Cellulolytic Potential Research Articles

Cellulolytic potential of fungi in wood degradation from an old house at the Medina of Fez

During the history of civilizations, advanced wood decay results from exposure to various agents for long periods of time. Bio-deterioration, under the influence of living organisms like fungi, can cause massive damage to historical monuments. In this work, we found that fungi participating in wood degradation share a single strategy for degrading wood polymers by secreting enzymes that break down the main constituents of wood such as cellulose, hemicellulose and lignin. WhilePenicillium commune, Penicillium granulatum andPenicillium chrysogenum showed the highest cellulase productivity and are therefore the most destructive for timber, other fungal species participate also in this biodegradation includingPenicillium crustosum, Penicillium expansum Cladosporium cladosporioides and a cellulotic specieThielavia hyalocarpa that we describe here for the first time.

Mutagenesis and Genotypic Characterization of Aspergillus niger FCBP-02 for Improvement in Cellulolytic Potential

Cellulase is a collective term that encompasses enzymes which catalyze reactions that participate in the degradation of insoluble cellulose to soluble carbohydrates. In the present study, production of extra cellular cellulases by a filamentous fungus, Aspergillus niger FCBP-02, was studied in solid-state fermentation (SSF) as well as in submerged fermentation (SmF). Trials were conducted to evaluate the effect of mutagenesis by UV irradiation (5-40 min) and ethyl methane sulfonate (EMS) treatment (50-300 microg mL(-1)) to obtain hyper active cellulase enzyme producers among the potential strains. The enzyme activity assays of parental and mutant strains clearly revealed significantly higher cellulase activity of mutant A-Ch-5.5 (96 Units mL(-1)), followed by A-UV-5.6 (71 Units mL(-1)) with respect to the wild strain of A. niger FCBP-02 (53.7 Units mL(-1)). The profile of genetic variability among wild and mutant derivatives was scrutinized through RAPD-PCR. The expression pattern of mutants exhibited that the mutants were isogenic variants of the wild type and the out performance of the mutants could be attributed to the change in genetic make up.

Efficacy of bacterial seed treatments for the control of Fusarium verticillioides in maize

Maize colonization by the phytopathogenic fungi Fusarium verticillioides leads to economical and food quality losses and also implicates potential health risks. In order to control this fungal species different strategies are being considered. In the present work we investigated the in situ effects of the use of two in vitro proven bacterial biological control agents against Fusarium verticillioides, using maize seedlings grown in greenhouse conditions. The treatment of maize seeds with Fusarium verticillioides 107 spores ml−1 negatively affected the length of the stem and the weight of the root systems of resultant seedlings, and also reduced the numbers of non-rhizospheric organisms with ammonification and nitrification activities. The addition of Bacillus amyloliquefaciens or Microbacterium oleovorans, at a concentration of 109 colony forming units ml−1, to the seeds significantly reduced Fusarium verticillioides count at the root inner tissues of resultant seedlings. When testing the impact of bacterial treatments on soil populations, no alterations with respect to control numbers of organisms with nitrification, ammonification or cellulolytic potentials were observed. Culturable bacterial richness and diversity calculated at the rhizoplane and root inner tissues of maize seedlings neither changed in bacterized treatments when compared to control treatment. Our study showed that the Fusarium verticillioides in vitro proven antagonists, Bacillus amyloliquefaciens and Microbacterium oleovorans (at 109 colony forming units ml−1), were also effective at greenhouse conditions without causing major changes in culturable rhizospheric and endophytic microbial richness and diversity.

Insight into Trichoderma reesei’s genome content, organization and evolution revealed through BAC library characterization

Trichoderma reesei is an important industrial fungus known for its ability to efficiently secrete large quantities of protein as well as its wide variety of biomass degrading enzymes. Past research on this fungus has primarily focused on extending its protein production capabilities, leaving the structure of its 33 Mb genome essentially a mystery. To begin to address these deficiencies and further our knowledge of T. reesei’s secretion and cellulolytic potential, we have created a genomic framework for this fungus. We constructed a BAC library containing 9216 clones with an average insert size of 125 kb which provides a coverage of 28 genome equivalents. BAC ends were sequenced and annotated using publicly available software which identified a number of genes not seen in previously sequenced EST datasets. Little evidence was found for repetitive sequence in T. reesei with the exception of several copies of an element with similarity to the Podospora anserina transposon, PAT. Hybridization of 34 genes involved in biomass degradation revealed five groups of co-located genes in the genome. BAC clones were fingerprinted and analyzed using fingerprinted contigs (FPC) software resulting in 334 contigs covering 28 megabases of the genome. The assembly of these FPC contigs was verified by congruence with hybridization results.

Biodegradation and bioconversion of cellulose wastes using bacterial and fungal cells immobilized in radiopolymerized hydrogels

