Production of thermostable pullulanase by Clostridium thermosulfurogenes SV2 in solid-state fermentation: optimization of enzyme leaching conditions using response surface methodology

The optimization of parameters for the effective leaching of thermostable pullulanase from Clostridium thermosulfurogenes SV2-fermented bran was carried out using response surface methodology based on the central composite rotatable design. The design contains a total of 54 experimental trials with the first 32 organized in a fractional factorial design and experimental trials from 33-40 and 51-54 involving the replication of the central points. The design was employed by selecting solvent to wheat bran ratio (S/BB), process temperature, solvent pH, shaking (RPM) and contact time (h) as model factors. Among the five independent variables studied, the S/BB, solvent pH and shaking were found to be significant. S/BB ratio of 9.0, 200 RPM shaking and solvent pH 6.0 were identified as optimum for the leaching of thermostable pullulanase from the strain SV2-fermented bran.     

Optimization of enzymatic hydrolysis of mango kernel starch by response surface methodology

Amyloglucosidase (EC 3.2.1.3) from Aspergillus niger was employed for the saccharification of mango (Mangifera indica Linn) kernel starch. Response surface methodology based on a three-level three-factor Box-Behnken design was employed to optimize the important process variables such as substrate concentration (137.5-412.5 mg), enzyme concentration (4-12 mg) and temperature (35-55 °C). The sugar yield increased with both enzyme concentration and temperature, and decreased with substrate concentration. The response surface model indicated optimum conditions (substrate, 137.5 mg; enzyme, 12 mg; temperature, 55 °C) for obtaining 0.4851 mg sugar/mg substrate, which was also verified experimentally.     

Use of Box–Behnken design of experiments in the production of manganese peroxidase by Phanerochaete chrysosporium (MTCC 767) and decolorization of crystal violet

Biodegradation of crystal violet by Phanerochaete chrysosporium (MTCC 767) was experimentally determined and Box-Behnken design methods and analysis of variance have been applied to these experimental biodegradation studies. Response surface methodology was used in the identification of significant effects and interaction for the biodegradation studies. This methodology assumes and identifies the principal experimental variables and their interactions which have the greatest effect on biodegradation. The experimental values are in good agreement with predicted values, and the correlation coefficient was found to be 0.9997.     

Colour removal from bagasse-based pulp mill effluent using a white rot fungus

Colour removal of pulp plant effluent was studied using white rot fungus, Trametes (Coriolus) versicolor. The batch experiments were carried out using fungus in the form of mycelial pellets. In the present investigation, the effect of pH, concentrations of glucose (substrate), initial effluent colour and ammonium chloride (nutrient) on colour removal efficiency were studied. It was found that the maximum colour removal efficiency of 82.5% was obtained with an optimal glucose and ammonium chloride concentrations of 15 g/l and 0.5 g/l respectively at a pH of 4.5 without diluting the effluent.     

Solid-state production of polygalacturonase by Aspergillus sojae ATCC 20235

The effect of solid substrates, inoculum and incubation time were studied using response surface methodology (RSM) for the production of polygalacturonase enzyme and spores in solid-state fermentation using Aspergillus sojae ATCC 20235. Two-stage optimization procedure was applied using D-optimal and face-centered central composite design (CCD). Crushed maize was chosen as the solid substrate, for maximum polygalacturonase enzyme activity based on D-optimal design. Inoculum and incubation time were determined to have significant effect on enzyme activity and total spore (p<0.01) based on the results of CCD. A second order polynomial regression model was fitted and was found adequate for individual responses. All two models provided an adequate R(2) of 0.9963 (polygalacturonase) and 0.9806 (spores) (p<0.001). The individual optimum values of inoculum and incubation time for maximum production of the two responses were 2 x 10(7) total spores and 5-6 days. The predicted enzyme activity (30.55 U/g solid) and spore count (2.23 x 10(7)spore/ml) were very close to the actual values obtained experimentally (29.093 U/g solid and 2.31 x 10(7)spore/ml, respectively). The overall optimum region considering the two responses together, overlayed with the individual optima. Solid-state fermentation provided 48% more polygalacturonase activity compared to submerged fermentation under individually optimized conditions.     

