Exopolysaccharide production and mycelial growth in an air-lift bioreactor using Fomitopsis pinicola

For effective exopolysaccharide production and mycelial growth by a liquid culture of Fomitopsis pinicola in an air-lift bioreactor, the culture temperature, pH, carbon source, nitrogen source, and mineral source were initially investigated in a flask. The optimal temperature and pH for mycelial growth and exopolysaccharide production were 25degrees C and 6.0, respectively. Among the various carbon sources tested, glucose was found to be the most suitable carbon source. In particular, the maximum mycelial growth and exopolysaccharide production were achieved in 4% glucose. The best nitrogen sources were yeast extract and malt extract. The optimal concentrations of yeast extract and malt extract were 0.5 and 0.1%, respectively. K2HPO4 and MgSO4 x 7H2O were found to be the best mineral sources for mycelial growth and exopolysaccharide production. In order to investigate the effect of aeration on mycelial growth and exopolysaccharide production in an air-lift bioreactor, various aerations were tested for 8 days. The maximum mycelial growth and exopolysaccharide production were 7.9 g/l and 2.6 g/l, respectively, at 1.5 vvm of aeration. In addition, a batch culture in an air-lift bioreactor was carried out for 11 days under the optimal conditions. The maximum mycelial growth was 10.4 g/l, which was approximately 1.7-fold higher than that of basal medium. The exopolysaccharide production was increased with increased culture time. The maximum concentration of exopolysaccharide was 4.4 g/l, which was about 3.3-fold higher than that of basal medium. These results indicate that exopolysaccharide production increased in parallel with the growth of mycelium, and also show that product formation is associated with mycelial growth. The developed model in an air-lift bioreactor showed good agreement with experimental data and simulated results on mycelial growth and exopolysaccharide production in the culture of F pinicola.     

Enzymes involved in carbohydrate metabolism and their role on exopolysaccharide production in Streptococcus thermophilus

The role of the enzymes uridine-5'-diphospho-(UDP) glucose pyrophosphorylase and UDP galactose 4-epimerase in exopolysaccharide production of Gal- ropy and non-ropy strains of Streptococcus thermophilus in a batch culture was investigated. Growth of the ropy and non-ropy strains was accompanied by total release of the galactose moiety from lactose hydrolysis in modified Bellinker broth with lactose as the only carbon source. This was associated with a greater exopolysaccharide production by the ropy strain. The polymer produced by both strains in cultures with lactose or glucose as carbon sources contained glucose, galactose and rhamnose, indicating that glucose was used as a carbon source for bacterial growth and for exopolysaccharide formation. UDP-glucose pyrophosphorylase activity was associated with polysaccharide production during the first 12 h in a 20 h culture in the ropy strain, but not in the non-ropy strain. UDP-galactose 4-epimerase was not associated with exopolysaccharide synthesis in any strain. The evidence presented suggests that the glucose moiety from lactose hydrolysis is the source of sugar for heteropolysaccharide synthesis, due to a high UDP-glucose pyrophosphorylase activity.     

Enzymes involved in carbohydrate metabolism and their role on exopolysaccharide production in Streptococcus thermophilus

The role of the enzymes uridine-5'-diphospho-(UDP) glucose pyrophosphorylase and UDP galactose 4-epimerase in exopolysaccharide production of Gal⁻ ropy and non-ropy strains of Streptococcus thermophilus in a batch culture was investigated. Growth of the ropy and non-ropy strains was accompanied by total release of the galactose moiety from lactose hydrolysis in modified Bellinker broth with lactose as the only carbon source. This was associated with a greater exopolysaccharide production by the ropy strain. The polymer produced by both strains in cultures with lactose or glucose as carbon sources contained glucose, galactose and rhamnose, indicating that glucose was used as a carbon source for bacterial growth and for exopolysaccharide formation. UDP-glucose pyrophosphorylase activity was associated with polysaccharide production during the first 12 h in a 20 h culture in the ropy strain, but not in the non-ropy strain. UDP-galactose 4-epimerase was not associated with exopolysaccharide synthesis in any strain. The evidence presented suggests that the glucose moiety from lactose hydrolysis is the source of sugar for heteropolysaccharide synthesis, due to a high UDP-glucose pyrophosphorylase activity.     

