Studies on cultivation and biological activities ofPleurotus nebrodensis inzenga

Environmental factors affecting mycelial growth and exo-polysaccharide production fromPleurotus nebrodensis Inzenga (PN) and biological activities of PN extractsin vitro were studied. The culture conditions for effective mycelial growth and exo-polysaccharide production were found to be 25 ‡C, 5% of inoculum size, and an initial pH from 6.5 to 7.0. When 5% of glucose was used, the maximum mycelial growth and exo-polysaccharide concentrations were 8.3 and 3.07 g/L, respectively. Among the various nitrogen sources, the mycelial growth and exo-polysaccharide production were very strong when polypeptone was used. In the case of the minerals sources, K₂HPO₄ and MgSO₄·7H₂O were found to best support for mycelial growth and exo-polysaccharide production. Under optimal conditions and methods, the maximum mycelial growth and exo-polysaccharide production were obtained after 10 days of culture, 12.84 and 4.85 g/L, respectively, in a jar fermentor. The effects of the PN extracts on the viability of three human cancer cell lines and antioxidant activity were investigatedin vitro. When cancer cells of the lung (A549), cervical region (HeLa) and colon (KM12C) were incubated at 6 mg/mL of the PN ethanol extracts, the viabilities of the HeLa and KM12C cells were slightly decreased. However, the growth of the A549 cells was remarkably inhibited when the PN ethanol extract was over 4 mg/mL. The antioxidant activity showed 46.2% at 40 μL, which was about 3.2 fold higher than that of the PN methanol extract.     

Enhancement of polysaccharide production by optimization of culture conditions in shake flask submerged cultivation of Grifola umbellata

Grifola umbellata (Fries) is a traditional Chinese medicinal mushroom. In this paper, the effects of cultural condition on the mycelial growth and exopolysaccharide (EPS) production were studied. Glucose was the best carbon source for mycelia growth and EPS production. Yeast extract was the best nitrogen source for mycelia growth, whereas skim milk as nitrogen source can remarkably improve EPS production. The optimal medium constituents for EPS production were as follows: glucose 3%, skim milk 0.2%, 0.1% KH₂PO₄, 0.1% MgSO₄, 7H₂O and 0.005% vitamin B1. The initial pH value of 5 was the most efficient to EPS production. The G. umbellata culture with skim milk as nitrogen source displayed a much higher specific EPS yield of 112.35 mg/g, accounting for a 4.36 times increase compared to that with combined nitrogen source medium.     

新型气升式反应器中浸没/非浸没周期对水母雪莲细胞培养的影响

应用新型周期浸没气升式反应器，进行了水母雪莲细胞的浸没悬浮和非浸没静置交替循环培养，研究了培养过程中的关键条件?浸没/非浸没周期对水母雪莲细胞生长、黄酮合成及细胞聚集体调控的影响，发现5 min/4 h为最佳的循环周期. 在此循环周期下，当通气量为40 L/h、接种量(干重DW)为2~3 g/L时，经20 d的培养，细胞聚集体分布最佳, 细胞干重达9.6 g/L，黄酮类化合物的含量及黄酮类化合物的产量分别可达35.2 mg/g (DW)及338 mg/L.     

Application of statistical experimental design for optimization of physiological factors and their influences on production of pullulan by <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> HP-2001 using an orthogonal array method

Physiological factors for the production of pullulan by A. pullulans HP-2001 were optimized using orthogonal array method and their influences were compared using Qualitek-4 software. The analysis of variance (ANOVA) indicated that the most important factor for cell growth was yeast extract, whereas that for production of pullulan was glucose. The optimal conditions for cell growth were found to be 100.0 g/L glucose, 10.0 g/L yeast extract, and initial pH of 6.0, whereas those for the production of pullulan were 100.0 g/L glucose, 2.5 g/L yeast extract, and initial pH of 5.5. Among four mineral salts in the medium, potassium phosphate (K₂HPO₄) was found to be the most important factor for cell growth as well as production of pullulan. Next important salt for cell growth was (NH₄)₂SO₄, whereas that for production of pullulan was NaCl. The optimal concentrations of K₂HPO₄, NaCl, MgSO₄·7H₂O, and (NH₄)₂SO₄ for cell growth were 7.5, 1.00, 0.1, and 1.20 g/L, respectively, whereas those for production of pullulan were 2.5, 0.25, 0.8, and 0.30 g/L. The expected cell growth and the production of pullulan by A. pullulans HP-2001 under these optimized conditions were 12.61 and 11.49 g/L, respectively.     

