Simultaneous and comparative assessment of parent and mutant strain of Rhizobium meliloti for nutrient limitation and enhanced polyhydroxyalkanoate (PHA) production using optimization studies

Nutrient limitation conditions, optimization and comparison of polyhydroxyalkanoate (PHA) yields and biomass production by parent and mutant strains of Rhizobium meliloti were investigated. Complex interactions among concentrations of sucrose (5–55 g/l), urea (0.05–0.65 g/l) inoculum (10–250 ml/l) and K₂HPO₄ (0.5–2 g/l), were studied using central composite rotatable design (CCRD) experiments. Phosphate-limiting medium (0.33 g K₂HPO₄/l) in the presence of excess carbon (sucrose 42.5 g/l) results in more production of PHA (2.2 g/l) in the parent strain. In comparison, the mutant strain required moderate levels of sucrose (30 g/l), along with excess of phosphate (1 g/l) for high PHA content of cell biomass (80%) and PHA yield (3.3 g/l). Optimised PHA production (biomass 4.8 g/l and PHA 3.09 g/l) by the parent strain occurred at: sucrose 51.58 g/l, urea 0.65 g/l, K₂HPO₄ 0.48 g/l and inoculum 10 ml/l. In the mutant strain, higher yields of biomass (9.05 g/l) and PHA (5.66 g/l) were obtained in Optimised medium containing: sucrose 55 g/l, urea 0.65 g/l, K₂HPO₄ 1.0 g/l and inoculum 150.58 ml/l.     

高效杀蚊苏云金芽孢杆菌BRC-LLP29的发酵优化

苏云金芽孢杆菌BRC-LLP29为新型高效杀蚊菌株,应用快速有效的数学统计方法对其杀蚊毒力的发酵培养进行优化.通过单因素筛选确定最佳碳源为葡萄糖、麦芽糖、可溶性淀粉,氮源为typetone、大豆蛋白胨、干酪素;最佳金属离子为Mg²⁺、Al³⁺.采用二水平Placlkett-Burman设计对影响毒力的8因素进行显著性筛选,获得培养基成分中3个重要影响因子:葡萄糖、干酪素和Al₂（SO₄）₃;运用爬坡路径法对这3种因子进行试验,获得3种重要因子的最适浓度范围;通过响应面分析法得到3个重要因子的交互作用和最佳条件,确定BRC-LLP29菌株最佳毒力水平的发酵培养基为:葡萄糖19.8g/L、干酪素28.4g/L、Al₂（SO₄）₃1.2g/L、MgSO₄ 2g/L、K₂HPO₄ 3g/L、CaCO₃ 0.5g/L,优化后毒力水平达到致死率61.11%,与响应面数学模型的预测值只有5.91%的误差.发酵条件优化结果表明:发酵温度为31°,发酵初始pH为7.0,摇瓶装量为40mL/250mL三角瓶,每瓶的接种量为3.5%,发酵72h,对致倦库蚊最终致死率达到最高为83.33%.     

Optimization of culture conditions for production of cis-epoxysuccinic acid hydrolase using response surface methodology

Response surface methodology, which allows for rapid identification of important factors and optimization of them to enhance enzyme production, was employed here to optimize culture conditions for the production of cis-epoxysuccinic acid hydrolase from Bordetella sp. strain 1–3. In the first step, a Plackett–Burman design was used to evaluate the effects of nine variables (yeast extract, cis-epoxysuccinic acid, KH₂PO₄, K₂HPO₄ · 3H₂O, MgSO₄ · 7H₂O, trace minerals solution, culture volume, initial pH and incubation time) on the enzyme production. Yeast extract, cis-epoxysuccinic acid and KH₂PO₄ had significant influences on cis-epoxysuccinic acid hydrolase production and their concentrations were further optimized using central composite design and response surface analysis. A combination of adjusting the concentration of yeast extract to 7.8 g/l, cis-epoxysuccinic acid to 9.8 g/l, and KH₂PO₄ to 1.12 g/l would favor maximum cis-epoxysuccinic acid hydrolase production. An enhancement of cis-epoxysuccinic acid hydrolase production from 5.6 U/ml to 9.27 U/ml was gained after optimization.     

