Formation of the exopolysaccharide ethapolan by <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. IMV B-7005 on a fumarate-glucose mixture

Our studies enabled us to intensify the synthesis of the microbial exopolysaccharide (EPS) ethapolan produced by Acinetobacter sp. IMV B-7005 grown on a mixture of fumarate (an energy-excessive substrate) and glucose (an energy-deficient substrate). Supplementing glucose-containing medium with sodium (potassium) fumarate at a molar ratio of 4: 1 resulted in a 1.3–2.2-fold increase of the EPS amount synthesized and in a 1.3–2-fold increase of the EPS yield relative to the biomass compared to cultivation on monosubstrates. The conversion of the carbon of both substrates to EPS was the highest if the carbon/nitrogen ratio in the cultivation medium was 70.5 and inoculum grown on glucose monosubstrate was used.     

Intensification of the exopolysaccharide synthesis by Acinetobacter sp. on an ethanol-glucose mixture: aspects related to biochemistry and bioenergetics

The possibility of intensifying the synthesis of microbial exopolysaccharides (EPS) by a strain of Acinetobacter sp. grown on a mixture of two substrates nonequivalent in terms of bioenergetics (ethanol + glucose) was shown. Based on theoretical calculations of the energy requirements for biomass and EPS synthesis from the energy-deficient substrate (glucose), the supplementary concentration of the energy-excessive substrate (ethanol) was determined that prevents the loss of glucose carbon that occurs when glucose is oxidized to CO2 to obtain energy for the processes of constructive metabolism. This made it possible to increase the efficiency of conversion of the substrate carbon to EPS. The introduction of ethanol into glucose-containing medium at a molar ratio of 3.1:1 allowed the amount of the EPS synthesized to be increased 1.8- to 1.9-fold; their yield relative to biomass increased 1.4- to 1.7-fold, and the yield of EPS relative to the substrate consumed increased 1.5- to 2-fold as compared to growth of the producer on single substrates. These results form the basis for the development of new technologies for obtaining secondary metabolites of practical value with the use of mixed growth substrates.     

Intensification of Exopolysaccharide Synthesis by Acinetobacter sp. on an Ethanol–Glucose Mixture: Aspects Related to Biochemistry and Bioenergetics

The possibility of intensifying the synthesis of microbial exopolysaccharides (EPS) by a strain of Acinetobacter sp. grown on a mixture of two substrates nonequivalent in terms of bioenergetics (ethanol + glucose) was shown. Based on theoretical calculations of the energy requirements for biomass and EPS synthesis from the energy-deficient substrate (glucose), the supplementary concentration of the energy-excessive substrate (ethanol) was determined that prevents the loss of glucose carbon that occurs when glucose is oxidized to CO₂ to obtain energy for the processes of constructive metabolism. This made it possible to increase the efficiency of conversion of the substrate carbon to EPS. The introduction of ethanol into glucose-containing medium at a molar ratio of 3.1 : 1 allowed the amount of the EPS synthesized to be increased 1.8- to 1.9-fold; their yield relative to biomass increased 1.4- to 1.7-fold, and the yield of EPS relative to the substrate consumed increased 1.5- to 2-fold as compared to growth of the producer on single substrates. These results form the basis for the development of new technologies for obtaining secondary metabolites of practical value with the use of mixed growth substrates.     

Intensification of synthesis of the exopolysaccharide ethapolan by Acinetobacter sp. 12S grown on a mixture of substrates

Enhanced synthesis of the exopolysaccharide (EPS) ethapolan by Acinetobacter sp. 12S was observed when the bacterium was grown on a mixture of two energetically nonequivalent substrates (ethanol and glucose) taken in a molar proportion of 3.1:1. The efficiency of carbon transformation into EPSs was maximum when sodium ions were absent in the medium, the concentration of nitrogen source was reduced to 0.3-0.45 g/l, and the inoculum was grown on ethanol. Such conditions provided an increase in the maximum specific growth rate and its attainment in earlier cultivation terms. Molasses as a substitution for glucose was inefficient. The activities of the key enzymes of C2-metabolism in Acinetobacter sp. 12S cells grown on the substrate mixture was 1.1 to 1.7 times lower than they were during growth on ethanol alone. The activity of isocitrate lyase in cells grown on the substrate mixture declined to an even greater extent (by 4 to 7 times), indicating that the role of the glyoxylate cycle in such cells is insignificant.     