Optimization of a medium and the role of its various components for the biosynthesis of Mycobacterium cyaneum exopolysaccharide

The effect of some components of the medium on the growth of Mycobacterium cyaneum B-646 and on the biosynthesis of an exoglycan by the culture was studied. A medium in which the M. cyaneum M variant produced up to 2.2 g of the exopolysaccharide per litre, which was nearly 4 times more than in the original medium, was proposed. The new medium differed from the original one in an elevated content of carbon, iron and potassium (11.2, 5.0 and 1.4 times, respectively) and in a lower phosphorus content (6.7 times). The exopolysaccharide produced by the culture in this medium contained glucose, galactose, fucose and uronic acid. Therefore, its monomeric composition did not depend on the medium used for growing the culture which produced the exopolysaccharide.     

Influence of nutritional conditions on the mycelial growth and exopolysaccharide production in Paecilomyces sinclairii

AIMS: The objective of the study was to optimize the submerged culture conditions for the production of exopolysaccharide from Paecilomyces sinclairii. METHODS AND RESULTS: The optimal temperature and initial pH for exopolysaccharide production by Paecilomyces sinclairii in shake flask culture were found to be 30 degrees C and 6.0, respectively. Sucrose (60 g l(-1)) and corn steep powder (10 g l(-1)) were the most suitable carbon and nitrogen source for exopolysaccharide production. CONCLUSIONS: Under optimal culture medium, the maximum exopolysaccharide concentration in a 5-l stirred-tank fermenter indicated 7.4 g l(-1), which was approximately three times higher than that in basal medium. The maximum specific growth rates (micro max) and yield coefficient (Y(P/S)) in the optimal culture medium was 0.16 h(-1) and 0.19, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimal culture conditions reported in this article can be widely applied to the processes for submerged cultures of other mushrooms.     

Effect of culture medium pH on bacterial gellan production.

The effect of the initial pH of the culture medium used in the production of the exopolysaccharide gellan by the bacterium Pseudomonas species ATCC 31461, when glucose or corn syrup served as the carbon source, was investigated. With glucose as the carbon source, exopolysaccharide formation was highest after 72 h of growth when the initial pH of the culture medium was 6.8 to 7.4. Polysaccharide production by the bacterial cells grown on corn syrup for 72 h was maximal when the initial pH of the medium was 7.0 or 7.2. Cell weights of the strain after 72 h tended to be higher for the glucose-grown cells than for the corn syrup-grown cells.     

Growth and exopolysaccharide production by Azotobacter vinelandii in media containing phenolic acids

Azotobacter vinelandii was cultured in chemically defined, nitrogen-free media supplemented with either 4-hydroxyphenylacetic, 4-hydroxybenzoic or protocatechuic acids at different concentrations. Under these conditions, biomass, exopolysaccharide production and consumption of the carbon sources were investigated. Results obtained throughout this study showed that 4-hydroxyphenylacetic acid yielded the highest growth levels measured as biomass, and exopolysaccharide production, independently of the concentration of the carbon source tested. 4-Hydroxybenzoic acid also supported appreciable growth and exopolysaccharide recovery by A. vinelandii. Protocatechuic acid, however, only allowed a very small production of biomass and exopolysaccharide by the strain investigated. Under given conditions, more than 26% of the carbon source supplied was converted to exopolysaccharide in cultures of A. vinelandii .     

Optimization of submerged culture requirements for the production of mycelial growth and exopolysaccharide by Cordyceps jiangxiensis JXPJ 0109

AIMS: The objective of the present study was to investigate the optimal culture requirements for mycelial growth and exopolysaccharide production by Cordyceps jiangxiensis JXPJ 0109 in submerged culture. METHODS AND RESULTS: The effects of medium ingredients (i.e. carbon and nitrogen sources, and growth factor) and other culture requirements (i.e. initial pH, temperature, etc.) on the production of mycelia and exopolysaccharide were observed using a one-factor-at-a-time method. More suitable culture requirements for mycelial growth and exopolysaccharide production were proved to be maltose, glycerol, tryptone, soya bean steep powder, yeast extract, medium capacity 200 ml in a 500-ml flask, agitation rate 180 rev min(-1), seed age 4-8 days, inoculum size 2.5-7.5% (v/v), etc. The optimal temperatures and initial pHs for mycelial growth and exopolysaccharide production were at 26 degrees C and pH 5 and at 28 degrees C and pH 7, respectively, and corresponding optimal culture age were observed to be 8 and 10 days respectively. According to the primary results of the one-factor-at-a-time experiments, the optimal medium for the mycelial growth and exopolysaccharide production were obtained using an orthogonal layout method to optimize further. Herein the effects of medium ingredients on the mycelial growth of C. jiangxiensis JXPJ 0109 were in the order of yeast extract > tryptone > maltose > CaCl2 > glycerol > MgSO4 > KH2PO4 and the optimal concentration of each composition was 15 g maltose (food-grade), 10 g glycerol, 10 g tryptone, 10 g yeast extract, 1 g KH2PO4, 0.2 g MgSO4, and 0.5 g CaCl2 in 1 l of distilled water, while the order of effects of those components on exopolysaccharide production was yeast extract > maltose > tryptone > glycerol > KH2PO4 > CaCl2 > MgSO4, corresponding to the optimal concentration of medium was as follows: 20 g maltose (food-grade), 8 g glycerol, 5 g tryptone, 10 g yeast extract, 1 g KH2PO4, and 0.5 g CaCl2 in 1 l of distilled water. CONCLUSIONS: Under the optimal culture requirements, the maximum exopolysaccharide production reached 3.5 g l(-1) after 10 days of fermentation, while the maximum production of mycelial growth achieved 14.5 g l(-1) after 8 days of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the submerged culture requirements for mycelial growth and exopolysaccharide in C. jiangxiensis, and this two-step optimization strategy in this study can be widely applied to other microbial fermentation processes.     

