Methylcellulose effect on cell proliferation and glucose utilization in chemically defined medium in large stationary cultures

Three different established strains of mammalian cells were grown in chemically defined medium in large cultures. The degree of proliferation of cells of an established strain from human skin in large stationary cultures was significantly greater in the presence of methylcellulose (medium NCTC 135M) than in its absence (medium NCTC 135). The relatively fragile cells of a derivative of monkey kidney LLCMK2 strain were carried in large stationary cultures through 11 transfer generations during 152 days. The presence of methylcellulose was associated with higher cell population levels, proliferation rates, and cell viability. Cells of this strain utilized glucose at an extremely high rate; during two representative periods the rate averaged 1.2 mg/10⁶ cells/day in cultures on medium 135M and 1.9 mg in medium 135. In a 53-day experiment with mouse fibroblast 2071-L cells, the cells in suspension culture during the first 28 days went through the normal lag, logarithmic plateau, and initial decline phases in medium 135M, and then were transferred to large stationary cultures, where they proliferated for 7 days at uniformly high rates in both medium 135 and medium 135M. It appeared that cells of strain 2071-L in such stationary cultures had no need for Methocel as a protective agent. Glucose utilization rates while these cells were carried in large stationary cultures averaged 2–4 times the rates while they were in suspension cultures: about 0.8 and 0.2 mg/10⁶ cells/day, repectively.     

d(−)lactic acid production by suspended and aggregated continuous cultures of Bacillus laevolacticus

Summary A new method of d(–)lactic acid production based on the aggregated form of growth of Bacillus laevolacticus in continuous culture in an anaerobic gaslift reactor is presented. With glucose as the substrate a bacterial dry weight of 25 g·1^–1 and a lactic acid production rate of 13 g·1^–1·h^–1 was attained. In conventional glucose-limited chemostat cultures elevated levels of lactic acid stimulated specific lactic acid production while the formation rates of other end-products remained unaffected. In glucose-limited aggregated cultures lactic acid positively influenced the aggregation of cells, improving the volumetric production rate. It is concluded that lactic acid itself is a positive effector in the optimisation of lactic acid production with aggregated cultures of B. laevolacticus.Offprint requests to: J. P. de Boer     

Enhanced production of lactic acid by an adapted strain of <Emphasis Type="Italic">Lactobacillus</Emphasis><Emphasis Type="Italic">delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis>

Lactobacillus delbrueckii subsp. lactis strains were developed having increased activity, by gradually acclimatizing the bacteria to acidic conditions over repeated batch culture. Cells from one batch culture were used as the inoculum for the subsequent batch culture and thereby an adapted strain of Lactobacillus was obtained showing improved lactic acid productivity, cell growth and total glucose utilization. Furthermore, the acclimatized cells used significantly less nitrogen for a given level of lactic acid production, which is significant from an industrial point of view. The developed procedure decreases fermentation time and nutrient use, leading to reduced operation costs, while providing a lactic acid yield superior to previously reported methods.     

Fructose as a carbohydrate source yields stable pH and redox parameters in microcarrier cell culture

The pH and cytosolic NADH/NAD⁺ redox potential in microcarrier cultures of Madin-Darby canine kidney cells remain within physiological range when fructose is substituted for glucose in medium formulation. This difference is accounted for by the low rate of lactic acid production in cultures utilizing fructose as a primary carbohydrate source.     

Recovery of cells in vitro from the effects hypoxia and hyperoxia

The cytotoxic effect of high, as well as low, oxygen tension of proliferation and metabolism of Low line cells in culture is reversible even after several days of exposure provided the cells are returned to 95% air + 5% CO₂ environment. This suggests that the activity of certain mechanisms within the cells may have been altered or in other ways inhibited by the abnormal environments but are quite rapidly regenerated once the adverse condition is removed. The cells tolerate a low O₂ exposure for at least 20 days while continuous exposure to high O₂ atmosphere results in degeneration and death after 7–10 days. Both glucose utilization and lactic acid production are elevated in cultures exposed to either low or high O₂ tensions, although they are markedly higher in the latter condition. When cell so exposed are returned to an air + 5% CO₂ atmosphere, rate of glucose uptake and lactic acid formation soon approaches that found in control cultures.     

