a metabolic trait associated with specific cell lines. Taken together, the results presented in these experiments provide a means to advance industrial CHO cell culture process control and development.     

Decreasing glycolysis increases sensitivity to mitochondrial inhibition in primary cultures of renal proximal tubule cells

Summary We have previously shown that shaking the culture plates (SHAKE) of rabbit renal proximal tubule cells (RPTC) to maintain adequate aeration increased aerobic metabolism and decreased the induction of glycolysis compared to RPTC cultured under standard conditions (STILL). However, glycolysis in SHAKE RPTC remained elevated compared to glycolysis in proximal tubules in vivo. In the present study the contribution of culture medium sugar composition and concentration to glycolytic metabolism was assessed in RPTC. SHAKE and STILL RPTC cultured in 5 mM glucose contained lactate levels equivalent to the respective SHAKE and STILL RPTC cultured in standard culture medium which contains 17.5 mM glucose. Similarly, the activity of lactate dehydrogenase was unchanged by lowering the medium glucose concentration. Substituting 5 mM galactose for 5 mM glucose in the culture medium significantly reduced the lactate content of both SHAKE and STILL RPTC but had no effect on lactate dehydrogenase activity. Cell growth was equivalent under all culture conditions. Sensitivity to mitochondrial inhibition was determined for each culture condition by measuring cell death after exposure to the respiratory inhibitor antimycin A. The results showed a hierarchy of sensitivity to antimycin A (5 mM galactose SHAKE >5 mM glucose SHAKE >17.5 mM glucose SHAKE = 17.5 mM glucose STILL), which was generally inversely correlated with the level of glycolysis as measured by lactate content (17.5 mM glucose STILL >17.5 mM glucose SHAKE = 5 mM glucose SHAKE >5 mM galactose SHAKE).     

Effects of Thymol on Ruminal Microorganisms

Thymol (5-methyl-2-isopropylphenol) is a phenolic compound that is used to inhibit oral bacteria. Because little is known regarding the effects of this compound on ruminal microorganisms, the objective of this study was to determine the effects of thymol on growth and lactate production by the ruminal bacteria Streptococcus bovis JB1 and Selenomonas ruminantium HD4. In addition, the effect of thymol on the in vitro fermentation of glucose by mixed ruminal microorganisms was investigated. Neither 45 nor 90 μg/ml of thymol had any significant effect on growth or lactate production by S. bovis JB1, but 180 μg/ml of thymol completely inhibited growth and lactate production. In the case of S. ruminantium HD4, 45 μg/ml of thymol had little effect on growth and lactate production; however, 90 μg/ml of thymol completely inhibited growth of S. ruminantium HD4. Thymol also decreased glucose uptake by whole cells of both bacteria. When mixed ruminal microorganisms were incubated in medium that contained glucose, 400 μg/ml of thymol increased final pH and the acetate to propionate ratio and decreased concentrations of methane, acetate, propionate, and lactate. In conclusion, thymol was a potent inhibitor of glucose fermentation by S. bovis JB1 and S. ruminantium HD4. Even though thymol treatment decreased methane and lactate concentrations and increased final pH in mixed ruminal microorganism fermentations of glucose, concentrations of acetate and propionate were also reduced. Received: 13 May 2000 / Accepted: 14 June 2000     

Enzymatic regulation of glucose catabolism by Lactobacillus plantarum in response to pH shifts in a chemostat

Summary The influence of medium pH on the regulation of glucose catabolism by Lactobacillus plantarum 8014 was examined in anaerobic chemostat cultures. When L. plantarum was grown in a chemostat at pH 5.5, and the pH shifted to pH 7.5, acetate was produced in addition to lactate and acetoin. After the shift, acetate kinase and NAD-dependent lactate dehydrogenase activities increased while the acetoin dehydrogenase and alpha-acetolactate synthase activities decreased. The high acetate kinase activity together with low acetoin dehydrogenase and alpha-acetolactate synthase activities may explain why L. plantarum made more acetate at the expense of acetoin in response to alkaline conditions.Offprint requests to: T.J. Montville     

