Optimization of medium by orthogonal matrix method for submerged mycelial culture and exopolysaccharide production in Collybia maculata

Optimization of submerged culture conditions for the production of mycelial growth and exopolysaccharides (EPSs) by Collybia maculata was investigated. The optimum temperature and the initial pH for EPS production in a shake-flask culture of C. maculata were found to be 20°C and 5.5, respectively. Among the various medium’s constituents examined, glucose, Martone A-1, K₂HPO₄, and CaCl₂ were the most suitable carbon, nitrogen, and mineral sources for EPS production, respectively. The optimum concentration of the medium’s ingredients determined using the orthogonal matrix method was as follows: 30 g/L of glucose, 20 g/L of Martone A-1, 1g/L of K₂HPO₄, and 1g/L of CaCl₂. Under the optimized culture conditions, the maximum concentration of EPSs in a 5-L stirred-tank reactor was 2.4 g/L, which was approximately five times higher than that in the basal medium. A comparative fermentation result showed that the EPS productivity in an airlift reactor was higher than that in the stirred-tank reactor despite the lower mycelial growth rate. The specific productivities and the yield coefficients in the airlift reactor were higher than those in the stirred-tank reactor even though the volumetric productivities were higher in the stirred-tank reactor than in the airlift reactor.     

Fungal morphology in submerged cultures and its relation to glucose oxidase excretion by recombinant Aspergillus niger

The effect of culture conditions such as medium composition and shear stress on the fungal pellet morphology in shake-flask cultures and its relation to glucose oxidase (GOD) excretion by recombinant Aspergillus niger NRRL 3 (GOD 3–18) was investigated. It was shown that culture conditions resulting in the formation of smaller fungal pellets with an increased mycelial density result in higher yields of exocellular GOD. The pellets obtained in shake-flask cultures showed distinct layers of mycelial density with only the thin outer layer consisting of a dense mycelial network. The performance of the recombinant strain and the process of pellet formation was also analyzed during batch cultivation in a stirred-tank bioreactor. It was shown that the process of pellet formation occurred in two steps: (1) aggregation of free spores to spore clusters with subsequent germination and formation of small aggregates surrounded by a loose hyphal network, and (2) aggregation of the primary aggregates to the final full-size pellets. The fungal pellets formed during bioreactor cultivation were smaller, did not show large differences in mycelial density, and were more efficient with respect to the production of exocellular GOD. The decreasing pellet size also correlated with an increased mycelial density, indicating an improvement of the transport of nutrients to the inner parts of the pellet.     

Synthesis and characterization of a novel extracellular polysaccharide by Rhodotorula glutinis

The aim of this work was to characterize an exopolysaccharide by Rhodotorula glutinis KCTC 7989 and to investigate the effect of the culture conditions on the production of this polymer. The extracellular polysaccharide (EPS) produced from this strain was a novel acidic heteropolysaccharide composed of neutral sugars (85%) and uronic acid (15%). The neutral sugar composition was identified by gas chromatography as mannose, fucose, glucose, and galactose in a 6.7:0.2:0.1:0.1 ratio. The molecular weight of purified EPS was estimated to be 1.0−3.8×10⁵ Dalton, and the distribution of the molecular weight was very homogeneous (polydispersity index =1.32). The EPS solution showed a characteristic of pseudoplastic non-Newtonian fluid at a concentration >2.0% in distilled water. The maximum EPS production was obtained when the strain was grown on glucose (30 g/L). Ammonium sulfate was the best suitable nitrogen source for EPS production. The highest yield of EPS was obtained at a carbon to nitrogen ratio of 15. The EPS synthesis was activated at the acidic range of pH 3.0–5.0 and increased when the pH of the culture broth decreased naturally to <2.0 during the fermentation. When the yeast was grown on glucose (30 g/L) and ammonium sulfate (2 g/L) at 22°C at an initial pH of 4.0, EPS production was maximized (4.0 g/L), and the glucose-based production yield coefficient and carbon-based production yield coefficient were 0.30 g of EPS/g of glucose and 0.34 g (carbon of EPS)/g (carbon of glucose), respectively.     