Utilization of light for the assimilation of organic matter in Chlorella sp. VJ79

Chlorella sp. strain VJ79 was isolated from a total heterotrophic count of a wastewater collector. It grows autotrophically, heterotrophically, and mixotrophically on a variety of organic substrates. Glucose and serine promote a mixotrophic growth from which the yield is higher than the sum of autotrophic and heterotrophic yields, but serine assimilation requires light. The interaction of glucose and light was studied in proliferating and nonproliferating cells by respirometry (IRGA and Warburg) and growth experiments. Glucose inhibits the photosynthetic CO(2) fixation ten-fold and modifies the pigmentary system as it does in heterotrophic cultures. Light inhibits glucose uptake and assimilation, but under mixotrophic conditions maximal utilization of glucose is obtained. Mutants defective in autotrophic growth were isolated by mutagenesis with nitrosoguanidine. They show a degenerated pigmentary system and a mixotrophic growth yield equal to that of the heterotrophic growth. The analysis of the mixotrophic system shows that light energy, dissipated during autotrophic growth, is used under mixotrophic conditions. From the increase in growth, the increase in photosynthetic efficiency can be calculated as ca. sixfold.     

Cell density and proliferation modulate collagen synthesis and procollagen mRNA levels in arterial smooth muscle cells.

Collagen synthesis and procollagen mRNA levels were determined and compared in (1) sparse, rapidly proliferating smooth muscle cells (SMC); (2) postconfluent, density-arrested SMC; and (3) sparse, nonproliferating (mitogen-deprived) rabbit arterial SMC. Collagen synthesis per SMC was decreased by 70% in postconfluent versus proliferating cells. However, relative collagen synthesis, expressed as the percentage of total protein synthesis, increased from 3.7% in sparse cultures to approximately 7% in postconfluent cultures. Slot blot analyses demonstrated that the relative steady state alpha 1(I) and alpha 1(III) procollagen mRNA levels were also increased in postconfluent cultures when compared to sparse cultures. As with collagen synthesis per cell, the mRNA levels per cell for types I and III procollagen in postconfluent cells, determined by densitometry of blots, were likewise approximately half that found in sparse, proliferating cells. In a separate study to determine if cell-cell contact was necessary for eliciting these changes in collagen synthesis, we determined collagen synthesis in mitogen-deprived and proliferating SMC cultures at low density. Mitogen-deprived cultures synthesized only 10% the amount of collagen produced (per cell) by proliferating cultures in 10% fetal bovine serum. Relative collagen synthesis in proliferating and nonproliferating cultures was 5.0 and 8.3%, respectively. These results demonstrate elevated collagen synthesis, per cell, by proliferating cultures compared with nonproliferating cultures, regardless of whether cells were rendered quiescent by density arrest or by mitogen deprivation. Results also suggest a pretranslational mechanism for the regulation of collagen synthesis in rabbit aortic smooth muscle cells.     

Acid Sensitivity of a Mutant of Lactococcus lactis subsp. lactis C2 with Reduced Membrane-bound ATPase Activity

Mutants with reduced membrane-bound ATPase activities were isolated from Lactococcus lactis subsp. lactis C2 as spontaneous neomycin-resistant mutants. Characteristics of the representative mutant, No. 1016–51, were compared with the parental strain in cultures using a jar fermentor with the pH controlled at various values. At pH 6.5, the fermentation patterns, i.e., glucose consumption, growth, and lactic acid production, of both strains appeared identical. At pH 4.5, however, the levels of growth, lactic acid production, and the amounts of lactic acid produced per cell after the culture for 24 h decreased to 60, 36, and 60% of the parental strain, respectively. During the cultures at pH 6.5, no differences were found in viabilities between both strains even after 80 h. On the other hand, at pH 4.0, the viable count of the strain No. 1016–51 in a 72-h culture decreased to less than 1% of that of the zero time, while the parental strain maintained its original viability. Therefore, it was concluded that the membrane-bound ATPase is essential for this organism to survive at low pH, probably through its function of proton pumping for maintaining cytoplasmic pH levels.     

