A new combinational statistical approach for cellulase optimization in Aspergillus nidulans

Augmented cellulase production by Bacillus subtilis strain MU S1 using different statistical experimental designs.

Aims: To isolate fungi capable of simultaneous production of amylase and cellulase
 Study Design: The experiment was carried out in aseptic conditions, data were subjected to one way Analysis of Variance (ANOVA) and the means were separated using Least Significance Deference (LSD).
 Place and Duration of Study: Department of Microbiology, University of Nigeria, Nsukka, Enugu State, Nigeria, between August 2010 and July 2013.
 Methodology: Plantain peels and infected bark of the stump of a tree were collected for fungi isolation. A 1 g sample each was weighed separately and added to 10 ml distilled water, 1 ml of each of the diluents was plated out on Emerson’s yeast phosphate soluble starch (YPSs) medium. After growth, colonies were picked and subcultured several times for purity. The isolates were characterized and identified based on colony morphology and microscopic examination. They were later screened for amylase and cellulase activities by growing them on various concentrations of starch and carboxymethyl cellulose
 Results: Amylolytic and cellulosic fungi were isolated from plantain peels and infected bark of a tree. The isolates were identified as Rhizopus and Fusarium sp and they were able to produce amylase and cellulase simultaneously. The enzyme activities were determined on various concentrations of starch and carboxymethyl cellulose (CMC) in liquid medium. At 2 % starch + 0 % CMC, enzymes activities were 127.44 U/ml and 144.59 U/ml glucoamylase and 356.43 U/ml and 263.63 U/ml cellulase for Rhizopus and Fusarium sp respectively. When the organisms were grown on a solid medium (koji) supplemented with various concentrations of CMC, there was an increase in cellulase activities as CMC increased. Cellulase activity of 1902.02 U/g was recorded by Rhizopus sp at 1.5 g CMC supplementation and 1481.18 U/g was from Fusarium sp at 1 g CMC supplementation, but highest glucoamylase activities of 322.68 U/g and 302.12 U/g were recorded for Rhizopus and Fusarium spp respectively at 1 g CMC supplementation. Glucoamylase activities were not significantly affected by CMC supplementation in solid state culture.
 Conclusion: The result of the study showed that the isolates were able to produce amylase and cellulase enzymes in appreciable quantities and therefore can find usefulness in industrial application of these enzymes.

Utilization of agricultural wastes for production of useful metabolites requires hydrolysis using both cellulase and amylase enzymes. We isolated Aspergillus fumigatus IB-A1 and evaluated its ability to simultaneously produce both cellulase and amylase. Although the isolate could produce both cellulase and amylase from either soluble starch or carboxymethyl cellulose, amylase activity was higher with soluble starch while cellulase activity was higher when carboxymethyl cellulose was used as the sole carbon source. With a mixture of carboxymethyl cellulose and soluble starch, both the amylase and cellulase activities increased with increase in the ratio of soluble starch. The optima ratio of carboxymethyl cellulose to soluble starch for cellulase and amylase activities were 0.7:0.3, and 0.4 to 0.6 respectively. For practical application, the optimum ratio of carboxymethyl cellulose to soluble starch in the production medium depends on the relative composition of cellulose and starch in the substrate to be hydrolyzed. The isolate was also able to efficiently produce both amylase and cellulase from cassava peel. With 10 g/L cassava peel, the cellulase and amylase activities were 6.122± 0.320 U/ml/min and 4.342± 0.210 U/ml/min respectively. When the cells were immobilized on loofa sponge and subjected to alternating air phase- liquid phase culture, cellulase and amylase production from cassava peel increased to 8.106± 0.620 U/ml/min and 5.206± 1.24 U/ml/min respectively. The optimum ratio of the air phase to the liquid phase was 3 hours of air phase to 21 hours of liquid phase.

