Rapid Isolation of the Trichoderma Strain with Higher Degrading Ability of a Filter Paper and Superior Proliferation Characteristics Using Avicel Plates and the Double-Layer Selection Medium

The cost of cellulase is still a problem for bioethanol production. As the cellulase of Trichoderma reesei is applicable for producing ethanol from cellulosic materials, the cellulase productivity of this fungus should be increased. Therefore, we attempted to develop a system to isolate the strain with higher degrading ability of a filter paper and superior proliferation characteristics among the conidia treated with the mitotic arrester, colchicine. When green mature conidia of T. reesei RUT C-30 were swollen, autopolyploidized, and incubated in the double-layer selection medium containing Avicel, colonies appeared on the surface earlier than the original strain. When such colonies and the original colony were incubated on the Avicel plates, strain B5, one of the colonies derived from the colchicine-treated conidia, showed superior proliferation characteristics. Moreover, when strain B5 and the original strain were compared in the filter paper degrading ability and the cellulose hydrolyzing activity, strain B5 was also superior to the original strain. It was suspected that superior proliferation characteristics of strain B5 reflects higher filter paper degrading ability. Thus, we concluded that the Trichoderma strain with higher degrading ability of a filter paper and superior proliferation characteristics can be isolated using Avicel plates and the double-layer selection medium.     

Endocellulase and exocellulase activities of two Streptomyces strains isolated from a forest soil

Two Streptomyces strains, M7a and M23, from a Brazilian forest soil were evaluated for the cellulase production of their superna tants after growth in a microcrystalline cellulose medium, using carboxy methylcellulose and filter paper as substrates at different temperatures and pH values. Endoglucanase and exoglucanase activities were compared to a commercial Trichoderma reesei cellulase using fluorogenic conjugated substrates Similar specific activities were observed for the enzyme preparations of strain M23 and T. reesei. For M7a the activities were about seven times higher than those obtained for T. reesei. Extracellular or cell-associated cellobiase activities were not detected in both strains.

     

Cellulase production by Trichoderma reesei using sawdust hydrolysate

Sawdust hydrolysates were investigated for their ability to support cell growth and cellulase production, and for potential inhibition of Trichoderma reesei Rut C30. Simultaneous fermentations were conducted to compare the hydrolysate-based media with the controls having equivalent concentrations of glucose and Avicel cellulose. Six hydrolysates differing in the boiling durations in the hydrolysis procedure were evaluated. The hydrolysates were found to support cell growth and induce active cellulase synthesis. The maximum specific cellulase production rate was 0.046 filter paper units (FPU)/(g of cells · h) in the hydrolysate-based systems, much higher than that (0.017 FPU/[g of cells · h]) in the controls.     

Dynamics of cellulase production by glucose grown cultures of Trichoderma reesei Rut-C30 as a response to addition of cellulose

An economic process for the enzymatic hydrolysis of cellulose would allow utilization of cellulosic biomass for the production of easily fermentable low-cost sugars. New and more efficient fermentation processes are emerging to convert this biologic currency to a variety of commodity products with a special emphasis on fuel ethanol production. Since the cost of cellulase production currently accounts for a large fraction of the estimated total production costs of bioethanol, a significantly less expensive process for cellulase enzyme production is needed. It will most likely be desirable to obtain cellulase production on different carbon sources—including both polymeric carbohydrates and monosaccharides. The relation between enzyme production and growth profile of the microorganism is key for designing such processes. We conducted a careful characterization of growth and cellulase production by the soft-rot fungus Trichoderma reesei. Glucosegrown cultures of T. reesei Rut-C30 were subjected to pulse additions of Solka-floc (delignified pine pulp), and the response was monitored in terms of CO₂ evolution and increased enzyme activity. There was an immediate and unexpectedly strong CO₂ evolution at the point of Solka-floc addition. The time profiles of induction of cellulase activity, cellulose degradation, and CO₂ evolution are analyzed and discussed herein.     

