Depolymerization of cellulose to glucose requires the synergistic action of three key cellulases, endoglucanase (E.C. 3.2.1.4), exoglucanase (E.C. 3.2.1.176) (E.C. 3.2.1.91) and β-glucosidase (E.C. 3.2.1.21). They belong to the glycoside hydrolase (GH) family and catalyze the hydrolysis of glyosidic linkages depolymerizing cellulose to fermentable sugars. Cellulases are naturally produced by a wide spectrum of bacteria and fungi. These enzymes usually exist as cellulosomes attached to the cell wall of bacteria but are secreted into environments in fungi. They exist either as monomers or multimers with each monomer having a simple architecture containing a cellulose binding domain (CBD) and a catalytic domain (CD) interlinked by a linker peptide. Thermophilic, mesophilic and psychrophilic cellulases are quite different in their structures and amino acid compositions. Post-translational modifications such as glycosylation contribute to enzyme function, multiplicity and stability. Recent advances in recombinant DNA technology allow fast identification of novel cellulase genes, large scale production of cellulases and their genetic modifications to make tailor-made enzymes for various applications. Cellulases have been traditionally used in food processing and textile industries. The rapid depletion of fossil fuels and production of cheap fermentable sugars from abundant renewable resources have increased the demand for cellulases in lignocellulose-based biorefinery. However, the lack of a better understanding of the mechanisms of individual cellulases, their synergistic actions and their high prices are the major bottlenecks yet to be overcome for large scale commercial applications of cellulases in lignocellulose-based biorefinery.

Microbial cellulases: Engineering, production and applications

Among invertebrate fungal pathogens, Beauveria bassiana has assumed a key role in management of numerous arthropod agricultural, veterinary and forestry pests. Beauveria is typically deployed in one or more inundative applications of large numbers of aerial conidia in dry or liquid formulations, in a chemical paradigm. Mass production is mainly practiced by solid-state fermentation to yield hydrophobic aerial conidia, which remain the principal active ingredient of mycoinsecticides. More robust and cost-effective fermentation and formulation downstream platforms are imperative for its overall commercialization by industry. Hence, where economics allow, submerged liquid fermentation provides alternative method to produce effective and stable propagules that can be easily formulated as dry stable preparations. Formulation also continues to be a bottleneck in the development of stable and effective commercial Beauveria-mycoinsecticides in many countries, although good commercial formulations do exist. Future research on improving fermentation and formulation technologies coupled with the selection of multi-stress tolerant and virulent strains is needed to catalyze the widespread acceptance and usefulness of this fungus as a cost-effective mycoinsecticide. The role of Beauveria as one tool among many in integrated pest management, rather than a stand-alone management approach, needs to be better developed across the range of crop systems. Here, we provide an overview of mass-production and formulation strategies, updated list of registered commercial products, major biocontrol programs and ecological aspects affecting the use of Beauveria as a mycoinsecticide.

The production and uses of Beauveria bassiana as a microbial insecticide.

Solid state fermentation for production of microbial cellulases: Recent advances and improvement strategies.

Geopolitical concerns (unstable supply of gasoline, environmental pollution, and regular price hikes), economic, and employment concerns have been prompting researchers, entrepreneurs, and policy makers to focus on harnessing the potential of lignocellulosic feedstock for fuel ethanol production and its commercialization. The carbohydrate skeleton of plant cell walls needs to be depolymerised into simpler sugars for their application in fermentation reactions as a chief carbon source of suitable ethnologic strains for ethanol production. The role of cellulolytic enzymes in the degradation of structural carbohydrates of the plant cell wall into ready-to-fermentable sugar stream is inevitable. Cellulase synergistically acts upon plant cell wall polysaccharides to release glucose into the liquid media. Cellulase predominantly dominates all the plant cell wall degrading enzymes due to their vast and diverse range of applications. Apart from the major applications of cellulases such as in detergent formulations, textile desizing, and development of monogastric feed for ruminants, their role in biorefinery is truly remarkable. This is a major area where new research tools based upon fermentation based formulations, biochemistry, and system biology to expedite the structure-function relationships of cellulases including cellulosomes and new designer enzymatic cocktails are required. In the last two decades, a considerable amount of research work has been performed on cellulases and their application in biomass saccharification. However, there are still technical and economic impediments to the development of an inexpensive commercial cellulase production process. Advancements in biotechnology such as screening of microorganisms, manipulation of novel cellulase encoding traits, site-specific mutagenesis, and modifications to the fermentation process could enhance the production of cellulases. Commercially, cheaper sources of carbohydrates and modified fermentation conditions could lead to more cost-effective production of cellulases with the goal to reduce the cost of ethanol production from lignocellulosics. Implementation of integrated steps like cellulase production and cellulase mediated saccharification of biomass in conjunction with the fermentation of released sugars in ethanol in a single step so called consolidated bio-processing (CBP) is very important to reduce the cost of bioethanol. This paper aims to explore and review the important findings in cellulase biotechnology and the forward path for new cutting edge opportunities in the success of biorefineries.

