ABSTRACT Lignocellulose degrading enzymes were produced by solid substrate fermentation (SSF) wit... more ABSTRACT Lignocellulose degrading enzymes were produced by solid substrate fermentation (SSF) with Trichoderma and Gliocladium spp. on various agricultural by-products, such as spent brewing grain, corn fiber and wheat straw/wheat bran mixture. In a 10 day SSF, the hypercellulolytic mutant Trichoderma reesei Rut C30 produced 31 Filter Paper Unit (FPU)/g dry weight (DW) cellulase activity and 650 International Unit (IU)/g DW xylanase activity on corn fiber (corn seed hull), 55 FPU/g DW cellulase and 2400 IU/g DW xylanase activity on spent brewing grain. In a much faster 3 day fermentation, the wild strain Trichoderma hamatum TUB F-105 produced 7 FPU/g DW cellulase activity and 7600 IU/g DW xylanase activity on extracted (starch-removed) corn fiber. Gliocladium sp. TUB F-498 secreted 11 FPU/g DW cellulase activity in a 6 day SSF on a wheat straw-wheat bran 9:1 mixture. The hydrolyzing potential of SSF enzymes on native autologous substrates is substrate specific and does not always coincide with their filter paper activity. The F-498 and F-105 enzymes, at 5.0 FPU/g cellulose enzyme load hydrolyzed corn fiber and spent brewing grain more efficiently than the Rut C30 enzyme, although the Rut C30 enzyme hydrolyzed pure cellulose (Solka Floc) faster and more efficiently. The F-498 enzyme required lower enzyme loads for the hydrolysis of corn fiber and spent brewing grain than Rut C30. Due to adequate hydrolyzing potential, much reduced production cost and direct applicability, SSF enzymes are excellent candidates for enzymatic agrobiotechnological processes.
We have used isolates of Trichoderma spp. collected in South-East Asia, including Taiwan and West... more We have used isolates of Trichoderma spp. collected in South-East Asia, including Taiwan and Western Indonesia, to assess the genetic and metabolic diversity of endemic species of Trichoderma. Ninety-six strains were isolated in total, and identified at the species level by analysis of morphological and biochemical characters (Biolog system), and by sequence analysis of their internal transcribed spacer regions 1 and 2 (ITS1 and 2) of the rDNA cluster, using ex-type strains and taxonomically established isolates of Trichoderma as reference. Seventy-eight isolates were positively identified as Trichoderma harzianum/Trichoderma inhamatum (37 strains) Trichoderma virens (16 strains), Trichoderma spirale (8 strains), Trichoderma koningii (3 strains), Trichoderma atroviride (3 strains), Trichoderma asperellum (4 strains), Hypocrea jecorina (anamorph: Trichoderma reesei; 2 strains), Trichoderma viride (2 strains), Trichoderma hamatum (1 strain), and Trichoderma ghanense (1 strain). Analysis of biochemical characters revealed that T. virens, T. spirale, T. asperellum, T. koningii, H. jecorina, and T. ghanense formed clearly defined clusters, thus exhibiting speciesspecific metabolic properties. In biochemical character analysis T. atroviride and T. viride formed partially overlapping clusters, indicating that these two species may share overlapping metabolic characteristics. This behavior was even more striking with T. harzianum/T. inhamatum where genotypes defined on the basis of ITS1 and 2 sequences overlapped significantly with adjacent genotypes in the biochemical character analysis, and four strains from the same location (Bali, Indonesia) even clustered with species from section Longibrachiatum. The data indicate that the T. harzianum/T. inhamatum group represents species with high metabolic diversity and partially unique metabolic characteristics. Nineteen strains yielded three different ITS1/2 sequence types which were not alignable with any known species. They were also uniquely characterized by morphological and biochemical characters and therefore represent three new taxa of Trichoderma.
