Optimisation of extracellular tannase production from <Emphasis Type="Italic">Paecilomyces variotii</Emphasis>

The tannase production by Paecilomyces variotii was confirmed by high performance thin layer chromatography (HPTLC), and substrate specificity of the tannase was determined by zymogram analysis in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE). A clear band of activity observed after electrophoresis of culture filtrate in non-denaturing gels indicated the production of extracellular tannase by P. varoitii. HPTLC analysis revealed that gallic acid was the enzymatic degradation product of tannic acid during the fermentation process. The optimum condition for tannase production was at 72 h of incubation in shaking condition and addition of 1.5% tannic acid, 1% glucose and 0.2% sodium nitrate at temperature of 35°C and pH of 5–7. The production of extracellular tannase from Paecilomyces variotii was investigated under optimized conditions in solid-state fermentation (SSF), submerged fermentation (SmF) and liquid surface fermentation (LSF) processes. The maximum extracellular tannase production was obtained within 60 h of incubation under SSF followed by SmF and LSF.     

Fungal cultures of tar bush and creosote bush for production of two phenolic antioxidants (Pyrocatechol and Gallic acid)

‘Tar bush’ and ‘creosote bush’ were substrates of fungal cultivation for tannase production and gallic acid and pyrocatechol accumulation. Aspergillus niger GH1 grew similarly on both plant materials under solid state culture conditions, reaching maximal levels after 4 d. Fungal strain degraded all tannin content of creosote bush after 4 d of fermentation and >75 % of tar bush after 5 d. Higher level of tannase activity was detected in tar bush fermentation. Biotransformation of tannins to gallic acid was high (93 % in creosote bush and 89 % in tar bush). Pyrocatechol was released poorly. Kinetic parameters of tannin conversion were calculated.     

Statistical optimization of alkaline xylanase production from <Emphasis Type="Italic">Streptomyces violaceoruber</Emphasis> under submerged fermentation using response surface methodology

Response surface methodology employing central composite design (CCD) was used to optimize fermentation medium for the production of cellulase-free, alkaline xylanase from Streptomyces violaceoruber under submerged fermentation. The design was employed by selecting wheat bran, peptone, beef extract, incubation time and agitation as model factors. A second-order quadratic model and response surface method showed that the optimum conditions for xylanase production (wheat bran 3.5 % (w/v), peptone 0.8 % (w/v), beef extract 0.8 % (w/v), incubation time 36 h and agitation 250 rpm) results in 3.0-fold improvement in alkaline xylanase production (1500.0 IUml⁻¹) as compared to initial level (500.0 IUml⁻¹) after 36 h of fermentation, whereas its value predicted by the quadratic model was 1347 IUml⁻¹. Analysis of variance (ANOVA) showed a high coefficient of determination (R²) value of 0.9718, ensuring a satisfactory adjustment of the quadratic model with the experimental data. The economical and cellulase-free nature of xylanase would enhance its applicability in pulp and paper industry.     

Alginate production by <Emphasis Type="Italic">Pseudomonas mendocina</Emphasis> in a stirred draft fermenter

In this study alginate production by Pseudomonas mendocina in a laboratory-scale fermenter was investigated. In the experiments the effect of temperature (25–31°C) and agitation (500–620 rev min⁻¹) at a constant air flow of 10 v/v/h were evaluated in relation to the rate of glucose bioconversion to alginate using response surface methodology (RSM). The fermenter configuration was also adapted to a system with a screw mixer and draft tube, due to the change in rheological characteristics of the fermentation broth. The adjusted model indicates a temperature of 29.1°C and agitation of 553 rev min⁻¹ for optimum alginate synthesis. In this fermentation system a Y _p/s of 44.8% was achieved. The alginate synthesized by P. mendocina showed a partially acetylated pattern as previously reported for alginates obtained from other Pseudomonas spp and Azotobacter vinelandii.     

