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 of inulo-oligosaccharides from inulin by recombinant E. coli containing endoinulinase activity

To utilize intracellular endoinulinase for inulo-oligosaccharide (IOS) production from inulin, the endoinulinase gene (inu1) of Pseudomonas sp. was successfully cloned into the plasmid pBR322 by using EcoRI restriction endoinulinase and E. coli HB101 as a host strain. The endoinulinase from E. coli HB101/pKMG50 was constitutively expressed, showing similar reaction modes as compared to those of the original strain. However, some critical differences existed in optimal reaction conditions and oligosaccharide compositions between the two products catalyzed by the native enzyme of original strain and those by intact cells from recombinant cells. The IOS compositions produced by recombinant E. coli were quite different due to the diffusional restriction of the substrate and products within the cell wall. Optimal reaction conditions for batchwise production of IOS were as follow : optimum temperature, 55v°C; pH, 7.5; substrate concentration, 100 g/l inulin; enzyme dosage, 20 units/g substrate. Continuous production of IOS from inulin was also carried out at 50v°C using a bioreactor packed with the recombinant cells immobilized on calcium alginate gel. The optimal feed concentration and the feed flow rate were 100 g/l inulin and 0.6 h^у as a superficial space velocity, respectively. Under the optimum operation conditions, continuous production of IOS was successfully performed with productivity of 166.7 g/l·h for 15 days at 50v°C without significant loss of initial activity.     

Hydrolysis of lactose in whey permeate by immobilized β-galactosidase from Penicillium notatum

A new low-cost β-galactosidase (lactase) preparation for whey permeate saccharification was developed and characterized. A biocatalyst with a lactase activity of 10 U/mg, a low transgalactosylase activity and a protein content of 0.22 mg protein/mg was obtained from a fermenter culture of the fungus Penicillium notatum. Factors influencing the enzymatic hydrolysis of lactose, such as reaction time, pH, temperature and enzyme and substrate concentration were standardized to maximize sugar yield from whey permeate. Thus, a 98.1% conversion of 5% lactose in whey permeate to sweet (glucose-galactose) syrup was reached in 48 h using 650 β-galactosidase units/g hydrolyzed substrate. After the immobilization of the acid β-galactosidase from Penicillium notatum on silanized porous glass modified by glutaraldehyde binding, more than 90% of the activity was retained. The marked shifts in the pH value (from 4.0 to 5.0) and optimum temperatures (from 50°C to 60°C) of the solid-phase enzyme were observed and discussed. The immobilized preparation showed high catalytic activity and stability at wider pH and temperature ranges than those of the free enzyme, and under the best operating conditions (lactose, 5%; β-galactosidase, 610–650 U/g lactose; pH 5.0; temperature 55°C), a high efficiency of lactose saccharification (84–88%) in whey permeate was achieved when lactolysis was performed both in a batch process and in a recycling packed-bed bioreactor. It seems that the promising results obtained during the assays performed on a laboratory scale make this immobilizate a new and very viable preparation of β-galactosidase for application in the processing of whey and whey permeates.     

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.     

Production and purification of α-amylase from hydrogen producing Enterobacter cloacae IIT-BT 08

Enterobacter cloacae IIT-BT08 was found to produce both !-amylase and hydrogen in a batch system using soluble starch as substrate. Incubation time, temperature, pH and substrate concentration for the maximum !-amylase activity (130 U/ml) were 8 h, 37 °C, 6.00 and 10 g/l of soluble potato starch respectively. However, the optimum temperature and pH for the crude !-amylase activity were 60 °C and 4 respectively. The maximum rate of hydrogen production was observed at 10th h of fermentation and corresponding hydrogen yield was 7.6 mmol H₂/g soluble potato starch.     