Canadian Journal of Botany-revue Canadienne De Botanique, 2003
... Phialides 26 verticillatae, ampulliformes, 4.5 7.0 × 3.45.4 µm. Conidia breviter cylindrac... more ... Phialides 26 verticillatae, ampulliformes, 4.5 7.0 × 3.45.4 µm. Conidia breviter cylindracea, 4.05.8 × 2.33.2 µm, vel in culturis veteribus usque ad 10.5 × 4.2 µm. Simile Trichodermati longipili, sed conidia magis lata et per sequentem ITS12 et tef1α distinguibile. ...
Single-parameter optimization was carried out for the production of chitinase using a soil isolat... more Single-parameter optimization was carried out for the production of chitinase using a soil isolate belonging to Trichoderma harzianum in solid-state fermentation. Maximum chitinase activity (3.18 U/gds) was obtained after 96 h of incubation at 30 • C when wheat bran moistened (65.7%) with salt solution was supplemented with colloidal chitin (1%, w/w) and yeast extract (2%, w/w) used as the substrate. The inoculum contains 4 × 10 7 spores of T. harzianum (TUBF 781). The growth of the fungus on wheat bran particle was visualized by a scanning electron microscope. Chitinase activity was measured as the amount of N-acetyl glucosamine (NAG) liberated in mol/min under reaction conditions.
Trichoderma brevicompactum, a new species, was isolated from soil or tree bark in North, Central ... more Trichoderma brevicompactum, a new species, was isolated from soil or tree bark in North, Central and South America, including the Caribbean Islands, and southwestern and southeastern Asia. Morphological and physiological characters, the internal transcribed spacer regions of the rDNA cluster (ITS1-5.8SrDNA-ITS2) and partial sequences of translation elongation factor 1-alpha (tef1) are described. Trichoderma brevicompactum is characterized by a pachybasium-type morphology, morphologically resembling other small-spored species referable to Trichoderma section Pachybasium but with essentially subglobose conidia. It is most closely related phylogenetically to Hypocrea lutea, from which it differs in morphological and physiological characters.
To screen Streptomyces isolates for transglutaminase (TGase) production in solid-state fermentati... more To screen Streptomyces isolates for transglutaminase (TGase) production in solid-state fermentation (SSF) on various substrates. Streptomyces mobaraensis NRRL B-3729, Streptomyces paucisporogenes ATCC 12596 and Streptomyces platensis NRRL 2364 strains were screened for extracellular TGase production in SSF on different substrates. High-protein-content beans, peas and lentils proved to be the best substrates. Good TGase production was obtained on liver kidney beans and green mung beans in a 4- to 6-day SSF. Temperature optima of the enzymes varied between 45 to 50 degrees C. Molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) indicated similar size ( approximately 37 kDa) for all three enzymes. TGase was the dominating protein band on SDS PAGE for two Streptomyces strains in SSF extracts. Other enzymes were present in smaller quantities. Streptomyces mobaraensis NRRL B-3729, S. paucisporogenes ATCC 12596 and S. platensis NRRL 2364 strains were successfully propagated under SSF conditions on crushed/milled liver kidney bean and green mung bean to obtain good level of TGase. Owing to much reduced production cost and direct applicability, SSF TGase without downstream processing (cheap in situ enzyme, crude enzyme) may be an excellent candidate for some nonfood applications.
Journal of Industrial Microbiology & Biotechnology, 2002
The effect of applying Lactobacillus buchneri, alone or in combination with Lactobacillus plantar... more The effect of applying Lactobacillus buchneri, alone or in combination with Lactobacillus plantarum, at ensiling, on the aerobic stability of wheat and corn silages was studied in 50 -l plastic containers. Treatments comprised control ( no additives ) , L. plantarum, L. buchneri and a combination of L. plantarum + L. buchneri. After 3 months of storage, the wheat silages treated with L. buchneri had higher acetic acid contents than the control or L. plantarum -treated silages, and were free of mold, whereas the top layers of the control or L. plantarum -treated silages were moldy. In an aerobic stability test the L. buchneri -treated silages were stable, whereas those treated with L. plantarum deteriorated. In the corn silages the effects of L. buchneri were not as clear and the top layer was moldy in all silages. However, L. buchneri also improved the aerobic stability of the corn silage, as indicated by lower yeast numbers, less CO 2 production and stable pH. It is concluded that L. buchneri has a potential as a silage additive that protects the silage upon aerobic exposure. The 50 -l plastic containers can serve as an appropriate model to test silage additives before conducting full -scale farm experiments.