Evaluation of Metal Ions and Surfactants Effect on Cell Growth and Exopolysaccharide Production in Two-Stage Submerged Culture of Cordyceps militaris

During the two-stage submerged fermentation of medicinal mushroom Cordyceps militaris, it was found that K⁺, Ca²⁺, Mg²⁺, and Mn²⁺ were favorable to the mycelial growth. The EPS production reached the highest levels in the media containing Mg²⁺ and Mn²⁺. However, Ca²⁺ and K⁺ almost failed to increase significantly exopolysaccharides (EPS) production. Sodium dodecyl sulfate (SDS) significantly enhanced EPS production compared with that of without adding SDS when SDS was added on static culture stage of two-stage cultivation process. The presence of Tween 80 in the medium not only simulated mycelial growth but also increased EPS production. By response surface methods (RSM), EPS production reached its peak value of 3.28?g/L under optimal combination of 27.6?mM Mg²⁺, 11.1?mM Mn²⁺, and 0.05?mM SDS, which was 3.76-fold compared with that of without metal ion and surfactant. The results obtained were useful in better understanding the regulation for efficient production of EPS of C. militaris in the two-stage submerged culture.     

Two-stage pH Control Mode in Batch Fermentation of a Novel Bioflocculant from Corynebacterium Glutamicum

The effect of pH of the fermentation medium on cell growth and the production of a novel bioflocculant(named REA-11) by Corynebacterium glutamicum CCTCC M201005 were investigated. The maximum biomass(2.23 g/L) and flocculating activity(142.2 U/mL) were simultaneously obtained at the 14th hour when the pH value of the culture medium was maintained at 7.0 during the whole fermentation process. The production of REA-11 kept on a trend of increase till the later phase of fermentation process, which resulted in the ultimate flocculating activity of the culture broth to enhance to nearly 100 U/mL at pH 6.0. A two-stage pH control mode was adopted in REA-11 production in which the pH value of the culture medium was controlled at 7.0 during the first 14 h, then decreased to 6. 0 that was maintained until the end of the fermentation process. With the two-stage pH control mode, the maximum flocculating activity reached 178. 8 U/mL which was 30% higher than that obtained under the condition of pH 7.0 and the biomass enhanced about 15%. Compared with the fermentation process without pH control, REA-11 production and cell growth via the two-stage pH control mode increased 80% and 25 %, respectively.     

Production of l-lactic acid byRhizopus oryzae in a bubble column fermenter

Two distinctive forms of growth (mycelial filamentous and mycelial pellets) ofRhizopus oryzae were obtained by manipulating the initial pH of the medium with the controlled addition of CaCO₃ in a bubble fermenter. In the presence of CaCO₃, diffused filamentous growth was obtained when the initial pH of the substrate was 5.5. In the absence of CaCO3, mycelial pellet growth was obtained when the initial pH was 2.0. The fermentation study indicated that the mycelial growth has a shorter lag period before the onset of acid formation. Both physical forms of growth ofRhizopus exhibited a high yield of L-lactic acid in the bubble fermenter when the initial glucose concentration exceeded 70 g/L. A final lactic acid concentration of 62 g/L was produced by the filamentous form ofRhizopus from 78 g/L glucose after 27 h. This showed a weight yield of 80% of glucose consumed, with an average specific productivity of 1.46 g/h/g. Similarly, the pellet form ofRhizopus produced a final lactic acid concentration of 66 g/L from 76 g/L glucose after 43 h, with a weight yield of 86% and an average specific productivity of 1.53 g/h/g.     

Effects of polysaccharide elicitors from endophytic Fusarium oxysporium Dzf17 on growth and diosgenin production in cell suspension culture of Dioscorea zingiberensis

Three polysaccharides, namely exopolysaccharide (EPS), water-extracted mycelial polysaccharide (WPS) and sodium hydroxide-extracted mycelial polysaccharide (SPS), were prepared from the endophytic fungus Fusarium oxysporium Dzf17 isolated from the rhizomes of Dioscorea zingiberensis. The effects of the time of addition and polysaccharide concentration on the growth and diosgenin accumulation in cell suspension culture of D. zingiberensis were studied. Among them, WPS was found to be the most effective polysaccharide. When WPS was added to the medium at 20 mg/L on the 25th day of culture, the cell dry weight was increased 1.34-fold, diosgenin content 2.85-fold, and diosgenin yield 3.83-fold in comparison to those of control. EPS and SPS showed moderate and relatively weak enhancement effects on cell growth and diosgenin accumulation, respectively. The dynamics of cell growth and diosgenin accumulation when WPS was added to the medium at 20 mg/L on the 25th day of culture were investigated, and results showed that dry weight of cells reached a maximum value on day 30 but the maximum diosgenin content was achieved on day 31.     