Improvement of rifamycin B production using mutant strains of Amycolatopsis mediterranei

Two mutant strains of Amycolatopsis mediterranei VA17 and VA18 were isolated using physical (UV) and chemical (NTG) mutagens gave high rifamycin B than the parent type when grown in the same fermentation medium with a pH of 7.2, temperature 32v°C for a period of 12 days. The cultural conditions of both mutant strains are similar to the parent strain except temperature which was higher by 4v°C. By this mutation and selection study, rifamycin B production was improved from 1400 mg/l to 2450 mg/l.     

Culture conditions for the production of esterase from Lactobacillus casei CL96

Culture conditions in growth and esterase production by a newly isolated Lactobacillus casei CL96 were investigated using a dextrose-free MRS medium supplemented with different sugars in a 2 l fermentor at different pHs (4.0-9.0) and temperatures (20-50°C). The optimal growth was obtained in basal MRS medium containing 1% (w/v) lactose at pH 7.0 and 30°C. The maximal esterase production was obtained intracellularly during the late logarithmic phase, but during the stationary phase, the esterase activity was released in the culture medium. The enzyme activity was maximal at pH 7.0 and 37°C. Among various substrates (C₂-C₁₆) tested, the highest activity was towards C₆ and C₈. Though the enzyme was produced constitutively, the tributylin induced the enzyme production by 2.5 fold. L. casei CL96 esterase was very active at neutral pH and ambient temperature and might be suitable for biotechnological applications in the dairy industry.     

Production and partial purification of invertase using <Emphasis Type="Italic">Cympopogan caecius</Emphasis> leaf powder as substrate

The present study investigates the efficiency of Aspergillus niger to produce invertase, an industrially important enzyme by using powdered stem of Cympopogan caecius (Lemon grass) as sole substrate and sole carbon source for the microorganism. The molecular weight of invertase was estimated to be 66–70 kDa by sodium do decyl sulphate poly acrylamide gel electrophoresis (SDS PAGE). The production of the enzyme was studied at different pH scales ranging from pH 4.0 to 7.0 at a constant temperature of 30°C and 2% substrate concentration. The maximum production of invertase (specific activity −0.0516 μk/mg protein) was obtained at pH 5.5 at 30°C temperature, and incubation for 48 h. The activity was found to be stable at pH 5.5 for 30 min. The enzyme was found to be stable in the temperature range of 20–55°C. The effect of divalent metal ions Cu²⁺, Fe²⁺, Co²⁺ on the activity of the enzyme invertase showed that these ions affected the activity by a certain factor. The study can be further industrially exploited in a country-like India where lemon grass is found in plenty and can be used as substrate for enzyme production. Moreover, the preparation of the substrate is also a simple process.     

Microsite characteristics of Muhlenbergia richardsonis (Trin.) Rydb., an alpine C4 grass from the White Mountains, California

C₄ plants are uncommon in cold environments and do not generally occur in the alpine tundra. In the White Mountains of California, however, the C₄ grass Muhlenbergia richardsonis is common in the alpine zone at 3,300-3,800 m, with the highest population observed at 3,960 m (13,000 feet) above sea level. This is the highest reported C₄ species in North America and is near the world altitude limit for C₄ plants (4,000-4,500 m). Above 3,800 m, M. richardsonis is largely restricted to southern slope aspects, with greatest frequency on southeast-facing slopes. In open tundra, M. richardsonis formed prostrate mats with a mean height of 2.5 cm. Neighboring C₃ grasses were two to three times taller. Because of its short stature, leaf temperature of M. richardsonis was greatly influenced by the boundary layer of the ground, rising over 20°C above air temperature in full sun and still air and over 10°C above air temperature in full sun and wind velocity of 1-4 m s^-1. Thus, although air temperatures did not exceed 15°C, midday leaf temperatures of M. richardsonis were routinely between 25°C and 35°C, a range favorable to C₄ photosynthesis. At night, leaf temperature of M. richardsonis was often 5-12°C below air temperature, resulting in regular exposure to subzero temperatures and frosting of the leaves. No visible injury was associated with exposure to freezing night temperatures. The presence of M. richardsonis in the alpine zone demonstrates that C₄ plants can tolerate extreme cold during the growing season. The localization to microsites where leaf temperatures can exceed 25°C during the day, however, indicates that even when cold tolerant, C₄ plants still require periods of high leaf temperature to remain competitive with C₃ species. In this regard, the prostrate growth form of M. richardsonis compensates for the alpine climate by allowing sufficient heating of the leaf canopy during the day.     