Physicochemical properties of the microbial exopolysaccharide ethapolan synthesized on a mixture of growth substrates

Some physicochemical properties of the microbial exopolysaccharide (EPS) ethapolan synthesized by Acinetobacter sp. 12S depended on whether the producer was grown on a mixture of ethanol and glucose or on single substrates. Irrespective of the carbon source in the nutrient medium, the contents of carbohydrates, pyruvic acid, uronic acids, and mineral components in the EPS remained unchanged. The EPS were also identical in their monosaccharide composition: the molar ratio of glucose, mannose, galactose, and rhamnose was 3:2:1:1. EPS with a higher proportion of fatty acids was synthesized during growth on the mixture of ethanol and glucose. Average molecular weight and the proportion of high-molecular (over two million) fractions were greater in ethapolan produced on the substrate mixture. In the presence of 0.1 M KCl, after transformation into the H+ form, and in the Cu(2+)-glycine system, solutions of these EPS showed higher viscosity than solutions of EPS synthesized on single substrates. The reasons for the improved rheological properties of the EPS produced on the substrate mixture are discussed.     

Physicochemical Properties of the Microbial Exopolysaccharide Ethapolan Synthesized on a Mixture of Growth Substrates

Some physicochemical properties of the microbial exopolysaccharide (EPS) ethapolan synthesized by Acinetobacter sp. 12S depended on whether the producer was grown on a mixture of ethanol and glucose or on a single substrate. Irrespective of the carbon source in the nutrient medium, the contents of carbohydrates, pyruvic acid, uronic acids, and mineral components in the EPS remained unchanged. The EPS were also identical in their monosaccharide composition: the molar ratio of glucose, mannose, galactose, and rhamnose was 3 : 2 : 1 : 1. EPS with a higher content of fatty acids was synthesized during growth on the mixture of ethanol and glucose. The average molecular mass and the content of high-molecular (M > 2 MDa) fractions were greater in ethapolan produced on the substrate mixture. In the presence of 0.1 M KCl, after transformation into the H⁺ form, and in the Cu²⁺–glycine system, solutions of these EPS showed higher viscosity than solutions of EPS synthesized on single substrates. The reasons for the improved rheological properties of the EPS produced on the substrate mixture are discussed.     

Regulation of reductive production of succinate under anaerobic conditions in baker's yeast

When baker's yeast grown aerobically on ethanol as a carbon source was anaerobically cultured in a medium containing glucose, the activity of a cytoplasmic fumarate reductase irreversibly catalyzing the conversion of fumarate to succinate increased, reaching about 3 times the original activity after 12 h, while the activity of succinate dehydrogenase was almost lost after 10 h. These results indicate that the citrate cycle is partially modified to become a reductive pathway leading to succinate during the anaerobic cultivation. In non-proliferating cells grown anaerobically on glucose, the rates of accumulating succinate and pyruvate were decreased and increased, respectively, with increasing concentrations of L-aspartate or NH4Cl in the medium containing glucose as a substrate. These changes were accompanied with increase in the cellular content of aspartate, an inhibitor of pyruvate carboxylase that is involved in supplying the intermediates of the citrate cycle, and pyruvate, a substrate of the enzyme. The aminotransferase inhibitor, aminooxyacetate, prevented the changes in succinate accumulation and cellular aspartate following the addition of NH4Cl. The addition of L-glutamate caused a marked increase in the rate of succinate accumulation without changing the cellular content of aspartate. Neither L-glutamate nor L-aspartate had the ability to produce succinate. The rate of glucose consumption was not changed upon adding these nitrogen compounds. Similar findings were also observed in experiments using proliferating cells. This report presents evidence that in cells containing a large amount of the fumarate reductase, the production of succinate from glucose is regulated by the cellular level of aspartate through the pyruvate carboxylase reaction and that glutamate regulates the succinate production by a mechanism distinct from that involved in the regulation by L-aspartate.     