Exopolysaccharide production by the epipelic diatom Cylindrotheca closterium: effects of nutrient conditions

During the stationary phase of a batch culture of the epipelic diatom Cylindrotheca closterium, accumulation of exopolysaccharides and intracellular carbohydrates was observed. When nitrogen was added to the culture in the stationary phase, growth was resumed and the accumulation of exopolysaccharides was delayed. This indicated that nitrogen depletion caused cessation of growth, and stimulated exopolysaccharide accumulation. Exopolysaccharide accumulation was also stimulated when cells were either resuspended in medium lacking N or P, or when they were inoculated in medium with low concentrations of N or P. Growth was not immediately affected by low N or P concentrations. S depletion only resulted in exopolysaccharide accumulation when growth was affected. Si or Fe depletion did not stimulate exopolysaccharide accumulation, even when growth rates were lowered. Apparently, stimulation of exopolysaccharide accumulation is dependent on the type of nutrient depletion. Intracellular storage carbohydrates did not accumulate when cells were incubated at low N or P concentrations. Cells grown with ammonium as nitrogen source produced more carbohydrates (both extracellular and intracellular) than cells grown with nitrate as nitrogen source, indicating that both exopolysaccharides and intracellular carbohydrates accumulated as a result of overflow metabolism.     

Accumulation of Soybean Lectin-Binding Polysaccharide During Growth of Rhizobium japonicum as Determined by Hemagglutination Inhibition Assay

A hemagglutination inhibition assay was used to estimate the presence of soybean lectin-binding polysaccharide in whole culture, culture supernatant, and isolated exopolysaccharide of Rhizobium japonicum USDA 138. The occurrence of 0.1 to 0.2 μg of lectin-binding polysaccharide could be detected within 2 h with a 0.5-ml total sample. Lectin-binding polysaccharide was detected in all preparations during both exponential and stationary growth phases. The formation of lectin-binding polysaccharide was not, whereas that of total exopolysaccharide was, markedly affected by culture conditions.     

Purification and characterization of adhesive exopolysaccharides from Pseudomonas putida and Pseudomonas fluorescens

In this study, the adhesive exopolysaccharides of strains of Pseudomonas putida and P. fluorescens, both isolated from freshwater epilithic communities, were examined with regard to their chemical composition, biosynthesis, and their role in adhesion. Electron microscopy showed that both strains were enrobed in fibrous glycocalyces and that these structures were involved in attachment of the cells to a solid surface and as structural matrices in the microcolony mode of growth. In batch culture experiments most of the extracellular polysaccharide of both strains was found to be soluble in the growth medium rather than being associated with bacterial cells. Exopolysaccharide was synthesized during all phases of growth, but when growth was limited by exhaustion of the carbon source, exopolysaccharide synthesis ceased whereas exopolysaccharide synthesis continued for some time after cessation of growth in nitrogen-limited cultures. Exopolysaccharide from both strains was isolated and purified. Pseudomonas putida synthesized an exopolysaccharide composed of glucose, galactose, and pyruvate in a ratio of 1:1:1; the P. fluorescens polymer contained glucose, galactose, and pyruvate in a ratio of 1:1:0.5, respectively. Polymers from both strains were acetylated to a variable degree.     