Journal of Industrial Microbiology and Biotechnology, 1999
The effect of applying Lactobacillus buchneri (LB), alone or in combinations with L. plantarum (L... more The effect of applying Lactobacillus buchneri (LB), alone or in combinations with L. plantarum (LP) and yeasts at ensiling, on the ensiling fermentation and aerobic stability of wheat and sorghum silages was studied under laboratory conditions. Treatments comprised LB, LP, yeasts, LB + yeasts, LP + yeasts, LB + LP and B-589 (a lactic acid bacterial strain isolated from wheat silage in Israel) alone. The treatments were also applied to sterilized aqueous extracts of wheat which were incubated at 30°C for 10 days. The pH of all treatments was below 4.0 already on day 4 of the experiment. Silages treated with LB had higher acetic acid concentrations than those treated with LP: 32-34 vs 16-18, and 28-34 vs 4-7 g kg −1 in the experiments with wheat and sorghum, respectively. Similar results were obtained in wheat extracts. In the aqueous phase, marked differences in pH decrease were noticed among the treatments: 4.4 in LB, 6.0 in the yeast, and 3.7 in LP and B-589 (from day 3 and onwards). In both crops LB resulted in aerobically stable silages when applied alone or with LP and yeasts, whereas LP resulted in unstable silages upon aerobic exposure; the stability of the LB-treated silages is attributed to the higher acetic acid concentrations. The isolated strain (B-589) did not exhibit any advantage with regard to aerobic stability.
Extra-cellular chitinase production by a chitinolytic fungus Trichoderma harzianum TUBF 966 using... more Extra-cellular chitinase production by a chitinolytic fungus Trichoderma harzianum TUBF 966 using submerged fermentation was studied. Colloidal chitin (1.5% w/v) was used as sole carbon source. Maximum chitinase production (14.7 U/ml) was obtained when fermentation was carried out at 30 °C for 96 h using 72 h old mycelium in a medium containing colloidal chitin 1.5% (w/v) as carbon source and 0.42 (% w/v) peptone as nitrogen source (pH 5.5). Supplementation of additional carbon sources (0.75% w/v) showed no further enhancement in chitinase production while supplementation of nitrogen sources (0.42% w/v) such as peptone and tryptone in the fermentation medium showed a marked increase in production. The process parameters that controlled chitinase production by the fungus were studied and presented here.
Aims: The purpose of this work was to study the effect of temperature on the ensiling process and... more Aims: The purpose of this work was to study the effect of temperature on the ensiling process and aerobic stability of corn and wheat silages. Methods and Results: The crops were ensiled in 1á5 l anaerobic jars, with and without an inoculant, at room or elevated temperatures (37±41°C). After two months of ensiling, the silages were subjected to an aerobic stability test at room and elevated (33°C) temperature. The results indicate that ensiling at elevated temperatures resulted in higher pH values, less lactic acid and higher losses. The silages which were stored at elevated temperatures were more susceptible to aerobic spoilage than those stored at room temperature, especially when the test was performed at elevated temperature. Conclusions: High temperatures are detrimental to both the ensiling process and the aerobic stability of silages. Signi®cance and Impact of the Study: The ®ndings of the current study suggest that in a warm climate, special care should be taken during silage making and storage in order to avoid heating as much as possible. In addition, in a warm climate, silages are more susceptible to aerobic deterioration and therefore, special care should be taken during unloading.