Exopolysaccharide production and peculiarities of C6-metabolism in Acinetobacter sp. grown on carbohydrate substrates

An Acinetobacter sp. strain grown on carbohydrate substrates (mono- and disaccharides, molasses, starch) was shown to synthesize exopolysaccharides (EPS). Glucose catabolism proved to proceed via the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways. Pyruvate entered the tricarboxylic acid cycle due to pyruvate dehydrogenase activity. Pyruvate carboxylation by pyruvate carboxylase was the anaplerotic reaction providing for the synthesis of intermediates for the constructive metabolism of Acinetobacter sp. grown on C6-substrates. The C6-metabolism in Acinetobacter sp. was limited by coenzyme A. Irrespective of the carbohydrate growth substrate (glucose, ethanol), the activities of the key enzymes of both C2- and C6-metabolism was high, except for the isocitrate lyase activity in glucose-grown bacteria. Isocitrate lyase activity was induced by C2-compounds (ethanol or acetate). After their addition to glucose-containing medium, both substrates were utilized simultaneously, and an increase was observed in the EPS synthesis, as well as in the EPS yield relative to biomass. The mechanisms responsible for enhancing the EPS synthesis in Acinetobacter sp. grown on a mixture of C2- and C6-substrates are discussed.     

Exopolysaccharide production by a new Halomonas strain CRSS isolated from saline lake Cape Russell in Antarctica growing on complex and defined media

A haloalkalophilic Halomonas strain CRSS, isolated from salt sediments in Antarctica, produced exocellular polysaccharides (EPS) up to 2.9 g g(-1) dry cells. Acetate was the most efficient carbon source for EPS production. The composition of media strongly affected the nature of the polymers; a mannan and a xylo-mannan, were obtained when cells were grown on complex media. Acetate was the most efficient carbon source for EPS production and in presence of this substrate, a new polysaccharide, a fructo-glucan, was produced. The EPS fraction was composed by glucose, fructose, glucosamine and galactosamine in relative proportions of 1:0.7:0.3:trace.     

Succination of protein thiols during adipocyte maturation: a biomarker of mitochondrial stress

Although obesity is a risk factor for development of type 2 diabetes and chemical modification of proteins by advanced glycoxidation and lipoxidation end products is implicated in the development of diabetic complications, little is known about the chemical modification of proteins in adipocytes or adipose tissue. In this study we show that S-(2-succinyl)cysteine (2SC), the product of chemical modification of proteins by the Krebs cycle intermediate, fumarate, is significantly increased during maturation of 3T3-L1 fibroblasts to adipocytes. Fumarate concentration increased > or =5-fold during adipogenesis in medium containing 30 mm glucose, producing a > or =10-fold increase in 2SC-proteins in adipocytes compared with undifferentiated fibroblasts grown in the same high glucose medium. The elevated glucose concentration in the medium during adipocyte maturation correlated with the increase in 2SC, whereas the concentration of the advanced glycoxidation and lipoxidation end products, N(epsilon)-(carboxymethyl)lysine and N(epsilon)-(carboxyethyl)lysine, was unchanged under these conditions. Adipocyte proteins were separated by one- and two-dimensional electrophoresis and approximately 60 2SC-proteins were detected using an anti-2SC polyclonal antibody. Several of the prominent and well resolved proteins were identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry. These include cytoskeletal proteins, enzymes, heat shock and chaperone proteins, regulatory proteins, and a fatty acid-binding protein. We propose that the increase in fumarate and 2SC is the result of mitochondrial stress in the adipocyte during adipogenesis and that 2SC may be a useful biomarker of mitochondrial stress in obesity, insulin resistance, and diabetes.     

