Production of Aspergillus xylanase by lignocellulosic waste fermentation and its application

Strains of Aspergillus terreus and A. niger, known to produce xylanase with undetectable amounts of cellulase, were studied for xylanase (EC 3.2.1.8) production on various lignocellulosic substrates using solid state fermentation. Of the lignocellulosic substrates used, wheat bran was the best for xylanase production. The effects of various parameters, such as moistening agent, level of initial moisture content, temperature of incubation, inoculum size and incubation time, on xylanase production were studied. The best medium for A. terreus was wheat bran moistened with 1:5 Mandels and Strenberg mineral solution containing 0.1% tryptone, at 35 degrees C, and at inoculum concentration 2x107-2x108 spores 5 g-1 substrate; for A. niger, the best medium was wheat bran moistened with 1:5 Mandels and Strenberg mineral solution containing 0.1% yeast extract, at 35 degrees C, and at an inoculum concentration of 2x107-2x108 spores 5 g-1 substrate. Under these conditions, A. terreus produced 68.9 IU ml-1 of xylanase, and A. niger, 74.5 IU ml-1, after 4 d of incubation. A crude culture filtrate of the two Aspergillus strains was used for the hydrolysis of various lignocellulosic materials. Xylanase preparations from the two strains selectively removed the hemicellulose fraction from all lignocellulosic materials tested.     

Individual and combined effects of physicochemical parameters on ellagitannin acyl hydrolase and ellagic acid production from ellagitannin by <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

The individual and interactive effects of physicochemical parameters on ellagitannin acyl hydrolase activity and ellagic acid production by Aspergillus oryzae using ellagitannins from acorn fringe of oak as substrate were studied. Ellagitannins concentration, incubation time were identified as important physicochemical parameters influencing the enzyme synthesis and the production accumulation, and the substrate concentration with initial pH was determined to has an interactive effect on the enzyme synthesis, while ellagitannins concentration and initial pH with incubation time were found to have interactions on the production accumulation. Furthermore, the parameters were optimized by quadratic programming. Under optimum condition, the fermentation run lasted 84 h with 4 g L⁻¹ ellagitannins concentration, yielding 17.7% ellagic acid. However, the maximum enzyme activity was obtained in 96 h with 5 g L⁻¹ substrate concentration. The research demonstrated a possible way to develop an efficient approach for recovery of higher added-value product (ellagic acid) from forestry byproduct (acorn fringe of oak).     

海洋侧孢短芽孢杆菌Brevibacillus laterosporus Lh-1 产抗菌肽R-1的培养条件优化

采用Plackett-Burman(PB)设计和响应面分析(RSM)方法, 对一株海洋侧孢短芽孢杆菌Lh-1产抗菌肽R-1的发酵条件进行了优化。研究中数据的统计和分析均使用MINITAB 15.0。在研究中, 首先使用了Plackett-Burman (PB)设计对影响Lh-1产抗菌肽的15个因素进行了筛选, 得到了影响产量的显著因素为：葡萄糖、蛋白胨和氯化钙。在此基础上采用响应面法对该3个显著因素的最佳水平范围进行研究, 得到的最佳浓度为 15.72 g/L 葡萄糖、6.01 g/L 蛋白胨和3.29     

Optimization of fermentation parameters for production of ethanol from kinnow waste and banana peels by simultaneous saccharification and fermentation

A study was taken up to evaluate the role of some fermentation parameters like inoculum concentration, temperature, incubation period and agitation time on ethanol production from kinnow waste and banana peels by simultaneous saccharification and fermentation using cellulase and co-culture of Saccharomyces cerevisiae G and Pachysolen tannophilus MTCC 1077. Steam pretreated kinnow waste and banana peels were used as substrate for ethanol production in the ratio 4:6 (kinnow waste: banana peels). Temperature of 30°C, inoculum size of S. cerevisiae G 6% and (v/v) Pachysolen tannophilus MTCC 1077 4% (v/v), incubation period of 48 h and agitation for the first 24 h were found to be best for ethanol production using the combination of two wastes. The pretreated steam exploded biomass after enzymatic saccharification containing 63 gL⁻¹ reducing sugars was fermented with both hexose and pentose fermenting yeast strains under optimized conditions resulting in ethanol production, yield and fermentation efficiency of 26.84 gL⁻¹, 0.426 gg ⁻¹ and 83.52 % respectively. This study could establish the effective utilization of kinnow waste and banana peels for bioethanol production using optimized fermentation parameters.     