Cellulase production on bagasse pretreated with hot water

Because pretreatment of biomass with hot water only in differential flow systems offers very digestible cellulose and potentially less inhibition by liquid hydrolysate, solids and liquid hydrolysate from bagasse pretreated with hot water were fed to a batch cellulase production system using the Rut C30 strain of Trichoderma reesei to determine the suitability of these substrates for cellulase production. The organism was found to be sensitive to inhibitors in the liquid hydrolysate but could be adapted to improve its tolerance. In addition, filtering of the material reduced inhibitory effects. The organism was also sensitive to some component in the solids, and they had to be washed heavily to achieve good growth and cellulase production rates. Even then, a lag was found before enzyme production would commence on pretreated solids whereas no such lag was experienced with Solka Floc. However, once enzyme production began, as high and even somewhat greater cellulase productivities were realized with washed pretreated solids. Adding lignin to Solka Floc delayed enzyme production, suggesting that lignin or other materials in the lignin solids could cause the lag observed for pretreated bagasse, but more studies are needed to resolve the actual reason for this delay.     

Polykaryon formation using a swollen conidium of Trichoderma reesei

The cellulolytic fungus, Trichoderma has oval and mononucleate conidia. When these conidia are incubated in a liquid medium, they begin to swell and their shape becomes spherical followed by an increase in inner space. In such swollen conidia, it is possible to produce a larger autopolyploid nucleus using a mitotic arrester compared with the case of the original conidia. In this study, polykaryon formation was attempted using these swollen conidia. Dried mature green conidia of Trichoderma reesei QM6a (IFO 31326) were incubated in Mandel's medium in order to swell. The swollen conidia were treated with a mitotic arrester, colchicine, for autopolyploidization. After autopolyploidization, polykary on formation was carried out using the swollen conidia. After the treatment, multiple smaller nuclei whose diameter was almost the same as that of the original strain were generated from an autopolyploid nucleus in a swollen conidium. A cellulase hyperproducer without decrease in growth rate could be selected using such swollen conidia.     

Diminished Respirative Growth and Enhanced Assimilative Sugar Uptake Result in Higher Specific Fermentation Rates by the MutantPichia stipitis FPL-061

A mutant strain ofPichia stipitis, FPL-061, was obtained by selecting for growth on L-xylose in the presence of respiratory inhibitors. The specific fermentation rate of FPL-061, was higher than that of the parent,Pichia stipitis CBS 6054, because of its lower cell yield and growth rate and higher specific substrate uptake rate. With a mixture of glucose and xylose, the mutant strain FPL-061 produced 29.4 g ethanol/L with a yield of 0.42 g ethanol/g sugar consumed. By comparison, CBS 6054 produced 25.7 g ethanol/L with a yield of 0.35 gJg. The fermentation was most efficient at an aeration rate of 9.2 mmoles O₂ L^-1 h^-1. At high aeration rates (22 mmoles O₂ L^-1 h^-1), the mutant cell yield was less than that of the parent. At low aeration rates, (1.1 to 2.5 O₂ L^-1 h^-1), cell yields were similar, the ethanol formation rates were low, and xylitol accumulation was observed in both the strains. Both strains respired the ethanol once sugar was exhausted. We infer from the results that the mutant, P.stipitis FPL-061, diverts a larger fraction of its metabolic energy from cell growth into ethanol production.     

Cellulase Production by Pink Pigmented Facultative Methylotrophic Strains (PPFMs)