Effect of Thiosulfate on the Photosynthetic Growth of Rhodopseudomonas palustris

Cell yields of Rhodopseudomonas palustris grown photoheterotrophically in pyruvate-mineral salts medium were increased by the photooxidation of added thiosulfate. However, thiosulfate had no effect on cell yields of cultures grown aerobically in darkness, although thiosulfate was also oxidized. The presence of thiosulfate increased photosynthetic cell yields on a variety of other organic substrates. Growth of cells in thiosulfate-containing medium, or the addition of thiosulfate to cells grown in thiosulfate-free medium, induced the formation of a thiosulfate-oxidizing system which quantitatively photooxidized thiosulfate to sulfate. R. palustris grew photoautotrophically with thiosulfate as an oxidizable substrate. Large amounts of supplemental bicarbonate carbon were incorporated when cells were grown photosynthetically in pyruvate-thiosulfate medium. Cells harvested after photoautotrophic or photoheterotrophic growth in fumarate-thiosulfate medium fixed (14)CO(2) at an 8- to 10-fold greater rate when provided with thiosulfate. The evolution of (14)CO(2) from pyruvate-1-(14)C during photoassimilation by R. palustris was greatly suppressed by the presence of thiosulfate. The increase in photoheterotrophic cell yields of R. palustris caused by the oxidation of thiosulfate may result from assimilation of substrate carbon which is normally evolved as carbon dioxide.     

Extracellular polysaccharide production byRhizobium ciceri from Turkey

The ability of theRhizobium ciceri, to produce extracellular polysaccharides (EPS) was investigated. Tested carbon and nitrogen sources influced EPS synthesis whenR. ciceri Rc5 was grown in a chemically defined medium. Mannitol was the most efficient carbon source among the six sources tested (sucrose, glucose, arabinose, fructose, xylose and rhamnose) and sodium nitrate was the most efficient nitrogen source among the two tested (ammonium sulphate and glycine). High amounts of EPS (1182.0 and 1015 μg ml−1, respectively) were produced by the Rc5 strain in mannitol and sodium nitrate respectively, which was accompanied by a great increase in the production compared to the control.     

Exopolysaccharide production by a new Halomonas strain CRSS isolated from saline lake Cape Russell in Antarctica growing on complex and defined media

A haloalkalophilic Halomonas strain CRSS, isolated from salt sediments in Antarctica, produced exocellular polysaccharides (EPS) up to 2.9gg^-1 dry cells. Acetate was the most efficient carbon source for EPS production. The composition of media strongly affected the nature of the polymers; a mannan and a xylo-mannan, were obtained when cells were grown on complex media. Acetate was the most efficient carbon source for EPS production and in presence of this substrate, a new polysaccharide, a fructo-glucan, was produced. The EPS fraction was composed by glucose, fructose, glucosamine and galactosamine in relative proportions of 1:0.7:0.3:trace.Revisions requested; Revisions received 6 September 2004     

Effect of mixed carbon substrate on exopolysaccharide production of cyanobacterium Nostoc flagelliforme in mixotrophic cultures

The effects of organic carbon sources on cell growth and exopolysaccharide (EPS) production of dissociated Nostoc flagelliforme cells under mixotrophic batch culture were investigated. After 7?days of cultivation, glycerol, acetate, sucrose, and glucose increased the final cell density and final EPS concentrations, and mixotrophic growth achieved higher biomass concentrations. The increase in cell growth was particularly high when glucose was added as the sole carbon source. On the other hand, EPS production per dry cell weight was significantly enhanced by adding acetate. For more effective EPS production, the effects of the mixture of glucose and acetate were investigated. Increasing the ratio of glucose to acetate resulted in higher growth rate with BG-11 medium and higher EPS productivity with BG-11₀ medium (without NaNO₃). When the medium was supplemented with a mixture of glucose (4.0?g?L^?1) and acetate (2.0?g?L^?1), 1.79?g?L^?1 biomass with BG-11 medium and 879.6?mg?L^?1 of EPS production with BG-11₀ medium were achieved. Adopting this optimal ratio of glucose to acetate established in flask culture, the culture was also conducted in a 20-L photobioreactor with BG-11 medium for 7?days. A maximum biomass of 2.32?g?L^?1 was achieved, and the EPS production was 634.6?mg?L^?1.     