The realm of cellulases in biorefinery development

Microbial-processing of fruit and vegetable wastes for production of vital enzymes and organic acids: Biotechnology and scopes

Spores of Aspergillus sp. SU14 were treated repeatedly and sequentially with Co(60) γ-rays, ultraviolet irradiation, and N-methyl-N'-nitro-N-nitrosoguanidine. One selected mutant strain, Aspergillus sp. SU14-M15, produced cellulase in a yield 2.2-fold exceeding that of the wild type. Optimal conditions for the production of cellulase by the mutant fungal strain using solid-state fermentation were examined. The medium consisted of wheat-bran supplemented with 1% (w/w) urea or NH(4)Cl, 1% (w/w) rice starch, 2.5 mM MgCl(2), and 0.05% (v/w) Tween 80. Optimal moisture content and initial pH was 50% (v/w) and 3.5, respectively, and optimal aeration area was 3/100 (inoculated wheat bran/container). The medium was inoculated with 25% 48 hr seeding culture and fermented at 35℃ for 3 days. The resulting cellulase yield was 8.5-fold more than that of the wild type strain grown on the basal wheat bran medium.

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Improvement of Fungal Cellulase Production by Mutation and Optimization of Solid State Fermentation

The production of conidia of entomopathogenic Beauveria bassiana by solid-state fermentation was studied for the development of a biocontrol agent against aphid Myzus persicae. The optimal conditio...

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Optimization of Solid-State Fermentation for Improved Conidia Production of Beauveria bassiana as a Mycoinsecticide.

Cellulose, which is the most abundant renewable resource, is a polysaccharide composed of β-D-glucopyranosyl units joined by 1,4-glycosidic bonds (Gardner and Blackwell, 1974; Kolpak and Blackwell, 1976). Because cellulose can be utilized to produce ethanol, it is a promising alternative energy source for the production of fossil fuels. Cellulose is degraded by cellulases to reducing sugars and fermented by yeast or bacteria to ethanol (Duff and Murray, 1996).

Cellulases are produced by various microorganisms including Trichoderma sp., Chrysosporium sp., Fusarium sp., Sclerotium sp., Phanerochaete sp., Aspergillus sp., Schizophyllum sp. and Bacillus sp. (Selby and Maitland, 1967; Wood and Phillips, 1969; Toyama and Ogawa, 1975; Sternberg, 1976; Fan et al., 1987; Duff and Murray, 1996). Three major types of cellulases, endoglucanases (EC 3.2.1.4), exo-cellobiohydrolases (EC 3.2.1.91) and β-glucosidases (EC 3.2.1.21), have been identified to date (Coughlan, 1985; Coughlan and Ljungdahl, 1988).

A reduction in the cost of cellulase production, an improvement in cellulase activity and an increase in sugar yields are all vital to reducing the processing costs of bioethanol from cellulosic substrates (Zhang et al., 2006). During ethanol production from lignocellulosics, cellulases play a very important role in the cellulose digestion process and so far the cost of cellulases is very expensive due to the large amounts required for cellulose digestion (Duff and Murray, 1996; Himmel et al., 1997; Schell et al., 2003; Sun and Cheng, 2002). Therefore, the improvement of microbial strains for the over-production of cellulases has attracted attention in the commercial fermentation process.

The use of different mutagenic agents for strain improvement was demonstrated by Parekh et al. (2000). Additionally, treatment of F. oxysporum with ultraviolet (UV) followed by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) was used to improve carboxymethyl cellulase (CMCase) production (Kuhad et al., 1994). Moreover, Chand et al. (2005) used simultaneous treatment with NTG, ethidium bromide and UV or NTG combined with ethidium bromide to create mutant fungi that produced more CMCase and filter paper cellulase (FPase) than wild type fungi.

The purpose of this study was to screen microbial strains for the selection of a strain producing high level of cellulase and to improve the microbial strain further by mutation. In this study, γ-rays of Co60, UV and NTG were repeatedly and sequentially used to mutate the fungal strain to induce the hyper-production of cellulase.

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Fungal strain improvement for cellulase production using repeated and sequential mutagenesis.

In recent years, there has been a resurgence of interest in the use of fungi for the control of insect pests. This revival of interest has led to the large-scale production of several promising fungi candidates, and also to the marketing of the first commercial mycoinsecticides. Beauveria bassiana (Balsamo) Vuillemin is a fungus with a broad natural distribution; its potential to control more than 70 insect pests has been responsible for a substantial increase in interest in the large-scale production of the fungus for applications in the field (Thomas et al., 1987). Moreover, this fungus also appears to be innocuous to most non-target organisms.

