Microbial production of extra-cellular phytase using polystyrene as inert solid support

Aspergillus ficuum TUB F-1165 and Rhizopus oligosporus TUB F-1166 produced extra-cellular phytase during solid-state fermentation (SSF) using polystyrene as inert support. Maximal enzyme production (10.07 U/g dry substrate (U/gds) for A. ficuum and 4.52 U/gds for R. oligosporus) was observed when SSF was carried out with substrate pH 6.0 and moisture 58.3%, incubation temperature 30 degrees C, inoculum size of 1.3 x 10(7) spores/5 g substrate, for 72 h for A. ficuum and with substrate pH 7.0 and moisture 58.3%, incubation temperature 30 degrees C, inoculum size of 1 x 10(6) spores/5 g substrate for 96 h for R. oligosporus. Results indicated scope for production of phytase using polystyrene as inert support.     

Influence of cultivating conditions on the alpha-galactosidase biosynthesis from a novel strain of Penicillium sp. in solid-state fermentation

AIMS: The work is intended to achieve optimum culture conditions of alpha-galactosidase production by a mutant strain Penicillium sp. in solid-state fermentation (SSF). METHODS AND RESULTS: Certain fermentation parameters involving incubation temperature, moisture content, initial pH value, inoculum and load size of medium, and incubation time were investigated separately. The optimal temperature and moisture level for alpha-galactosidase biosynthesis was found to be 30 degrees C and 50%, respectively. The range of pH 5.5-6.5 was favourable. About 40-50 g of medium in 250-ml flask and inoculum over 1.0 x 10(6) spores were suitable for enzyme production. Seventy-five hours of incubation was enough for maximum alpha-galactosidase production. Substrate as wheat bran supplemented with soyabean meal and beet pulp markedly improved the enzyme yield in trays. CONCLUSIONS: Under optimum culture conditions, the alpha-galactosidase activity from Penicillium sp. MAFIC-6 indicated 185.2 U g(-1) in tray of SSF. SIGNIFICANT AND IMPACT OF THE STUDY: The process on alpha-galactosidase production in laboratory scale may have a potentiality of scaling-up.     

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.     

Media and Fermentation Processes for the Rapid Production of High Concentrations of Stable Blastospores of the Bioinsecticidal Fungus Paecilomyces fumosoroseus

In shake flask and fermentor studies, various media components and culture inocula were tested to improve P. fumosoroseus spore production rates, yield and stability. To evaluate inoculum potential and inoculum scale-up for fermentor studies, conidia and liquid culture-produced spores of various strains of P. fumosoroseus were compared as inoculum. Inoculation of liquid cultures with blastospores at concentrations of at least 1×10⁶ spores mL^-1 resulted in the rapid production of high concentrations of blastospores (∼1×10⁹ spores mL^-1, 48 h fermentation time) for all strains tested. The rapid germination rate of blastospores (90% after 6 h incubation) compared to conidia (>90% after 16 h incubation) and the use of higher inoculum rates reduced the fermentation time from 96 to 48 h for maximal spore yields. A comparison of various complex nitrogen sources showed that liquid media supplemented with acid hydrolyzed casein or yeast extract supported the production of high concentrations of blastospores that were significantly more desiccation-tolerant (79-82% survival after drying) when compared to blastospores produced in media supplemented with other nitrogen sources (12-50% survival after drying). For rapid spore production, requirements for trace metals and vitamin supplementation were dependent on the type of hydrolyzed casein used in the medium. Fermentor studies with two strains of P. fumosoroseus showed that high concentrations (1.3-1.8×10⁹ spores mL^-1) of desiccation-tolerant blastospores could be produced in 48-h fermentations. These studies have demonstrated that the infective spores of various strains of the fungal bioinsecticide Paecilomyces fumosoroseus can be rapidly produced using deep-tank, liquid culture fermentation techniques.     