ABSTRACT The effects of applying Lactobacillus plantarum and Lact. amylovorus at ensiling on whea... more ABSTRACT The effects of applying Lactobacillus plantarum and Lact. amylovorus at ensiling on wheat silage stored at 25 and41 °C was studied under laboratory conditions. The inoculants were applied at 106 cfu g−1.Silages with no additives served as controls. Three jars per treatment were sampled on days 2, 8 and 60 after ensiling, for chemical and microbiological analyses. After the ensiling period, the silages were subjected to an aerobic stability test. The control and Lact. plantarum inoculated wheat fermented faster at 25 than at 41 °C, whereas silages inoculated with Lact. amylovorus fermented faster at 41 °C. This was apparent from the rate of pH decrease and from the contents of residual sugars and lactic acid in the final silages. The numbers of lactobacilli in the control and Lact. plantarum silages at 41 °C after 2 and 8 days of ensiling were lower than in the corresponding silages at 25 °C. For the Lact. amylovorus silage the opposite held true. The control silages at both temperatures and the Lact. plantarum silage at 41 °C were the most stable silages under aerobic exposure.
We have used isolates of Trichoderma spp. collected in South-East Asia, including Taiwan and West... more We have used isolates of Trichoderma spp. collected in South-East Asia, including Taiwan and Western Indonesia, to assess the genetic and metabolic diversity of endemic species of Trichoderma. Ninety-six strains were isolated in total, and identified at the species level by analysis of morphological and biochemical characters (Biolog system), and by sequence analysis of their internal transcribed spacer regions 1 and 2 (ITS1 and 2) of the rDNA cluster, using ex-type strains and taxonomically established isolates of Trichoderma as reference. Seventy-eight isolates were positively identified as Trichoderma harzianum/Trichoderma inhamatum (37 strains) Trichoderma virens (16 strains), Trichoderma spirale (8 strains), Trichoderma koningii (3 strains), Trichoderma atroviride (3 strains), Trichoderma asperellum (4 strains), Hypocrea jecorina (anamorph: Trichoderma reesei; 2 strains), Trichoderma viride (2 strains), Trichoderma hamatum (1 strain), and Trichoderma ghanense (1 strain). Analysis of biochemical characters revealed that T. virens, T. spirale, T. asperellum, T. koningii, H. jecorina, and T. ghanense formed clearly defined clusters, thus exhibiting speciesspecific metabolic properties. In biochemical character analysis T. atroviride and T. viride formed partially overlapping clusters, indicating that these two species may share overlapping metabolic characteristics. This behavior was even more striking with T. harzianum/T. inhamatum where genotypes defined on the basis of ITS1 and 2 sequences overlapped significantly with adjacent genotypes in the biochemical character analysis, and four strains from the same location (Bali, Indonesia) even clustered with species from section Longibrachiatum. The data indicate that the T. harzianum/T. inhamatum group represents species with high metabolic diversity and partially unique metabolic characteristics. Nineteen strains yielded three different ITS1/2 sequence types which were not alignable with any known species. They were also uniquely characterized by morphological and biochemical characters and therefore represent three new taxa of Trichoderma.
Aspergillus fumigatus is an anamorphic euascomycete mold with a ubiquitous presence worldwide. De... more Aspergillus fumigatus is an anamorphic euascomycete mold with a ubiquitous presence worldwide. Despite intensive work to understand its success as a pathogen infecting immunosuppressed patients, the population dynamics and recent evolutionary history of A. fumigatus remain understudied. We examined patterns of genetic variation at three intergenic loci for 70 natural isolates from Europe, North America, South America, Asia, Africa, and Australia. The same loci were used to analyze within-population genetic variation for 33 isolates obtained from five geographic locations. Neither data set detected evidence of population differentiation or found any association between the genetic and geographic distances among these isolates. No evidence for genetic differentiation within the two A. fumigatus mating types was detected. The genetic diversity of A. fumigatus, contrasted with that of its close teleomorphic relatives, Neosartorya fischeri and Neosartorya spinosa, is remarkably low.
... Phialides 26 verticillatae, ampulliformes, 4.5 7.0 × 3.45.4 µm. Conidia breviter cylindrac... more ... Phialides 26 verticillatae, ampulliformes, 4.5 7.0 × 3.45.4 µm. Conidia breviter cylindracea, 4.05.8 × 2.33.2 µm, vel in culturis veteribus usque ad 10.5 × 4.2 µm. Simile Trichodermati longipili, sed conidia magis lata et per sequentem ITS12 et tef1α distinguibile. ...