Optimization of cyclodextrin production from sago starch

Cyclodextrin (CD) is synthesized by bacterial cyclodextrin glycosyltransferase (CGTase) and is widely used in food, pharmaceutical, cosmetic, and agricultural industries. In this study, Bacillus circulans CGTase was partially purified by ammonium sulfate precipitation at 50-70% saturation. The optimum pH and temperature for CD production from sago starch were found to be in the ranges of 4.5-5.0 and 55-60 degrees C, respectively. beta-CD was the predominant product, constituting 65% of all CD products. The beta-CD produced using partially purified and crude CGTase were compared and found to have no significant difference in yield and productivity. The appropriate proportion of CGTase to sago starch for beta-CD production was determined by response surface methodology. The most appropriate enzyme:substrate ratio was 50 U g sago starch(-1) CGTase and 60 g l(-1) sago starch.     

Influence of varying sources of dietary electrolytes on the performance of broilers reared in a high temperature environment

Experiment using 297 broiler chicks, kept from 1 to 42 days under cyclic heat stress (minimum 29.3 °C and maximum 38 °C), was conducted to investigate the relative efficacies of three sodium (NaHCO₃, Na₂CO₃, Na₂SO₄), and potassium (KHCO₃, K₂CO₃, K₂SO₄) supplements at an identical dietary electrolyte balance (DEB) 250 mEq/kg and two chloride supplements (CaCl₂ and NH₄Cl) at DEB 50 mEq/kg. The desired levels of DEB were achieved by substitution of individual mineral supplement at the expense to builder's sand in the basal starter (DEB 185.6 mEq/kg) and finisher (DEB 174 mEq/kg) diet containing only salt as a source of Na and Cl. Each diet (one basal and eight experimental) was fed to three experimental units having 11 chicks each. The growth performances in K supplements, except KHCO₃, were poorer than those of Na supplements. The NaHCO₃, Na₂CO₃, Na₂SO₄ and NH₄Cl increased body weight gain, feed intake and improved feed to gain ratio than those of K₂CO₃ and K₂SO₄ after 42 days of age. These supplements also increased the water intake and resulted in lowered body temperature as well as reduced mortality. Very poor performance in CaCl₂ supplement was associated with disturbed calcium:phosphorus and very low blood pH (7.19). Better performance was noted with bicarbonate than that with carbonate and sulfate sources. The KHCO₃ accentuate the respiratory alkalosis by increasing blood HCO₃ contents (26.09 mmol/l) and gave poorer performance than NaHCO₃. Blood lymphocytes, hemoglobin, hematocrit and electrolyte balance in heat stressed broilers were better improved by NH₄Cl, NaHCO₃, Na₂CO₃, Na₂SO₄ and KHCO₃ than those of CaCl₂, K₂CO₃ and K₂SO₄ supplements. Present findings emphasized the importance of considering metabolisable anions supplemented in association with cations while balancing the dietary electrolyte equations. That is why DEB equation cannot be used to predict the relative benefits of different mineral supplements.     

Production of cyclodextrin by poly-lysine fused Bacillus macerans cyclodextrin glycosyltransferase immobilized on cation exchanger

Bacillus macerans cyclodextrin glycosyltransferase (CGTase) fused with 10 lysine residues at its C-terminus (CGTK10ase) was immobilized onto a cation exchanger by ionic interaction and used to produce -cyclodextrin (CD) from soluble starch. Poly-lysine fused immobilization increased the V_m of the immobilized CGTase by 40% without a change in K_m. The activation energies of thermal deactivation (E_a) were 41.4, 28.1, and 25.9 kcal mol⁻¹, respectively, for soluble wild-type (WT) CGTase, soluble CGTK10ase, and immobilized CGTK10ase, suggesting destabilization of CGTase by poly-lysine fusion and immobilization onto a cation exchanger. Maximum -CD productivity of 539.4 g l⁻¹ h⁻¹ was obtained with 2% soluble starch solution which was constantly fed at a flow rate of 4.0 ml min⁻¹ (D = 240 h⁻¹) in a continuous operation mode of a packed-bed reactor. The operational half-life of the packed-bed enzyme reactor was estimated 12 days at 25 °C and pH 6.0.     