Production of tannase from wood-degrading fungus using as substrate plant residues: purification and characterization

In the present study Lenzites elegans, Schizophyllum commune, Ganoderma applanatum and Pycnoporus sanguineus (wood-degrading fungi) were assayed for their tannase producing potential in culture media containing plant residues or/and tannic acid as carbon source. Aspergillus niger was used as positive control for tannase production. We also carried out the isolation, purification and characterization of the enzyme from the fungi selected as the major productor. The highest fungal growth was observed in A. niger and L. elegans in the media containing tannic acid + glucose + plant residues (Fabiana densa). A. niger and L. elegans reached the highest extracellular tannase production in a medium containing tannic acid + F. densa and in a medium supplemented with glucose + tannic acid + F. densa. The produced enzyme by L. elegans was purified by DEAE-Sepharose. Km value was 5.5 mM and relative molecular mass was about 163,000. Tannase was stable at a pH range 3.0–6.0 and its optimum pH was 5.5. The enzyme showed an optimum temperature of 60°C and was stable between 40 and 60°C. This paper is the first communication of tannase production by wood-degrading fungi. Fermentation technology to produce tannase using plant residues and xylophagous fungi could be very important in order to take advantage of plant industrial waste.     

Pestalotiopsis mangiferae isolated from cocoa leaves and concomitant tannase and gallic acid production

The enzyme tannase is of great industrial and biotechnological importance for the hydrolysis of vegetable tannins, reducing their undesirable effects and generating products for a wide range of processes. Thus, the search for new microorganisms that permit more stable tannase production is of considerable importance. A strain of P. mangiferae isolated from cocoa leaves was selected and investigated for its capacity to produce tannase enzymes and gallic acid through submerged fermentation. The assessment of the variables affecting tannase production by P. mangiferae showed that tannic acid, ammonium nitrate and temperature were the most significant (8.4 U/mL). The variables were analyzed using Response Surface Methodology - RSM (Box-Behnken design), with the best conditions for tannase production being: 1.9% carbon source, 1% nitrogen source and temperature of 23 °C. Tannase activity doubled (16.9 U/mL) after the optimization process when compared to the initial fermentation. A pH of 7.0 was optimal for the tannase and it presented stability above 80% with pH between 4.0 and 7.0 after 2h of incubation. The optimal temperature was 30 °C and activity remained at above 80% at 40–60 °C after 1 h. Production of gallic acid was achieved with 1% tannic acid (0.9 mg/mL) and P. mangiferae had not used up the gallic acid produced by tannic acid hydrolysis after 144 h of fermentation. A 5% tannic acid concentration was the best for gallic acid production (1.6 mg/mL). These results demonstrate P. mangiferae’s potential for tannase and gallic acid production for biotechnological applications.     

Production of tannase by Aspergillus niger Aa-20 in submerged and solid-state fermentation: influence of glucose and tannic acid

Tannase production by Aspergillus niger Aa-20 was studied in submerged (SmF) and solid-state (SSF) fermentation systems with different tannic acid and glucose concentrations. Tannase activity and productivity were at least 2.5 times higher in SSF than in SmF. Addition of high tannic acid concentrations increased total tannase activity in SSF, while in SmF it was decreased. In SmF, total tannase activity increased from 0.57 to 1.03 IU/mL, when the initial glucose concentration increased from 6.25 to 25 g/L, but a strong catabolite repression of tannase synthesis was observed in SmF when an initial glucose concentration of 50 g/L was used. In SSF, maximal values of total tannase activity decreased from 7.79 to 2.51 IU when the initial glucose concentration was increased from 6.25 to 200 g/L. Kinetic results on tannase production indicate that low tannase activity titers in SmF could be associated to an enzyme degradation process which is not present in SSF. Tannase titers produced by A. niger Aa-20 are fermentation system-dependent, favoring SSF over SmF. Journal of Industrial Microbiology & Biotechnology (2001) 26, 296–302. Received 07 July 2000/ Accepted in revised form 15 February 2001     

Optimization of Lipase Production from <Emphasis Type="Italic">Aspergillus terreus</Emphasis> by Response Surface Methodology and Its Potential for Synthesis of Partial Glycerides Under Solvent Free Conditions

Aspergillus terreus produces lipase 7.01 IU/ml in 96 h after optimization by one variable at a time method. Using the significant factors i.e. corn oil (A), sodium nitrate (B), casein (C), agitation rate (D) and incubation period (E) RSM was carried out resulting in 19.65 IU/ml from the combination +1(A), −1(B), −1(C), +1(D) and 0(E). The interactions between sodium nitrate, casein and agitation with corn oil were most significant. Scale up of production from 250 ml shake flask to 30 l bioreactor resulted in increased productivity of 0.52 IU/ml/h as against 0.2 IU/ml/h obtained in shake flasks. This lipase could carryout solvent free synthesis of partial glycerides of oleic acid with 96% efficiency in 12 h.     