Macrophage-stimulating activity of exo-biopolymer from cultured rice bran with <Emphasis Type="Italic">Monascus pilosus</Emphasis>

To find a new use of rice bran, five fungi were examined for the production of exo-biopolymer with macrophage-stimulating activity from rice bran. Among the exo-biopolymers produced from the cultures, Monascus pilosus had the most potent macrophage stimulating activity in a liquid culture rather than in a solid culture. In order to improve the yield of exo-biopolymer with macrophage-stimulating activity, a suitable medium for exo-biopolymer was tested in submerged culture of M. pilosus. The highest amount of exo-biopolymer (13.9 mg/mL) was obtained in a medium containing rice bran as an only carbon source followed by media with additional maltose and sucrose (13.8 and 13.7 mg/mL, respectively). The addition of peptone resulted in the production of high amount of exo-biopolymer (15.1 mg/mL), meanwhile the addition of ammonium chloride resulted in 264.0 μg/mL of glucosamine content. Among eight different kinds of inorganic salts tested, potassium phosphate (0.1%) was the most effective inorganic salt for the mycelial growth and exo-biopolymer production. Therefore the optimal medium composition was as follows (g/L): 20 g of rice bran, 5 g of peptone, and 1 g of KH₂PO₄. The optimal culture pH and time for mycelial growth and exo-biopolymer production was pH 5.0 and 25°C, respectively. The maximum exo-biopolymer (20.1 mg/mL) was observed at the fourth day of cultivation. Exo-biopolymer, a crude polysaccharide fraction, mainly contained neutral sugar (81.8%) with considerable amounts of uronic acid (18.2%). Component sugar analysis showed that the active fraction consisted mainly of arabinose, galactose, glucose, which was digested from starch of rice bran during cultivation, and uronic acid (molar ratio; 0.8:1.0:0.7:0.8).     

Optimization of l -(+)-lactic acid production using pelletized filamentous Rhizopus oryzae NRRL 395

Lactic acid is used as a food additive for flavor and preservation and a precursor in the development of poly-lactic acid, a product used to make biodegradable plastics and textiles. Rhizopus oryzae NRRL 395 is known to be a strain that produces optically pure l-(+)-lactic acid. The morphology of Rhizopus cultures is complex, forming filamentous, clumps, and pellet mycelia. Different morphology growth has significant effects on lactic acid production. In bioreactors, the filamentous or clump mycelia increase the viscosity of the medium, wrap around impellers, and block the nutrient transportation, leading to a decrease in production efficiency and bioreactor performance. Growing fungi in pellet form can significantly improve these problems. In this study, factors that affect lactic acid production in pelletized flask cultures using R. oryzae NRRL 395 were investigated in detail. Completely randomized designs were used to determine the influence of culture temperature, time, concentration of glucose, and inoculum size. Lactic acid fermentation using clump and pellet morphologies were performed in a 5 L fermentor at the optimal values obtained from flask culture. Finally, fed-batch culture was used to enhance the lactate concentration in broth. The final lactate concentration of fed-batch culture reached 92 g/L. The data presented in the article can provide useful information on optimizing lactic acid production using alternative source materials.     

Effect of environmental factors and carbohydrate on gellan gum production

Submerged culture fermentation studies were carried out in batch mode for optimizing the environmental parameters and carbon source requirement by Pseudomonas elodea for the production of gellan gum. The maximum production of gellan gum was obtained with 16-h-old culture and 8% inoculum at 30°C and pH 7.0 after 52 h of incubation (6.0 g/L). Of the various carbon sources tested, 2% sucrose, glucose, and soluble starch yielded considerably high amounts of gellan. Studies on the concentration of various carbohydrates on gellan gum production indicated that the optimum concentration of glucose and starch was 3%, whereas for sucrose it was 4%. The addition of glucose in the medium above 3% had a detrimental effect on gellan yield. The investigation of intermediate two-step addition of glucose under identical conditions of fermentation showed an enhanced production of gellan (8.12 g/L) as compared with the control (6.0 g/L). To optimize the recovery of gellan from fermented broth, different solvents were tested for precipitation of gellan gum. Among the various solvents tested, tetrahydrofuran gave better recovery of gellan (82%) as compared with the conventional solvent isopropanol (49%).     