Production of holocellulase by a newly isolated marine Bacillus species via submerged fermentation technique at varying cultivation conditions was investigated. Enzyme production was optimized by altering one variable per time. Moreover effect of lignocellulosic saccharides in inducing cellulase and xylanase production was also investigated. The 16S ribosomal Deoxyribonucleic Acid (16S rDNA) gene sequence analysis exhibited 98% sequence similarity of the isolate with other Bacillus species in the gene bank and was deposited with the accession number KX524510. Optimal xylanase and cellulase production was attained at pH 7, temperature 30C and agitation speed of 50 and 150 rpm. Furthermore, maximum xylanase and cellulase production were both achieved at 60 h corresponding to the late stationary growth stage, with activity of 16.6 and 0.061 U mL -1 respectively. Xylanase production was maximally induced by beechwood xylan, xylose and arabinose with activities of 13.59, 8.78 and 1.90 (U mL -1 ) respectively; while cellulase production was induced by carboxymethyl cellulose only and no cellulase activity was detected in the culture supernatant of the other carbon sources tested. Optimization increased cellulase and xylanase yields being 0.006-0.061 and 0.23-16.6 (U mL -1 ) from unoptimized to optimized respectively. The results of the study suggest the bacterial strain to be a proficient producer of cellulase and xylanase with potentials in biotechnological application.

Pretreated lignocellulosic residues are suitable substrates for cellulases production by filamentous fungi. In the current work, plantain rachis was pretreated with sequential acid and alkali and then used as the main carbon source for cellulases production. First, a full 23 factorial design and response surface methodology (RSM), based on central composite rotatable design (CCRD), were carried out to cellulases production media optimization from plantain rachis by Penicillium oxalicum. The cellulases production was evaluated in flasks and bioreactor scale; in parallel, the addition of possible cellulases inductors was evaluated in flasks: molasses, beer bran, oat bran, and wheat bran. Results from statistical analyses with a level of confidence of 95% demonstrated that the concentration of ammonium sulfate must be kept at 1.625 g/L. The optimum urea and yeast extract concentrations were 0.560 g/L and 0.250 g/L, respectively. Cellulases volumetric productivities were higher in instrumented bioreactor than in flasks: 78.03% for exoglucanase, 10.87% for endoglucanase, 1.58% for β‐glucosidase, and 44.36% for FPU. Therefore, P. oxalicum was able to produce cellulases from plantain rachis in flasks and bioreactor, and molasses was the additional inductor that presented an increment in cellulases activities: endoglucanase 15%, exoglucanase 81%, and β‐glucosidase 55%.

This study describes the isolation and characterization of a novel fungus, Aspergillus flavus BS1 and its cellulolytic activities with special emphasis on endoglucanase production. Preliminary screening studies showed that A. flavus BS1 was a potent strain for the production of cellulase. To study the cellulolytic activities in detail by submerged fermentation (SmF), productions of endoglucanase, exoglucanase, and β-glucosidase were estimated from the basal salt medium (BSM) supplemented with 1 % carboxy methyl cellulose (CMC). CMC medium supported the maximum yield of endoglucanase (2,793 U/ml) on day 5 of incubation at 28 °C and 150 rpm, which was higher than that obtained with naturally available supplements (flour) from banana, tapioca, potato, or banana peel. During cellulase production by solid-state fermentation, 10 % (w/w) tapioca flour in sawdust (teak wood) moisturized with BSM (1:2, w/v) supported maximum cellulase yield (5,408 U/g dry substrate) on day 3 at 28 °C, which was 2-fold higher than that obtained during SmF. The active cellulase was qualitatively estimated by polyacrylamide gel electrophoresis (PAGE). Native-PAGE (0.25 % CMC impregnated on the 10 % gel) activity staining with congo-red showed a clear zone for CMCase activity, whereas SDS-PAGE showed a distinct band. In conclusion, this study showed that A. flavus strain BS1 is a potent strain for the production of cellulase on lignocellulosic media, the hot enzyme for bioethanol production from the lignocellulosic biomass by SSF.

A five-level-four-factor central composite rotary design (CCRD) was employed in combination with response surface methodology (RSM) to optimize the process variables for the production of a novel copolymer consisting of short-chain-length (SCL) and long-chain-length (LCL) PHA units, i.e., P(3HB-3HV-3HHD-3HOD) copolymer in Pseudomonas aeruginosa MTCC 7925. The four variables involved in this study were ethanol, glucose, ammonium nitrate (NH(4)NO(3)), and potassium dihydrogen phosphate (KH(2)PO(4)). A second-order polynomial equation was obtained by multiple regression analysis using RSM. The statistical analyses of the results showed that all the four variables had significant impact on the copolymer yield. The model predicted a maximum yield of 81.1% of dry cell weight (dcw) on setting the concentrations of ethanol and glucose at 1.5 and 1.1%, and KH(2)PO(4) and NH(4)NO(3) at 2.79 and 1.86 g/L, respectively. Verification of the predicted value resulted into a yield of 77.6% (dcw). This novel copolymer exhibited comparable material properties with polypropylene (PP) and low density polyethylene (LDPE), thus advocating its potential applications in various fields.