Influence of phosphate on rhamnose-containing exopolysaccharide rheology and production by Klebsiella I-714

Physiological conditions enhancing rhamnose-containing polysaccharide synthesis by Klebsiella I-714 were studied in batch culture (0.3-l and 2-l bioreactors). The four carbon sources tested, sucrose, sorbitol, Neosorb and Cerelose, allowed exopolysaccharide production. Larger amounts of polymer were produced when high carbon/nitrogen ratios and complex nitrogen sources were used. Exopolysaccharide synthesis was greatest at 30 °C, which was a suboptimal growth temperature. A reduction in the phosphate content of the medium enhanced rhamnose-containing polysaccharide production. When the initial carbon source concentration was augmented, byproducts other than exopolysaccharide were formed. Rhamnose-containing polysaccharide rheology can be modulated by changing the phosphate content of the medium. Received: 11 April 1997 / Received revision: 19 June 1997 / Accepted: 23 June 1997     

Variation in composition and yield of exopolysaccharides produced by Klebsiella sp. strain K32 and Acinetobacter calcoaceticus BD4

The exopolysaccharides produced by Klebsiella sp. strain K32 and Acinetobacter calcoaceticus BD4 under different growth conditions have been analyzed for sugar composition. The first use of ion chromatography for the quantitative determination of microbial exopolysaccharide composition is reported. Klebsiella sp. strain K32 produced a polymer composed of rhamnose, galactose, and mannose early in its fermentation. The composition of the polymer varied markedly depending on the growth stage of the organism. Klebsiella sp. strain K32 grown in a fermentor produced a polymer which was rich in mannose during early exponential growth in a complex medium, but in the late stationary phase it did not contain detectable levels of mannose. The rhamnose present in the polymer increased from 12 to 55% over the course of growth, whereas galactose decreased from 63 to 45%. A. calcoaceticus BD4 produced a polymer containing rhamnose, glucose, mannose throughout its growth and stationary phase. Klebsiella sp. strain K32 and A. calcoaceticus BD4 were grown on various carbon sources in shake flasks. The polymer yield and composition from both organisms were found to vary with the carbon source. The exopolysaccharide with the highest mannose composition was obtained by using rhamnose as a carbon source for both organisms. These and other data suggest that regulatory changes caused by growth on different substrates result in either the production of a different distribution of polymers or a change in exopolysaccharide structure.     

Variation in composition and yield of exopolysaccharides produced by Klebsiella sp. strain K32 and Acinetobacter calcoaceticus BD4.

The exopolysaccharides produced by Klebsiella sp. strain K32 and Acinetobacter calcoaceticus BD4 under different growth conditions have been analyzed for sugar composition. The first use of ion chromatography for the quantitative determination of microbial exopolysaccharide composition is reported. Klebsiella sp. strain K32 produced a polymer composed of rhamnose, galactose, and mannose early in its fermentation. The composition of the polymer varied markedly depending on the growth stage of the organism. Klebsiella sp. strain K32 grown in a fermentor produced a polymer which was rich in mannose during early exponential growth in a complex medium, but in the late stationary phase it did not contain detectable levels of mannose. The rhamnose present in the polymer increased from 12 to 55% over the course of growth, whereas galactose decreased from 63 to 45%. A. calcoaceticus BD4 produced a polymer containing rhamnose, glucose, mannose throughout its growth and stationary phase. Klebsiella sp. strain K32 and A. calcoaceticus BD4 were grown on various carbon sources in shake flasks. The polymer yield and composition from both organisms were found to vary with the carbon source. The exopolysaccharide with the highest mannose composition was obtained by using rhamnose as a carbon source for both organisms. These and other data suggest that regulatory changes caused by growth on different substrates result in either the production of a different distribution of polymers or a change in exopolysaccharide structure.     

New culture media to suppress exopolysaccharide production by Rhizobium japonicum

Summary Secretion of exopolysaccharide by Rhizobium japonicum strain USDA 110 ws measured during growth on media comprising 12.5 mM MES [2-(N-morpholino)ethanesulfonic acid] buffer, pH 5.9; 5 mM NH₄Cl; trace elements; and 25 or 100 mM of one of 23 different carbon sources. Significant amounts of exopolysaccharide were produced on all aldopentoses, polyols, organic acids, and sugar acids tested, but little or none was secreted during growth on aldohexoses or amino acids. Media suitable for routine slime-free culture of several. R. japonicum strains were made by using 100 mM d-galactose or 25 mM l-aspartate as the carbon source.     

Influence of nutritional conditions on exopolysaccharide production by submerged cultivation of the medicinal fungus <Emphasis Type="Italic">Shiraia bambusicola</Emphasis>

Maltose and yeast extract were the most favourable carbon and nitrogen sources for exopolysaccharide production by submerged culture of Shiraia bambusicola WZ-003, and initial maltose and yeast extract concentrations were at 30 and 3 g l⁻¹, respectively. Plant oils could increase the mycelial growth and exopolysaccharide production in tested concentration. K⁺ and Mg²⁺ could enhance the mycelial growth and exopolysaccharide biosynthesis. The optimal cultivation temperature and initial pH were found to be 26°C and 6.0, respectively. Exopolysaccharide concentration reached 0.53 g l⁻¹ in 15-l fermenter under optimal nutritional conditions.     