Pilot-scale production of bacterial cellulose by a spherical type bubble column bioreactor using saccharified food wastes

Bacterial cellulose (BC) was produced by Acetobacter xylinum KJ1 in a modified airlift-type bubble column bioreactor, which had a low shear stress and high oxygen transfer rate (k _L a). Saccharified food wastes (SFW) were used as the BC production medium due to its low cost. An aeration rate of 1.2 vvm (6 L/min) was tentatively determined as the optimal aeration condition in a 10 L spherical type bubble column bioreactor, by analysis of the oxygen transfer coefficient. When 0.4% agar was added, the BC production reached 5.8 g/L, compared with 5.0 g/L in the culture without the addition of agar. The BC productivity was improved by 10% in the addition of 0.4% agar into the SFW medium. Then, by conversion of a linear velocity of 0.93 cm/sec, from the relationship between the linear velocity and oxygen transfer rate, 1.0 vvm (30 L/min) was determined as an optimal aeration condition in a 50 L spherical type bubble column reactor. Using SFW medium, with the addition of 0.4% agar, and air supplied of 1.0 vvm, 5.6 g/L BC was produced in the 50 L spherical type bubble column bioreactor after 3 days of cultivation, which was similar to that produced in the 10 L bioreactor. In conclusion, the addition of agar, a viscous polysaccharide, into SFW medium is effective for the production of BC, and this scale-up method is very useful for the mass production in a 50 L spherical type bubble column bioreactor by decreasing the shear stress and increasing the k _L a.     

Optimization of medium components for D-ribose production by transketolase-deficient <Emphasis Type="Italic">Bacillus subtilis</Emphasis> NJT-1507

Statistical experimental designs were used to optimize the composition of culture media for the production of D-ribose by Bacillus subtilis. A fractional factorial design 2^(5-2) was used to determine medium components that significantly affected D-ribose production. The concentrations of glucose and (NH₄)₂SO₄ were the significant factors. Central composite design and response surface methodology were then used to estimate the quadratic response surface and determine the factor levels for maximum production of D-ribose. Finally, the optimal medium composition was obtained (g/L): glucose, 172.75; (NH₄)₂SO₄, 13.2; yeast powder, 4; corn steep liquor, 8 and MnSO₄, 0.5. This optimization strategy increased D-ribose production from 73.21 g/L to 88.57 g/L, an increase of 22% compared with the original conditions. The D-ribose production yield to glucose concentration was also enhanced from 0.37 g/g to 0.52 g/g. Confirmatory experiments were also performed to demonstrate the accuracy of the model. Under the optimal medium using ammonia to control pH in a 5 L fermenter, the D-ribose yield was increased to 95.28 g/L after 3 days of cultivation at 37 °C.     

Optimization of medium for phenylalanine ammonia lyase production in <Emphasis Type="Italic">E. coli</Emphasis> using response surface methodology

A culture medium for phenylalanine ammonia lyase (PAL) production in E. coli was developed following preliminary studies by means of response surface methodology (RSM). The medium components having significant effect on the production were first identified by using a fractional factorial design. Then, central composite design (CCD) was used to optimize the medium constituents and explain the combined effects of four medium constituents: glucose, yeast extract, (NH₄)₂HPO₄ and MgSO₄. A quadratic model was found to fit the PAL production. CCD revealed that the optimum values of the test variables for PAL production were glucose 28.2 g/L, yeast extract 5.01 g/L, (NH₄)₂HPO₄ 7.02 g/L and MgSO₄ 1.5 g/L. PAL production of 62.85 U/g, which was in agreement with the prediction, was observed in the verification experiment. In comparison to the production of basal medium, 1.8-fold increase was obtained.     

Suspension culture of Nicotiana tabacum cells in a rotary-drum bioreactor

Suspension cultures of Nicotiana tabacum (tobacco) were studied using a rotary-drum bioreactor and the effects of various operating factors on culture growth were investigated. Conditions of oxygen concentration, aeration and drum rotation rates required for optimum culture growth rate were determined. The results were compared with those obtained in an air-lift bioreactor with a draft tube. The culture growth rates were higher in the rotary-drum bioreactor than in the air-lift bioreactor under the same conditions of aeration. The rotary-drum bioreactor was superior to the air-lift bioreactor in respect of oxygen mass transfer and the level of shear damage to N. tabacum cells.     

