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.     

Optimal conditions of in vitro mycelial growth of <Emphasis Type="Italic">Lentinus strigosus</Emphasis>, an edible mushroom isolated in the Brazilian Amazon

The protocols of in vitro cultivation described in the literature for mushrooms are usually correlated with temperate climate habitat, but it is necessary to study protocols for species of tropical climates. In this article, we collected, isolated, and evaluated the conditions of in vitro mycelial growth of Lentinus strigosus and correlated these with the characteristics of its habitat. These results indicate, as optimal conditions of in vitro mycelial growth for L. strigosus, the use of 35°C for incubation, initial pH from 5 to 7, without illumination, Sabouraud dextrose agar medium, and agitation for culture in liquid medium.     

Effects of carbon and nitrogen sources,carbon-to-nitrogen ratio,and initial pH on the growth of nematophagous fungus <Emphasis Type="Italic">Pochonia chlamydosporia</Emphasis> in liquid culture

The effects of carbon and nitrogen sources, carbon-to-nitrogen ratio (C:N) and initial pH value on the growth and sporulation of the nematophagous fungus Pochonia chlamydosporia in liquid culture were examined. Among the 21 carbon sources and 15 nitrogen compounds tested, the optimal carbon and nitrogen sources for mycelial growth were sweet potato and L-tyrosine, and for sporulation were sweet potato and casein peptone. A C:N ratio of 10:1 at pH 3.7 gave the maximum yield of conidia and a C:N ratio of 40:1 at pH 6.8 gave the maximum biomass. The initial pH value had a significant effect on mycelial growth and conidial production, with the optimal ranges being 3.5–4.5 for sporulation and 5–6 for growth. Maximum conidial production was obtained at an initial pH of 4.0 and the maximum biomass at pH 6.0. The results also showed that the final pH after 7 days cultivation was always higher than the initial value. The variability in growth and sporulation of seven strains of P. chlamydosporia in liquid culture was also compared and discussed.     

Use of Phanerochaete chrysosporium biomass for the removal of textile dyes from a synthetic effluent

Summary The use of Phanerochaete chrysosporium biomass for the removal of Reactofix Golden Yellow from aqueous solution and eight textile dyes (four azo and four anthraquinone) from a synthetic effluent (0.6 g/l) at different pH, temperature and biomass concentrations was studied. Adsorption was maximum at pH 2.0 and 40 °C using 2.45 g mycelial biomass. The rate constant of adsorption was 1.95×10⁻¹/min for Reactofix Golden Yellow and 1.64×10⁻¹/min for synthetic effluent. In both cases, the equilibrium data fitted well in the Langmuir but not the Freundlich model of adsorption, and the adsorption was biphasic. Adsorption decreased the COD of Reactofix Golden Yellow and synthetic effluent by 54 and 57%, respectively. Desorption (80–84%) of dyes from P. chrysosporium mycelial surface occurred as the pH increased from 2 to 10.     

Comparison of conditions for mycelial growth of <Emphasis Type="Italic">Lepista sordida</Emphasis> causing fairy rings on <Emphasis Type="Italic">Zoysia matrella</Emphasis> turf to those on <Emphasis Type="Italic">Agrostis palustris</Emphasis> turf

Symptoms of fairy rings caused by Lepista sordida have been reported on Zoysiagrass (Zoysia spp.) turf maintained at fairway height (2 cm), but not on bentgrass (Agrostis spp.) maintained at putting green height (0.5 cm). The mycelia of this fungus inhabit primarily the upper 0–2 cm layer of the soil extending into the thatch. To compare conditions for the mycelial growth in Z. matrella turf to those in A. palustris turf, we examined the effects of nutrients, temperature, water potential, and pH in the field as well as in the laboratory. Greater growth of the mycelia was observed in medium that included hot water extracts from soil of the 0–1 cm zone in Z. matrella turf compared to that from A. palustris. The upper soil layer in Z. matrella turf contained more organic matter from clippings than that in A. palustris. The temperature and water potential of the 0–2 cm soil zone in Z. matrella turf were also more favorable for the mycelial growth. The soil pH values of this zone in Z. matrella turf were less favorable compared to A. palustris but within the range for accelerating mycelial growth. Part of this study was presented orally at the 46th meeting of the Mycological Society of Japan in 2002     

Mycelial elongation and sporulation of two fungi on amended media in light or dark