Improvement in raw sago starch degrading enzyme production from Acremonium sp. endophytic fungus using carbon and nitrogen sources

Attempts were made to improve the growth of endophytic fungus Acremonium sp. and its raw sago starch degrading enzyme (RSSDE) production using different nitrogen and carbon sources at varying pH values and temperatures. It was observed that growth and enzyme activity levels were highest with peptone and sodium nitrate as the nitrogen sources and raw sago starch as the carbon source of which the optimum concentrations were 0.5 g/l, 3 g/l, and 20 g/l, respectively. Cell growth and RSSDE production reached their optimum at pH 5.0 and incubation temperature of 30 degrees C. Under these conditions, the enzyme production was significantly increased by 19- to 22-folds compared to the activity obtained in the original basal medium.     

Sago starch as a low-cost carbon source for exopolysaccharide production by Lactobacillus kefiranofaciens

Low-cost sago starch was used as a carbon source for production of the exopolysaccharide kefiran by Lactobacillus kefiranofaciens. A simultaneous saccharification and fermentation process of sago starch for kefiran production was evaluated. Factors affecting the process such as an initial pH, temperature, starch concentration, including a mixture of α-amylase and glucoamylase were determined. The highest kefiran concentration of 0.85 g/l was obtained at the initial pH of 5.5, temperature of 30 °C, starch concentration of 4% and mixed-enzymes with activity of 100 U/g-starch. The use of a mixture of α-amylase and glucoamylase could enhance the productivity compared to the use of α-amylase alone. The optimal ratio of α-amylase to glucoamylase of 60:40 gave the highest kefiran production rate of 11.83 mg/l/h. This study showed that sago starch could serve as a low-cost substrate for kefiran production.     

漏斗多孔菌液体菌种培养基及其栽培条件

为实现漏斗多孔菌资源化利用,设计单因素试验,以菌丝生物量、菌球密度和菌球直径为指标,获得漏斗多孔菌Polyporus arcularius液体菌种培养基配方为马铃薯（去皮）200g、玉米粉20.0g、蛋白胨5.0g、KH₂PO₄ 3.0g、K₂HPO₄ 1.0g,MgSO₄·7H₂O 1.5g,初始pH 5.0,并优化培养条件为装液量250mL/500mL,接种量2%,培养温度32℃,转速170r/min,种龄6d;依上述条件制备液体菌种,以固体菌种为对照,进行出菇试验,调查农艺性状,试验结果表明液体菌种接种后,菌袋满袋时间提前7d,子实体原基产生时间提前8d,头潮菇鲜重提高22.5%;配方试验结果表明,以细木屑为主料的配方产量最高,头潮菇鲜重为270.6g/袋。     

Effect of water content on the gelatinisation temperature of sago starch

Differential scanning calorimetry (DSC) has been used to study gelatinisation phenomena of sago starch. Two endothermic transitions were observed for starch heated in the presence of a limited amount of water (starch/water=37–50%w/w). These transitions appear to be due to co-operative effects of water-mediated melting of starch crystallites, remaining crystallites and/or amylopectin crystallites. At a water content of 50%, evidence of M₁ endotherm was observed and 85°C represents the effective T_m at the end of melting of native sago starch. The effect of starch concentration on the shape of these two endotherms was studied for sago starch. The experimental data were treated thermodynamically by applying equations describing phase transition of semi-crystalline polymers. The T⁰_m value obtained by extrapolation to v₁=0 was 390.6 K for sago.     