Effects of temperature, pH and additives on the activity of tannase produced by a co-culture of Rhizopus oryzae and Aspergillus foetidus

Summary Tannase was produced by modified solid-state fermentation (MSSF) of tannin rich substrates by a co-culture of the two filamentous fungi, Rhizopus oryzae and Aspergillus foetidus. The enzyme thus produced was then partially purified by solvent precipitation and DEAE-Sephadex column chromatography. A study on the effects of temperature and pH was made on the activity of tannase so purified. The optimum values of incubation time, reaction temperature and pH for tannase activity were 5 min, 40 °C and 5.0 respectively. The half-life period thermal stability and kinetic constants (K _m 0.21 mM, V _max 4.9×10⁻² M min^-1 at 40 °C) of this tannase were determined and the effects of different metal ions, surfactants, chelators, denaturants and inhibitors on the enzyme activity were also studied.     

Large-scale production of tannase using the yeast <Emphasis Type="Italic">Arxula adeninivorans</Emphasis>

Tannase (tannin acyl hydrolase, EC 3.1.1.20) hydrolyses the ester and depside bonds of gallotannins and gallic acid esters and is an important industrial enzyme. In the present study, transgenic Arxula adeninivorans strains were optimised for tannase production. Various plasmids carrying one or two expression modules for constitutive expression of tannase were constructed. Transformant strains that overexpress the ATAN1 gene from the strong A. adeninivorans TEF1 promoter produce levels of up to 1,642 U L⁻¹ when grown in glucose medium in shake flasks. The effect of fed-batch fermentation on tannase productivity was then investigated in detail. Under these conditions, a transgenic strain containing one ATAN1 expression module produced 51,900 U of tannase activity per litre after 142 h of fermentation at a dry cell weight of 162 g L⁻¹. The highest yield obtained from a transgenic strain with two ATAN1 expression modules was 31,300 U after 232 h at a dry cell weight of 104 g L⁻¹. Interestingly, the maximum achieved yield coefficients [Y(P/X)] for the two strains were essentially identical.     

Production of tannase by <Emphasis Type="Italic">Aspergillus niger</Emphasis> HA37 growing on tannic acid and Olive Mill Waste Waters

Production of tannase (tannin acyl hydrolase, EC 3.1.1.20) by Aspergillus nigerHA37 on a synthetic culture medium containing tannic acid at different concentrations has been studied. Maximal enzymatic activity increased according to the initial concentration of tannic acid; respectively 0.6, 0.9 and 1.5 enzyme activity units (EU) ml⁻¹ medium in the presence of 0.2%, 0.5% and 1% of tannic acid. Tannase production by A. niger HA37 on fourfold diluted olive mill waste waters (OMWW) as substrate, was between 0.37 and 0.65 EU ml⁻¹. Enzyme production on the diluted OMWW remained globally stable during more than 30 h. Growth of A. niger HA37 on OMWW was correlated with about 70% degradation of phenolic compounds present in the waste. This strain has therefore the capacity to degrade complex wastewaters which cause environmental damage to aquatic streams.     

Optimization of fermentation medium for triterpenoid production from <Emphasis Type="Italic">Antrodia camphorata</Emphasis> ATCC 200183 using artificial intelligence-based techniques

In this study, alteration in morphology of submergedly cultured Antrodia camphorata ATCC 200183 including arthroconidia, mycelia, external and internal structures of pellets was investigated. Two optimization models namely response surface methodology (RSM) and artificial neural network (ANN) were built to optimize the inoculum size and medium components for intracellular triterpenoid production from A. camphorata. Root mean squares error, R ², and standard error of prediction given by ANN model were 0.31%, 0.99%, and 0.63%, respectively, while RSM model gave 1.02%, 0.98%, and 2.08%, which indicated that fitness and prediction accuracy of ANN model was higher when compared to RSM model. Furthermore, using genetic algorithm (GA), the input space of ANN model was optimized, and maximum triterpenoid production of 62.84 mg l⁻¹ was obtained at the GA-optimized concentrations of arthroconidia (1.78 × 10⁵ ml⁻¹) and medium components (glucose, 25.25 g l⁻¹; peptone, 4.48 g l⁻¹; and soybean flour, 2.74 g l⁻¹). The triterpenoid production experimentally obtained using the ANN–GA designed medium was 64.79 ± 2.32 mg l⁻¹ which was in agreement with the predicted value. The same optimization process may be used to optimize many environmental and genetic factors such as temperature and agitation that can also affect the triterpenoid production from A. camphorata and to improve the production of bioactive metabolites from potent medicinal fungi by changing the fermentation parameters.     