Botrytis allii andCollectotrichum dematium are onion pathogens which can infect in the field and cause decay in storage. Some phenolics can hinder development of these fungi, but the effect of cytokinins is not clear. Cytokinins (kinetin or 6-benzyladenine) or phenolics (caffeic or chlorogenic acids) were added to agar at concentrations of 0 to 10^–3 M. Cultures were continuously irradiated with fluorescent light or maintained in the dark for 6 days. On unamended media, final mycelial elongation was 45 or 17.8 mm and sporulation was 28 or 10.6 × 10⁴ spores/ml forBotrytis andColletotrichum, respectively. ForBotrytis, mycelial elongation was slightly (5%) but significantly increased and sporulation increased by 21% by incubation on phenolics as compared to cytokinins. Mycelial extension ofColletotrichum was not affected by amendment. Sporulation ofColletotrichum on kinetin was 16 to 28% greater than on the other amendments. As amendments concentration increased elongation of mycelia of both fungi decreased. Sporulation ofBotrytis increased by 60% as amendment concentration increased from 0 to 10^–5 M and then decreased 25% at 10^–3 M. As amendment concentration increased from 0 to 10^–3 M, sporulation ofColletotrichum increased by 45%. Incubation in light increased mycelial extension 3 to 17% forBotrytis andColletotrichum respectively, and sporulation was increased approximately 78% for both fungi. These compounds do not appear to inhibit development of theseBotrytis orColletotrichum species in culture.     

Optimization of process parameters for the production of carbonyl reductase by <Emphasis Type="Italic">Candida viswanathii</Emphasis> in a laboratory-scale fermentor

The effect of pH, aeration and mixing on the growth and production of carbonyl reductase by Candida viswanathii was investigated in a 6.6-l fermentor. Controlling the pH at 8.0 had a very significant effect on the enzyme production. Aeration and agitation influenced the dissolved oxygen concentration which in turn affected growth as well as enzyme production. A maximum carbonyl reductase activity (53 Umg⁻¹) was attained in 24 h under the optimal cultivation conditions of controlled pH at 8.0, aeration rate 1 vvm and an agitation speed of 250 rpm at 25°C. The enzyme activity was twice as high (56 Umg⁻¹) in the fermentor as compared to a shake flask. Further, the duration of growth and enzyme production in the fermentor was shortened. Cells cultivated under the optimized conditions were used for the preparative scale reduction of N, N-dimethyl-(3-keto)-2-thienyl-propanamine to (S)-N, N-dimethyl-(3-hydroxy)-2-thienyl-propanamine, a key intermediate in the production of the important antidepressant drug (S)-duloxetine.     

Mycelial cultivation of <Emphasis Type="Italic">Phellinus linteus</Emphasis> using cheese-processing waste and optimization of bioconversion conditions

A medicinal mushroom, Phellinus linteus, was successfully cultivated using a cheese-processing waste, whey, and the optimal bioconversion conditions for the maximum mycelial growth rate was also estimated through solid-state cultivation experiments. Response surface analysis with a face-centered design (center point replication = 5) was applied to statistically approximate the simultaneous effects of the three variables, i.e., substrate concentration (10–30 g lactose l⁻¹), temperature (20–30°C), and pH (4–6), on the mycelial growth rate of P. linteus. The following is a partial cubic model where η is the mycelial growth rate (K _r ) and x _k is the corresponding variable term (k = substrate concentration, temperature, and pH in order): η = −23.8 + 8.67 × 10⁻² x ₁ + 1.48x ₂ + 1.77x ₃ + 8.00 × 10⁻⁴ x ₁ x ₂ + 7.25 × 10⁻² x ₁ x ₃ + 5.13 × 10⁻² x ₂ x ₃ −1.28 × 10⁻² x ₁² –3.18 × 10⁻² x ₂². −2.64 × 10⁻¹ x ₃² −3.28 × 10⁻³ x ₁ x ₂ x ₃ + 4.68 × 10⁻⁴ x ₁² x ₂. The produced response surface model proved to be significant (r ² > 0.99, P-value <0.0001, coefficient of variation <5%) to describe the explored space. Temperature was found to be the most significant factor of dominant effects on the mycelial growth rate, and other variables such as temperature², pH, pH², and (substrate concentration² × temperature) also showed significant effects on the model output. The maximum mycelial growth rate was predicted to be 2.80 mm d⁻¹ at 29.7 g lactose l⁻¹, 26.2°C, and pH 5. Our results proved a good potential of whey to serve as an alternative growth medium for cultivating P. linteus mycelia. This may provide another potential for managing this nutrient-rich waste in a cost-effective way.     