Conditions that promote production of lactic acid byZymomonas mobilis in batch and continuous culture

This study documents the similar pH-dependent shift in pyruvate metabolism exhibited byZymomonas mobilis ATCC 29191 and ATCC 39676 in response to controlled changes in their steady-state growth environment. The usual high degree of ethanol selectivity associated with glucose fermentation by Z.mobilis is associated with conditions that promote rapid and robust growth, with about 95% of the substrate (5% w/v glucose) being converted to ethanol and CO₂, and the remaining 5% being used for the synthesis of cell mass. Conditions that promote energetic uncoupling cause the conversion efficiency to increase to 98% as a result of the reduction in growth yield (cell mass production). Under conditions of glucose-limited growth in a chemostat, with the pH controlled at 6.0, the conversion efficiency was observed to decrease from 95% at a specific growth rate of 0.2/h to only 80% at 0.042/h. The decrease in ethanol yield was solely attributable to the pH-dependent shift in pyruvate metabolism, resulting in the production of lactic acid as a fermentation byproduct. At a dilution rate (D) of 0.042/h, decreasing from pH 6.0 to 5.5 resulted in a decrease in lactic acid from 10.8 to 7.5 g/L. Lactic acid synthesis depended on the presence of yeast extract (YE) or tryptone in the 5% (w/v) glucose-mineral salts medium. At D = 0.15/h, reduction in the level of YE from 3 to 1 g/L caused a threefold decrease in the steady-state concentration of lactic acid at pH 6. No lactic acid was produced with the same mineral salts medium, with ammonium chloride as the sole source of assimilable nitrogen. With the defined salts medium, the conversion efficiency was 98% of theoretical maximum. When chemostat cultures were used as seed for pH-stat batch fermentations, the amount of lactic acid produced correlated well with the activity of the chemostat culture; however, the mechanism of this prolonged induction     

Biosynthesis of poly-β-hydroxybutyrate and exopolysaccharides on azotobacter chroococcum strain 6B utilizing simple and complex carbon sources

Coproduction of poly-β-hydroxybutyrate (PHB) and exopolysaccharides (EPS) was investigated with Azotobacter chroococcum strain 6B isolated from soil samples. The bacterium was cultured using various carbon sources solely or with 0.1 g/L of ammonium sulfate. Ammonium addition resulted in reduced PHB and EPS production with glucose, fructose, and sucrose media, but cellular mass remained constant except for sucrose. Protein was nearly twofold higher in ammonium-grown cultures. Glucose and fructose alone biosynthesized high amounts of EPS (maximum 2.1 and 1.1 g/L, respectively, at 72 h), whereas PHB was accumulated only in glucose-grown cells. Sucrose almost did not produce EPS. Conversely, PHB content was the highest obtained from all experimented conditions (1.1 g/L at 48 h, 40% cell dry wt). When a complex carbon source such as sugar cane molasses was utilized, PHB was accumulated concomitant with EPS production from the initial time to 48 h (0.75 g/L, 37% cell dry wt and 0.6 g/L, respectively), and then PHB decayed at 72 h (0.2 g/L). On the other hand, EPS continued to be biosynthesized (1.1 g/L, 72 h). PHB fractions of total intra- and extracellular biopolymers were calculated. Sucrose-modified Burk’s medium without ammonium addition is suggested as a medium capable of diverting the carbon source for the production of intracellular PHB rather than EPS with A. chroococcum 6B.     