In this review the state of the art of lignocellulose bioconversion by solid substrate fermentati... more In this review the state of the art of lignocellulose bioconversion by solid substrate fermentation (SSF) is presented. The most important lignocellulolytic fungi and their properties are described, and their application in novel solid state bioreactors with on-line process control is discussed. The most important bioconversion products, biofuels, enzymes, animal feeds, biofertilizers, biopesticides, biopromoters, secondary metabolites, and the economy of their production by SSF is discussed. The use of SSF in the pulp and paper industry and in integrated crop management is illustrated.
Xylanase production by seven fungal strains was investigated using concentrated spent sulphite li... more Xylanase production by seven fungal strains was investigated using concentrated spent sulphite liquor (SSLc), xylan and D-xylose as carbon substrates. An SSLcbased medium induced xylanase production at varying levels in all of these strains, with Aspergillus oryzae NRRL 3485 and Aspergillus phoenicis ATCC 13157 yielding activities of 164 and 146 U ml −1 , respectively; these values were higher than those obtained on xylan or D-xylose with the same fungal strains. The highest xylanase activity of 322 U ml −1 was obtained with Aspergillus foetidus ATCC 14916 on xylan. Electrophoretic and zymogram analysis indicated three xylanases from A. oryzae with molecular weights of approximately 32, 22 and 19 kDa, whereas A. phoenicis produced two xylanases with molecular weights of about 25 and 21 kDa. Crude xylanase preparations from these A. oryzae and A. phoenicis strains exhibited optimal activities at pH 6.5 and 5.0 and at 65 and 55°C, respectively. The A. oryzae xylanolytic activity was stable at 50°C over the pH range 4.5-10. The crude xylanase preparations from these A. oryzae and A. phoenicis strains had negligible cellulase activity, and their application in the biobleaching of hardwood pulp reduced chlorine dioxide consumption by 20-30% without sacrificing brightness.
ABSTRACT Traditional ensiling of plant material by anaerobic lactic acid fermentation was combine... more ABSTRACT Traditional ensiling of plant material by anaerobic lactic acid fermentation was combined with enzymatic hydrolysis (ENLAC for short) with cell wall degrading enzymes (hemicellulases, cellulases, and pectinases) to increase fiber digestibility or to increase the recovery of cell content from plants. Such findings were made using 0.015% (w/w, wet basis) Phylacell® enzyme preparation by ENLAC of corn and corn-sorghum mixtures, but not of forage grasses. Addition to alfalfa of a mixture of cell wall degrading enzymes, such as NOVO Viscozyme® together with NOVO Celluclast® each at 0.2–1.0% (w/w, wet basis), resulted in more rapid ensiling and improvement of rumen digestibility of silage by 20%. After 20 d of ensiling at 25 °C when the same enzymes were added to alfalfa at the 1.0% level, protein recovery by pressing increased by 35%, β-carotene recovery by 80%, and chlorophyll/xanthophyll recovery by 30%. ENLAC with the same enzymes also increased the recovery of sclareol from muscatel sage by 400%.
Selection of suitable strains for biotechnological purposes is frequently a random process suppor... more Selection of suitable strains for biotechnological purposes is frequently a random process supported by high-throughput methods. Using chitinase production by Hypocrea lixii/Trichoderma harzianum as a model, we tested whether fungal strains with superior enzyme formation may be diagnosed by DNA bar codes. We analyzed sequences of two phylogenetic marker loci, internal transcribed spacer 1 (ITS1) and ITS2 of the rRNA-encoding gene cluster and the large intron of the elongation factor 1-alpha gene, tef1, from 50 isolates of H. lixii/T. harzianum, which were also tested to determine their ability to produce chitinases in solid-state fermentation (SSF). Statistically supported superior chitinase production was obtained for strains carrying one of the observed ITS1 and ITS2 and tef1 alleles corresponding to an allele of T. harzianum type strain CBS 226.95. A tef1-based DNA bar code tool, TrichoCHIT, for rapid identification of these strains was developed.