Cultured trout liver cells: Utilization of substrates and response to hormones

Summary The characterization of a recently established system for the short-term culture of rainbow trout (Oncorhynchus mykiss) liver cells in chemically defined medium has been extended to studies on the metabolic competence of the cells and the characterization of their response to hormones. Three areas of metabolism have been addressed: a) the utilization of the exogenously added substrates fructose, lactate, glucose, dihydroxyacetone, and glycerol for glucose and lactate formation; b) the effects of the pancreatic hormones insulin and glucagon on cellular glucose formation, lactate formation, and fatty acid synthesis; and c) the effects of insulin and dexamethasone on the estradiol-dependent production of vitellogenin. Incubation of trout liver cells with fructose, lactate, glucose, dihydroxyacetone, or glycerol resulted in enhanced rates of cellular glucose and lactate production. Substrate-induced effects usually were more clearly expressed after extended (20 h) than after acute (5 h) culture periods. Addition of the hormones insulin or glucagon caused dose-dependent alterations in the flux of substrates to glucose and lactate. Rates of de novo synthesis of fatty acids from [¹⁴C]acetate were stimulated by insulin and inhibited by glucagon during acute and extended incubation periods. Treatment of liver cells isolated from male trout for 72 h with estradiol induced vitellogenin production and secretion into the medium. However, the addition of insulin or dexamethasone drastically reduced this estrogen-induced vitellogenesis. These results indicate that trout liver cells cultured in defined medium maintain central metabolic pathways, including glycolysis, gluconeogenesis, lipogenesis, and vitellogenesis as well as their responsiveness to various hormones, for at least 72 h. This cell culture system should provide an excellent model to further characterize metabolic processes in fish liver.     

Selecting for lactic acid producing and utilising bacteria in anaerobic enrichment cultures

Lactic acid-producing bacteria are important in many fermentations, such as the production of biobased plastics. Insight in the competitive advantage of lactic acid bacteria over other fermentative bacteria in a mixed culture enables ecology-based process design and can aid the development of sustainable and energy-efficient bioprocesses. Here we demonstrate the enrichment of lactic acid bacteria in a controlled sequencing batch bioreactor environment using a glucose-based medium supplemented with peptides and B vitamins. A mineral medium enrichment operated in parallel was dominated by Ethanoligenens species and fermented glucose to acetate, butyrate and hydrogen. The complex medium enrichment was populated by Lactococcus, Lactobacillus and Megasphaera species and showed a product spectrum of acetate, ethanol, propionate, butyrate and valerate. An intermediate peak of lactate was observed, showing the simultaneous production and consumption of lactate, which is of concern for lactic acid production purposes. This study underlines that the competitive advantage for lactic acid-producing bacteria primarily lies in their ability to attain a high biomass specific uptake rate of glucose, which was two times higher for the complex medium enrichment when compared to the mineral medium enrichment. The competitive advantage of lactic acid production in rich media can be explained using a resource allocation theory for microbial growth processes.     

Effect of varying citrate levels on C4 compound formation and on enzyme levels in Leuconostoc mesenteroides subsp. cremoris grown in continuous culture

Summary The effects of citrate on diacetyl, acetoin and 2,3-butylene glycol (2,3-BG) production by Leuconostoc mesenteroides subsp. cremoris grown in continuous culture at pH 5.2 were studied. In glucose alone end-product production agreed with the theoretical stoichiometry. In the presence of citrate, lactate and acetate production was higher than the theoretical stoichiometry from glucose. Lactate production was constant when the initial citrate concentration was increased whereas ethanol production strongly decreased. In the absence of citrate, citrate lyase (CL) exhibited weak activity. Diacetyl reductase (DR) and acetoin reductase (AR) exhibited basal activity. When varying citrate concentrations ranging from 10 to 75 mm were added to glucose broth, DR, AR, lactate dehydrogenase, NADH oxidase and alcohol dehydrogenase decreased as the initial citrate concentration increased suggesting that they were partly repressed by citrate. In contrast, CL increased and the specific citrate utilization rate also increased in the same way, indicating no saturation of the first step of citrate metabolism. Acetate kinase (AK) was slightly higher in the presence of citrate and increased when the initial citrate concentration increased. This result was correlated with an increase of acetate from the acetyl phosphate pathway. More ATP was produced in the presence of citrate, which could explain the increase in biomass formation. Citrate bioconversion into diacetyl, acetoin and 2,3-BG increased as the initial citrate increased. Correspondence to: C. Diviès     

Enhanced cutinase production of Thermobifida fusca by a two-stage batch and fed-batch cultivation strategy

In this study, cutinase production by Thermobifida fusca WSH03-11 was investigated with mixed short-chain organic acids as co-carbon sources to demonstrate the possibility of producing high value-added products from organic wastes. T. fusca WSH03-11 was cultured with different combinations of butyrate, acetate, and lactate with a purpose of increasing cutinase activity. The optimum proportion of butyrate, acetate, and lactate was 4:1:3. In batch cultivation, acetate and lactate were consumed quickly, while the consumption of butyrate was depressed in the presence of acetate with a concentration higher than 0.5 g/L. Based on these results, a two-stage batch and fed-batch cultivation strategy was proposed: a batch culture with acetate and lactate as the co-carbon sources in the first 10 h, and then a fed-batch culture with a constant butyrate feeding rate of 12 mL/h during 11∼20 h. By this two-stage cultivation strategy, cutinase activity, dry cell weight, and consumption rate of butyrate were increased by 70%, 103.4%, and 4.3-fold, respectively, compared to those of the batch cultivation. These results provided a novel and efficient way to produce high value-added products from organic wastes.     