An optimized method for Aspergillus niger spore production on natural carrier substrates

Aspergillus niger spores have wide ranging applications in the fermentation industry as well as in wastewater treatment. We present an optimized method for production of A. niger spores on natural substrates such as rice, split pea, and millet. The specific productivity (number of spores per gram of dry substrate) was 31-fold greater and volumetric productivity was 750-fold greater compared to agar slopes. The important process variables were incubation temperature, moisture content, and inoculum quantity. We find that the optimal condition for total spore count is different from the viable spore count for millet. The optimum lies in a narrow region defined by the process parameters. Of the three substrates tested split pea gave the highest specific spore productivity of 3.1 x 10(10) spores per gram of dry substrate. This is the first report of systematic study on the effect of process parameters on spore viability. The method of A. niger spore production on natural substrate appears advantageous as compared to the currently practiced method in terms of scale-up, cost, and ease of operation.     

Xylanase production in solid state fermentation by Aspergillus niger mutant using statistical experimental designs

The initial moisture content, cultivation time, inoculum size and concentration of basal medium were optimized in solid state fermentation (SSF) for the production of xylanase by an Aspergillus niger mutant using statistical experimental designs. The cultivation time and concentration of basal medium were the most important factors affecting xylanase activity. An inoculum size of 5 x 10(5) spores/g, initial moisture content of 65%, cultivation time of 5 days and 10 times concentration of basal medium containing 50 times concentration of corn steep liquor were optimum for xylanase production in SSF. Under the optimized conditions, the activity and productivity of xylanase obtained after 5 days of fermentation were 5,071 IU/g of rice straw and 14,790 IU l(-1) h(-1), respectively. The xylanase activity predicted by a polynomial model was 5,484 IU/g of rice straw.     

Screening,selection and development of Bacillus subtilis apr-IBL04 for hyper production of macromolecule alkaline protease

Bacillus subtilis microbe is commonly found in soil and produces proteases on nitrogen and carbon-containing sources and increases the fertility rate by degrading nitrogenous organic materials. The present study was aimed to develop hyper producing mutant strain of B. subtilis for the production of proteases, to improve the process variables by the response surface methodology (RSM) under central composite design (CCD) and the production of protease by the particular mutant strain in a liquid state fermentation media. The mutation of the strain was carried out using ethidium bromide. Pure B. subtilis strain was collected and screened for hyper-production of protease. The production of protease by mutant B. subtilis strain was optimized by varying temperature, inoculum size, pH and incubation time under liquid state fermentation. The CCD model were found to be reliable with r² of 0.999. The maximum enzyme activity of B. subtilis IBL-04 mutant with 3 mL/100 mL inoculum size, 72 h fermentation time, pH 8, and 45 °C temperature was developed with enzyme activity 631.09 U/mL, indicates 1–7-fold increase in enzyme activity than the parent strain having 82.32 U/mL activity. These characteristics render its potential use in industries for pharmaceutical and dairy formulation.     

Improved polygalacturonase production from Bacillus sp. MG-cp-2 under submerged (SmF) and solid state (SSF) fermentation

AIMS: To investigate the effect of amino acids, vitamins and surfactants on polygalacturonase production from Bacillus sp. MG-cp-2 under submerged (SmF) and solid state fermentation (SSF). METHODS AND RESULTS: Bacillus sp. MG-cp-2 was isolated from the outer covering of the seeds of Celastrus paniculatus. Out of the various surfactants, amino acids and vitamins, Tween-60, DL-serine and folic acid maximally enhanced polygalacturonase production by 2.7-fold (240.0 U x ml(-1)), 4.0-fold (360.0 U x ml(-1)) and 3.8-fold (342.0 U x ml(-1)) respectively, under submerged fermentation (SmF). In solid state fermentation (SSF), Tween-80, pyridoxine and DL-ornithine monohydrochloride induced highest enzyme production up to 1.73-fold (6956.5 U x g(-1)), 5.3-fold (21224.4 U x g(-1)) and 5.74-fold (23076.9 U x g(-1)), respectively. CONCLUSION: Amino acids and their analogues, vitamins and surfactants effect significantly polygalacturonase production by Bacillus sp. MG-cp-2 when grown under submerged (SmF) and solid state fermentation (SSF) conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides useful information about regulation of polygalacturonase biosynthesis in Bacillus sp. MG-cp-2, which appears to be an interplay of nutritional and physical factors. Alkaline polygalacturonase from Bacillus sp. MG-cp-2 will be extremely useful in the treatment of alkaline pectic waste waters from vegetable and fruit processing industries and in degumming of bast fibres.     