ABSTRACT Lignocellulose degrading enzymes were produced by solid substrate fermentation (SSF) wit... more ABSTRACT Lignocellulose degrading enzymes were produced by solid substrate fermentation (SSF) with Trichoderma and Gliocladium spp. on various agricultural by-products, such as spent brewing grain, corn fiber and wheat straw/wheat bran mixture. In a 10 day SSF, the hypercellulolytic mutant Trichoderma reesei Rut C30 produced 31 Filter Paper Unit (FPU)/g dry weight (DW) cellulase activity and 650 International Unit (IU)/g DW xylanase activity on corn fiber (corn seed hull), 55 FPU/g DW cellulase and 2400 IU/g DW xylanase activity on spent brewing grain. In a much faster 3 day fermentation, the wild strain Trichoderma hamatum TUB F-105 produced 7 FPU/g DW cellulase activity and 7600 IU/g DW xylanase activity on extracted (starch-removed) corn fiber. Gliocladium sp. TUB F-498 secreted 11 FPU/g DW cellulase activity in a 6 day SSF on a wheat straw-wheat bran 9:1 mixture. The hydrolyzing potential of SSF enzymes on native autologous substrates is substrate specific and does not always coincide with their filter paper activity. The F-498 and F-105 enzymes, at 5.0 FPU/g cellulose enzyme load hydrolyzed corn fiber and spent brewing grain more efficiently than the Rut C30 enzyme, although the Rut C30 enzyme hydrolyzed pure cellulose (Solka Floc) faster and more efficiently. The F-498 enzyme required lower enzyme loads for the hydrolysis of corn fiber and spent brewing grain than Rut C30. Due to adequate hydrolyzing potential, much reduced production cost and direct applicability, SSF enzymes are excellent candidates for enzymatic agrobiotechnological processes.
We have used isolates of Trichoderma spp. collected in South-East Asia, including Taiwan and West... more We have used isolates of Trichoderma spp. collected in South-East Asia, including Taiwan and Western Indonesia, to assess the genetic and metabolic diversity of endemic species of Trichoderma. Ninety-six strains were isolated in total, and identified at the species level by analysis of morphological and biochemical characters (Biolog system), and by sequence analysis of their internal transcribed spacer regions 1 and 2 (ITS1 and 2) of the rDNA cluster, using ex-type strains and taxonomically established isolates of Trichoderma as reference. Seventy-eight isolates were positively identified as Trichoderma harzianum/Trichoderma inhamatum (37 strains) Trichoderma virens (16 strains), Trichoderma spirale (8 strains), Trichoderma koningii (3 strains), Trichoderma atroviride (3 strains), Trichoderma asperellum (4 strains), Hypocrea jecorina (anamorph: Trichoderma reesei; 2 strains), Trichoderma viride (2 strains), Trichoderma hamatum (1 strain), and Trichoderma ghanense (1 strain). Analysis of biochemical characters revealed that T. virens, T. spirale, T. asperellum, T. koningii, H. jecorina, and T. ghanense formed clearly defined clusters, thus exhibiting speciesspecific metabolic properties. In biochemical character analysis T. atroviride and T. viride formed partially overlapping clusters, indicating that these two species may share overlapping metabolic characteristics. This behavior was even more striking with T. harzianum/T. inhamatum where genotypes defined on the basis of ITS1 and 2 sequences overlapped significantly with adjacent genotypes in the biochemical character analysis, and four strains from the same location (Bali, Indonesia) even clustered with species from section Longibrachiatum. The data indicate that the T. harzianum/T. inhamatum group represents species with high metabolic diversity and partially unique metabolic characteristics. Nineteen strains yielded three different ITS1/2 sequence types which were not alignable with any known species. They were also uniquely characterized by morphological and biochemical characters and therefore represent three new taxa of Trichoderma.