Protein-based complex medium design for recombinant serine alkaline protease production

This work reports on the design of a complex medium based on simple and complex carbon sources, i.e. glucose, sucrose, molasses, and defatted-soybean, and simple and complex nitrogen sources, i.e. (NH₄)₂HPO₄, casein, and defatted-soybean, for serine alkaline protease (SAP) production by recombinant Bacillus subtilis carrying pHV1431::subC gene. SAP activity was obtained as 3050 U cm⁻³ with the initial defatted-soybean concentration C_soybean^o=20 kg m⁻³ and initial glucose concentration C_G^o=8 kg m⁻³; whereas, addition of the inorganic nitrogen source (NH₄)₂HPO₄ decreased SAP production considerably. Further increase in SAP production (3850 U cm⁻³) was obtained when sucrose was replaced with glucose at C_sucrose^o=15 kg m⁻³ and C_soybean^o=20 kg m⁻³. Nevertheless, when molasses was replaced with sucrose, the maximum activity was obtained with molasses having 10 kg m⁻³ initial sucrose concentration and C_soybean^o=15 kg m⁻³as 2130 U cm⁻³; moreover, when casein was replaced with defatted-soybean SAP production decreased considerably (ca. 250 U cm⁻³). Thereafter, the effects of inorganic ionic compounds were investigated; and except phosphate, inorganic compounds supplied from defatted-soybean were found to be sufficient for the bioprocess. The highest SAP activity was obtained as 5350 U cm⁻³ in the medium that contained (kg m⁻³): C_soybean^o=20, C_sucrose^o=15, C_Na2HPO4^o=0.021, and C_NaH2PO4^o=2.82, that was 6.5-fold higher than that of the SAP produced in the defined medium. By using the designed complex medium, oxygen transfer characteristics of the bioprocess were investigated; and, Damköhler number that is the oxygen transfer limitation increases with the cultivation time until t=14 h; and, at t>20 h both mass transfer and biochemical reaction resistances were effective. Overall oxygen transfer coefficient varied between 0.010 and 0.044 s⁻¹; volumetric oxygen uptake rate varied between 0.001 and 0.006 mol m⁻³ s⁻¹; and specific oxygen uptake rate varied between 0.0001 and 0.0022 mol kg⁻¹ DW s⁻¹ throughout the bioprocess.     

Optimization of submerged culture conditions for mycelial polysaccharide production in Cordyceps pruinosa

Cordyceps pruinosa is an entomogenous fungus noteworthy for its various bioactivities. The influence of synthetic medium and cultural conditions on polysaccharides production was investigated in shake flask culture. In the present study, optimal medium and submerged culture conditions were investigated using an orthogonal layout. Media and cultural conditions including potato starch 2% (w/v), sucrose 2.5%, soybean 0.5%, beef extract 0.5%, yeast extract 0.1%, KCl 0.02%, K₂HPO₄ 0.1%, MgSO₄·7H₂O 0.05%, pH 7.0, inoculum size 5%, medium capacity 50 ml/250 ml flask, dispersant 15 beads, culture time 7 days were employed. In fermentation medium, sucrose, beef extract and yeast extract were replaced with molasses of sucrose, groundnut and Vitamin B complex, respectively. Under optimal culture conditions, the yield of polysaccharides production was 9.51 g l⁻¹ after 54 h of fermentation in a 25 l fermenter, which was approximately twice as high as that in shake flask cultures. In addition the entire period of fermentation was shorted to around 1/4 of flask culture time (9 days). Thus, it will meet closely the requirements of industrial fermentation scale of polysaccharides production in C. pruinosa.     

Use of response surface methodology to optimize culture medium for production of lipase with Candida sp. 99-125

Response surface methodology (RSM) was employed to optimize culture medium for production of lipase with Candida sp. 99-125. In the first step, a Plackett–Burmen design was used to evaluate the effects of different components in the culture medium. Soybean oil, soybean powder and K₂HPO₄ have significant influences on the lipase production. The concentrations of three factors were optimized subsequently using central composite designs and response surface analysis. The optimized condition allowed the production of lipase to be increased from 5000 to 6230 IU/ml in shake flask system. The lipase fermentation in 5 l fermenter reached 9600 IU/ml.     