Pink pigmented facultative methylotrophs (PPFM) isolated from water samples of Cooum and Adyar rivers in Chennai and soil samples of forests located in various districts of Tamil Nadu, India were screened for cellulase production using carboxymethylcellulose agar (CMC agar) medium. The strains showed wide variations in the production of clearing zones around the colonies on CMC agar medium flooded with Congo red. CMCase and filter paper assays were used to quantitatively measure the cellulase activity of 13 PPFM strains. Among the strains, Methylobacterium gregans, MNW 60, MHW 109, MSF 34, and MSF 40 showed cellulolytic activity ranging from 0.73 to 1.16 U mL⁻¹ with wide temperature (35–65°C) and pH (5 to 8) tolerance. SDS-PAGE analysis of the crude enzyme of PPFM strain MNW 60 exhibited several protein bands, and zymogram analysis revealed two dimeric cellulase bands with molecular mass of ~92 and 42 kDa. Scanning electron microscopic studies revealed significant morphological differences between the cells grown in normal and CMC amended medium. The strain MNW 60 was identified as Methylobacterium sp. based on biochemical, physiological, and morphological analyses, and the methylotrophic nature was authenticated by the presence of mxaF gene, encoding methanol dehydrogenase as a key indicator enzyme of methylotrophs, with 99% similarity to Methylobacterium lusitanum. With the 16S ribosomal RNA sequence showing 97% similarity to M. lusitanum strain MP2, this can be proposed as a novel taxon of the genus Methylobacterium. The study forms the first detailed report on the extracellular cellulase production by pink pigmented Methylobacterium sp., and it is expected that this might be the basis for further studies on cellulase production by PPFMs to explore the molecular mechanism, strain improvement, and large-scale cellulase production for its application.     

Effect of Different Carbon Sources on Cellulase Production by Marine Strain Microbulbifer hydrolyticus IRE-31-192

Cellulase is an important enzyme that can be used to breakdown lignocellulose into glucose. Microbulbifer hydrolyticus IRE-31(ATCC 700072) is a kind of marine bacterium, which could grow in high salinity medium and has fast-strong growth ability. In this study, a novel strain was screened from Microbulbifer hydrolyticus IRE-31 through mutations to produce cellulase. The effect of different carbon sources on the growth as well as on the production of cellulase of the new strain was studied. Carboxymethyl-cellulase (CMCase) activity selected to represent cellulase was proven to be effectively promoted while xylose, galactose, and melibiose as well as glucose were used as carbon sources. When xylose and glucose were chosen to be further investigated, 472.57 U/L and 266.01 U/L CMCase activity were obtained from 30 g/L glucose and 10 g/L xylose, respectively. These results clarified the effect of different carbon sources on the production of cellulase, which laid a good foundation for the further research in the production of cellulase by marine bacteria.

     

Random mutagenesis and media optimisation for hyperproduction of cellulase from Bacillus species using proximally analysed Saccharum spontaneum in submerged fermentation

This study deals with the isolation of novel mutant of Bacillus and optimisation of media for the hyperproduction of cellulase. Cellulase-producing Bacillus PC-BC6 was subjected to physical and chemical mutagenesis to enhance the cellulolytic potential. Later, mutagenesis isolates were screened both qualitatively and quantitatively. Among all the tested isolates, Bacillus N3 yielded maximum (CMCase 1250 IU/mL/min and FPase 629 IU/mL/min) activity. The Bacillus N3 strain exhibited 1.7-fold more enzyme production as compared with the parental strain. Proximate analysis of untreated and pretreated Saccharum spontaneum was carried out to improve cellulase production. Three different media were tested for the production of cellulase, among which M2 medium containing MgSO₄, pretreated S. spontaneum, K₂HPO₄, (NH₄)₂SO₄ and peptone was found to be the best for maximum enzyme production by mutant Bacillus N3.     

Cloning, characterization, and expression of a new cry2Ab gene from Bacillus thuringiensis strain 14-1