Optimization of tannase production by Aspergillus niger in solid-state packed-bed bioreactor

Tannin acyl hydrolase, also known as tannase, is an enzyme with important applications in the food, feed, pharmaceutical, and chemical industries. However, despite a growing interest in the catalytic properties of tannase, its practical use is very limited owing to high production costs. Several studies have already demonstrated the advantages of solid-state fermentation (SSF) for the production of fungal tannase, yet the optimal conditions for enzyme production strongly depend on the microbial strain utilized. Therefore, the aim of this study was to improve the tannase production by a locally isolated A. niger strain in an SSF system. The SSF was carried out in packed-bed bioreactors using polyurethane foam as an inert support impregnated with defined culture media. The process parameters influencing the enzyme production were identified using a Plackett–Burman design, where the substrate concentration, initial pH, and incubation temperature were determined as the most significant. These parameters were then further optimized using a Box-Behnken design. The maximum tannase production was obtained with a high tannic acid concentration (50 g/l), relatively low incubation temperature (30°C), and unique low initial pH (4.0). The statistical strategy aided in increasing the enzyme activity nearly 1.97-fold, from 4,030 to 7,955 U/l. Consequently, these findings can lead to the development of a fermentation system that is able to produce large amounts of tannase in economical, compact, and scalable reactors.     

Optimization of submerged culture condition for the production of mycelial biomass and exopolysaccharides by Agrocybe cylindracea

The optimization of submerged culture conditions and nutritional requirements was studied for the production of exopolysaccharide (EPS) from Agrocybe cylindracea ASI-9002 using the statistically based experimental design in a shake flask culture. Both maximum mycelial biomass and EPS were observed at 25 degrees C. The optimal initial pH for the production of mycelial biomass and EPS were found to be pH 4.0 and pH 6.0, respectively. Subsequently, optimum concentration of each medium component was determined using the orthogonal matrix method. The optimal combination of the media constituents for mycelial growth was as follows: maltose 80 g/l, Martone A-1 6 g/l, MgSO4 x 7H2O 1.4 g/l, and CaCl2 1.1 g/l; for EPS production: maltose 60 g/l, Martone A-1 6 g/l, MgSO4 x 7H2O 0.9 g/l, and CaCl2 1.1 g/l. Under the optimal culture condition, the maximum EPS concentration achieved in a 5-l stirred-tank bioreactor indicated 3.0 g/l, which is about three times higher than that at the basal medium.     

Optimization of simultaneously enzymatic fructo- and inulo-oligosaccharide production using co-substrates of sucrose and inulin from Jerusalem artichoke

Prebiotic substances are extracted from various plant materials or enzymatic hydrolysis of different substrates. The production of fructo-oligosaccharide (FOS) and inulo-oligosaccharide (IOS) was performed by applying two substrates, sucrose and inulin; oligosaccharide yields were maximized using central composite design to evaluate the parameters influencing oligosaccharide production. Inulin from Jerusalem artichoke (5–15% w/v), sucrose (50–70% w/v), and inulinase from Aspergillus niger (2–7 U/g) were used as variable parameters for optimization. Based on our results, the application of sucrose and inulin as co-substrates for oligosaccharide production through inulinase hydrolysis and synthesis is viable in comparative to a method using a single substrate. Maximum yields (674.82?mg/g substrate) were obtained with 5.95% of inulin, 59.87% of sucrose, and 5.68 U/g of inulinase, with an incubation period of 9?hr. The use of sucrose and inulin as co-substrates in the reaction simultaneously produced FOS and IOS from sucrose and inulin. Total conversion yield was approximately 67%. Our results support the high value-added production of oligosaccharides using Jerusalem artichoke, which is generally used as a substrate in prebiotics and/or bioethanol production.     