Regulation of hydrogenase activity in enterobacteria.

Proteus vulgaris, Escherichia coli, and Citrobacter freundii cells were devoid of hydrogenase activity when grown on complex medium or minimal medium plus glucose in the presence of saturating levels of dissolved oxygen. Anaerobically grown cells had appreciable hydrogenase activity. Cells grown anaerobically in the presence of CO (an inhibitor of hydrogenase) or nitrate (an electron acceptor) lacked hydrogenase activity. To make hydrogenase essential for anaerobic growth, cells were grown on fumarate, a nonfermentable carbon source. P. vulgaris and C. freundii evolved H2 gas under these conditions, and the hydrogenase-specific activity was 8 to 10 times greater than that in cells grown on glucose. Cell growth was inhibited by CO, and the cells grew but lacked hydrogenase activity when grown in the presence of nitrate. E. coli grew on fumarate plus H2, and the specific activity was five times greater than that in cells grown on glucose. Thus, hydrogenase activity is inducible and is expressed maximally when the enzyme is essential for cellular growth. Under conditions of growth where the enzyme would not be catalytically active, cells contain little active hydrogenase. Under anaerobic conditions where the enzyme is not essential for growth, the level of hydrogenase activity is intermediate.     

Physiological approach to extracellular polysaccharide production by Lactobacillus rhamnosus strain C83

Extracellular polysaccharide production was studied by growing strain C83 of Lactobacillus rhamnosus in a chemically defined medium containing various carbon sources (glucose, fructose, mannose or maltose) at different concentrations. Mannose or a combination of glucose + fructose were by far the most efficient carbon sources. An optimization of growth medium composition led to an improvement in both growth and EPS production. When the strain was cultured at a lower temperature the EPS production increased. A temperature shift, at the end of the exponential growth phase, did not enhance the EPS production. The EPS synthesis by strain C83 was growth-related. The isolated slime had a sugar composition, as shown by three different methods, of glucose and galactose with a ratio of 1 : 1.     

Optimization of water absorbing exopolysaccharide production on local cheap substrates by Bacillus strain CMG1403 using one variable at a time approach

Optimum culture conditions, and carbon and nitrogen sources for production of water absorbing exopolysaccharide by Bacillus strain CMG1403 on local cheap substrates were determined using one variable at a time approach. Carbon source was found to be sole substrate for EPS biosynthesis in the presence of yeast extract that supported the growth only and hence, indirectly enhanced the EPS yield. Whereas, urea only coupled with carbon source could enhance the EPS production but no effect on growth. The maximum yield of EPS was obtained when Bacillus strain CMG1403 was grown statically in neutral minimal medium with 25% volumetric aeration at 30°C for 10 days. Under these optimum conditions, a maximum yield of 2.71±0.024, 3.82±0.005, 4.33±0.021, 4.73±0.021, 4.85±0.024, and 5.52±0.016 g/L culture medium was obtained with 20 g (sugar) of sweet whey, glucose, fructose, sucrose, cane molasses and sugar beet the most efficient one respectively as carbon sources. Thus, the present study showed that under optimum culture conditions, the local cheap substrates could be superior and efficient alternatives to synthetic carbon sources providing way for an economical production of water absorbing EPS by indigenous soil bacterium Bacillus strain CMG1403.     

Cultivation of Escherichia coli with mixtures of 3-phenylpropionic acid and glucose: steady-state growth kinetics.