Ammonium fumarate production by free or immobilised Rhizopus arrhizus in bench‐ and laboratory‐scale bioreactors

Ammonium fumarate production from glucose‐based media by Rhizopus arrhizus NRRL 1526 with mycelial growth controlled by phosphorus limitation exhibited mixed‐growth‐associated product formation kinetics, with growth‐associated production related to secondary mycelial growth only. The contribution of the primary mycelial growth phase was minimised by resorting to prolonged batch production using free mycelia under intermittent glucose feeding or repeated batch production using immobilised mycelia. The metabolic activity of free or immobilised mycelia was limited by fumarate accumulation or by oxygen diffusion phenomena, respectively. For batch cultures in a 15 dm³ stirred bioreactor the peripheral impeller speed (v_I) was increased from 1.88 to 3.3 m s^?1, and the fumarate yield coefficient on glucose increased from 0.25 ± 0.01 to 0.42 ± 0.02 g g^?1, while the malate yield coefficient on fumarate (Y_M/F) reduced from 0.46 ± 0.01 to 0.14 ± 0.01 g g^?1. With a net increase in the fumarate‐to‐malate ratio from 2 to 6.5, a v_I value of 3.3 m s^?1 gave the best fermentation performance and provided a basis for further scale‐up studies. © 2002 Society of Chemical Industry     

Growth promotive conditions for enhanced eritadenine production during submerged cultivation of Lentinus edodes

BACKGROUND: Mycelium of the medicinal mushroom shiitake, Lentinus edodes, is a potential source for production of the blood cholesterol reducing compound eritadenine. To increase the mycelial biomass and in turn the production of eritadenine, a potential growth promoting substance in the form of a water extract of distillers dried grains with solubles (DDGS) was added to the culture media. RESULTS: The hot water extract of DDGS was shown to considerably increase the growth of shiitake mycelia in bioreactor cultivations; the mycelial yield was 2–3 times higher than in the control, and the highest final biomass concentration obtained was 3.4 g L^?1. Further, by using shake flask cultures as inoculums the bioreactor cultivation time could be reduced by 1 week for some of the experiments. The highest final titer of eritadenine in the present study was 25.1 mg L^?1, which was about 2 times higher than in the control, and was also obtained when a water extract of DDGS was added to the culture medium. CONCLUSION: It was demonstrated that a water extract of DDGS promoted the growth of shiitake mycelia in bioreactor cultivations, along with enhanced eritadenine production. Copyright © 2012 Society of Chemical Industry     

Optimization of arachidonic acid production by Mortierella alpina Wuji-H4 isolate

A fungal isolate Wuji-H4 with a dense-lobe rosette growth pattern on malt extract agar was identified as Mortierella alpina Peyronel. It was capable of producing 504 mg/L of arachidonic acid (AA) in the screening medium. Its AA content accounted for 42.4% of the total fatty acids. The AA yield was raised to 1,817 mg/L by a step-by-step approach, which uncovered that the preferred carbon source, nitrogen source, and temperature for fungal growth and lipid production were soluble starch, urea, and 24°C, respectively. Productivity was further optimized by exploiting the interactions between the constituents of the medium by the response surface method. A partial factorial design, followed by steepest ascent analysis, was carried out to locate the general vicinity of the optimal level of each nutrient. The response surface of AA production in this optimal region was then approximated with a full quadratic equation obtained from a three-factor/five-level central composite rotatable design. Maximum AA yield was predicted to occur in a medium that contained 99.7 g/L of soluble starch, 12.6 g/L of yeast extract, and 3.0 g/L of KH₂PO₄. Upon verification, the average experimental yield of AA (3,885 mg/L) was not significantly different from the predicted AA yield (3,940 mg/L), indicating that the response surface method had succeeded in exploiting the AA production potential of this new fungal isolate.     

霍山石斛类原球茎在气升式反应器中补料培养合成多糖

在10 L气升式反应器中进行了霍山石斛类原球茎悬浮培养. 研究了接种量、通气量、碳、磷等营养成分对霍山石斛类原球茎生长和多糖合成的影响. 在接种量为100 g/L(鲜重)、通气量为0.5 L/min时,30 g/L的蔗糖、2.5 mmol/L的磷酸盐有利于类原球茎生长,而0.312 mmol/L的磷酸盐有利于多糖积累. 根据霍山石斛类原球茎生长和多糖积累的动力学特性,提出了两步培养方式,采用补料策略,确定了最佳补料时间. 结果表明,采用两步培养,生物量达46.7 g/L(干重),是一步培养的1.5倍,多糖产率达8.15 g/L,是一步培养的2.6倍.     