Efforts to improve potential control agents often center around an appropriate mass-production method for the suitably large-scale production of the infective propagules. The mass production of insect-pathogenic fungi is a necessary prerequisite for any large-scale field application employing these fungi. The most frequently utilized technique for the cultivation of fungal spores is either a surface culture with a solid substrate, such as moistened wheat bran, millet or rice, or a submerged culture with a liquid medium (Feng et al., 2000). Submerged cultivation may have the advantage that fungi can be rapidly generated using conventional deep-tank fermentors, and the scale-up of this process is relatively easy. Submerged cultivation also has a number of highly desirable attributes: they are cheaper, particularly on a very large scale; environmental factors (pH, pO2, pCO2, nutrient levels) can be more readily controlled, and growth can be easily monitored (Bartlett and Jaronski, 1988). Harvesting from submerged fermentations is also made substantially easier, as the spores can be readily collected and concentrated via centrifugation, filtration, etc., and then dried.

The entomopathogenic hyphomycete fungi are generally easy to grow on a large scale, and can be cultivated on cheap media in submerged cultures. Depending on the strain, medium, and culture parameters, the fungal biomass is increased via vegetative growth forming either hyphal filaments, often with copious branching, or various forms of flocs and pellets of mycelia (Brown et al., 1988). The majority of fungal isolates are also capable of forming single cells via schizolytic separation at the septa or mechanical fragmentation of the hyphae, and can also be generated from the hyphae by yeast-like budding (Rombach, 1989; Jackson et al., 1990). Hyphal cells, which are also referred to as hyphal bodies or blastospores, are usually the only type of propagules produced in liquid fermentation schemes (Thomas et al., 1987; Jenkins et al., 1993).

Previously, we isolated the entomopathogenic B. bassiana KK5, which exhibits particularly high infectivity to aphids. Despite its high levels of infectivity, the large-scale application of this fungus for the control of aphids in the field is possible only in cases in which the fungus can be grown and can produce high spore titers economically, on readily available substrates. The primary objective of the present study was to evaluate the influences of different carbon and nitrogen sources and main culture parameters on blastospore yields in submerged fermentation cultures of the B. bassiana KK5 isolate. First, the effects of differing pH values, temperatures, medium compositions, and C/N ratios were optimized in shake flasks. Sporulation was then optimized using different substrates. Finally, blastospore production was carried out using these optimized parameters.

Production of Blastospore of Entomopathogenic Beauveria bassiana in a Submerged Batch Culture

A selected fungal strain, for production of the raw-starchdigesting enzyme by solid-state fermentation, was improved by two repeated sequential exposures to gamma-irradiation of Co60, ultraviolet, and four repeated treatments with Nmethyl- N'-nitrosoguanidine. The mutant strain Aspergillus sp. XN15 was chosen after a rigorous screening process, with its production of the raw-starch-digesting enzyme being twice that of usual wild varieties cultured under preoptimized conditions and in an unsupplemented medium. After 17 successive subculturings, the enzyme production of the mutant was stable. Optimal conditions for the production of the enzyme by solid-state fermentation, using wheat bran as the substrate, were accomplished for the mutant Aspergillus sp. XN15. With the optimal fermentation conditions, and a solid medium supplemented with nitrogen sources of 1% urea and 1% NH4NO3, 2.5 mM CoSO4, 0.05% (v/w) Tween 80, and 1% glucose, the mutant Aspergillus sp. XN15 produced the raw-starch-digesting enzyme in quantities 19.4 times greater than a typical wild variety. Finally, XN15, through simultaneous saccharification and fermentation of a raw rice corn starch slurry, produced a high level of ethanol with Yp/s of 0.47 g/g.

Improvement of a Fungal Strain by Repeated and Sequential Mutagenesis and Optimization of Solid-State Fermentation for the Hyper-Production of Raw-Starch-Digesting Enzyme

Tuan Anh Pham

Papers

Improvement of Fungal Cellulase Production by Mutation and Optimization of Solid State Fermentation

Optimization of Solid-State Fermentation for Improved Conidia Production of Beauveria bassiana as a Mycoinsecticide.

Fungal strain improvement for cellulase production using repeated and sequential mutagenesis.

Production of Blastospore of Entomopathogenic Beauveria bassiana in a Submerged Batch Culture

Improvement of a Fungal Strain by Repeated and Sequential Mutagenesis and Optimization of Solid-State Fermentation for the Hyper-Production of Raw-Starch-Digesting Enzyme