This study was aimed at isolation and screening of fungal species associated with Artemisia annua Plantation soils from one of the under studied areas in Plateau state, Nigeria for cellulase activity. A total of thirteen fungal species were isolated from various locations within the A. annua Plantation and were screened for cellulase production. Agar plate assay was carried out using basal medium supplemented with 1% Carboxymethylcellulose (CMC) powder and staining with 0.1% Congo red solution after the incubation period. Among these species, Aspergillus fumigatus, A. niger and A. terreus were more predominant and were recorded as cellulase producing species. They have shown to possess cellulose degrading ability and exhibited maximum zones of hydrolysis on Carboxymethylcellulose medium and were selected for enzyme assay using submerged fermentation (SmF). Enzyme production was analyzed by Dinitrosalicylic acid (DNSA) methods and the enzymes assayed for were CMCase (β-1,4-endoglucanase), β-glucosidase and FPase (total cellulose) using Carboxymethylcellulose, cellulose acetate and Filter paper as substrates respectively. The highest cellulase activity was observed on the 3 rd day in A. niger with enzyme production of 0.045IU/ml and 0.040IU/ml on CMC and filter paper media respectively. A . fumigatus had high enzyme activity of 0.037IU/ml and 0.025IU/ml on filter paper and cellulose acetate media respectively. Highest enzyme production of 0.034IU/ml was recorded for A. terreus on the 3 rd day on cellulose acetate medium. These fungal species could be employed specially to perform in situ environmental applications involving cellulose biodegradation of wastes.

Background: Sawdust or wood cheeping is lignocellulosic biomass that has enormous prospects. Sawdust (having 50% carbon) releasing from saw milling is not properly processed but dumped that causes the environmental pollution. One of the most notable sources of pollution is open air burning of such wastes. Air pollution is a matter of serious concern in Bangladesh. The overall aim of this study was to produce more healthy air by converting sawdust into economically valuable products. Materials and Methods: A potential cellulolytic bacteria (FFS1) was isolated from sawdust and identified based on the cultural, morphological and biochemical test as well as 16S rDNA analysis. It was studied for cellulolytic potential such as optimum condition for cellulase production and cellulase activities. Cellulase production by the bacteria was optimized using different environmental and nutritional factors such as incubation period, pH, temperature, carbon and nitrogen sources. Activity of the crude cellulase was also determined by using same factors along with metal ions, inhibitors and reductants. Results: In this study a cellulolytic bacteria (FFS1) was locally isolated from sawdust and identified as Lysinibacillus fusiformis (Accession No MG 930050) based on the cultural, morphological and biochemical test as well as 16S rDNA analysis. It was evaluated for its cellulolytic potential using different physicochemical parameters. Cellulase production required pH 7.5, temperature 300C-400C whereas cellulase activity required pH 9.5, temperature 350C. CMC and Beef Extract were best as Carbon and nitrogen sources for both the cellulase production and activity respectively. Cellulase activity was strongly inactivated by Ag+ and Fe3+ ions whereas detergent SDS also inhibited the cellulase activity. As the enzyme produced by the bacteria showed highest specificity to CMC as substrate it may be an Endo-β-1,4-Glucanase. Therefore, in optimized condition the bacteria will be the important tool for future use if its cellulase is further characterized. Conclusion: Locally isolated cellulolytic bacteria Lysinibacillus fusiformis have the potential for practical use for bioremediation through degradation of huge amount of sawdust

One of the leading limiting factors for wider industrial production and commercialization of microbial biopesticides refers to the high costs of cultivation media. The selection of alternative sources of macronutrients crucial for the growth and metabolic activity of the producing microorganism is a necessary phase of the bioprocess development. Gaining a better understanding of the influence of the medium composition on the biotechnological production of biocontrol agents is enabled through bioprocess modelling and optimization. In the present study, after the selection of optimal carbon and nitrogen sources, two modelling approaches were applied to mathematically describe the behavior of the examined bioprocess—the production of biocontrol agents effective against aflatoxigenic Aspergillus flavus strains. The modelling was performed using four independent variables: cellulose, urea, ammonium sulfate and dipotassium phosphate, and the selected response was the inhibition-zone diameter. After the comparison of the results generated by the Response Surface Methodology (RSM) and the Artificial Neural Network (ANN) approach, the first model was chosen for the further optimization step due to the better fit of the experimental results. As the final investigation step, the optimal cultivation medium composition was defined (g/L): cellulose 5.0, ammonium sulfate 3.77, dipotassium phosphate 0.3, magnesium sulfate heptahydrate 0.3.