Production of extracellular xylanases byPenicillium janthinellum

Xylanase production byPenicillium janthinellum using 10–100 mM of 2,2-dimethylsuccinate (DMS) buffer, in a range of pH 4.5-6.0 was studied. The enzyme activity was enhanced using oat xylan as the carbon source. Under these conditions a culture produced 1.14 Μmol/ min (11.4 U/mL or 84.4 U/mg) of Β-xylanase after 5 d of growth in a 10-mM buffer solution at pH 4.5. Protease was absent in the DMS buffer except when 100 mM phosphate buffer at pH 6.0 was used (4 U/mL). Β-Xylosidase was only found at a pH of 4.5 in all the buffer concentrations. At a 50 mM DMS buffer concentration at pH 4.5 Β- xylanases were induced by both oat and birch xylans, having a greater effect with oat spelt xylans. Electrophoretic analyses showed that the birchwood xylan induction exhibited different proteins profiles. No Β-xylosidase or Β- glucosidase was induced until d 5. The Β-xylanases were rapidly inactivated at 50‡C, however, birch xylanase appeared to be more stable than oat xylanase. Using oat xylan as an inductor, theΒ-xylosidase andΒ-glucosidase were 85 and 91 U/L, respectively, on d 7. The xylanase produced by induction from sugar cane bagasse hydrolyzate was used for pulp biobleaching. A 20% decrease on the Kappa value in Kraft pulp using the culture extract was obtained. These selective growth conditions led us to modulate the xylanase production for pulp delignification.     

Mushroom Polysaccharides and Lipids Synthesized in Liquid Agitated and Static Cultures. Part I: Screening Various Mushroom Species

The effect of four synthetic media containing glucose (initial concentration 30?g?l^?1) on mycelial growth, exopolysaccharides (EPS) and cellular lipids production was examined in 11 mushroom species after 12 and 16?days of culture in static- and shake-flasks. Fatty acid analysis of cellular lipids produced was also performed. Agitation had a positive effect on biomass production, glucose consumption and lipid biosynthesis. Media that favoured the production of biomass were not suitable for EPS biosynthesis and vice versa. Biomass values varied from ??1.0?g?l^?1 (Lentinula edodes) to ??19?g?l^?1 (Pleurotus ostreatus), while the highest EPS quantity achieved ranged between 1.6 and 1.8?g?l^?1 (for Ganoderma lucidum and L. edodes, respectively). Quantities of total cellular lipids varied between 2.5 and 18.5?% w/w, in dry mycelial mass for the fungi tested. Lipid in dry weight values were influenced by the medium composition. Cellular lipids presented noticeable quantities of poly-unsaturated fatty acids like linoleic acid. Compared to most of the mushrooms tested, lipids of Volvariella volvacea were more saturated. The ability of several mushroom species of our study to produce in notable quantities the above-mentioned added-value compounds renders these fungi worthy for further investigations.     

Biosynthesis of Unsaturated Fatty Acids via Mortierella Isabellina Cultivated in a Medium Containing Butanol

IntroductionThe biosynthesis of unsaturated fatty acidshasattracted more attention in recent years. Forexample,linoleic and linolenic acids are importantmaterials for pharmaceutical and food industries.Besides,they can be used to synthesize paint,printing ink and surfactant,etc. A number ofmicroorganisms have been studied as potentialcommercial sources to produce unsaturated fattyacids[1] .Agricultural and industrial products,by-products,etc. have been employed in thecultivation of those orga…     

UO22+对某些细胞毒性的初探

建立了由多种金属离子和小分子配体组成的多相细胞液热力学平衡模型,模拟研究了UO22+在组织液和细胞液的形态。体外培养了SD大鼠成骨细胞和人肾小管上皮细胞,通过体外细胞生长抑制实验探索了UO22+对成骨细胞及肾小管上皮细胞的毒性。研究表明,在细胞液中,当各形态UO22+物质总浓度[U]=8.4×10-6mol/L时,当pH为6.0～6.5时,UO22+主要以固相(UO2)3(PO4)2存在,当pH为6.8～7.5时,UO22+主要以水溶性[UO2(CO3)3]4-存在;当[U]=1.3×10-3mol/L时,在整个细胞液pH范围内,固相(UO2)3(PO4)2占主导地位。体外细胞生长抑制实验表明,UO22+对成骨细胞的生长具有抑制作用,能显著降低肾小管上皮细胞的存活率,具有明显的细胞毒性。     

Biosorption of copper(II) and cadmium(II) by a novel exopolysaccharide secreted from deep-sea mesophilic bacterium