Effect of pH,agitation and aeration on hyaluronic acid production byStreptococcus zooepidemicus

Summary The optimum pH for both the rate of production and yield of hyaluronic acid (HA) byStreptococcus zooepidemicus from glucose medium was 6.7±0.2 under anaerobic conditions. High agitation rates (600 rpm) gave superior results compared to 300 rpm. Aeration of the culture (0.3 VVM) improved the HA yield, but not the rate of production and lead to some acetate and CO₂ being formed, in addition to lactate and HA.     

Interaction of lipogenic substrates in porcine adipose tissue in vitro

• 1.1. Porcine adipose tissue was incubated with radiolabeled glucose, acetate or lactate. Saturation curves indicated that lactate > glucose > acetate in providing two-carbon units for fatty-acid synthesis.
• 2.2. Competition between individual substrates indicated that lactate was the best lipogenic substrate.
• 3.3. Incubation of all three substrates at concentrations observable in serum indicated that at 5.56mM, glucose was the preferred lipogenic substrate in the presence of 0.1 mM acetate and 1.0 mM lactate.
• 4.4. At elevated concentrations (18.52mM glucose, 1.0 mM acetate and 10.0 mM lactate), acetate and lactate were preferred to glucose as lipogenic substrates.

     

End Products of Glucose Fermentation by Brochothrix thermosphacta

Anaerobically, Brochothrix thermosphacta fermented glucose primarily to l-lactate, acetate, formate, and ethanol. The ratio of these end products varied with growth conditions. Both the presence of acetate and formate and a pH below about 6 increased l-lactate production from glucose. Small amounts of butane-2,3-diol were also produced when the pH of the culture was low (相似文献   

Anoxybacillus kamchatkensis sp. nov., a novel thermophilic facultative aerobic bacterium with a broad pH optimum from the Geyser valley, Kamchatka

A facultative aerobic, moderately thermophilic, spore forming bacterium, strain JW/VK-KG4 was isolated from an enrichment culture obtained from the Geyser valley, a geothermally heated environment located in the Kamchatka peninsula (Far East region of Russia). The cells were rod shaped, motile, peritrichous flagellated stained Gram positive and had a Gram positive type cell wall. Aerobically, the strain utilized a range of carbohydrates including glucose, fructose, trehalose, proteinuous substrates, and pectin as well. Anaerobically, only carbohydrates are utilized. When growing on carbohydrates, the strain required yeast extract and vitamin B₁₂. Anaerobically, glucose was fermented to lactate as main product and acetate, formate, ethanol as minor products. Aerobically, even in well-aerated cultures (agitated at 500 rpm), glucose oxidation was incomplete and lactate and acetate were found in culture supernatants as by-products. Optimal growth of the isolate was observed at pH^{25 C} 6.8–8.5 and 60°C. The doubling times on glucose at optimal growth conditions were 34 min (aerobically) and 40 min (anaerobically). The G+C content was 42.3 mol% as determined by T_m assay. Sequence analysis of the 16S rRNA gene indicated an affiliation of strain JW/VK-KG4 with Anoxybacillus species. Based on its morphology, physiology, phylogenetic relationship and its low DNA-DNA homology with validly published species of Anoxybacillus, it is proposed that strain JW/VK-KG4 represents a new species in the genus Anoxybacillus as A. kamchatkensis sp. nov. The type strain for the novel species is JW/VK-KG4^T (=DSM 14988, =ATCC BAA-549). The GenBank accession number for the 16S rDNA sequence is AF510985.     

Parameters Affecting Solvent Production by Clostridium pasteurianum

The effect of pH, growth rate, phosphate and iron limitation, carbon monoxide, and carbon source on product formation by Clostridium pasteurianum was determined. Under phosphate limitation, glucose was fermented almost exclusively to acetate and butyrate independently of the pH and growth rate. Iron limitation caused lactate production (38 mol/100 mol) from glucose in batch and continuous culture. At 15% (vol/vol) carbon monoxide in the atmosphere, glucose was fermented to ethanol (24 mol/100 mol), lactate (32 mol/100 mol), and butanol (36 mol/100 mol) in addition to the usual products, acetate (38 mol/100 mol) and butyrate (17 mol/100 mol). During glycerol fermentation, a completely different product pattern was found. In continuous culture under phosphate limitation, acetate and butyrate were produced only in trace amounts, whereas ethanol (30 mol/100 mol), butanol (18 mol/100 mol), and 1,3-propanediol (18 mol/100 mol) were the major products. Under iron limitation, the ratio of these products could be changed in favor of 1,3-propanediol (34 mol/100 mol). In addition, lactate was produced in significant amounts (25 mol/100 mol). The tolerance of C. pasteurianum to glycerol was remarkably high; growth was not inhibited by glycerol concentrations up to 17% (wt/vol). Increasing glycerol concentrations favored the production of 1,3-propanediol.     