Production of thermostable pullulanase by Clostridium thermosulfurogenes SV2 in solid-state fermentation: optimization of nutrients levels using response surface methodology

The optimization of nutrient levels for the production of thermostable pullulanase by Clostridium thermosulfurogenes SV2 in solid-state fermentation (SSF) was carried out using response surface methodology based on the central composite rotatable design. The design contains a total of 54 experimental trials with the first 32 organized in a fractional factorial design and experimental trials from 33-40 and 51-54 involving the replication of the central points. The design was employed by selecting potato starch, magnesium chloride, ferrous sulfate, corn steep liquor and pearl millet flour as model factors. Among the five independent variables studied, except magnesium chloride, all the nutrients were found significant. 16.5% potato starch, 2.5% corn steep, 0.015% ferrous sulfate and 14% pearl millet flour have been found optimal for the production of thermostable pullulanase. The strain SV2 produced 10% more pullulanase in the nutritionally optimized solid-state fermentation medium containing only four nutrients.     

Enhanced Biosynthesis of Sulfide Oxidase by Arthrobacter Species Using Response Surface Methodology

Response surface methodology was used to develop a fermentation medium for the enhanced biosynthesis of a novel sulfide oxidase by Arthrobacter species strain FR‐3. The interactive effect of the medium components – such as glucose as the carbon source, and tryptone and yeast extract as the nitrogen source – was evaluated by a 2³‐factorial central composite statistical design. Glucose and yeast extract were found to be the more influential medium constituents compared to tryptone since they had lower coefficients of linear effect, P‐values (< 0.02). The optimal fermentation medium components for the enhanced production of sulfide oxidase were recorded as glucose (8.98 g/L), tryptone (10.62 g/L) and yeast extract (7.3 g/L). Optimization of the medium constituents increased the experimental enzyme yield by 54 % compared to the unoptimized medium. This is the first report on the overproduction of sulfide oxidase by using response surface methodology.     

Optimization of temperature and initial pH and kinetic analysis of tartaric acid production by Gluconobacter suboxydans

Response surface methodology was applied for the simultaneous optimization of initial pH and temperature of fermentation for the production of tartaric acid by Gluconobacter suboxydans. A temperature of 32.8v°C and an initial pH of 6.05 was found to be most suitable. Kinetic analysis for the growth of Gluconobacter suboxydans and product formation was done and a logistic model was used to describe the growth of Gluconobacter suboxydans while the Leudeking-Piret model explained the product formation. Parameters of the model were evaluated and tartaric acid formation was found to be non-growth associated.     

Extraction of tylosin from its aqueous solutions by Rotating Film-pertraction

Removal of tylosin from its dilute aqueous solutions was studied applying the Rotating Film-pertraction technique. Non-toxic and practically water insoluble alcohols as n-octanol and n-decanol were used as intermediate, membrane liquid. A 0.001 mol/l solution of HCl was used as a stripping phase. Effects of temperature, feed phase pH-value and solute concentration on antibiotic distribution coefficients were obtained for both systems. It was found that the first two parameters have important effect, while the solute concentration in the studied range (0.02-0.1 g/l) did not modify these coefficients. Results obtained show that the first pertraction step - solute extraction from the feed - is a very fast process, while the second one - the alcohol stripping - is relatively slow, in particular when octanol was used as membrane phase. A mathematical model was proposed and used for evaluation of corresponding local mass transfer coefficients.     