Production of phosphatidylcholine containing conjugated linoleic acid mediated by phospholipase A2

Esterification of lysophosphatidylcholine (LPC) with conjugated linoleic acid (CLA) was carried out using porcine pancreatic phospholipase A₂ (PLA₂). PLA₂ only slightly synthesized phosphatidylcholine containing CLA (CLA-PC) at 2.6% by the addition of water. Addition of formamide in place of water markedly increased the yield of CLA-PC. In addition, synthesis of CLA-PC by PLA₂ was affected by the amount of substrate CLA and PLA₂ in the reaction system. Under optimal reaction conditions using 11 mg LPC, 18 mg CLA, 550 mg glycerol, 50 μL formamide, 3.3 × 10⁴ U PLA₂, and 0.3 μmol CaCl₂ at 37 °C for 6 h, the reaction yield of CLA-PC reached 65 mol%. Furthermore, addition of protein such as albumin and casein suppressed the decrease of CLA-PC yield after 6 h. PLA₂ exhibited the highest activity for the 10t,12c-CLA isomer among four CLA isomers (9c,11t-CLA, 9c,11c-CLA, 9t,11t-CLA and 10t,12c-CLA), whereas that for 9c,11c-CLA was the lowest. These results showed that the present esterification system for LPC and CLA by PLA₂ is effective for producing CLA-PC.     

Immobilization on Eupergit C of cyclodextrin glucosyltransferase (CGTase) and properties of the immobilized biocatalyst

The extreme thermophilic cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. was covalently attached to Eupergit C. Different immobilization parameters (incubation time, ionic strength, pH, ratio enzyme/support, etc.) were optimized. The maximum yield of bound protein was around 80% (8.1 mg/g support), although the recovery of β-cyclodextrin cyclization activity was not higher than 11%. The catalytic efficiency was lower than 15%. Results were compared with previous studies on covalent immobilization of CGTase.

The enzymatic properties of immobilized CGTase were investigated and compared with those of the soluble enzyme. Soluble and immobilized CGTases showed similar optimum temperature (80–85 °C) and pH (5.5) values, but the pH profile of the immobilized CGTase was broader at higher pH values. The thermoinactivation of the CGTase coupled to Eupergit C was slower than the observed with the native enzyme. The half-life of the immobilized enzyme at 95 °C was five times higher than that of the soluble enzyme. The immobilized CGTase maintained 40% of its initial activity after 10 cycles of 24 h each. After immobilization, the selectivity of CGTase (determined by the ratio CDs/oligosaccharides) was notably shifted towards oligosaccharide production.     

古尼虫草胞内多糖高产培养基优化研究

以古尼虫草Cordyceps gunnii胞内多糖产量为目标,利用单因素法筛选古尼虫草产胞内多糖的最适碳源、氮源和无机盐,运用正交试验筛选最佳培养基组合,用最佳培养基研究古尼虫草产胞内多糖的发酵动力学。结果表明：古尼虫草产胞内多糖最佳培养基为葡萄糖35g/L、蛋白胨15g/L、硫酸锌1g/L、KH₂PO₄ 1g/L、K₂HPO₄ 0.5g/L,用最佳培养基获得胞内多糖（5.169±0.274）g/L,产量是优化前的1.81倍;动力学研究表明,144h是古尼虫草胞内多糖最佳培养时间,此时产量最高为（6.794±0.221）g/L,是目前报道古尼虫草胞内多糖的最高产量。     