Optimization of monoclonal antibody production: combined effects of potassium acetate and perfusion in a stirred tank bioreactor

To increase the yield of monoclonal antibody in a hybridoma culture, it is important to optimize the combination of several factors including cell density, antibody productivity per cell, and the duration of the culture. Potassium acetate enhances the production of antibodies by cells but sometimes depresses cell density. The production of anti-(human B-type red blood cell surface antigen) antibody by Cp9B hybridoma was studied. In batch cultures, potassium acetate inhibited Cp9B cells growth and decreased the maximal cell density but the productivity of antibody per cell was increased. The balance of the two effects resulted in a slight decline of antibody production. In a stirred tank bioreactor, the inhibitory effect of potassium acetate on cell density was overcome by applying the perfusion technique with the attachment of a cell-recycling apparatus to the bioreactor. In such a reactor, potassium acetate at 1 g l^-1 did not cause a decrease in the cell density, and the antibody concentration in the culture supernatant was increased from 28 μg ml^-1 to 38 μg ml^-1. Potassium acetate also suppressed the consumption of glucose and the accumulation of lactate in batch cultures, but the glucose and lactate levels were kept stable by applying the perfusion technique in the stirred tank bioreactor. This revised version was published online in July 2006 with corrections to the Cover Date.     

A nuclear magnetic resonance study of the glucose metabolism of Hymenolepis diminuta exposed to histamine and serotonin in vitro.

The direct effects of the inflammatory mediators, histamine (HI) and serotonin (SE), on the glucose metabolism of Hymenolepis diminuta in vitro were studied by analyzing the excretory products from culture media, containing D-1-13C-glucose and various concentrations of HI and/or SE, by 1H-nuclear magnetic resonance (n.m.r.) spectroscopy. The results revealed that HI markedly accelerated the glycolysis process by increasing the amount of lactate production. The increased glycolytic activity was reflected in a concentration-dependent increase in glucose uptake. Excretion of acetate was also stimulated by HI. A low concentration of SE significantly increased succinate, acetate and lactate excretions, whereas a high concentration had little effect on lactate production and significantly decreased succinate and acetate excretions. A combination of HI and SE treatment at a low concentration had no significant effect, but at a high concentration showed an additive effect, with an increase in lactate production, a decrease in succinate production and an increase in glucose uptake. Thus this work confirms that HI and SE directly influence, albeit differently, energy metabolism of the tapeworm H. diminuta.     

Comparison of aerobic and anaerobic growth of Lactobacillus plantarum in a glucose medium

The growth rate of Lactobacillus plantarum in a complex medium with 55.6 mM glucose decreased during aerobic incubation (relative to anaerobic incubation). The decrease occurred much earlier than an increase in the rate of oxygen utilization by the culture which led to H₂O₂ accumulation. The concentration of H₂O₂ accumulated in the medium was easily tolerated by the culture and elimination of the H₂O₂ did not prevent the decrease in growth rate. Increased O₂ utilization was accompanied by a switch in metabolism which resulted in acetate rather than lactate accumulation in aerobic cultures.Abbreviation MRSG Man, Rogosa and Sharpe (1960). Medium modified as in Materials and methods with glucose as fermentation substrate     

Effect of dilution rate and carbon availability on Bifidobacterium breve fermentation

Bifidobacterium breve NCFB 2257 was grown in glucose-limited and nitrogen (N)-limited chemostats at dilution rates (D) from 0.04 to 0.60 h^–1, to study the effect of nutrient availability on carbohydrate metabolism. The results showed that D had little effect on fermentation product formation, irrespective of the form of nutrient limitation. However, marked differeces were observed in the distribution of fermentation products, that were attributable to glucose availability. In glucose-limited cultures, formate and acetate were the principal end-products of metabolism. Lactate was never detected under these growth conditions. In contrast, lactate and acetate were mainly formed when glucose was in excess, and formate was not produced. These results are explained by the metabolic fate of pyruvate, which can be dissimilated by either phosphoroclastic cleavage to acetyl phosphate and formate, or alternatively, it may be reduced to lactate. Enzymic studies were made to establish the mechanisms that regulated pyruvate metabolism. The data demonstrated that control was not exercised through regulation of the synthesis and activity of lactate dehydrogenase (LDH), phosphofructokinase or alcohol dehydrogenase. It is possible however, that there was competition for pyruvate by LDH and the phosphoroclastic enzyme, which would determine the levels of lactate and formate produced respectively. These results demonstrate the metabolic flexibility of B. breve, which preferentially uses lactate as an electron sink during N-limited growth, whereas under energy-limitation, carbon flow is directed towards acetyl phosphate to maximise ATP synthesis. Correspondence to: B. A. Degnan