Efficient conversion of lactic acid to butanol with pH-stat continuous lactic acid and glucose feeding method by Clostridium saccharoperbutylacetonicum

In order to achieve high butanol production by Clostridium saccharoperbutylacetonicum N1-4, the effect of lactic acid on acetone–butanol–ethanol fermentation and several fed-batch cultures in which lactic acid is fed have been investigated. When a medium containing 20 g/l glucose was supplemented with 5 g/l of closely racemic lactic acid, both the concentration and yield of butanol increased; however, supplementation with more than 10 g/l lactic acid did not increase the butanol concentration. It was found that when fed a mixture of lactic acid and glucose, the final concentration of butanol produced by a fed-batch culture was greater than that produced by a batch culture. In addition, a pH-controlled fed-batch culture resulted in not only acceleration of lactic acid consumption but also a further increase in butanol production. Finally, we obtained 15.5 g/l butanol at a production rate of 1.76 g/l/h using a fed-batch culture with a pH-stat continuous lactic acid and glucose feeding method. To confirm whether lactic acid was converted to butanol by the N1-4 strain, we performed gas chromatography–mass spectroscopy (GC-MS) analysis of butanol produced by a batch culture during fermentation in a medium containing [1,2,3-¹³C₃] lactic acid as the initial substrate. The results of the GC-MS analysis confirmed the bioconversion of lactic acid to butanol.     

Recombinant Escherichia coli engineered for production of L-lactic acid from hexose and pentose sugars

Recombinant Escherichia coli have been constructed for the conversion of glucose as well as pentose sugars into L-lactic acid. The strains carry the lactate dehydrogenase gene from Streptococcus bovis on a low copy number plasmid for production of L-lactate. Three E. coli strains were transformed with the plasmid for producing L-lactic acid. Strains FBR9 and FBR11 were serially transferred 10 times in anaerobic cultures in sugar-limited medium containing glucose or xylose without selective antibiotic. An average of 96% of both FBR9 and FBR11 cells maintained pVALDH1 in anaerobic cultures. The fermentation performances of FBR9, FBR10, and FBR11 were compared in pH-controlled batch fermentations with medium containing 10% w/v glucose. Fermentation results were superior for FBR11, an E. coli B strain, compared to those observed for FBR9 or FBR10. FBR11 exhausted the glucose within 30 h, and the maximum lactic acid concentration (7.32% w/v) was 93% of the theoretical maximum. The other side-products detected were cell mass and succinic acid (0.5 g/l). Journal of Industrial Microbiology & Biotechnology (2001) 27, 259–264. Received 05 November 2000/ Accepted in revised form 03 July 2001     

Role of Sodium Carboxymethyl Cellulose and Hydroxyethyl Starch in Hematopoietic Cell Line Cultures

Sodium carboxymethyl cellulose and hydroxyethyl starch were found to support rapid growth of two hematopoietic cell lines. The polymers were not metabolized by the cells. In the presence of these compounds, lower rates of glucose utilization and lactic acid production were observed. The uptake of glucose by the cells decreased as the concentration of the polymer in the medium was increased. These results indicate that sodium carboxymethyl cellulose and hydroxyethyl starch probably protect the cells against physical stress in suspended cultures.     

Production of lactic acid from date juice extract with free cells of single and mixed cultures of Lactobacillus casei and Lactococcus lactis

The production of lactic acid from date juice by single and mixed cultures of Lactobacillus casei and Lactococcus lactis was investigated. In the present conditions, the highest concentration of lactic acid (60.3 g l⁻¹) was obtained in the mixed culture system while in single culture fermentations of Lactobacillus casei or Lactococcus lactis, the maximum concentration of lactic acid was 53 and 46 g l⁻¹, respectively. In the case of single Lactobacillus casei or Lactococcus lactis, the total percentage of glucose and fructose utilized were 82.2; 94.4% and 93.8; 60.3%, respectively, whereas in the case of mixed culture, the total percentage of glucose and fructose were 96 and 100%, respectively. These results showed that the mixed culture system gave better results than single cultures regarding lactic acid concentration, and sugar consumption.     