Variations in exopolysaccharide production by Rhizobium tropici

Rhizobium tropici, a legume-symbiont soil bacterium, is known for its copious production of exopolysaccharide (EPS). Many aspects of this organism’s growth and EPS production, however, remain uncharacterized, including the influence of environment and culturing conditions upon EPS. Here, we demonstrate that R. tropici EPS chemical composition and yield differ when grown with different substrates in a defined minimal medium in batch culture. Exopolysaccharide was quantified from R. tropici grown using arabinose, glucose, sucrose, mannitol, fructose, or glutamate as a sole carbon source. All tested substrates produced plenteous amounts of exopolysaccharide material. Variations in pH and carbon-to-nitrogen (C/N) ratio also resulted in assorted cell growth and exopolysaccharide production differences. We found that optimizing the C/N ratio has a greater impact upon R. tropici EPS production than upon R. tropici growth. A maximum EPS yield of 4.08 g/L was realized under optimized conditions, which is large even in comparison with other known rhizobia. We provide evidence that the chemical composition of R. tropici EPS can vary with changes to the growth environment. The composition of glucose-grown EPS contained rhamnose-linked residues that were not present in arabinose-grown EPS.     

Influence of culture conditions on exopolysaccharide production by Lactobacillus rhamnosus strain C83

The influence of culture conditions on growth and exopolysaccharide (EPS) production by Lactobacillus rhamnosus strain C83 was investigated using fermentor batch cultures. A temperature shift (from 37 to 25 °C) at the beginning of the exponential growth phase (0–5 h) enhanced EPS production. Furthermore, an optimal environmental pH of 6·2–7·2 improved the performance of strain C83 and partially anaerobic culture conditions (pO₂ from 0 to 10%) triggered EPS over-production by the cells. The sugar composition of this polymer was independent of culture conditions, such as carbon source, medium composition, temperature, pH, pO₂ and growth phases. In all cases, galactose and glucose were the principal components.     

Optimization of Nutritional Factors for Exopolysaccharide Production by Submerged Cultivation of the Medicinal Mushroom Oudemansiella radicata

Summary Effects of nutritional factors on exopolysaccharide production by submerged cultivation of the medicinal mushroom Oudemansiella radicata were investigated in shake flasks. Sucrose and peptone were optimal carbon and nitrogen sources for cell growth and exopolysaccharide production. The exopolysaccharide production was increased with an increase in initial sucrose concentration within the range of 10–40 g l⁻¹ and initial peptone concentration within the range of 1–3 g l⁻¹. To enhance further exopolysaccharide production, the effect of carbon/nitrogen ratios was studied using central composite design (CCD) and response surface analysis. The maximum exopolysaccharide production of 2.67 ± 0.15 g l⁻¹ was achieved in medium with optimized carbon and nitrogen sources, i.e. 39.3 g sucrose l⁻¹ and 3.16 g peptone l⁻¹ in the same cultivation conditions. The information obtained is helpful for the hyperproduction of exopolysaccharide by submerged cultivation of O. radicata on a large scale.     

Production of an exopolysaccharide bioflocculant by Sorangium cellulosum

AIMS: To isolate a new exopolysaccharide bioflocculant produced by the myxobacterium Sorangium cellulosum NUST06, and to characterize its chemical composition and expolysaccharide production relative to carbon source. METHODS AND RESULTS: Exopolysaccharide levels and biomass production by S. cellulosum NUST06 were analysed relative to carbon source. Glucose in the medium at a level of 3 g l(-1) completely inhibited cell growth and exopolysaccharide production, but low concentrations of glucose (1-2 g l(-1)) could stimulate cell utilization of starch. The chemical composition and flocculating activity of the NUST06 exopolysaccharide was investigated. The flocculant comprised 38.3% proteins and 58.5% carbohydrates, of which glucose, mannose and glucuronic acid were present at 51.3%, 39.2% and 10.5%, respectively. The flocculating activity of the NUST06 flocculant depended strongly on cations. CONCLUSIONS: It is feasible to produce an exopolysaccharide bioflocculant by the strain NUST06 in a mineral salts medium using starch as a carbon source. SIGNIFICANCE AND IMPACT OF THE STUDY: This strain may be advantageous for commercial bioflocculant production and may enrich existing knowledge of myxobacteria.