Bacillus thuringiensis is the major source for transfer of genes to impart insect resistance in transgenic plants. Cry2A proteins of B. thuringiensis are promising candidates for management of resistance development in insects owing to their difference from the currently used Cry1A proteins, in structure and insecticidal mechanism. The cry2Ab gene was found to lack a functional promoter and, hence, is cryptic in nature. The cry2Ab7 gene was cloned from a new indigenous B. thuringiensis strain, 14-1. Nucleotide sequencing of the cry2Ab gene cloned from B. thuringiensis strain 14-1 revealed an open reading frame of 1902 bp. The deduced amino acid sequence of Cry2Ab of B. thuringiensis strain 14-1 showed a variation in three amino acid residues in comparison to the holotype sequence, Cry2Ab1. Expression of the newly cloned cry2Ab gene was studied in an acrystalliferous strain of B. thuringiensis (4Q7) by fusing the cry2Ab gene downstream of cry2Aa promoter and orf1+orf2 sequences. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a spore-crystal mixture obtained from transformants of B. thuringiensis strain 4Q7 showed production of Cry2Ab protein of about 65 kDa. Alkali solubilized Cry2Ab7 protein showed toxicity against Helicoverpa armigera neonates.     

[14C]-Cellulase-humic complexes that are stable in soil

[¹⁴C]-Cellulase was extracted from the culture medium ofTrichoderma viride and an attempt made to complex it with humic acid by adsorption. The result showed that the humic acid extracted from soil does not form a stable complex with [¹⁴C]-cellulase. In contrast, the flocculation of humic acid by 0.025M Ca²⁺ in the presence of the cellulase resulted in the formation of stable humic-cellulase complexes. DEAE cellulose chromatography of cellulase-humic complex revealed that cellulase could not be separated from the humic acid. Enzyme activity was only eluted along with humic acid upon increasing gradient concentration from 1.0 to 1.5M NaCl. Furthermore, in order to test its stability, the enzyme-humic complex was incorporated into fresh soil for 90 d. During this period the enzyme-humic complex remained stable. The cellulase-humic complex was then extracted from soil. Fractionation of the extract on DEAE cellulose and G100 sephadex revealed that cellulase activity could not be separated from humic acid and was again eluted in the form of enzyme-humic complex. This confirmed the stability of cellulase-humic complex in soil.     

Overproduction of Glucoamylase by a Deregulated Mutant of a Thermophilic Mould <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis>

Thermomucor indicae-seudaticae, a glucoamylase-producing thermophilic mould, was mutagenised using nitrous acid and gamma (⁶⁰Co) irradiation in a sequential manner to isolate deregulated mutants for enhanced production of glucoamylase. The mutants were isolated on Emerson YpSs agar containing a non-metabolisable glucose analogue 2-deoxy-d-glucose (2-DG) for selection. The preliminary screening for glucoamylase production using starch–iodine plate assay followed by quantitative confirmation in submerged fermentation permitted the isolation of several variants showing varying levels of derepression and glucoamylase secretion. The mutant strain T. indicae-seudaticae CR19 was able to grow in the presence of 0.5 g l⁻¹ 2-DG and produced 1.8-fold higher glucoamylase. As with the parent strain, glucoamylase production by T. indicae-seudaticae CR19 in 250-ml Erlenmeyer flasks attained a peak in 48 h of fermentation, showing higher glucoamylase productivity (0.67 U ml⁻¹ h⁻¹) than the former (0.375 U ml⁻¹ h⁻¹). A large-scale cultivation in 5-l laboratory bioreactor confirmed similar fermentation profiles, though the glucoamylase production peak was attained within 36 h attributable to the better control of process parameters. Although the mutant grew slightly slow in the presence of 2-DG and exhibited less sporulation, it showed faster growth on normal Emerson medium with a higher specific growth rate (0.138 h⁻¹) compared to the parent strain (0.123 h⁻¹). The glucoamylase produced by both strains was optimally active at 60 °C and pH 7.0 and displayed broad substrate specificity by cleaving α-1,4- and α-1,6-glycosidic linkages in starch, amylopectin, amylose and pullulan. Improved productivity and higher specific growth rate make T. indicae-seudaticae CR19 a useful strain for glucoamylase production.     