Immobilization of Bacillus licheniformis L-Arabinose Isomerase for Semi-Continuous L-Ribulose Production

Bacillus licheniformis L-arabinose isomerase (BLAI) with a broad pH range, high substrate specificity, and high catalytic efficiency for L-arabinose was immobilized on various supports. Eupergit C, activated-carboxymethylcellulose, CNBr-activated agarose, chitosan, and alginate were tested as supports, and Eupergit C was selected as the most effective. After determination of the optimum enzyme concentration, the effects of pH and temperature were investigated using a response surface methodology. The immobilized BLAI enzyme retained 86.4% of the activity of the free enzyme. The optimal pH for the immobilized BLAI was 8.0, and immobilization improved the optimal temperature from 50 °C (free enzyme) to a range between 55 and 65 °C. The half life improved from 2 at 50 °C to 212 h at 55 °C following immobilization. The immobilized BLAI was used for semi-continuous production of L-ribulose. After 8 batch cycles, 95.1% of the BLAI activity was retained. This simple immobilization procedure and the high stability of the final immobilized BLAI on Eupergit C provide a promising solution for large-scale production of L-ribulose from an inexpensive L-arabinose precursor.     

Temperature-dependent pH regulation in stenothermal Antarctic and eurythermal temperate eelpout (Zoarcidae): an in-vivo NMR study

Temperature-dependent adjustments of intracellular pH are thought to play a major role in the maintenance of protein function. Comparative studies were carried out in two species from the same fish family (Zoarcidae), the stenothermal Antarctic eelpout (Pachycara brachycephalum) and the eurythermal eelpout (Zoarces viviparus), to find out whether pH regulation is modified by temperature in the closely related species and to what extent the respective pattern differs between eurytherms and stenotherms. Previous invasive studies had compared individual animals sampled at various temperatures and suggested that a decrease in intracellular pH (pHi) values occurs at rising temperatures, as predicted by the alpha-stat hypothesis of acid-base regulation. The present study used non-invasive in vivo ³¹P-NMR spectroscopy in non-anaesthetized, unrestrained fish for long-term online recordings in individual specimens. Control spectra obtained at T=0°C for P. brachycephalum and at 12°C for Z. viviparus indicated low stress conditions, as well as a high stability of energy and acid-base status over time periods longer than 1 week. Temperature changes had no influence on the concentration of high-energy phosphates like phosphocreatine or ATP. Temperature-induced pH changes were monitored continuously in a range between 0 and 6°C for polar, and 12 and 18°C for temperate eelpout. A pHi change of around -0.015 pH units/°C was observed within both species, in accordance with the alpha-stat hypothesis; however, extrapolation to the same temperature revealed different set points of pH regulation in the two species. These findings confirm that an alpha-stat pattern of pH regulation can be found in stenothermal Antarctic animals, at set points deviating from an alpha-stat pattern, however, in a between-species comparison.     

Verticillium lecanii spore production in solid-state and liquid-state fermentations