The influence of substrate on the functional response of an avian granivore and its implications for farmland bird conservation

Few studies to date have considered the effect of substrate on the functional response of an organism feeding on prey of varying visibility. Intake rates of lone captive canaries, Serinus canarius L., were measured at varying seed densities on patches of either earth or short grass (<1 cm). Experiment 1, using pale seeds, found intake rates were significantly higher and search times significantly lower on earth than on grass. Two measures of crypticity (contrast in light reflectance as measured using a spectrophotometer and an experiment with humans) found pale seeds to be more visible on earth. The results from experiment 1 could be explained by this difference in crypsis. Experiment 2 used identical seeds to those in experiment 1 except they were dyed to match their backgrounds. The two measures of crypticity both found that black seeds were less visible on earth than green seeds were on grass. However, intake rates were still significantly higher on earth than grass. Seed colour preference, vegetation impeding movement, and differences in vigilance rates or seed accessibility could not explain this result. We discuss three other potentially explanatory mechanisms, the most likely of which was the greater surface area needed for scanning created by the structure of grass. Crucially, regardless of the mechanism(s) involved, many vegetated substrates share similar properties with grass (structural complexity and shiny surfaces which reflect light) and so the outcome of our findings are likely to extend to many natural situations. Conservationists wishing to encourage granivorous birds should consider enhancing food accessibility by providing uniform substrates, such as bare earth, for them to forage on. In addition, behaviour-based models should incorporate the effects of habitat into their equations of the functional response.     

Optimization of methane fermentation from effluent of bio-hydrogen fermentation process using response surface methodology

The individual effects and interactive effects of substrate concentration, ratio of inoculum to substrate, Ca(2+) concentration on the methane yield from the effluent of bio-hydrogen fermentation of food waste were investigated in this study. A central composite design (CCD) and response methodology (RSM) were employed in designing the experiments, in order to determine the optimum conditions for methane fermentation. The experiment results showed that the effects of substrate concentration, ratio of inoculum to substrate, Ca(2+) concentration were statistically significant at 5% level. The interactive effect of substrate concentration and ratio of inoculum to substrate was significant, however the interactive effect of substrate concentration and Ca(2+) concentration, ratio of inoculum to substrate and Ca(2+) concentration were found to be insignificant at 5% level. A maximum yield of 565.76 ml CH(4)/g VS(added) was estimated under the optimum conditions for substrate concentration 7.77 g of VS/l, inoculum to substrate ratio of 2.81 and calcium concentration of 380.82 mg/l. Verification experiment of the estimated optimum conditions confirmed that the RSM was useful for optimizing the methane yield from effluent of bio-hydrogen fermentation of food waste.     

Periodic operation of bioreactors for autocatalytic reactions with Michaelis-Menten kinetics

The performance of an isothermal tubular bioreactor carrying out autocatalytic reactions obeying Michaelis-Menten Kinetics is analyzed for improvement in the average yield of product B. Under steady-state condition, the reactor is shown to exhibit input multiplicities in the yield of B with the mean residence time. Simulation results show that a significant improvement in the average yield of B is obtained under feed substrate concentration cycling. The two values of mean residence time giving identical yield under conventional steady-state operation is shown to give distinctly different behaviour under periodic operation. The lower value of the residence time gives improved average yield of B. The performances of the reactor with power law kinetics and that with the Michaelis-Menten kinetics show distinct average yield under periodic operation even though steady-state operation gives identical yield.     

Bioindicator production with <Emphasis Type="Italic">Bacillus atrophaeus</Emphasis>’ thermal-resistant spores cultivated by solid-state fermentation

Bacillus atrophaeus’ spores are used in the preparation of bioindicators to monitor the dry heat, ethylene oxide, and plasma sterilization processes and in tests to assess sterilizing products. Earlier production methods involved culture in chemically defined medium to support sporulation with the disadvantage of requiring an extended period of time (14 days) besides high cost of substrates. The effect of cultivation conditions by solid-state fermentation (SSF) was investigated aiming at improving the cost–productivity relation. Initial SSF parameters such as the type of substrate were tested. Process optimization was carried out using factorial experimental designs and response surface methodology in which the influence of different variables—particle size, moisture content, incubation time, pH, inoculum size, calcium sources, and medium composition—was studied. The results have suggested that soybean molasses and sugarcane bagasse are potential substrate and support, respectively, contributing to a 5-day reduction in incubation time. Variables which presented significant effects and optimum values were mean particle size (1.0 mm), moisture content (93%), initial substrate pH (8.0), and water as a solution base. The high-yield spore production was about 3 logs higher than the control and no significant difference in dry heat resistance was observed.     