Alpha-galactosidase production by Aspergillus oryzae in solid-state fermentation

Comparisons were made for alpha-galactosidase production using red gram plant waste (RGPW) with wheat bran (WB) and other locally available substrates using the fungus Aspergillus oryzae under solid-state fermentation (SSF). RGPW proved to be potential substrate for alpha-galactosidase production as it gave higher enzyme titers (3.4 U/g) compared to WB (2.7 U/g) and other substrates tested. Mixing WB with RGPW (1:1, w/w) resulted enhanced alpha-galactosidase yield. The volume of moistening agent in the ratio of 1:2 (w/v), pH 5.5 and 1 ml (1 x 10(6) spores) of inoculum volume and four days incubation were optimum for alpha-galactosidase production. Increase in substrate concentration (RGPW+WB) did not decrease enzyme yield in trays.     

Phytase production by a thermophilic mould Sporotrichum thermophile in solid state fermentation and its potential applications

Phytase production by a thermophilic mould Sporotrichum thermophile Apinis was investigated in solid state fermentation (SSF) using sesame oil cake as the substrate. Scanning electron microscopy of the fermented sesame oil cake revealed a dense growth of the mould with abundant conidia. Glucose, ammonium sulphate and incubation period were identified as the most significant factors by Plackett-Burman design. The optimum values of the critical components determined by central composite design of response surface methodology for the maximum phytase production were glucose 3%, ammonium sulphate 0.5% and incubation period 120 h. An overall 2.6-fold improvement in phytase production was achieved due to optimization. Highest enzyme production (348.76 U/g DMR) was attained at a substrate bed depth of 1.5 cm in enamel coated metallic trays. The enzyme liberated inorganic phosphate from wheat flour and soymilk with concomitant dephytinization and liberation of soluble inorganic phosphate.     

Optimization of the production of aroma compounds by Kluyveromyces marxianus in solid-state fermentation using factorial design and response surface methodology

Studies were carried out for the production of aroma compounds in solid-state fermentation using factorial design and response surface methodology (RSM) experiments. Five agro-industrial residues were evaluated as substrate for cultivating a strain of Kluyveromyces marxianus. The results proved the feasibility of using cassava bagasse and giant palm bran (Opuntia ficus indica) as substrates to produce fruity aroma compounds by the yeast culture. In order to test the influence of the process parameters on the culture to produce volatile compounds, two statistical experimental designs were performed. The parameters studied were initial substrate pH, addition of glucose, cultivation temperature, initial substrate moisture and inoculum size. Using a 2(5) factorial design, addition of glucose and initial pH of the substrate was found statistically significant for aroma compounds production on palm bran. Although this experimental design showed that addition of glucose did not have a significant role with cassava bagasse, 2(2) factorial design revealed that glucose addition was significant at higher concentrations. Head-space analysis of the culture by gas chromatography showed the production of nine and eleven compounds from palm bran and cassava bagasse, respectively, which included alcohols, esters and aldehyde. In both the cases, two compounds remained unidentified and ethyl acetate, ethanol and acetaldehyde were the major compounds produced. Esters produced were responsible for the fruity aroma in both the cases. With palm bran, ethanol was the compound produced in highest concentration, and with cassava bagasse (both supplemented with 10% glucose), ethyl acetate was produced at highest concentration, accumulating 418 and 1395μmoll(-1) head-spaceg(-1) substrate in 72h, respectively.     