Verticillium lecanii has been recognized as an entomopathogen with high potential in biological control of pests. Two types of cultivation methods, the solid-state fermentation (SSF) and the liquid-state fermentation (LSF), were examined for V. lecanii. In SSF, the substrate types including rice, rice bran, rice husk, and the mixtures of these components were tested. The results showed that both cooked rice with appropriate water addition and rice bran gave significantly higher spore production of 1.5 2 10⁹ spores/g substrate and 1.4 2 10⁹ spores/g substrate, respectively. In LSF, SMAY liquid medium was used as a base, and the effects of environmental conditions on the spore production of V. lecanii were investigated. From the time course study, on the 9th day the spore yield reached 1.2 2 10⁹ spores/ml of broth at 24v°C, 150 rpm for this strain. A series of medium volumes in the shaker-flask have been tested for the requirement of aeration. The largest surface aeration test, one tenth of the medium volume in the shaker-flask for cultivation, gave the highest spore count. The optimal pH value was tested and the initial pH 5 in the SMAY medium produced a high spore density. Finally, V. lecanii spores from SSF and LSF were different in size, shape, and size distribution; while mean spore length from SSF was 6.1 7m, and mean spore length from LSF was 5.0 7m.     

Clearance rates in the Arctic bivalves <Emphasis Type="Italic">Hiatella arctica</Emphasis> and <Emphasis Type="Italic">Mya </Emphasis>sp.

Filtration was studied in two Arctic clams, Hiatella arctica and Mya sp., collected in Young Sound, Northeast Greenland. Clearance rates were determined as a function of ambient temperature and algal cell concentration, using the clearance method and feeding with a unicellular flagellate. For both species, clearance rates increased with increasing temperature from <у up to 4-8°C. At higher temperatures, filtration ceased and the clams closed their valves. Clearance rates were also determined in temperate specimens of H. arctica collected on the west coast of Sweden. For these specimens, clearance rates increased with increasing temperature from 0 to 18-20°C. When weight-specific clearance rates were compared between the two populations and between species, there were no differences at 1°C. Clearance rates in Arctic H. arctica were maximal at algal cell concentrations corresponding to 2.5-8 µg chlorophyll a l^-1. Temperature compensation in Arctic bivalves is discussed and it is concluded that adaptations to constant low temperatures consist of a lower minimum temperature, for active filtration. Low clearance rates due to low temperatures did not seem to limit growth, under the prevailing conditions in Young Sound.     

A statistical approach for optimization of R-phycoerythrin extraction from the red algae Gracilaria verrucosa by enzymatic hydrolysis using central composite design and desirability function

The aim of this research is to statistically optimize enzymatic hydrolysis parameters for the production of R-phycoerythrin (RPE) from red algae Gracilaria verrucosa. Six independent variables, incubation temperature, incubation time, ratio of buffer to raw material, cellulase loading, xylanase loading, and pH, were selected for response surface methodology studies. A central composite design was employed to maximize RPE production. A mathematical model with high determination coefficient (R ²?=?0.86) was developed and could be employed to optimize RPE extraction. The optimal extraction conditions of RPE were determined as follows: incubation temperature (48°C), incubation time (6?h), ratio of buffer to raw material (20 w/v), cellulase loading (15%), xylanase loading (5%), and pH (6.5). Under this optimal condition, the experimental yield of RPE was 6.25?mg?g^?1. Based on the result of response surface methodology and desirability function approach study, total sugar, the main by-product in RPE extraction was considered as another response. A new optimal condition was predicted as follows: incubation temperature (30°C), incubation time (12?h), ratio of buffer to raw material (20, w/v), cellulase loading (15%), xylanase loading (5%), and pH (6). Under this condition, similar RPE levels were obtained while the concentration of total sugar decreased by 40%.     

Continuous production of inulo-oligosaccharides from chicory juice by immobilized endoinulinase

Several carrier materials were examined for endoinulinase immobilization. A polystyrene carrier material (UF93^®) gave the best immobilization capacity (217 units/g carrier) and operational stability. Carbohydrate compositions in the reaction product were quite similar irrespective of the support materials even though each carrier material has different pore structure associated with diffusional restriction. After immobilization the optimal pH for enzyme activity was shifted from 5.0 to 4.5, whereas optimal temperature (55v°C) was unaltered. Continuous production of inulo-oligosaccharides from chicory juice was carried out using the polystyrene-bound endoinulinase. The recommended operating conditions of the enzyme reactor for maximizing productivity were as follows: feed concentration, 100 g/l chicory juice; flow rate, as superficial space velocity 2.0 h^у; temperature, 55v°C. The enzyme reactor was run for 28 days at 55v°C achieving an oligosaccharide yield of 82% without any significant loss of initial enzyme activity, where the volumetric productivity was 200 g/l · h. Furthermore, there was no marked difference in operational stability between the two reactors fed with pure inulin solution and with chicory juice as a substrate even though chicory juice contains a lot of impurities.     