Effect of spore concentration on germination and autotropism inTrichoderma hamatum

Summary The effect of spore concentration on spore germination and germtube growth ofTrichoderma hamatum on water agar and on potato dextrose agar (PDA) was studied. Increasing inoculum size up to 10⁹ spores/plate on PDA and up to 10⁷ spores/plate on water agar shortened the incubation period required for germtubes emergence and increased germination rate. However, on water agar germination was inhibited at 10⁸ and was completely arrested at 10⁹ spores/plate. Inhibition in germination of 10⁷ spores/plate was observed on water agar when the plates were preincubated with 10⁹ spores/plate for 5 h or more. Addition of glucose and ammonium nitrate to the water agar medium allowed only 25% of the spores to germinate at 10⁹ as compared to 78% at 10⁷ spores/plate after 8 h of incubation. Addition of polysaccharides to the C+N supplemented medium, significantly increased germination up to 84% as compared to 100% on PDA, after 8 h of incubation. Germlings ofTrichoderma hamatum phialospores exhibited positive autotropism and anastamosis on both media. The phenomenon was positively related to inoculum size, being most pronounced at 10⁷ spores/plate.     

低温纤维素酶菌株CNY086发酵条件优化(Ⅱ)

本文是在前文(Ⅰ)确定菌株CNY086低温纤维素酶发酵培养基的基础上,通过单因素和正交实验研究温度、装液量、接种量及种龄对菌株CNY086低温纤维素酶发酵影响.最适发酵温度、装液量、接种量和种龄分别为15℃、250 mL/500 mL、15%、10 h.上述条件下CNY086菌株5 L发酵酶活力为104.36 U/mL.     

Optimization of amylase production by Aspergillus niger in solid-state fermentation using sugarcane bagasse as solid support material

Synthesis of amylase by Aspergillus niger strain UO-01 under solid-state fermentation with sugarcane bagasse was optimized by using response surface methodology and empirical modelling. The process parameters tested were particle size of sugarcane bagasse, incubation temperature and pH, moisture level of solid support material and the concentrations of inoculum, total sugars, nitrogen and phosphorous. The optimum conditions for high amylase production (457.82 EU/g of dry support) were particle size of bagasse in the range of 6–8 mm, incubation temperature and pH: 30.2°C and 6.0, moisture content of bagasse: 75.3%, inoculum concentration: 1 × 10⁷ spores/g of dry support and concentrations of starch, yeast extract and KH₂PO₄: 70.5, 11.59 and 9.83 mg/g of dry support, respectively. After optimization, enzyme production was assayed at the optimized conditions. The results obtained corroborate the effectiveness and reliability of the empirical models obtained.     

Solid state fermentation for the production of α-amylase from Penicillium chrysogenum using mixed agricultural by-products as substrate

Production of α-amylase from local isolate, Penicillium chrysogenum, under solid-state fermentation (SSF) was carried out in this study. Different agricultural by-products, such as wheat bran (WB), sunflower oil meal (SOM), and sugar beet oil cake (SBOC), were used as individual substrate for the enzyme production. WB showed the highest enzyme activity (750 U/gds). Combination of WB, SOM, and SBOC (1:3:1 w/w/w) resulted in a higher enzyme yield (845 U/gds) in comparison with the use of the individual substrate. This combination was used as mixed solid substrate for the production of α-amylase from P. chrysogenum by SSF. Fermentation conditions were optimized. Maximum enzyme yield (891 U/gds) was obtained when SSF was carried out using WB + SOM + SBOC (1:3:1 w/w/w), having initial moisture of 75%, inoculum level of 20%, incubation period of 7 days at 30°C. Galactose (1% w/w), urea and peptone (1% w/w), as additives, caused increase in the enzyme activity.     

Production of pectolytic enzymes by Aspergillus niger: effect of inoculum size and potassium hexacyanoferrate II-trihydrate