Cellulose represents the most abundant component of plant biomass on earth; it is degraded by cellulases, specific enzymes produced by microorganisms. However, cellulases of bacterial origin attract more interest due to their natural diversity and ability to inhabit a variety of niches, allowing the selection of cellulolytic strains resistant to environmental stresses. The screening of the cellulolytic activity of 398 bacteria isolated from various ecosystems in Algeria (cave, ruins, chott, thermal station, and rhizosphere of arid and semi-arid regions) was performed by the appearance of a hydrolysis zone on carboxymethylcellulose (CMC) medium. The cellulase activity on CMC (1%) broth allowed to select 26 strains among which 12 had the best activity (0.3 U/mL to 2.2 U/mL). Optimization of physicochemical parameters (salinity: 0–1 M NaCl; pH: 3, 4, 7, 9, and 11; temperature: 30, 45, and 50 °C; PEG8000: 0 and 30%) involved in growth and cellulose production showed that the majority of strains were mesophilic, neutrophilic, or alkali- tolerant and tolerant to 30% of PEG8000. The cellulase activity and stability under different stress allowed to retain five strains, which the most efficient. Based on the 16S-rRNA sequencing results, they belonged to the genus Bacillus. The physicochemical properties of cellulases (crude extract) showed a CMCase active over a wide range of pH (4 to 11), optimal at 50 °C and 60 °C. The inhibiting salinity effect on the activity was not detected and was negligible on the enzymatic stability. The residual CMCase activity remained between 40 and 70% in a temperature range between 40 and 70 °C, was stable over a wide range of saline concentrations (0–2000 mM), and was weakly affected at 30% of PEG8000. The crude enzyme extract was able to hydrolyze both soluble and insoluble cellulosic substrates. The evaluation of the hydrolysis capacity of lignocellulosic waste revealed the ability of tested strains to degrade wheat bran, barley bran, and corncob. In addition, the enzyme showed significant multi-stress resistance on solid and liquid media. By these characteristics, these cellulolytic bacteria could be attractive to be used in various industrial and biotechnology applications.

Synthesis, optimization via response surface methodology, and structural properties of carboxymethylcellulose/curcumin/graphene oxide biocomposite films/coatings for the shelf-life extension of shrimp

This study aimed to optimize the parameters, including medium formulations and culture conditions, for submerged fermentation (SmF) systems using a mutant strain of Trichoderma sp., Mut-4. Optimization was performed using the one-factor-at-a-time (OFAT) method to enhance cellulase activity and productivity. Parameters such as the blending ratio of carbon sources, type of nitrogen source, and initial pH were evaluated for their effects on enzyme activity and productivity. The optimal conditions were determined to be as follows: a 3:1 Avicel-to-cellulose ratio, yeast extract as the nitrogen source, and an initial pH of 5.5. Under these conditions, cellulase production was initiated earlier, and the activity of all cellulase components, along with protein concentration, increased by 1.17- to 1.43-fold at the flask scale and by 1.3- to 2.0-fold at the reactor scale. These results demonstrate the superior activity and productivity of Mut-4 under optimized conditions, highlighting its potential for application in large-scale cellulase production.

The effects of acetylated potato starch and sodium carboxymethyl cellulose on the quality of instant fried noodles

Botryococcus braunii has an outstanding ability to produce lipid; however, it is a slow-growing green microalgae. Statistical optimization of growth media was performed to faster growth and to increase lipid concentration. The effect of media composition on the growth of B. braunii LB572 was examined using fractional factorial design and central composite design. The media components examined include sodium carbonate, potassium phosphate, calcium chloride, magnesium sulfate, ferric citrate, and sodium nitrate. The results indicated that potassium phosphate and magnesium sulfate were major impact factors. The optimum concentrations of potassium phosphate and magnesium sulphate were found to be 0.058 and 0.09 g/L, respectively, for growth and 0.083 and 0.1 g/L, respectively, for lipid production. These values were validated using bubble column photobioreactors. Lipid productivity increased to 0.19 g/L/day in lipid-optimized media, with an average biomass productivity of 0.296 g/L/day and 64.96% w/w. In growth-optimized media, lipid productivity was 0.18 g/L/day, with an average biomass productivity of 0.304 g/L/day and 59.56% w/w.