Studies on the production and application of cellulase from Trichoderma reesei QM-9414

High yielding mutant strain, Trichoderma reesei QM-9414, was employed for the cellulase enzyme production. Enzyme production conditions (pH, inoculum age and concentration, and organic supplements) were optimized. The ability of partially purified enzyme to hydrolyze various regionally abundant lignocellulosic raw materials was studied. Enzymatic hydrolysis conditions (temperature, pH, enzyme and substrate concentrations) were optimized. Temperature 50v°C, pH 4.5, enzyme concentration 40 FPU/g substrate and substrate concentration 2.5% were found to be optimum for the maximum yields of sugars. #-glucosidase supplementation was found to increase both the sugar yield and hydrolysis rate, and shorten the reaction time significantly.     

Production and microtopography of bog bryophytes: response to warming and water-table manipulations

Boreal peatlands, which contain a large fraction of the world's soil organic carbon pool, may be significantly affected by changes in climate and land use, with attendant feedback to climate through changes in albedo, fluxes of energy or trace gases, and soil carbon storage. The response of peatlands to changing environmental conditions will probably be dictated in part by scale-dependent topographic heterogeneity, which is known to interact with hydrology, vegetation, nutrients, and emissions of trace gases. Because the bryophyte community can contribute the majority of aboveground production in bogs, we investigated how microscale topography affects the response of bryophyte species production and cover to warming (using overhead infrared lamps) and manipulations of water-table height within experimental mesocosms. We removed 27 intact peat monoliths (2.1-m² surface area, 0.5-0.7 m depth) from a bog in northern Minnesota, USA, and subjected them to three warming and three water-table treatments in a fully crossed factorial design. Between 1994 and 1998, we determined annual production of the four dominant bryophyte taxa within three microtopographic zones (low, medium, and high relative to the water table). We also estimated species cover and calculated changes in topography and roughness of the bryophyte surface through time. Total production of all bryophytes, and production of the individual taxa Polytrichum strictum, Sphagnum magellanicum, and Sphagnum Section Acutifolia, were about 100% greater in low microtopographic zones than in high zones, and about 50% greater in low than in medium zones. Production of bryophytes increased along the gradient of increasing water-table heights, but in most years, total production of bryophytes was negatively correlated with height above the set water table only for the wettest water-table treatment. Although bryophyte production was unaffected by the warming treatments, the bryophyte surface flattened in proportion to the degree of warming. These results indicate that production of bryophytes is driven most strongly by the absolute and relative height of the bryophyte surface above the water table. Predicted changes in water-table height commensurate with changes in surface temperature may thus affect both production and superficial topography of bryophyte communities.     

Viable bacterial biomass and functional diversity in fresh and marine waters in the Canadian Arctic

Bacterial biomass and functional diversity in four marine and four freshwater samples, collected from Resolute Bay, Nunavut, Canada, were studied using fluorescent nucleic-acid staining and sole-carbon-source utilization. Viable microbial counts using the LIVE/DEAD BacLight Viability Kit estimated viable marine bacterial numbers from 0.7 to 1.8᎒⁶ cells/l, which were lower than viable bacterial numbers in freshwater samples (2.1-9.9᎒⁶ cells/l) (RCBD-ANOVA). Calculations of the Shannon-Wiener diversity index and average well colour development were based on substrate utilization in ECO-Biolog plates incubated at 4°C and 20°C for 38 and 24 days, respectively. The Shannon-Wiener diversity of the marine water samples was significantly greater ( x _H'=2.40ǂ.08, P <0.005; RCBD-ANOVA) than that of freshwater samples ( x _H'=1.20ǂ.00, P <0.005; RCBD-ANOVA). Differences in microbial diversity between fresh and marine water samples at 4°C ( x _4°C =2.01) and 20°C (x_20°C =2.31) were also detected by RCBD-ANOVA analysis. Interactions between water type and incubation temperature were not significant ( F =1.926, F _c=5.12). Principal component analysis revealed differences in metabolic substrate utilization patterns and, consequently, the microbial diversity between water types and samples.     