K88大肠杆菌菌毛蛋白发酵工艺条件优化研究

分别采用LB培养基、牛肉膏蛋白胨培养基、强化营养培养基、玉米浆培养基对大肠杆菌K88进行发酵培养,选出最适于大肠杆菌K88生长的玉米浆培养基;采用正交实验对玉米浆培养基的C／N、K2HPO4／KH2PO4、Mg^2＋的配比进行优化,筛选最适于大肠杆菌K88生长的营养配比;研究生长曲线、接种量以及菌体和菌毛生产量的相关性,根据实验结果优化发酵培养条件,确定菌种的最佳发酵工艺,以收获最多的K88菌毛蛋白。研究表明,K88大肠杆菌在玉米浆培养基C／N、K2HP04／KH2PO4的配比分别为5／11、1／1,Mg^2＋为0．1g/mL,pH值为7．2,转速为200r／min,接种量为4．5％的条件下发酵26个小时,菌体和菌毛生产量均达到高峰,同时得出菌毛蛋白产生量和菌体量成正相关。     

Itaconic acid production using sago starch hydrolysate by Aspergillus terreus TN484-M1

Sago starch was hydrolyzed using either chemical agents, or enzymes at various pH and concentrations. Hydrolysis using 5000 AUN/ml (0.5%, w/v) glucoamylase exhibited the highest itaconic acid yield up to 0.36 g/g sago starch, whereas hydrolysis using nitric acid at pH 2.0 yielded 0.35 g/g sago starch. The medium was optimized and the composition was (g/l) 140 sago starch, 1.8 corn steep liquor, 1.2 MgSO(4).7H(2)O and 2.9 NH(4)NO(3). When the optimal conditions of hydrolysis and medium composition were applied to itaconic acid production in a 3-l jar fermentor, the itaconic acid production was 48.2 g/l with a yield of 0.34 g/g sago starch. This was filtered from the cultured broth and 37.1g of itaconic acid was recovered with a purity of 97.2%. This result showed that sago starch could be converted to a value-added product with only a simple pretreatment.     

Purification and characterization of cyclodextrin β-glucanotransferase from novel alkalophilic bacilli

The discovery of novel bacterial cyclodextrin glucanotransferase (CGTase) enzyme could provide advantages in terms of its production and relative activity. In this study, eight bacterial strains isolated from soils of a biodiversity-rich vegetation in Egypt based on their hydrolyzing activity of starch, were screened for CGTase activity, where the most active strain was identified as Bacillus lehensis. Optimization process revealed that the using of rice starch (25 %) and a mixture of peptone/yeast extract (1 %) at pH 10.5 and 37 °C for 24 h improved the bacterial growth and enzyme activity. The bacterial CGTase was successively purified by acetone precipitation, gel filtration chromatography in a Sephadex G-100 column and ion exchange chromatography in a DEAE-cellulose column. The specific activity of the CGTase was increased approximately 274-fold, from 0.21 U/mg protein in crude broth to 57.7 U/mg protein after applying the DEAE-cellulose column chromatography. SDS-PAGE showed that the purified CGTase was homogeneous with a molecular weight of 74.1 kDa. Characterization of the enzyme exhibited optimum pH and temperature of 7 and 60 °C, respectively. CGTase relative activity was strongly inhibited by Mg²⁺, Zn²⁺, Al³⁺ and K⁺, while it was slightly enhanced by 5 and 9 % with Cu²⁺ and Fe²⁺ metal ions, respectively.     

Hydroxyapatite for use as an animal cell culture substratum obtained by an alternate soaking process

Hydroxyapatite (HAp), the major inorganic component of hard tissues in vivo, was formed in/on a PVA gel. The HAp formation ratio depended on the reaction cycle number, but was independent of the alternate soaking period per cycle. The Ca/P molar ratio of HAp formed at 10 reaction cycles was very close to the theoretical value of HAp, 1.67. CHO-K1 cell adhesion, proliferation and maximum cell density on HAp plates were better on plates formed at two or five reaction cycles using 200 mM CaCl₂ and 120 mM Na₂HPO₄ solutions than on plates formed under other conditions. Furthermore, the adhesion ratio of CHO-K1 cells on HAp plates formed at 10 reaction cycles was about 60% of those at two or five reaction cycles.