Canadian Journal of Botany-revue Canadienne De Botanique, 2003
... Phialides 26 verticillatae, ampulliformes, 4.5 7.0 × 3.45.4 µm. Conidia breviter cylindrac... more ... Phialides 26 verticillatae, ampulliformes, 4.5 7.0 × 3.45.4 µm. Conidia breviter cylindracea, 4.05.8 × 2.33.2 µm, vel in culturis veteribus usque ad 10.5 × 4.2 µm. Simile Trichodermati longipili, sed conidia magis lata et per sequentem ITS12 et tef1α distinguibile. ...
Single-parameter optimization was carried out for the production of chitinase using a soil isolat... more Single-parameter optimization was carried out for the production of chitinase using a soil isolate belonging to Trichoderma harzianum in solid-state fermentation. Maximum chitinase activity (3.18 U/gds) was obtained after 96 h of incubation at 30 • C when wheat bran moistened (65.7%) with salt solution was supplemented with colloidal chitin (1%, w/w) and yeast extract (2%, w/w) used as the substrate. The inoculum contains 4 × 10 7 spores of T. harzianum (TUBF 781). The growth of the fungus on wheat bran particle was visualized by a scanning electron microscope. Chitinase activity was measured as the amount of N-acetyl glucosamine (NAG) liberated in mol/min under reaction conditions.
Trichoderma brevicompactum, a new species, was isolated from soil or tree bark in North, Central ... more Trichoderma brevicompactum, a new species, was isolated from soil or tree bark in North, Central and South America, including the Caribbean Islands, and southwestern and southeastern Asia. Morphological and physiological characters, the internal transcribed spacer regions of the rDNA cluster (ITS1-5.8SrDNA-ITS2) and partial sequences of translation elongation factor 1-alpha (tef1) are described. Trichoderma brevicompactum is characterized by a pachybasium-type morphology, morphologically resembling other small-spored species referable to Trichoderma section Pachybasium but with essentially subglobose conidia. It is most closely related phylogenetically to Hypocrea lutea, from which it differs in morphological and physiological characters.
To screen Streptomyces isolates for transglutaminase (TGase) production in solid-state fermentati... more To screen Streptomyces isolates for transglutaminase (TGase) production in solid-state fermentation (SSF) on various substrates. Streptomyces mobaraensis NRRL B-3729, Streptomyces paucisporogenes ATCC 12596 and Streptomyces platensis NRRL 2364 strains were screened for extracellular TGase production in SSF on different substrates. High-protein-content beans, peas and lentils proved to be the best substrates. Good TGase production was obtained on liver kidney beans and green mung beans in a 4- to 6-day SSF. Temperature optima of the enzymes varied between 45 to 50 degrees C. Molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) indicated similar size ( approximately 37 kDa) for all three enzymes. TGase was the dominating protein band on SDS PAGE for two Streptomyces strains in SSF extracts. Other enzymes were present in smaller quantities. Streptomyces mobaraensis NRRL B-3729, S. paucisporogenes ATCC 12596 and S. platensis NRRL 2364 strains were successfully propagated under SSF conditions on crushed/milled liver kidney bean and green mung bean to obtain good level of TGase. Owing to much reduced production cost and direct applicability, SSF TGase without downstream processing (cheap in situ enzyme, crude enzyme) may be an excellent candidate for some nonfood applications.
Journal of Industrial Microbiology & Biotechnology, 2002
The effect of applying Lactobacillus buchneri, alone or in combination with Lactobacillus plantar... more The effect of applying Lactobacillus buchneri, alone or in combination with Lactobacillus plantarum, at ensiling, on the aerobic stability of wheat and corn silages was studied in 50 -l plastic containers. Treatments comprised control ( no additives ) , L. plantarum, L. buchneri and a combination of L. plantarum + L. buchneri. After 3 months of storage, the wheat silages treated with L. buchneri had higher acetic acid contents than the control or L. plantarum -treated silages, and were free of mold, whereas the top layers of the control or L. plantarum -treated silages were moldy. In an aerobic stability test the L. buchneri -treated silages were stable, whereas those treated with L. plantarum deteriorated. In the corn silages the effects of L. buchneri were not as clear and the top layer was moldy in all silages. However, L. buchneri also improved the aerobic stability of the corn silage, as indicated by lower yeast numbers, less CO 2 production and stable pH. It is concluded that L. buchneri has a potential as a silage additive that protects the silage upon aerobic exposure. The 50 -l plastic containers can serve as an appropriate model to test silage additives before conducting full -scale farm experiments.