Summary The effect of inoculum size and potassium hexacyanoferrate II-trihydrate, K₄[Fe(CN)₆]·3 H₂O (KHCF), on pectinase synthesis by Aspergillus niger in submerged conditions were studied. Experiments were performed in shake flasks and in a 10-1 stirred bioreactor. Spore concentrations in the range 10²–10⁸ spores/1 of substrate were tested. Enzyme activity measured by the Apple Juice Depectinizing Assay (AJDA) showed the highest values using the smallest inoculum. Higher spore concentrations led to a 25% or even up to a 50% reduction of activity. Polygalacturonase (PG) activity decreased similarly to AJDA activity with higher inoculum concentration. Pectinlyase (PL) showed the opposite relationship, while pectin esterase (PE) did not show any correlation with inoculum concentration. Experiments in the fermentor using a reduced inoculum of 10² spores/1 showed that the whole process was prolonged in comparison to 10⁸ spores/1 inoculum. A pronounced effect of KHCF on fungal morphology as well as on enzymatic activity was observed. With increased concentration the morphology gradually changed from loose pellets to smaller compact ones. The enzymatic activity was markedly improved. In the bioreactor the amount of biomass was reduced from about g/l to 8 g/l. The activities were improved in comparison to fermentations without KHCF as follows: AJDA from 68 to 112 units (U)/ml, viscosity reduction from 83% to 90%, PG from 0.8 to 3.3 U/ml, PE from 32 to 49 U/ml and PL from 0.05 to 0.12 U/ml. The fermentation time was reduced from 96 to 68 h. Offprint requests to: J. Friedrich     

Chitinase Production in Solid-State Fermentation by Enterobacter sp. NRG4 Using Statistical Experimental Design

The optimization of nutrient levels for chitinase production by Enterobacter sp. NRG4 in solid-state fermentation conditions (SSF) was carried out using response surface methodology (RSM) based on central composite design (CCD). The design was employed by selecting wheat bran-to-flake chitin ratio, moisture level, inoculum size, and incubation time as model factors. The results of first-order factorial design experiments showed that all four independent variables have significant effects on chitinase production. The optimum concentrations for chitinase production were wheat bran-to-flake chitin ratio, 1; moisture level, 80%; inoculum size, 2.6 mL; and incubation time, 168 h. Using this statistical optimization method, chitinase production was found to increase from 616 U · g⁻¹ dry weight of solid substrate to 1475 U · g⁻¹ dry weight of solid substrate.     

The influence of culture conditions on mycelial structure and cellulase production by Trichoderma reesei Rut C-30

The morphology of Trichoderma reesei Rut C-30, during submerged cultivations in shake flask, was examined. The influence of the size inoculum and the composition of the fermentation medium on the morphology and cellulase production was studied. Different inoculum sizes were studied but the significative change in fungus morphology was observed for spores concentration between 10(5) and 10(7) spores/ml (i.e. 10(2) and 10(4) spores/ml in pre-culture medium). In the medium without Tween 80, at low inoculum size, the majority of the pellets were large and well individualized, in contrast, at higher inoculation densities small flocs were obtained, with higher production of soluble protein and higher filter paper activity. It was found that the average pellet size seems to be inversely proportional to the inoculum size. Medium composition, namely Tween 80, also influences the morphology of T. reesei Rut C-30 and enzyme production. The presence of Tween 80 in fermentation medium inhibited the pellet formation of this strain.     

Statistical optimization for tannase production from <Emphasis Type="Italic">Aspergillus niger</Emphasis> under submerged fermentation

Statistically based experimental design was employed for the optimization of fermentation conditions for maximum production of enzyme tannase from Aspergillus niger. Central composite rotatable design (CCRD) falling under response surface methodology (RSM) was used. Based on the results of ‘one-at-a-time’ approach in submerged fermentation, the most influencing factors for tannase production from A. niger were concentrations of tannic acid and sodium nitrate, agitation rate and incubation period. Hence, to achieve the maximum yield of tannase, interaction of these factors was studied at optimum production pH of 5.0 by RSM. The optimum values of parameters obtained through RSM were 5% tannic acid, 0.8% sodium nitrate, 5.0 pH, 5 × 10⁷ spores/50mL inoculum density, 150 rpm agitation and incubation period of 48 h which resulted in production of 19.7 UmL⁻¹ of the enzyme. This activity was almost double as compared to the amount obtained by ‘one-at-a-time’ approach (9.8 UmL⁻¹).     