Optimization of some parameters influencingThermoascus aurantiacus growth: Effects of lignin-related compounds

Statistical designs were used to optimize some parameters affecting the growth rate of a Brazilian strain ofThermoascus aurantiacus. The mycelial growth rate was measured using the horizontal tube method. Temperature of incubation and initial pH were the major factors affecting the growth rate. They were optimal at 6.0 and 48°C, respectively. The maximum growth rate was obtained in solid Czapek modified medium containing 1.5% glucose and 38.4 mEq L^–1NaNO₃. Under these conditions, the growth rate ofT. aurantiacus was 5.16±0.10 mm h^–1. Lignin-related compounds such as tannins and extractive substances added at 0.1% (w/v) to the minimal Czapek medium increased growth rate 14% and 29%, respectively.     

Mycelial forms of <Emphasis Type="Italic">Pseudallescheria boydii</Emphasis> present ectophosphatase activities

Phosphatase activities were characterized in intact mycelial forms of Pseudallescheria boydii, which are able to hydrolyze the artificial substrate p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 41.41 ± 2.33 nmol p-NP per h per mg dry weight, linearly with increasing time and with increasing cell density. MgCl₂, MnCl₂ and ZnCl₂ were able to increase the (p-NPP) hydrolysis while CdCl₂ and CuCl₂ inhibited it. The (p-NPP) hydrolysis was enhanced by increasing pH values (2.5-8.5) over an approximately 5-fold range. High sensitivity to specific inhibitors of alkaline and acid phosphatases suggests the presence of both acid and alkaline phosphatase activities on P. boydii mycelia surface. Cytochemical localization of the acid and alkaline phosphatase showed electron-dense cerium phosphate deposits on the cell wall, as visualized by electron microscopy. The product of p-NPP hydrolysis, inorganic phosphate (Pi), and different inhibitors for phosphatase activities inhibited p-NPP hydrolysis in a dose-dependent manner, but only the inhibition promoted by sodium orthovanadate and ammonium molybdate is irreversible. Intact mycelial forms of P. boydii are also able to hydrolyze phosphoaminoacids with different specificity.     

Enhanced iturin A production by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and its effect on suppression of the plant pathogen <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The behavior of Streptomyces peucetius var. caesius N47 was studied in a glucose limited chemostat with a complex cultivation medium. The steady-state study yielded the characteristic constants μ _max over 0.10 h⁻¹, Y _XS 0.536 g g⁻¹, and m_S 0.54 mg g⁻¹ h⁻¹. The product of secondary metabolism, ɛ-rhodomycinone, was produced with characteristics Y _PX 12.99 mg g⁻¹ and m _P 1.20 mg g⁻¹ h⁻¹. Significant correlations were found for phosphate and glucose consumption with biomass and ɛ-rhodomycinone production. Metabolic flux analysis was conducted to estimate intracellular fluxes at different dilution rates. TCA, PPP, and shikimate pathway fluxes exhibited bigger values with production than with growth. Environmental perturbation experiments with temperature, airflow, and pH changes on a steady-state chemostat implied that an elevation of pH could be the most effective way to shift the cells from growing to producing, as the pH change induced the biggest transient increase to the calculated ɛ-rhodomycinone flux.     

Production of xylanase from a newly isolated <Emphasis Type="Italic">Penicillium</Emphasis> sp. ZH-30

A new strain of Penicillium sp. ZH-30 that produces xylanase was isolated from soil. According to the morphology and comparison of internal transcribed spacer (ITS) rDNA gene sequence, the strain Penicillium sp. ZH-30 was identified as a strain of Penicillium oxalicum. When xylan or wheat bran was used as substrate at 30°C for 3 days under submerged cultivation, xylanase production was 5.3 and 13.3 U ml⁻¹, respectively. The temperature and pH for optimum activity were 50°C and 5.0–6.0, respectively.     