Optimization of extracellular keratinase production by poultry farm isolate Scopulariopsis brevicaulis

A Scopulariopsis brevicaulis poultry farm isolate was chosen to study factors influencing keratinase production. The parameters were optimized by factorial design. The highest enzyme production by this fungus was obtained at pH 7.5, a temperature of 30 degrees C and a growth period of 5 weeks. The production of the enzyme was enhanced when the culture medium was supplemented with glucose (1%), sodium nitrate (2%), feather (1.5%) and CaCl(2) (1 mM). According to the responses from the experimental design, the effects of each variable were calculated, and the interactions between them were determined. The experimental values were found to be in accordance with the predicted values, the correlation coefficient is 0.9978.     

Application of statistically-based experimental designs for the optimization of eicosapentaenoic acid production by the diatom Nitzschia laevis

Statistically based experimental designs were applied to the optimization of medium components and environmental factors for eicosapentaenoic acid (EPA) production by the diatom Nitzschia laevis in heterotrophic conditions. First, the Plackett-Burman design was used to evaluate the effects of variables including medium components and environmental factors on cell growth and EPA production. Among these variables, NaCl, CaCl(2), PI metal solution, pH, and temperature were identified to have the significant effects (with confidence level > 90 %). Subsequently, the concentrations of NaCl, CaCl(2), PI metal solution as well as the values of pH and temperature were optimized using central composite design. The cell growth and EPA production were found to respectively correlate to NaCl, CaCl(2), pH, and temperature that could be represented by second-order polynomial models. The optimal values of the four parameters were determined by response surface and numerical analyses as 8 g/L NaCl, 0.10 g/L CaCl(2), pH 8.5 and 19.8 degrees C for cell dry weight (DW), and 14 g/L NaCl, 0.10 g/L CaCl(2), pH 8.5 and 18 degrees C for EPA production, respectively. The subsequent verification experiments confirmed the validity of the models. This optimization strategy led to a DW of 9 g/L, an EPA yield of 280 mg/L and an EPA productivity of 28 mg/L/d, respectively, which were considerably higher than those obtained in the previous studies.     

Preparation of stearoyl lactic acid ester catalyzed by lipases from Rhizomucor miehei and porcine pancreas optimization using response surface methodology

The esterification reaction between stearic acid and lactic acid using Rhizomucor miehei lipase and porcine pancreas lipase was optimized for maximum esterification using response surface methodology. The formation of the ester was found to depend on three parameters namely enzyme/substrate ratio, lactic acid (stearic acid) concentration and incubation period. The maximum esterification predicted by theoretical equations for both lipases matched well with the observed experimental values. In the case of R. miehei lipase, stearoyl lactic acid ester formation was found to increase with incubation period and lactic acid (stearic acid) concentrations with maximum esterification of 26.9% at an enzyme/substrate (E/S) ratio of 125 g mol⁻¹. In the case of porcine pancreas lipase, esterification showed a steady increase with increase in incubation period and lactic acid (stearic acid) concentration independent of the E/S ratios employed. In the case of PPL, a maximum esterification of 18.9% was observed at an E/S ratio of 25 g mol⁻¹ at a lactic acid (stearic acid) concentration of 0.09 M after an incubation period of 72 h. Received: 12 February 1999 / Received revision: 31 May 1999 / Accepted: 4 June 1999     

The effect of cultural conditions on the production of lipase by Acremonium strictum

Summary The optimum cultural conditions for the production of lipase byA. strictum under stationary condition are: period of incubation, 7 days; temperature, 30°C; xylose at a concentration of 2% (w/v) and 3.5% (w/v) soyabean meal as carbon and nitrogen sources respectively. Incorporation of 1% (v/v) of Tween 80 in culture medium enhanced enzyme production while the presence of fatty acids reduced both fungal growth and lipase production. The enzyme showed broad substrate specificity.     