The fate of pollutants in the environment is affected by the presence of easily degradable carbon sources. As a step towards understanding these complex interactions, a model system was explored: the degradation of mixtures of glucose (i.e., an easily degradable substrate) and 3-phenylpropionic acid (3ppa) (a model pollutant) by Escherichia coli ML 30 was studied systematically in carbon-limited continuous culture. The two substrates were always consumed simultaneously regardless of the dilution rate applied. Even at dilution rates higher than the maximum specific growth rate for 3ppa (0.35 +/- 0.05 h-1), the two carbon substrates were utilized together. When cells were grown at a constant dilution rate with different mixtures of 3ppa and glucose, in which 3ppa contributed between 5 and 90% of carbon substrate in the feed medium, the steady-state concentrations of 3ppa and glucose were approximately proportional to the ratio of the two substrates in the feed medium. When cells were cultivated at different dilution rates with a 1:1 mixture (based on carbon) of glucose and 3ppa, an overall maximum specific growth rate of 0.90 +/- 0.05 h-1 and a Monod substrate saturation constant for 3ppa (Ks) of 600 to 700 micrograms liter-1, similar to that measured during growth with 3ppa alone, fitted the experimentally determined steady-state 3ppa concentrations. However, due to the highly differing substrate affinity constants for 3ppa and glucose (Ks approximately 30 to 70 micrograms liter-1), the total steady-state carbon concentration in the culture at a constant dilution rate was determined mainly by the steady-state 3ppa carbon concentration, and it increased with increasing proportions of 3ppa in the feed medium.     

Links between morphology and physiology of Ganoderma lucidum in submerged culture for the production of exopolysaccharide

Ganoderma lucidum was grown in submerged culture in shake flasks on a medium containing peptone, yeast extract and glucose. In pre-cultures, inoculated from an agar-grown culture, morphological and metabolic events were linked: the pellets originally produced protuberances when glucose was present in the medium, although glucose was not consumed. The protuberances were then liberated into the medium as second-generation pellets, at which time glucose consumption began and the rate of exopolysaccharide (EPS) production increased. The synchrony between events was repeated in cultures fed with either glucose or peptone and yeast extract. In main cultures, inoculated from a 16-day-old pre-culture, the biomass concentration increased linearly, while glucose consumption and EPS production were initially slow but then accelerated. Protuberances were produced and liberated similarly to the pre-culture, but there was less synchrony amongst the pellets. When glucose was added to such a culture on day 10, an EPS concentration of 5.7 g L(-1) was achieved on day 13, this being the highest reliable EPS concentration yet reported for submerged culture of G. lucidum. We conclude that a greater understanding of the morphological and physiological events during the culture of G. lucidum will allow the proposal of culture strategies to improve EPS production.     

Influence of ε-caprolactam on growth and physiology of environmental bacteria

ε-Caprolactam was found to have an effect on ecologically important soil bacteria. It inhibited the growth of several Bacillus sp. and Rhizobium sp. but cells of Arthrobacter sp. were able to grow in the presence of caprolactam. Sphingomonas sp. lost its inherent capacity to produce extracellular polymer (EPS) if grown in medium containing caprolactam. In the case of raw domestic sewage, the diversity of native bacteria was diminished in presence of caprolactam. Polluted sea water yielded predominantly one type of caprolactam-degrading bacteria of the genus Achromobacter. These cells efficiently utilized up to 10 g caprolactam/L as the sole source of carbon and nitrogen in synthetic medium even in the presence of 20 g NaCl/L. Compared to cells of Arthrobacter sp., cells of Achromobacter sp. accumulated high amount of 6-aminocaproic acid due to degradation of caprolactam. When using caprolactam as sole source of carbon and nitrogen, Achromobacter cells showed unique physiological ability to produce EPS upon prolonged incubation in solid medium and in broth with low phosphate (C:N:P ratio 100:20:0.05). Hydrolyzed cell-free EPS had glucose as its major component though the only substrate provided in the medium for growth was caprolactam.