Optimization of submerged culture conditions for exopolysaccharide production in <Emphasis Type="Italic">Sarcodon aspratus</Emphasis> (Berk) S.lto TG-3

The effect of medium components (carbon, nitrogen, and mineral sources) and environmental factors (initial pH and temperature) for mycelial growth and exopolysaccharide (EPS) production in Sarcodon aspratus(Berk) S.lto TG-3 was investigated. The optimal temperature (25°C) and initial pH (5.0) for the EPS production in shake flask cultures of S. aspratus were determined using the two-dimensional contour plot. The most suitable carbon, nitrogen, and mineral sources for EPS production were glucose, yeast extract, CaCl₂ and KH₂PO₄, respectively. Notably, the EPS production was significantly enhanced by supplementation of calcium ion. Subsequently, the optimum concentration of glucose (30gl^–1), yeast extract (15gl^–1), CaCl₂ (1.1gl^–1), and KH₂PO₄ (1.2gl^–1) were determined using the orthogonal matrix method. The effects of nutritional requirement on the mycelial growth of S.aspratuswere in regular sequence of glucose>KH₂PO₄>yeast extract>CaCl₂, and those on EPS production were in the order of glucose>yeast extract>CaCl₂>KH₂PO₄. Under the optimal culture conditions, the maximum EPS concentration in a 5-l stirred-tank reactor was 2.68gl^–1 after 4days of fermentation, which was 6-fold higher than that at a basal medium. The two-dimensional contour plot and orthogonal matrix method allowed us to find the relationship between environmental factors and nutritional requirement by determining optimal operating conditions for maximum EPS production in S.asparatus. The statistical experiments used in this work can be useful strategies for optimization of submerged culture processes for other mushrooms.     

香菇液体发酵高产多糖菌株筛选

通过评价香菇野生菌株发酵产多糖性能,筛选高产香菇多糖菌株.以采自长白山野生香菇通过组织分离获得的6株菌株和2株人工栽培菌株为出发菌株,对不同发酵培养时间菌丝体生物量、胞内多糖含量、胞外多糖含量等进行测试分析,结果表明,8株菌株随着培养时间的延长,菌丝体生物量均有不同程度的增加,但胞内多糖含量和胞外多糖得率变化趋势不同,...     

Bioconversion of <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine into 2-phenylethanol by <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> in grape must cultures

A 2³ factorial design was employed to find the best conditions of pH, l-phenylalanine concentration and temperature for the production of 2-phenylethanol by Kluyveromyces marxianus CBS 6556. The cultivation was carried out on grape must, which contains a great amount of nitrogen compounds. Central composite design (CCD) was used for the analysis of treatment combinations. Results showed a second-degree polynomial regression model with good agreement of experimental data, with R ² = 0.92015 (p < 0.05). The maximum production of 2-phenylethanol was found at pH 7.0, temperature of 37 °C, and a concentration of 3.0 g of l-phenylalanine L⁻¹. Further experiments in bioreactors showed that oxygen concentration is also important to 2-phenylethanol production, with best results obtained at oxygen mass transfer rates of 2.0 h⁻¹.     

Production of thermostable and neutral glucoamylase using immobilized <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis>

Thermomucor indicae-seudaticae was immobilized in alginate, κ-carrageenan, agarose, agar, polyacrylamide and loofah (Luffa cylindrica) sponge (as such or coated with alginate/starch/Emerson YpSs agar), and used for the production of glucoamylase in submerged fermentation. The mycelium developed from alginate-immobilized sporangiospores secreted higher glucoamylase titres (22.7 U ml⁻¹) than those immobilized in other gel matrices and the freely growing mycelial pellets (18.5 U ml⁻¹). Loofah network provided a good support for mycelial growth, but the enzyme production was lower than that attained with alginate beads. Glucoamylase production increased with inoculum density and the optimum levels were achieved when 40 calcium alginate beads (∼5 × 10⁶ immobilized spores) were used to inoculate 50 ml production medium. The alginate bead inoculum displayed high storage stability at 4°C and produced comparable enzyme titres up to 120 days. The glucoamylase production by hyphae emerged from the immobilized sporangiospores was almost stable over eight batches of repeated fermentation. Scanning electron micrographs of alginate beads, after batch fermentation, revealed extensive mycelial growth inside and around the beads.     