Enhanced solid‐state citric acid bio‐production using apple pomace waste through surface response methodology

Aims: To evaluate the potential of apple pomace (AP) supplemented with rice husk for hyper citric acid production through solid‐state fermentation by Aspergillus niger NRRL‐567. Optimization of two key parameters, such as moisture content and inducer (ethanol and methanol) concentration was carried out by response surface methodology. Methods and Results: In this study, the effect of two crucial process parameters for solid‐state citric acid fermentation by A. niger using AP waste supplemented with rice husk were thoroughly investigated in Erlenmeyer flasks through response surface methodology. Moisture and methanol had significant positive effect on citric acid production by A. niger grown on AP (P < 0·05). Higher values of citric acid on AP by A. niger (342·41 g kg^?1 and 248·42 g kg^?1 dry substrate) were obtained with 75% (v/w) moisture along with two inducers [3% (v/w) methanol and 3% (v/w) ethanol] with fermentation efficiency of 93·90% and 66·42%, respectively depending upon the total carbon utilized after 144 h of incubation period. With the same optimized parameters, conventional tray fermentation was conducted. The citric acid concentration of 187·96 g kg^?1 dry substrate with 3% (v/w) ethanol and 303·34 g kg^?1 dry substrate with 3% (v/w) methanol were achieved representing fermentation efficiency of 50·80% and 82·89% in tray fermentation depending upon carbon utilization after 120 h of incubation period. Conclusions: Apple pomace proved to be the promising substrate for the hyper production of citric acid through solid‐state tray fermentation, which is an economical technique and does not require any sophisticated instrumentation. Significance and Impact of the Study: The study established that the utilization of agro‐industrial wastes have positive repercussions on the economy and will help to meet the increasing demands of citric acid and moreover will help to alleviate the environmental problems resulting from the disposal of agro‐industrial wastes.     

Substrate level modulation of the activity of phospholipase A2 in vitro by 12-O-tetradecanoylphorbol-13-acetate.

The action of porcine pancreatic phospholipase A2 towards fluorescent phospholipid analogs is either enhanced or suppressed by 4 beta-12-O-tetradecanoylphorbol-13- acetate (TPA), depending on the chemical structure of the substrate and the concentration of Ca2+. In the presence of nmolar Ca2+ concentrations increasing [TPA] enhanced by approx. 5-fold the rate of hydrolysis of the pyrene-labelled acidic alkyl-acyl phospholipid, 1-octacosanyl-2-[6- (pyrene-1-yl)] hexanoyl-sn-glycero-3-phosphatidylmethanol. Maximal effect was obtained at high TPA/substrate molar ratios approaching 1:2. In the presence of 4 mM CaCl2 maximal activation was reduced to approximately 1.5-fold. With the corresponding phosphatidylcholine derivative as a substrate increasing [TPA] reduced fatty acid release maximally by 90% both at low [Ca2+] as well as in the presence of 4 mM CaCl2. Essentially identical results were obtained using 4 alpha-TPA, a stereoisomer which does not activate protein kinase C.     

Optimization of novel and greener approach for the coproduction of uricase and alkaline protease in Bacillus licheniformis by Box–Behnken model

This study explores a novel concept of coproduction of uricase and alkaline protease by Bacillus licheniformis using single substrate in single step. Seven local bacterial strains were screened for uricase production, amongst which B. licheniformis is found to produce highest uricase along with alkaline protease. Optimization of various factors influencing maximum enzyme coproduction by B. licheniformis is performed. Maximum enzyme productivity of 0.386?U/mL uricase and 0.507?U/mL alkaline protease is obtained at 8?hr of incubation period, 1% (v/v) inoculum, and at 0.2% (w/v) uric acid when the organism is cultivated at 25°C, 180?rpm, in a media containing xylose as a carbon source, urea as a nitrogen source, and initial pH of 9.5. The statistical experimental design method of Box–Behnken was further applied to obtain optimal concentration of significant parameters such as pH (9.5), uric acid concentration (0.1%), and urea concentration (0.05%). The maximum uricase and alkaline protease production by B. licheniformis using Box–Behnken design was 0.616 and 0.582?U/mL, respectively, with 1.6- and 1.13-fold increase as compared to one factor at a time optimized media. This study will be useful to develop an economic, commercially viable, and scalable process for simultaneous production of uricase and protease enzymes.     