The biosorption behaviors and mechanisms of a novel exopolysaccharide (EPS), which is secreted by a mesophilic bacterium (namely Wangia profunda (SM-A87)) isolated from deep-sea sediment, for heavy metals Cu(II) and Cd(II) have been studied in this paper. The effects of SM-A87 EPS concentration, solution pH and ionic strength on the metal uptake were investigated by employing batch adsorption techniques, respectively. The optimum biosorption capacities were observed at pH 5.0 for Cu(II) with 48.0 mg/g and pH 6.0 for Cd(II) with 39.75 mg/g, respectively. Addition of salts decreased Cu(II) or Cd(II) uptake in the order of K⁺ < Na⁺ < Ca²⁺. Langmuir and Freundlich models were employed to describe the biosorption equilibrium data, indicating the favorable biosorption occurs and larger biosorption capacity and intensity for Cu(II) than for Cd(II). The biosorption kinetics for both metals can be well described by pseudo-second-order kinetic model, compared with pseudo-first-order and intraparticle diffusion kinetic models. The competitive biosorption was also studied, indicating that in two-component solution with different metal ratios, the selective biosorption of SM-A87 EPS for Cu(II) was much higher than for Cd(II). The Fourier transform infrared spectroscopy (FT-IR) analysis indicated possible functional groups (e.g., OH, COO and COC) of SM-A87 EPS involved in metal biosorption process, which indicated the potential of using SM-A87 EPS as an effective sorbent for Cu(II) or Cd(II) removal from water.     

Production of propionic acid using a xylose utilizingPropionibacterium

The kinetics ofP. acidipropionici (ATCC25562), a xylose-utilizing rumen microorganism, was studied to assess its use for propionic acid production from wood hydrolyzates. Propionic acid has been shown to have a stronger inhibitory effect than acetic acid, with the undissociated acid form being responsible for the majority of the inhibitory effect. Thus, in batch tests with pH controlled at 6.0, the propionic acid concentration reaches 25 g/L and the acetic acid 7 g/L. Xylose uptake rate is dependent on the specific growth rate and glucose concentration. An immobilized cell columnar reactor at very high product yields (80%) proved adequate for propionic production. At cell concentrations of 95 g/L with high product concentration, volumetric productivities of 2.7 g/L·h were obtained in ultrafiltration cell recycle systems.     

Study of operational variables in the submerged growth ofPleurotus ostreatus mushroom mycelium

Pleurotus ostreatus mushroom mycelium was cultivated in submerged culture in shake-flask experiments with acid extract from peat and yeast extract as nutrient sources. Different concentrations of water-diluted peat extract were tested in an attempt to overcome the effect of growth inhibitors apparently present in nondiluted peat extracts. The best results were obtained with a ratio of one part of peat extract diluted with one part of water. Several operating variables were studied to optimize the growth of mycelial biomass ofP. ostreatus. The best results produced approximately 5 g/L dry biomass with a yield of 60% and an efficiency of 33%. These results were obtained in 8 d at 5% (v/v) inoculum ratio, 28°C, pH of 5.0, and 150 rpm.     

Optimization of l-(+)-lactic acid production using pelletized filamentous Rhizopus oryzae NRRL 395

Lactic acid is used as a food additive for flavor and preservation and a precursor in the development of poly-lactic acid, a product used to make biodegradable plastics and textiles. Rhizopus oryzae NRRL 395 is known to be a strain that produces optically pure L: -(+)-lactic acid. The morphology of Rhizopus cultures is complex, forming filamentous, clumps, and pellet mycelia. Different morphology growth has significant effects on lactic acid production. In bioreactors, the filamentous or clump mycelia increase the viscosity of the medium, wrap around impellers, and block the nutrient transportation, leading to a decrease in production efficiency and bioreactor performance. Growing fungi in pellet form can significantly improve these problems. In this study, factors that affect lactic acid production in pelletized flask cultures using R. oryzae NRRL 395 were investigated in detail. Completely randomized designs were used to determine the influence of culture temperature, time, concentration of glucose, and inoculum size. Lactic acid fermentation using clump and pellet morphologies were performed in a 5 L fermentor at the optimal values obtained from flask culture. Finally, fed-batch culture was used to enhance the lactate concentration in broth. The final lactate concentration of fed-batch culture reached 92 g/L. The data presented in the article can provide useful information on optimizing lactic acid production using alternative source materials.