High level expression of a recombinant phospholipase C from <Emphasis Type="Italic">Bacillus cereus</Emphasis> in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Twenty-two Bacillus cereus strains were screened for phospholipase C (PLC, EC 3.1.4.3) activity using p-nitrophenyl phosphorylcholine as a substrate. Two strains (B. cereus SBUG 318 and SBUG 516) showed high activity at elevated temperatures (>70°C) at acidic pH (pH 3.5–6) and were selected for cloning and functional expression using Bacillus subtilis. The genes were amplified from B. cereus DNA using primers based on a known PLC sequence and cloned into the expression vector pMSE3 followed by transformation into B. subtilis WB800. On the amino acid level, one protein (PLC318) was identical to a PLC described from B. cereus, whereas PLC516 contained an amino acid substitution (E173D). PLC production using the recombinant strains was performed by an acetoin-controlled expression system. For PLC516, 13.7 U g⁻¹ wet cell weight was determined in the culture supernatant after 30 h cultivation time. Three purification steps resulted in pure PLC516 with a specific activity of 13,190 U mg⁻¹ protein.     

Xylanase production under solid-state fermentation and its characterization by an isolated strain of <Emphasis Type="Italic">Aspergillus foetidus</Emphasis> in India

Summary A locally isolated strain of Aspergillus foetidus MTCC 4898 was studied for xylanase (EC 3.2.1.8) production using lignocellulosic substrates under solid state fermentation. Corncobs were found as the best substrates for high yield of xylanases with poor cellulase production. The influence of various parameters such as temperature, pH, moistening agents, moisture level, nitrogen sources and pretreatment of substrates were evaluated with respect to xylanase yield, specific activity and cellulase production. Influence of nitrogen sources on protease secretion was also examined. Maximum xylanase production (3065 U/g) was obtained on untreated corncobs moistened with modified Mandels and Strenberg medium, pH 5.0 at 1 5 moisture levels at 30 °C in 4 days of cultivation. Submerged fermentation under the same conditions gave higher yield (3300 U/g) in 5 days of cultivation, but productivity was less. Ammonium sulphate fractionation yielded 3.56-fold purified xylanase with 76% recovery. Optimum pH and temperature for xylanase activity were found to be 5.3 and 50 °C respectively. Kinetic parameters like K_m and V_max were found to be 3.58 mg/ml and 570 μmol/mg/min. Activity of the enzyme was found to be enhanced by cystiene hydrochloride, CoCl₂, xylose and Tween 80, while significantly inhibited by Hg⁺⁺, Cu⁺⁺ and glucose. The enzyme was found to be stable at 40 °C. The half life at 50 °C was 57.53 min. However thermostability was enhanced by glycerol, trehalose and Ca⁺⁺. The crude enzyme was stable during lyophilization and could be stored at less than 0 °C.     

Cultivation ofLactobacillus crispatus KLB46 isolated from human vagina

Bacterial vaginosis can be treated by restoring the normal vaginal flora using lactobacilli.Lactobacillus crispatus KLB46 that was isolated from the human vagina has a strong antimicrobial activity and was grown in a batch and in a continuous fermentor. During batch cultivation, the maximum specific growth rate ofL. crispatus KLB 46 was 0.63 h⁻¹ and the highest viable cell count (1.9×10⁹ CFU/mL) was obtained at pH 5.5.L. crispatus KLB 46 did not grow well at either pH 3.5 or 7.5. During continuous cultivation, the highest viable cell count (1.53×10⁹ CFU/mL) was obtained at a dilution rate of 0.32 h⁻¹. However, the maximum productivity of viable cells was obtained at a dilution rate of 0.52 h⁻¹, and was 7.33×10¹¹ CFU L⁻¹ h⁻¹, that is approximately 5 times higher than that obtained from batch culture.     

Growth of Chlorella pyrenoidosa in wastewater from cassava ethanol fermentation

In a cycle tubular photobioreactor, Chlorella pyrenoidosa was cultured in undiluted wastewater from ethanol fermentation using cassava powder as raw material. The results showed that the optimum cultivation conditions were initial pH of 6.0, temperature at 27°C, continuous illumination at 3,000 lux, and cycle speed of 110 ml min⁻¹. Under these optimum conditions, after the logarithmic phase of batch cultivation with wastewater of pH 6.0, the reactor could be continuously operated with natural pH wastewater (3.8) as feed solution. By a dilution ratio of 0.17 day⁻¹, it could be operated stably for over 30 days in continuous cultivation. pH, removal rate of chemical oxygen demand, and biomass (cell dry weight) concentration ranged from 6.22 to 6.47, 72.21 to 76.32% and 3.55 to 3.73 g l⁻¹, respectively. After treatment, the wastewater could be used again in the process of ethanol fermentation.