Optimization of some parameters of solid state fermentation of wheat bran for protease production by a local strain of Rhizopus oryzae

Some parameters of the production of an alkaline protease by Rhizopus oryzae in the solid state fermentation of wheat bran were optimized. Using the optimum parameters of an inoculum age of 7 days, an incubation time of 9 days, an amount of CZAPEK ‐DOX (liquid medium) of 6 ml/g bran and an incubation temperature of 33°C, an activity of 50 U/g bran was achieved. The initial pH of the CZAPEK ‐DOX medium had little effect. Re‐incubation of mouldy bran with only fresh CZAPEK ‐DOX yielded 3 times total activity compared to single‐cycle fermentation. As for the effect of the amount CZAPEK ‐DOX medium, the water constituent contributed more to activity increase than did the salt component. The ARRHENIUS activation energies were 23 and 7.9 kcal/mole below and above the optimum of 33°C, respectively. In all the studies, along with protease production, variation of protein content and specific activity were also observed. Attempts were made to explain the effects and also gauge their implications for large‐scale production.     

Effects of inoculum concentration,temperature, and carbon sources on tannase production during solid state fermentation of cashew apple bagasse

In this study, the optimization of tannase production by solid state fermentation was investigated using cashew apple bagasse (CAB), an inexpensive residue produced by the cashew apple agroindustry, as a substrate. To accomplish this, CAB was enriched with 2.5% (w/w) tannic acid and 2.5% (w/w) ammonium sulphate and then moistened with water (60 mL/100 g of dry CAB). The influence of inoculum concentration (10⁴ to 10⁷ spores/g), temperature (20, 25, 30, and 35°C) and several additional carbon sources (glucose, starch, sucrose, maltose, analytical grade glycerol, and glycerol produced during biodiesel production) on enzyme production by Aspergillus oryzae was then evaluated. Supplementation with maltose and glycerol inhibited tannase synthesis, which resulted in lower enzyme activity. Starch and sucrose supplementation increased enzyme production, but decreased the enzyme productivity. The maximum tannase activity (4.63 units/g of dry substrate) was obtained at 30°C, using 10⁷ spores/g and 1.0% (w/v) sucrose as an additional carbon source.     

Enhanced production of raw starch degrading enzyme using agro-industrial waste mixtures by thermotolerant Rhizopus microsporus for raw cassava chip saccharification in ethanol production

In the present study, solid-state fermentation for the production of raw starch degrading enzyme was investigated by thermotolerant Rhizopus microsporus TISTR 3531 using a combination of agro-industrial wastes as substrates. The obtained crude enzyme was applied for hydrolysis of raw cassava starch and chips at low temperature and subjected to nonsterile ethanol production using raw cassava chips. The agro-industrial waste ratio was optimized using a simplex axial mixture design. The results showed that the substrate mixture consisting of rice bran:corncob:cassava bagasse at 8?g:10?g:2?g yielded the highest enzyme production of 201.6?U/g dry solid. The optimized condition for solid-state fermentation was found as 65% initial moisture content, 35°C, initial pH of 6.0, and 5?×?10⁶ spores/mL inoculum, which gave the highest enzyme activity of 389.5?U/g dry solid. The enzyme showed high efficiency on saccharification of raw cassava starch and chips with synergistic activities of commercial α-amylase at 50°C, which promotes low-temperature bioethanol production. A high ethanol concentration of 102.2?g/L with 78% fermentation efficiency was achieved from modified simultaneous saccharification and fermentation using cofermentation of the enzymatic hydrolysate of 300?g raw cassava chips/L with cane molasses.     

Bacillus megaterium spore germination is influenced by inoculum size

AIMS: The effect of spore density on the germination (time-to-germination, percent germination) of Bacillus megaterium spores on tryptic soy agar was determined using direct microscopic observation. METHODS AND RESULTS: Inoculum size varied from approximately 10(3) to 10(8) cfu ml(-1) in a medium where pH=7 and the sodium chloride concentration was 0.5% w/v. Inoculum size was measured by global inoculum size (the concentration of spores on a microscope slide) and local inoculum size (the number of spores observed in a given microscope field of observation). Both global and local inoculum sizes had a significant effect on time-to-germination (TTG), but only the global inoculum size influenced the percentage germination of the observed spores. CONCLUSIONS: These results show that higher concentrations of Bacillus megaterium spores encourage more rapid germination and more spores to germinate, indicating that low spore populations do not behave similarly to high spore populations. SIGNIFICANCE AND IMPACT OF THE STUDY: A likely explanation for the inoculum size-dependency of germination would be chemical signalling or quorum sensing between Bacillus spores.