Enzyme production, growth, and adaptation of T. reesei strains QM9414, L-27, RL-P37, and rut C-30 to conditioned yellow poplar sawdust hydrolysate

National Renewable Energy Laboratory (NREL) has developed a conditioning process that decreases acetic acid levels in pretreated yellow poplar hydrolysate. Trichoderma reesei is sensitive to acetic acid and this conditioning method has enabled applied cellulase production with hardwoods. T. reesei strains QM9414, L-27, RL-P37, and Rut C-30 were screened for growth on conditioned hydrolysate liquor. Tolerance to hydrolysate was found to be strain-dependent. Strain QM9414 was adapted to grow in 80% (v/v) conditioned hydrolysate (40 g/L of soluble sugars and 1.6 g/L acetic acid from pretreated poplar). However, enzyme production was highest at 20% (v/v) hydrolysateusing strain L-27. Cellulasetiters of 2–3 International Filter Paper Units (IFPU)/mL were achieved using pretreated yellow poplar liquors and solids as the sole carbon sources.     

Production of cellulase/β-glucosidase by the mixed fungi culture of Trichoderma reesei and Aspergillus phoenicis on dairy manure

A cellulase production process was developed by growing the fungi Trichoderma reesei and Aspergillus phoenicis on dairy manure. T. reesei produced a high total cellulase titer (1.7 filter paper units [FPU]/mL, filter paper activity) in medium containing 10 g/L of manure (dry basis [w/w]), 2 g/L KH₂PO₄, 2 mL/L of Tween-80, and 2mg/L of CoCl₂. However, β-glucosidase activity in the T. reesei-enzyme system was very low. T. reesei was then cocultured with A. phoenicis to enhance the β-glucosidase level. The mixed culture resulted in a relatively high level of total cellulase (1.54 FPU/mL) and β-glucosidase (0.64 IU/mL). The ratio of β-glucosidase activity to filter paper activity was 0.41, suitable for hydrolyzing manure cellulose. The crude enzyme broth from the mixed culture was used for hydrolyzing the manure cellulose, and the produced glucose was significantly (p<0.01) higher than levels obtained by using the commercial enzyme or the enzyme broth of the pure culture T. reesei.     

Invertase production on solid-state fermentation by Aspergillus niger strains improved by parasexual recombination

Invertase production by Aspergillus niger grown by solid-state fermentation was found to be higher than by conventional submerged fermentation. The haploid mutant strains Aw96-3 and Aw96-4 showed better productivity of various enzymes, as compared to wild-type parental strain A. niger C28B25. Here we use parasexual crosses of those mutants to increase further the productivity of invertase in solid-state fermentation. We isolated both a diploid (DAR2) and an autodiploid (AD96-4) strain, which were able to grow in minimal medium after mutation complementation of previously isolated haploid auxotrophic strains. Invertase production was measured in solid-state fermentation cultures, using polyurethane foam as an inert support for fungal growth. Water activity value (Aw) was adjusted to 0.96, since low Aw values are characteristic in some solid-state fermentation processes. Such diploid strains showed invertase productivity levels 5–18 times higher than levels achieved by the corresponding haploid strains. For instance, values for C28B25, Aw96-3, Aw96-4, DAR2, and AD96-4 were 441, 254, 62, 1324, and 2677 IU/(L·h), respectively. These results showed that genetic recombination, achieved through parasexual crosses in A. niger, results in improved strains with potential applications for solid-state fermentation processes.     

Production, purification, and characterization of a low-molecular-mass xylanase from Aspergillus sp. and its application in baking

An extracellular xylanase produced by a Mexican Aspergillus strain was purified and characterized. Aspergillus sp. FP-470 was able to grow and produce extracellular xylanases on birchwood xylan, oat spelt xylan, wheat straw, and corncob, with higher production observed on corncob. The strain also produced enzymes with cellulase, amylase, and pectinase activities on this substrate. A 22-kDa endoxylanase was purified 30-fold. Optimum temperature and pH were 60°C and 5.5, respectively, and isoelectric point was 9.0. The enzyme has good stability from pH 5.0 to 10.0 retaining >80% of its original activity within this range. Half-lives of 150 min at 50°C and 6.5 min at 60°C were found. K _m and activation energy values were 3.8 mg/mL and 26 kJ/mol, respectively, using birch wood xylan as substrate. The enzyme showed a higher affinity for 4-O-methyl-d-glucuronoxylan with a K _m of 1.9 mg/mL. The enzyme displayed no activity toward other polysaccharides, including cellulose. Baking trials were conducted using the crude filtrate and purified enzyme. Addition of both preparations improved bread volume. However, addition of purified endoxylanase caused a 30% increase in volume over the crude extract.     