Lactic acid fermentation by Lactobacillus casei in free cell form and immobilised on gluten pellets

A comparative study of the fermentation of a range of carbohydrate substrates, at various temperatures, was carried out using a commercial Lactobacillus casei strain in a free cell form and immobilised on gluten pellets. This strain required yeast extract, l-cysteine HCl and Mn²⁺ at 5, 0.5 and 0.1 g l^–1, respectively, for maximum growth and lactic acid production. Sugar fermentation using free cells showed preference in the order glucose, sucrose, fructose while lactose was poorly utilised. Optimum temperature for growth and lactic acid production over (18–30 h) was 43 °C. L. casei was successfully immobilised on gluten pellets and fermented glucose and sucrose in a shorter time (18 h) with increased lactic acid production (42 and 41 g l^–1 on glucose and sucrose, respectively).     

Isolation and characterization of a novel lactic acid bacterium for the production of lactic acid

We isolated a novel lactic acid bacterium from a Korean traditional fermented food, soybean paste. The newly isolated strain, dubbed RKY2, grew well on glucose, sucrose, galactose, and fructose, but it could not utilize xylose, starch, or glycerol. When the partially amplified 16S rDNA sequence (772 bp) of the strain RKY2 was compared with 10 reference strains, it was found to be most similar toLactobacillus pentosus JCM 1588^T, with 99.74% similarity. Therefore, the strain RKY2 was renamedLactobacillus sp. RKY2, which has been deposited in the Korean Collection for Type Cultures as KCTC 10353BP.Lactobacillus sp. RKY2 was found to be a homofermentative lactic acid bacterium, because its end-product from glucose metabolism was found to be mainly lactic acid. It could produce more than 90 g/L of lactic acid from MRS medium supplemented with 100 g/L of glucose, with 5.2 g L⁻¹ h⁻¹ of productivity and 0.95 g/g of lactic acid yield.     

Metabolism of cyclic AMP in non-pathogenic Mycobacterium smegmatis

The intracellular concentration of cyclic AMP reached a maximum in 3.5-day old cultures of Mycobacterium smegmatis grown in the presence of glycerol as the main source of carbon. Glucose-grown cells exhibited decreased cyclic AMP levels at all stages of growth. When M. smegmatis cells were incubated with various metabolites, pyruvate increased whereas glucose, citric acid, succinic acid and lactic acid decreased intracellular cyclic AMP levels. No cyclic AMP was detected in the incubation medium. The presence of a cyclic AMP-binding protein was demonstrated in cellfree extracts of M. smegmatis.     

Flour carbohydrate catabolism and metabolite production by sourdough lactic acid bacteria

The aim of this study was to assess the mode of carbohydrate catabolism by lactic acid bacteria isolated from traditional sourdoughs, as well as to study their effect on the metabolites produced. For this purpose, single cultures of the heterofermentative lactic acid bacteria Lactobacillus sanfranciscensis, Lactobacillus brevis, Weissella cibaria, and the homofermentative Lactobacillus paralimentarius and Pediococcus pentosaceus were grown in liquid media containing glucose, fructose, maltose and sucrose, either as a single carbon source or in combination with glucose. Carbon catabolism and the production of metabolites were determined by HPLC analysis. W. cibaria could ferment all carbon sources, L. sanfranciscensis, L. paralimentarius and P. pentosaceus could not ferment sucrose, while L. brevis could only ferment maltose. The presence of glucose did not influence the utilization of fructose and maltose by L. sanfranciscensis, while it repressed the fermentation of fructose, maltose and sucrose by W. cibaria, and fructose and maltose by L. paralimentarius and P. pentosaceus. Moreover, L. sanfranciscensis and L. brevis could obtain extra ATP through the reduction of fructose to mannitol, which favored the production of acetic acid against ethanol. The utilization of fructose as an electron acceptor has a decisive effect on the prevailing of L. sanfranciscensis and L. brevis in spontaneously fermented sourdough and in the scarce appearance of the other lactic acid bacteria studied.     

Pyruvate production by Enterococcus casseliflavus A-12 from gluconate in an alkaline medium

A newly isolated lactic acid bacterium, Enterococcus casseliflavus A-12, produced pyruvic acid (16 g/l) during aerobic culture in an alkaline medium containing sodium gluconate (50 g/l) as the carbon source. The production was dependent on the pH of the culture, the optimum initial pH being 10.0. With static culture, the organism produced lactic acid (2.7 g/l) from both gluconate and glucose. Pyruvate did not accumulate in growing cultures on glucose, but resting cells obtained from a culture on gluconate produced pyruvate from glucose as well as gluconate. The enzyme profiles of the organism, which grew on gluconate and glucose, suggested that gluconate was metabolized via the Entner-Doudoroff and Embdem-Meyerhof-Parnas pathways in aerobic culture, and that glucose was oxidized mainly via the latter pathway under both aerobic and anaerobic conditions. Gluconokinase, a key enzyme in the aerobic metabolism of gluconate, was partially purified from this strain and characterized.     