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.     

Tannase production by Aspergillus fumigatus MA under solid-state fermentation

A tannase yielding fungal culture identified as Aspergillus fumigatus MA was isolated from the effluent collected from a local small scale tannery. The fungal culture produced high yields of extracellular tannase under solid-state fermentation (SSF) using different agro forest residues such as Amla leaves (Phyllanthus emblica), Ber leaves (Zyzyphus mauritiana), Jamun leaves (Syzygium cumini), Jamoa leaves (Syzygium sp.) and Keekar leaves (Acacia nilotica). Among different substrates used, Jamun leaves yielded maximal extra-cellular production of tannase. Various parameters were studied to optimize the extracellular yield of tannase under SSF. The maximum yield of 174.32 U g⁻¹ was obtained at 25°C after 96 h of incubation at pH 5.0. The tap water was used as a moistening agent. A substrate to tap water ratio of 1:1 was found to best for tannase production. Supplementation of the medium with ammonium sulfate as nitrogen source had enhanced tannase production whereas glucose had decreased the enzyme production. This is the first report on production of tannase by Aspergillus fumigatus MA, giving a much higher yield of enzyme under SSF with Jamun leaves as the substrate.     

Production of cephamycin C by <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis> NT4 using solid-state fermentation

Cephamycin C is an extracellular broad spectrum β-lactam antibiotic produced by Streptomyces clavuligerus, S. cattleya and Nocardia lactamdurans. In the present study, different substrates for solid-state fermentation were screened for maximum cephamycin C production by S. clavuligerus NT4. The fermentation parameters such as substrate concentration, moisture content, potassium dihydrogen phosphate, inoculum size and ammonium oxalate were optimized by response surface methodology (RSM). The optimized conditions yielded 21.68 ± 0.76 mg gds⁻¹ of cephamycin C as compared to 10.50 ± 1.04 mg gds⁻¹ before optimization. Effect of various amino acids on cephamycin C production was further studied by using RSM, which resulted in increased yield of 27.41 ± 0.65 mg gds⁻¹.     

Use of lysozyme for treatment of bacterial contamination in <Emphasis Type="Italic">in vitro</Emphasis> shoot cultures of fruit plants

Summary Use of lysozyme was tested for treatment of bacterial contaminations in in vitro shoot cultures of quince (Cydonia oblonga) ‘BA 29’ and the hybrid (Prunus persica × P. amygdalus) rootstock ‘GF 677’. Shoots which had been contaminated for about 1 yr by Bacillus circulans and Sphingomonas paucimobilis were treated in liquid culture, at pH 4.5, with 9–36 mg ml⁻¹ egg white lysozyme (EWL), and compared to each other and to untreated cultures for their growth, proliferation, and number of bacterial colony-forming units in the tissues. EWL did not negatively affect shoot growth up to 18 mg ml⁻¹; furthermore, the proliferation rates of EWL-treated shoots were sometimes higher than those of controls. In contrast, the concentration of 36 mg ml⁻¹ had some deleterious effect on the regrowth capacity and shoot production of ‘GF 677’ at the first subculture to solid medium after EWL, treatments. EWL had a simple bacteriostatic effect against Sphingomonas paucimobilis; in contrast, it was effective at 18 mg ml⁻¹ in eliminating Bacillus circulans in both ‘BA 29’ and ‘GF 677’ cultures, after optimal treatment duration.     

Efficient and simple plant regeneration via organogenesis from leaf segment cultures of persimmon (Diospyros kaki thunb.)