The effect of Hordeum chilense and Triticum cytoplasms on anther culture response of tritordeum

With the aim of determining the effect of different cytoplasms on anther culture response, we analyzed anthers from different tritordeum lines with either Hordeum chilense, Triticum aestivum or T. turgidum cytoplasms for their response in vitro. The use of these tritordeum alloplasmic lines enabled cytoplasms belonging to two different genera to be compared for the first time. The results showed that the response from anthers of the tritordeum line containing T. turgidum cytoplasm was significantly lower than the response of the other two lines. This finding was encouraging from a practical point of view, since cytoplasms from both H. chilense and T. aestivum are preferred for the breeding of tritordeum.     

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).     

Scale down of recombinant protein production: a comparative study of scaling performance

A large bioreactor is heterogeneous with respect to concentration gradients of substrates fed to the reactor such as oxygen and growth limiting carbon source. Gradient formation will highly depend on the fluid dynamics and mass transfer capacity of the reactor, especially in the area in which the substrate is added. In this study, some production-scale (12 m³ bioreactor) conditions of a recombinant Escherichia coli process were imitated on a laboratory scale. From the large-scale cultivations, it was shown that locally high concentration of the limiting substrate fed to the process, in this case glucose, existed at the level of the feedpoint. The large-scale process was scaled down from: (i) mixing time experiments performed in the large-scale bioreactor in order to identify and describe the oscillating environment and (ii) identification of two distinct glucose concentration zones in the reactor. An important parameter obtained from mixing time experiments was the residence time in the feed zone of about 10 seconds. The size of the feed zone was estimated to 10%. Based on these observations the scale-down reactor with two compartments was designed. It was composed of one stirred tank reactor and an aerated plug flow reactor, in which the effect of oscillating glucose concentration on biomass yield and acetate formation was studied. Results from these experiments indicated that the lower biomass yield and higher acetate formation obtained on a large scale compared to homogeneous small-scale cultivations were not directly caused by the cell response to the glucose oscillation. This was concluded since no acetate was accumulated during scale-down experiments. An explanation for the differences in results between the two reactor scales may be a secondary effect of high glucose concentration resulting in an increased glucose metabolism causing an oxygen consumption rate locally exceeding the transfer rate. The results from pulse response experiments and glucose concentration measurements, at different locations in the reactor, showed a great consistency for the two feeding/pulse positions used in the large-scale bioreactor. Furthermore, measured periodicity from mixing data agrees well with expected circulation times for each impeller volume. Conclusions are drawn concerning the design of the scale-down reactor.     

Growth optimization of an ectomycorrhizal fungus with respect to pH and temperature in vitro, using Design of Experiments

The effect of external factors like pH of the medium, temperature and incubation period on the growth of the ectomycorrhizal fungus, Pisolithus tinctorius was studied as a function of the total fungal biomass produced, extracellular protein content and residual pH of the medium. The effect of the three parameters on the three responses was found to be significant and interdependent. Therefore, an attempt was made to optimize the growth of the fungus under varying conditions of pH, temperature and incubation period using the Box-Behnken Design of Experiments which is a second order model involving Response Surface Methodology and a second order quadratic equation. With this design expert, the optimum conditions of the three parameters that favoured the maximum growth of the fungus in vitro were found. The experimental values corroborated with the predicted values got from the model with the correlation coefficients 0.9349 for biomass, 0.9913 for protein and 0.9959 for final pH.