Insulin and (or) dexamethasone effects on leptin production and metabolic activities of ovine adipose tissue explants

The in vitro effects of insulin and/or dexamethasone (DEX) on leptin production were studied on adipose tissue (AT) from adult non-lactating, non-pregnant ewes. Perirenal AT explants were incubated for 2 or 4 days and leptin production was determined using a specific ovine RIA. The effects of these hormones were simultaneously measured on glucose and acetate utilisation and on lipogenic enzyme activities. A preliminary dose-response study showed a maximal leptin production by the addition in the incubation medium of 2 mIU x mL(-1) of insulin and 100 nM of DEX. By using these concentrations, insulin or DEX increased leptin production by ovine AT explants whatever the incubation duration and the effects of these two hormones were additive. Insulin also increased substrate utilisation as well as lipogenic enzyme activities while DEX decreased substrate utilisation and did not change the lipogenic enzyme activities. To conclude, leptin response to DEX is specific and largely independent of the overall metabolic or lipogenic activity.     

Cytochemical detection of catalase with 3,3'-diaminobenzidine. A quantitative reinvestigation of the optimal conditions

The influence of various parameters of fixation and incubation upon the oxidation of DAB by catalase have been analyzed. Crystalline beef liver catalase was fixed with different concentrations of glutaraldehyde and peroxidatic activity was determined spectrophotometrically using DAB as hydrogen donor. Although aldehyde fixation appeared to be important in elicitation of the peroxidatic activity of catalase, the final pigment production after 60 min incubation was optimal with the lowest concentration of glutaraldehyde (1%), after the shortest fixation period (30 min), and at the lowest temperature (5 degrees C) tested. Similarly cytochemical studies with rat kidney sections incubated for 10 min confirmed that the staining of peroxisomes in proximal tubules was strongest after the "mildest" fixation conditions. The pH and the temperature of incubation were closely interrelated, so that at room temperature (25 degrees C) the maximal pigment production was obtained at pH 10.5, but incubation at 45 degrees C gave the strongest staining at pH 8.5. The production of pigment increased with higher DAB concentrations which required larger amounts of H2O2 in the incubation medium. Cytochemical studies on renal peroxisomes were in agreement with these biochemical findings. The observations indicate that there are several options for the localization of catalase depending on the fixation and incubation conditions. Hence, these conditions should be selected according to the tissue and the purpose of the study. Examples for such selective applications are presented.     

Process optimization for the production of diosgenin with Trichoderma reesei

Based on the response surface methodology, an effective microbial system for diosgenin production from enzymatic pretreated Dioscorea zingiberensis tubers with Trichoderma reesei was studied. The fermentation medium was optimized with central composite design (3⁵) depended on Plackett–Burmann design which identified significant impacts of peptone, K₂HPO₄ and Tween 80 on diosgenin yield. The effects of different fermentation conditions on diosgenin production were also studied. Four parameters, i.e. incubation period, temperature, initial pH and substrate concentration were optimized using 4⁵ central composite design. The highest diosgenin yield of 90.57% was achieved with 2.67% (w/v) of peptone, 0.29% (w/v) of K₂HPO₄, 0.73% (w/v) of Tween 80 and 9.77% (w/v) of substrate, under the condition of pH 5.8, temperature 30 °C. The idealized incubation time was 6.5 days. After optimization, the product yield increased by 33.70% as compared to 67.74 ± 1.54% of diosgenin yield in not optimized condition. Scale-up fermentation was carried out in a 5.0 l bioreactor, maximum diosgenin yield of 90.17 ± 3.12% was obtained at an aeration of 0.80 vvm and an agitation rate of 300 rpm. The proposed microbial system is clean and effective for diosgenin production and thus more environmentally acceptable than the traditional acid hydrolysis.