Summary An efficient and simple plant regeneration system via organogenesis from leaf segments of persimmon (Diospyros kaki Thunb.) cultivars ‘Fuyu’ and ‘Nishimurawase’ has been developed. The regeneration capacity was influenced by the culture vessels, gelling agents, plant growth regulators, and light conditions. Leaf explants taken from in vitro shoots were cultured on a modified Murashige and Skoog medium (MS1/2N), for 16 wk without transfer to fresh medium. Adventious shoots appeared after 4 and 8 wk in culture of ‘Nishimurawase’ and ‘Fuyu’ tissues, respectively. The culture of leaf explants in Erlenmeyer flasks with medium containing 4 g l⁻¹ agar enhanced shoot formation in comparison to media with increased agar concentrations. Optimal shoot regeneration was obtained with 5 mg l⁻¹ (22.8 μM) zeatin and 0.1 mg l⁻¹ (0.05 μM) indole-3-butyric acid (IBA) for ‘Nishimurawase’, and 10 mg l⁻¹ (45.6 μM) zeatin and 0.1 mg l⁻¹ (0.05 μM) IBA for ‘Fuyn’. Shoot regeneration frequencies in both cultivars were 100%, and shoot numbers per explant reached up to 9.2 for ‘Nishimurawase’ and 2.2 for ‘Fuyu’. Dark incubation during the first 4–5 wk was the most effective condition to successfully influence shoot regeneration in both cultivars. While dark incubation was essential for adventitious shoot formation by ‘Fuyu’, it was only slightly beneficial to ‘Nishimurawase’. More than 80% of the regenerated shoots rooted within 4 wk on hormone-free MS1/2N demium after having been dipped for 30 s in 250 mg l⁻¹ (1.1. mM) IBA solution.     

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.     

Optimization of polyhydroxybutyrate production by <Emphasis Type="Italic">Bacillus</Emphasis> sp. CFR 256 with corn steep liquor as a nitrogen source

Polyhydroxyalkanotes (PHAs), the eco-friendly biopolymers produced by many bacteria, are gaining importance in curtailing the environmental pollution by replacing the non-biodegradable plastics derived from petroleum. The present study was carried out to economize the polyhydroxybutyrate (PHB) production by optimizing the fermentation medium using corn steep liquor (CSL), a by-product of starch processing industry, as a cheap nitrogen source, by Bacillus sp. CFR 256. Response surface methodology (RSM) was used to optimize the fermentation medium using the variables such as corn steep liquor (5–25 g l⁻¹), Na₂HPO₄ 2H₂O (2.2–6.2 g l⁻¹), KH₂PO₄ (0.5–2.5 g l⁻¹), sucrose (5–55 g l⁻¹) and inoculum concentration (1–25 ml l⁻¹). Central composite rotatable design (CCRD) experiments were carried out to study the complex interactions of the variables. The optimum conditions for maximum PHB production were (g l⁻¹): CSL-25, Na₂HPO₄ 2H₂O-2.2, KH₂PO₄ − 0.5, sucrose − 55 and inoculum − 10 (ml l⁻¹). After 72 h of fermentation, the amount of PHA produced was 8.20 g l⁻¹ (51.20% of dry cell biomass). It is the first report on optimization of fermentation medium using CSL as a nitrogen source, for PHB production by Bacillus sp.     

Tannase production by Paecilomyces variotii

Surface response methodology was applied to the optimization of the laboratory scale production of tannase using a lineage of Paecilomyces variotii. A preliminary study was conducted to evaluate the effects of variables, including temperature ( degrees C), residue (%) (coffee husk:wheat bran), tannic acid (%) and salt solutions (%) on the production of tannase during 3, 5 and 7 days of fermentation. Among these variables, temperature, residues and tannic acid had significant effects on tannase production. The variables were optimized using surface response methodology. The best conditions for tannase production were: temperature (29-34 degrees C); tannic acid (8.5-14%); % residue (coffee husk:wheat bran 50:50) and incubation time of 5 days. The supplementation of external nitrogen and carbon sources at 0.4%, 0.8% and 1.2% concentration on tannase production were studied in the optimized medium. Three different nitrogen sources included yeast extract, ammonia nitrate and sodium nitrate along with carbon source (starch) were studied. Only ammonia nitrate showed a significant effect on tannase production. After the optimization process, the tannase activity increased 8.6-fold.