Liquid fluidized bed starter culture ofTrichoderma reesei for cellulase production

A starter culture ofTrichoderma reesei (Rut-C30) prepared in a liquid fluidized bed reactor (LFBR) gave better growth and greater cellulase production in submerged fermentation than a conventional shake flask inoculum. The LFBR starter was prepared by first coatingT. reesei spores to 0.25 mm size corncob (1.0x10⁸g^-1) in a medium containing 1.0% corncob, 0.5 gL^-1 xylose and 0.1 gL^-1 lactose in a balanced salt solution, then fluidizing the particles in the LFBR for 36 h to allow germination of the spores, and covering the particles with an approx 30 μm thick biofilm. This biofilm that developed in constant adherence to the lignocellulosic carrier, apparently became well adapted to grow rapidly on insoluble cellulose substrates (Solca Floc), and had the enzymes of the cellulase complex induced for increased cellulase production. The LFBR starter used in a stirred tank reactor (STR) gave 15 gL^-1 biomass production and 6.5 IU mL^-1 overall cellulase activity with a volumetric productivity of 64 IU L^-1h^-1 in a 5 d fermentation, compared with a 7 d shake flask inoculum that gave 11 gL^-1 biomass and 3.2 IU mL^-1 cellulase activity, with a volumetric productivity of 31IU L^-1h^-1. The LFBR starter culture retained its viability in dry storage for 6–9 mo.     

Effect of pH on cellulase production of Trichoderma ressei RUT C30

Currently, the high market price of cellulases prohibits commercialization of the lignocellulosics-to-fuel ethanol process, which utilizes enzymes for saccharification of cellulose. For this reason research aimed at understanding and improving cellulase production is still a hot topic in cellulase research. Trichoderma reesei RUT C30 is known to be one of the best hyper producing cellulolytic fungi, which makes it an ideal test organism for research. New findings could be adopted for industrial strains in the hope of improving enzyme yields, which in turn may result in lower market price of cellulases, thus making fuel ethanol more cost competitive with fossil fuels. Being one of the factors affecting the growth and cellulase production of T. reesei, the pH of cultivation is of major interest. In the present work, numerous pH-controlling strategies were compared both in shake-flask cultures and in a fermentor. Application of various buffer systems in shake-flask experiments was also tested. Although application of buffers resulted in slightly lower cellulase activity than that obtained in non-buffered medium, β-glucosidase production was increased greatly.     

Expression and high-level secretion of Trichoderma reesei endoglucanase I in Yarrowia lipolytica

The endoglucanase I (EGI) from fungus Trichoderma reesei was cloned, expressed, and secreted from Yarrowia lipolytica using the XPR2 promoter. The signal sequence of EGI transferred from T. reesei was efficiently processed in the Y. lipolytica secretory pathway and directed the secretion of active EGI into the culture medium. However, the recombinant EGI produced from YLCSIn strain was hyperglycosylated and significantly larger than the native enzyme produced by the parent strain. The expression of EGI using XPR2 preproregion has caused secretion of modified proteins that still retained cellulase activity. This resulted from imprecise processing of the N-terminus of recombinant protein. While the batch culture produced 5 mg EGI/L from YLCSIn strain, the EGI yield was increased approx 20-fold when the fed-batch fermentation process strategy in combination with the high-cell density cultivation technique was employed. These results showed that the Y. lipolytica is a useful host organism for production of a large amount of large size heterologous proteins, especially when used in combination with high-cell density and fed-batch culture techniques.