Annually, great amounts of cellulose wastes, which could be measured in many billions of tons, are produced worldwide as residues from agricultural activities and industrial food processing. Consequently, the use of microorganisms in order to remove, reduce or ameliorate these potential polluting materials is a real environmental challenge, which could be solved by a focused research concerning efficient methods applied in biological degradation processes. In this respect, the scope of this chapter is to present the state of the art concerning the biodegradation of redundant cellulose wastes from agriculture and food processing by continuous enzymatic activities of immobilized bacterial and fungal cells as improved biotechnological tools and, also, to report on our recent research concerning cellulose wastes biocomposting to produce natural organic fertilizers and, respectively, cellulose bioconversion into useful products, such as: ‘single-cell protein’ (SCP) or ‘protein-rich feed’ (PRF). In addition, there are shown some new methods to immobilize microorganisms on polymeric hydrogels such as: poly-acrylamide (PAA), collagen-poly-acrylamide (CPAA), elastin-poly-acrylamide (EPAA), gelatin-poly-acrylamide (GPAA), and poly-hydroxy-ethyl-methacrylate (PHEMA), which were achieved by gamma polymerization techniques. Unlike many other biodegradation processes, these methods were performed to preserve the whole viability of fungal and bacterial cells during long term bioprocesses and their efficiency of metabolic activities. The immobilization methods of viable microorganisms were achieved by cellular adherence mechanisms inside hydrogels used as immobilization matrices which control cellular growth by: reticulation size, porosity degree, hydration rate in different colloidal solutions, organic and inorganic compounds, etc. The preparative procedures applied to immobilize bacterial and fungal viable cells in or on radiopolymerized hydrogels and, also, their use in cellulose wastes biodegradation are discussed in detail. In all such performed experiments were used pure cell cultures of the following cellulolytic microorganisms: Bacillus subtilis and Bacillus licheniformis from bacteria, and Pleurotus ostreatus, Pleurotus florida, and Trichoderma viride from fungi. These species of microorganisms were isolated from natural habitats, then purified by microbiological methods, and finally, tested for their cellulolytic potential. The cellulose biodegradation, induced especially by fungal cultures, used as immobilized cells in continuous systems, was investigated by enzymatic assays and the bioconversion into protein-rich biomass was determined by mycelial protein content, during such long time processes. The specific changes in cellular development of immobilized bacterial and fungal cells in PAA hydrogels emphasize the importance of physical structure and chemical properties of such polymeric matrices used for efficient preservation of their metabolic activity, especially to perform in situ environmental applications involving cellulose biodegradation by using immobilized microorganisms as long-term viable biocatalysts.

Cellulase Formation by Species ofTrichodermasect.Longibrachiatumand ofHypocreaspp. with Anamorphs Referable toTrichodermasect.Longibrachiatum

Kubicek, C. P., Bölzlbauer, U.M., Kovacs, W., Mach, R. L., Kuhls, K., Lieckfeldt, E., Börner, T., and Samuels, G. J. 1996. Cellulase formation by species ofTrichodermasect.Longibrachiatumand ofHypocreaspp. with anamorphs referable toTrichodermasect.Longibrachiatum. Fungal Genetics and Biology20,105–114. The cellulolytic potential of the wild-type strain ofTrichoderma reeseiwas compared to other members ofTrichodermasect.LongibrachiatumandHypocreaspp. that have anamorphs referable to that section. There was high diversity even within the same species (as defined by morphological and macromolecular characters). Differences, where notable, were more pronounced for carboxymethyl-cellulase activity than for filter paper activity. High cellulase activities were observed for several strains ofT. longibrachiatumandT. citrinoviride,whereasT. parceramosumformed only low levels of activity. Among the corresponding teleomorphs, most strains ofH. schweinitziiwere comparatively poor producers, whereas the highest percentage of high producers was found amongH. jecorinaisolates, and many strains were even more active than the parentT. reeseiQM 6a. Immunoblot analysis of corresponding culture filtrates of variousH. jecorinastrains showed that the three major cellulase proteins (cellobiohydrolase I, cellobiohydrolase II, and endoglucanase I) were present in culture filtrates and theirMrwas identical to that of the respectiveT. reeseiproteins. ELISA analysis demonstrated that these enzymes were also present in the same relative proportions in culture filtrates fromH. jecorinaandT. reesei.With the aid of primers, corresponding to conserved sequences in the cellobiohydrolase I-encoding genecbh1,a fragment of this gene was amplified from selected strains ofH. jecorina, T. reesei, T. longibrachiatum, T. citrinoviride,andH. schweinitzii.The fragments had the same size in all fungi. Cleavage of this fragment withHhaI produced a RFLP pattern which was identical inH. jecorinaandT. reesei,but different in the other species. In the latter, the RFLP pattern was also species specific. These results provide support for a close genetic similarity ofT. reeseiandH. jecorinacellulases. In the latter, an ascomycetous model system for cellulase biosynthesis is now availabale. The results further indicate that other anamorphs ofTrichodermasectionLongibrachiatumare promising sources of high cellulase production.

Cellulose digestion in insects

1. 1. Cellulose digestion has been demonstrated in the Thysanura (Lepismatidae), Orthoptera (Cryptocercidae, Blattidae). Isoptera (Mastotermitidae. Kalotermitidae. Hodotermitidae, Rhinotermitdae, Termitidae). Coleoptera (Buprestidae. Anobiidae, Scarabaeidae, Cerambycidae), and Hymenoptera (Siricidae). 2. 2. In all but the scarab beetles, cellulose digestion is brought about by a complex of three types of enzymes (C 1-cellulases, C x-cellulases, and cellobiases), as in fungi. 3. 3. Many insects are able to synthesize their own C x-cellulases and cellobiases, but few (if any) can synthesize C 1-cellulases. 4. 4. Insects compensate for their inability to synthesize C 1-cellulases by exploiting the cellulolytic potential of protozoa, bacteria, or fungi. 5. 5. The maintenance of permanent populations of hindgut protozoa, the maintenance of permanent populations of hindgut bacteria, and the ingestion of fungal cellulases are described as three distinct mechanisms by which insects have been shown to use the cellulolytic potential of microorganisms. 6. 6. A process in which ingested cellulolytic bacteria proliferate in one region of the gut at the expense of ingested cellulose, only to be digested and assimilated in a more posterior section, is a fourth possible mechanism by which insects might accomplish the digestion of cellulose with the help of microorganisms.