Plant frequency,stem and root characteristics,and CO<Subscript>2</Subscript> uptake for <Emphasis Type="Italic">Opuntia acanthocarpa</Emphasis>: elevational correlates in the northwestern Sonoran Desert

A common cylindropuntia in the northwestern Sonoran Desert, Opuntia acanthocarpa, was investigated for the following hypotheses: its lower elevational limit is set by high temperatures, so its seedlings require nurse plants; its upper elevational limit is set by freezing; spine shading is the least at intermediate elevations; and changes in plant size and frequency with elevation reflect net CO₂ uptake ability. For four elevations ranging from 230 m to 1,050 m, the mean height of O. acanthocarpa approximately doubled and its frequency increased 14-fold. Nurse plants were associated with only 4% of O. acanthocarpa less than 20 cm tall at the two lower elevations compared with 57% at 1,050 m, where putative freezing damage was especially noticeable, suggesting that nurse plants protect from low temperature damage. Spine shading of the stem doubled from the lowest to the highest elevation. Net CO₂ uptake, which followed a Crassulacean acid metabolism pattern, was maximal at day/night air temperatures of 25/15°C and was halved by 4 weeks of drought and by reducing the photosynthetic photon flux from 30 to 12 mol m^-2 day^-1. The root system of O. acanthocarpa was shallow, with a mean depth of only 9 cm for the largest plants. Root growth was substantial and similar for plants at 25/15°C and 35/25°C, decreasing over 70-fold at 15/5°C and 45/35°C. Based on cellular uptake of the vital stain neutral red, neither roots nor stems tolerated tissue temperatures below -5°C for 1 h while both showed substantial high temperature acclimation, roots tolerating 1 h at 61°C and stems 1 h at 70°C for plants grown at 35/25°C. The increase in height and frequency of O. acanthocarpa with elevation apparently reflected both a greater ability for net CO₂ uptake and greater root growth and hence water uptake. This species achieves its greatest ecological success at elevations where it becomes vulnerable to low temperature damage.     

PURIFICATION AND CHARACTERIZATION OF AN ALKALINE CELLULASE PRODUCED BY Bacillus subtilis (AS3)

An extracellular alkaline carboxymethycellulase (CMCase) from Bacillus subtilis was purified by salt precipitation followed by anion-exchange chromatography using DEAE-Sepharose. The cell-free supernatant containing crude enzyme had a CMCase activity of 0.34 U/mg. The purified enzyme gave a specific activity of 3.33 U/mg, with 10-fold purification and an overall activity yield of 5.6%. The purified enzyme displayed a protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular size of 30 kDa, which was also confirmed by zymogram analysis. The enzyme displayed multisubstrate specificity, showing significantly higher activity with lichenan and β-glucan as compared to carboxymethylcellulose (CMC), laminarin, hydroxyethylcellulose, and steam-exploded bagasse, and negligible activity with crystalline substrate such as Avicel and filter paper. It was optimally active at pH 9.2 and temperature 45°C. The enzyme was stable in the pH range 6–10 and retained 70% activity at pH 12. Thermal stability analysis revealed that the enzyme was stable in temperature range of 20°C to 45°C and retained more than 50% activity at 60°C for 30 min. The enzyme had a Km of 0.13 mg/ml and Vmax of 3.38 U/mg using CMC as substrate.     