Optimal culture condition for the production of phenyalanine ammonia lyase from <Emphasis Type="Italic">E. coli</Emphasis>

The effects of culturing conditions on phenylalanine ammonia lyase production by a recombinant E. coli strain were investigated by using a controlled fed-batch fermentation system. In a 5 L fermentor, the optimal composition of the batch medium was 2% glucose, 1% yeast extract, 0.7% K₂HPO₄, 0.8% KH₂PO₄, 0.5% (NH₄)₂SO₄, 0.1% MgSO₄·7H₂O. The optimal feed glucose solution was 50%. Glucose concentration and pH of the culture broth were maintained at about 2.0 g/L and 7.0 during the fed-batch phase, respectively. Following 24-h cultivation, 0.2 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG) was added and temperature was shifted from 37°C to 42°C to induce pal gene expression. Under optimal conditions, a high productivity of 300 U/g could be achieved after 48 h culture, and a cell density of OD₆₀₀ about 82 was obtained at 52 h culture at 500 r/m stirrer speed and 1 vvm, respectively.     

氮源对转基因小球藻异养生长及兔防御素表达的影响

在250 ml摇瓶中研究了氮源对转兔防御素(NP-1)基因小球藻的异养生长和NP-1表达的影响,结果表明,转NP-1基因小球藻异养培养的最适氮源为硝酸钾和酵母粉,二者的最佳浓度分别为0.9和9 g/L,藻细胞密度达5.11 g/L,是不添加硝酸钾时细胞密度的1.55倍,而NP-1表达量基本不变. 5 L生物反应器分批培养结果表明,转NP-1基因小球藻在含有硝酸钾和酵母粉两种混合氮源的培养基中培养时,硝酸钾被快速消耗而有机氮源充足,藻细胞内的叶绿素和蛋白质含量下降,但NP-1表达量基本不变.     

Improvement of Staphylococcus xylosus lipase production through optimization of the culture conditions

The Staphylococcus xylosus strain produces without induction an original lipase named S. xylosus lipase (SXL). Since considerable interest has been given to microbial lipases for biotechnology applications like detergents, food, drugs and pharmaceutical products, improvement of their production is of great importance to reduce the final cost. This goal could be reached through the optimization of several physicochemical culture conditions. Indeed, an appropriate medium was formulated for SXL production. It was composed of 17 g/L pancreatic digest of casein, 2.5 g/L glucose, 6 g/L yeast extract, 0.75 g/L ammonium sulfate corresponding to a C/N ratio of 6, 1 g/L K₂HPO₄ and 1 g/L KH₂PO₄. In such a medium, SXL production reached 42 U/mL. Moreover, the usefulness of such a medium for large‐scale production of SXL was also evidenced in an automated fully controlled 2.6‐L fermenter. It was shown that aeration of the medium, which strongly affected the growth, regulated the lipase synthesis by the produced cells. It was found that when using a dissolved oxygen saturation of the medium of 50%, the SXL production reached 62 U/mL.     

运用试验设计方法提高胞外杂合b-葡聚糖酶在重组大肠杆菌中的表达

A genetically engineered Escherichia coli JM109 harboring pLF3 was used to produce a hybrid extracellular β-glucanase. Starting with enzyme production medium, glycerol and yeast extract combined with NaNO3 were screened to be the most suitable carbon and nitrogen source, respectively. Analysis of six components of the enzyme production medium by employing statistical optimization methods such as Plackett-Burman design and steepest ascent showed that yeast extract was the only significant variable and its best concentration for enzyme production was 12g·L^-1. After optimization of the medium, 297.71U·ml^-1 of β-glucanase activity in the medium and 352350U·g^-1 of β-glucanase selectivity could be obtained, which were 14 and 72 folds higher than those obtained from original medium, respectively. Even higher enzyme activities were achieved by batch cultivations in a conventional stirred bioreactor on the optimized medium.     

Production of polysaccharides by submerged mycelial culture of entomopathogenic fungus Cordyceps takaomontana and their apoptotic effects on human neuroblastoma cells

The effect of medium components and environmental factors on the production of mycelial biomass for the preparation of intracellular polysaccharides (IPS) and exopolysaccharides (EPS) by Cordyceps takaomontan was investigated in submerged cultures. The optimal culture condition was as follows (g/l): glucose 30, yeast extract 4, KH₂PO₄ 0.46, K₂HPO₄ 1, and MgSO₄ 0.5, and 28 °C and pH 8. When the fungus was cultivated under various agitation and aeration conditions in a 5-l stirred-tank fermenter, the maximum mycelial biomass (10.7 g/l) and EPS production (1.9 g/l) was obtained at 300 rpm and 2 vvm, respectively. The inhibitory effect of both IPS and EPS on the growth of SK-N-SH cells was studied by treating the cells with crude IPS and EPS at concentrations of 0.5, 1, and 2 mg/ml for 24, 48, 72 h, respectively. The maximum inhibitory effect on apoptosis of SK-N-SH cells (64.6%) was observed when IPS was treated at a concentration of 2 mg/ml, for 72 h. The apoptosis of SK-N-SH cells induced by IPS was evidenced by comet assay, where the number of the comet cells increased by the IPS treated. These findings suggest that IPS may therefore have therapeutic potential against neuroblastoma cells.     