An automated flow injection analysis system for on-line monitoring of glucose and L-lactate during lactic acid fermentation in a recycle bioreactor

The study concerns on-line sequential analysis of glucose and L-lactate during lactic acid fermentation using a flow injection analysis (FIA) system. Enzyme electrodes containing immobilized glucose oxidase and L-lactate oxidase were used with an amperometric detection system. A 12-bit data acquisition card with 16 analog input channels and 8 digital output channels was used. The software for data acquisition was developed using Visual C++, and was devised for sampling every hour for sequential analyses of lactate and glucose. The detection range was found to be 2–100 g l^–1 for glucose and 1–60 g l^–1 for L-lactate using the biosensors. This FIA system was used for monitoring glucose utilization and L-lactate production by immobilized cells of Lactobacillus casei subsp. rhamnosus during a lactic acid fermentation process in a recycle batch reactor. After 13 h of fermentation, complete sugar utilization and maximal L-lactate production was observed. A good agreement was observed between analysis data obtained using the biosensors and data from standard analyses of reducing sugar and L-lactate. The biosensors exhibited excellent stability during continuous operation for at least 45 days.     

Lactobacillus pentosus CECT 4023 T co-utilizes glucose and xylose to produce lactic acid from wheat straw hydrolysate: Anaerobiosis as a key factor

Lactic acid is a versatile chemical that can be produced via fermentation of lignocellulosic materials. The heterolactic strain Lactobacillus pentosus CECT 4023 T, that can consume glucose and xylose, was studied to produce lactic acid from steam exploded wheat straw prehydrolysate. The effect of temperature and pH on bacterial growth was analyzed. Besides, the effect of oxygen on lactic acid production was tested and fermentation yields were compared in different scenarios. This strain showed very high tolerance to the inhibitors contained in the wheat straw prehydrolysate. The highest lactic acid yields based on present sugar, around 0.80 g g⁻¹, were obtained from glucose in presence of 25%, 50%, and 75% v v⁻¹ of prehydrolysate in strict anaerobiosis. Lactic fermentation of wheat straw hydrolysate obtained after enzymatic hydrolysis of the prehydrolysate yielded 0.39 g of lactic acid per gram of released sugars, which demonstrated the high potential of L. pentosus to produce lactic acid from hemicellulosic hydrolysates. Results presented herein not only corroborated the ability of L. pentosus to grow using mixtures of sugars, but also demonstrated the suitability of this strain to be applied as an efficient lactic acid producer in a lignocellulosic biorefinery approach. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2739, 2019     

Generalized model of the effect of pH on lactate fermentation and citrate bioconversion in Lactococcus lactis ssp. Lactis biovar. diacetylactis

An aroma-imparting mesophilic lactic starter (Lactococcus lactis ssp. lactis biovar. diacetylactis) was studied in batch culture in medium with 50 g·l^–1 lactose and 2 g·l^–1 citrate. The effect of pH on the physiology of growth and the production of flavour compounds was investigated with a mathematical model. The specific rates of growth and of lactose fermentation obeyed a law of non-competitive inhibition by lactic acid produced, inhibition increasing as the pH of the medium decreased. The pH thus acted indirectly by increasing the proportion of non-dissociated lactic acid, identified as the inhibiting form of lactic acid. The generalized model, taking into account the effect of pH, was tested using fermentations at pH controlled at different values (4.5–6.5), as well as with a fermentation conducted at non-regulated pH. These simulations supported the working hypotheses. The effect of pH on the fermentation of citric acid resulted in an increase in the maximal specific rate of citrate utilization, in the bioconversion yield, and in the constant of diacetyl and acetoin reduction at acid pH. The production of flavour compounds is a complex phenomenon resulting from the interaction of pH, citric acid concentration, and the physiological state of the cells. These results are discussed with respect to the use of this strain in the preparation of manufactured dairy products.