Pretreatment of Extruded Corn Stover with Polyethylene Glycol to Enhance Enzymatic Hydrolysis: Optimization, Kinetics, and Mechanism of Action

Due to the high potential of the extrusion technique for pretreatment of lignocellulosic substrates, several attempts have been conducted in previous studies to further increase the subsequent sugar yield from extrusion pretreatment. Examples include application of chemicals along with extrusion, such as alkali-extrusion and ethylene glycol-extrusion, or before extrusion, such as hot water extraction. In this study, a new sequential technique has been developed for pretreatment of corn stover (CS), which utilizes an initial extrusion pretreatment (155?rpm screw speed and temperatures of 90°C, 180°C and 180°C corresponding to feed, barrel and die zones, respectively at a reaction time of 45?C90?s) followed by pretreatment with polyethylene glycol 6,000 (PEG). In order to fully characterize the response for sugar yield over the range of surfactant treatment conditions assessed, response surface methodology was used. Treatment temperature, incubation time and PEG concentration were varied between 45?C55°C, 1?C4?h, 0.15?C0.6?g PEG/g glucan, respectively. Statistical analysis was conducted by fitting the glucose and xylose yields to a quadratic polynomial model. PEG concentration and temperature were found to be the most significant factors in surfactant pretreatment. The optimum condition resulted in 25.4% and 10.3% increase in glucose and xylose yield, respectively. Using the combination of 10.8?FPU/g glucan of Ctec₂ and 0.3?g PEG/g glucan, the glucose yield of extruded CS reached 98%. A yield was 64% resulted from application of similar amounts of Ctec and Htec. Decreased adsorption of enzyme to the lignocellulosic substrate as well as increased enzyme activity and reaction velocity indicated by kinetic parameter evaluation and nitrogen combustion analysis suggested an increased solubilization of cellulase in the presence of PEG. We propose that a non-productive adsorption of enzymes occur during hydrolysis not only due to lignin but also due to crystalline cellulose. Comparison of enzyme adsorptions and increase in sugar yields between Avicel and CS suggests the presence of other potential mechanisms of action for PEG in addition to increase of enzyme solubilization.     

Purification and characterization of an endoxylanase from the culture broth of <Emphasis Type="Italic">Bacillus cereus</Emphasis> BSA1

An extracellular xylanase from the fermented broth of Bacillus cereus BSA1 was purified and characterized. The enzyme was purified to 3.43 fold through ammonium sulphate precipitation, DEAE cellulose chromatography and followed by gel filtration through Sephadex-G-100 column. The molecular mass of the purified xylanse was about 33 kDa. The enzyme was an endoxylanase as it initially degraded xylan to xylooligomers. The purified enzyme showed optimum activity at 55°C and at pH 7.0 and remained reasonably stable in a wide range of pH (5.0–8.0) and temperature (40–65°C). The K _m and V _max values were found to be 8.2 mg/ml and 181.8 μmol/(min mg), respectively. The enzyme had no apparent requirement of cofactors, and its activity was strongly inhibited by Cu²⁺, Hg²⁺. It was also a salt tolerant enzyme and stable upto 2.5 M of NaCl and retained its 85% activity at 3.0 M. For stability and substrate binding, the enzyme needed hydrophobic interaction that revealed when most surfactants inhibited xylanase activity. Since the enzyme was active over wide range of pH, temperature and remained active in higher salt concentration, it could find potential uses in biobleaching process in paper industries.     

Production of Rifamycin using Amycolatopsis mediterranei (MTCC14)

Production of Rifamycin with Amycolatopsis mediterranei (MTCC14) was studied using carbon and locally available cheaper nitrogen sources. A. Mediterranei gave initial yield of 650 mg/l with standard fermentation medium. This was improved to 1400 mg/l by using various Carbon, Nitrogen sources and optimizing various cultural conditions. Glucose, Ammonium sulphate and combination of soya bean and pea nut meals were found to induce more Rifamycin production in 7 days with a pH of 7.2, temperature 28v°C.