Screening of <Emphasis Type="Italic">Candida utilis</Emphasis> and medium optimization for co-production of <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine and glutathione

An effective S-adenosylmethionine and glutathione enriching yeast mutant of Candida utilis CCTCC M 209298 was first screened from plates containing 0.5 g/L of DL-ethionine by complex mutagenesis with UV and γ-ray in this study. Medium components optimization for enhanced co-production of S-adenosylmethionine and glutathione by C. utilis CCTCC M 209298 was further carried out using response surface methodology. The significant factors influencing S-adenosylmethionine and glutathione co-production were selected by Plackett-Burman design as sucrose, KH₂PO₄ and L-methionine, and Box-Behnken design was applied for further optimization studies. Based on these approaches, the optimized concentrations on medium components for higher co-production of S-adenosylmethionine and glutathione were sucrose 35.4 g/L, (NH₄)₂SO₄ 10 g/L, KH₂PO₄ 12.3 g/L, MgSO₄·7H₂O, 0.05 g/L, CaCl₂ 0.05 g/L and L-methionine 4.6 g/L. The medium optimization by response surface methodology led to a total production of 589.3 mg/L on S-adenosylmethionine and glutathione, which was 2.4-fold increased compared with the medium without optimization.     

Mathematical model of growth and polyhydroxybutyrate production using microbial fermentation of Bacillus subtilis

Poly-3-hydroxybutyrate (PHB) has been extensively utilized as a biodegradable plastic. The value of substrate inhibition constant (K_I) was also established in shake flask cultures by varying the initial glucose concentration (20–160?g/L) in growth media. Excess carbon source adversely affected the growth of Bacillus subtilis cultures. The production kinetics of PHB was studied using batch fermentation strategy for B. subtilis culture. Batch cultivation was also performed in a 5-L stirred tank bioreactor to obtain a Biomass and PHB yield of 5.25 and 1.6?g/L, respectively. The kinetic data of biomass, PHB production, and substrate consumption was used to estimate the optimized values of the growth and product formation kinetic parameters. Optimal values of kinetic parameters (µ_m value of 0.325, K_S value of 10.53?g/L for glucose, Y value of 0.183?g/g of glucose, K_I value of 105?g/L, m value of 0.12?g/(g h), k₁ of 0.36?g/g, and k₂ value was 0.12?g/(g h)) and the initial values of biomass, substrate and PHB concentration (X?=?0.14?g/L, S?=?9.99?g/L, and P?=?0.13?g/L) were utilized to obtain a modified mathematical model for PHB production. Statistical validity of the mathematical model simulations were measured using F-test. The F-test showed that the statistical validity of the model was more than 95% when compared with experimentally obtained values. This model also predicted that the production of PHB using B. subtilis cultures is mainly growth associated.     

Optimization of Xylanase Production from Penicillium sp.WX-Z1 by a Two-Step Statistical Strategy: Plackett-Burman and Box-Behnken Experimental Design

The objective of the study was to optimize the nutrition sources in a culture medium for the production of xylanase from Penicillium sp.WX-Z1 using Plackett-Burman design and Box-Behnken design. The Plackett-Burman multifactorial design was first employed to screen the important nutrient sources in the medium for xylanase production by Penicillium sp.WX-Z1 and subsequent use of the response surface methodology (RSM) was further optimized for xylanase production by Box-Behnken design. The important nutrient sources in the culture medium, identified by the initial screening method of Placket-Burman, were wheat bran, yeast extract, NaNO(3), MgSO(4), and CaCl(2). The optimal amounts (in g/L) for maximum production of xylanase were: wheat bran, 32.8; yeast extract, 1.02; NaNO(3), 12.71; MgSO(4), 0.96; and CaCl(2), 1.04. Using this statistical experimental design, the xylanase production under optimal condition reached 46.50 U/mL and an increase in xylanase activity of 1.34-fold was obtained compared with the original medium for fermentation carried out in a 30-L bioreactor.