Journal of Industrial Microbiology and Biotechnology, 1999
The effect of applying Lactobacillus buchneri (LB), alone or in combinations with L. plantarum (L... more The effect of applying Lactobacillus buchneri (LB), alone or in combinations with L. plantarum (LP) and yeasts at ensiling, on the ensiling fermentation and aerobic stability of wheat and sorghum silages was studied under laboratory conditions. Treatments comprised LB, LP, yeasts, LB + yeasts, LP + yeasts, LB + LP and B-589 (a lactic acid bacterial strain isolated from wheat silage in Israel) alone. The treatments were also applied to sterilized aqueous extracts of wheat which were incubated at 30°C for 10 days. The pH of all treatments was below 4.0 already on day 4 of the experiment. Silages treated with LB had higher acetic acid concentrations than those treated with LP: 32-34 vs 16-18, and 28-34 vs 4-7 g kg −1 in the experiments with wheat and sorghum, respectively. Similar results were obtained in wheat extracts. In the aqueous phase, marked differences in pH decrease were noticed among the treatments: 4.4 in LB, 6.0 in the yeast, and 3.7 in LP and B-589 (from day 3 and onwards). In both crops LB resulted in aerobically stable silages when applied alone or with LP and yeasts, whereas LP resulted in unstable silages upon aerobic exposure; the stability of the LB-treated silages is attributed to the higher acetic acid concentrations. The isolated strain (B-589) did not exhibit any advantage with regard to aerobic stability.
Extra-cellular chitinase production by a chitinolytic fungus Trichoderma harzianum TUBF 966 using... more Extra-cellular chitinase production by a chitinolytic fungus Trichoderma harzianum TUBF 966 using submerged fermentation was studied. Colloidal chitin (1.5% w/v) was used as sole carbon source. Maximum chitinase production (14.7 U/ml) was obtained when fermentation was carried out at 30 °C for 96 h using 72 h old mycelium in a medium containing colloidal chitin 1.5% (w/v) as carbon source and 0.42 (% w/v) peptone as nitrogen source (pH 5.5). Supplementation of additional carbon sources (0.75% w/v) showed no further enhancement in chitinase production while supplementation of nitrogen sources (0.42% w/v) such as peptone and tryptone in the fermentation medium showed a marked increase in production. The process parameters that controlled chitinase production by the fungus were studied and presented here.
Aims: The purpose of this work was to study the effect of temperature on the ensiling process and... more Aims: The purpose of this work was to study the effect of temperature on the ensiling process and aerobic stability of corn and wheat silages. Methods and Results: The crops were ensiled in 1á5 l anaerobic jars, with and without an inoculant, at room or elevated temperatures (37±41°C). After two months of ensiling, the silages were subjected to an aerobic stability test at room and elevated (33°C) temperature. The results indicate that ensiling at elevated temperatures resulted in higher pH values, less lactic acid and higher losses. The silages which were stored at elevated temperatures were more susceptible to aerobic spoilage than those stored at room temperature, especially when the test was performed at elevated temperature. Conclusions: High temperatures are detrimental to both the ensiling process and the aerobic stability of silages. Signi®cance and Impact of the Study: The ®ndings of the current study suggest that in a warm climate, special care should be taken during silage making and storage in order to avoid heating as much as possible. In addition, in a warm climate, silages are more susceptible to aerobic deterioration and therefore, special care should be taken during unloading.
ABSTRACT The effects of applying Lactobacillus plantarum and Lact. amylovorus at ensiling on whea... more ABSTRACT The effects of applying Lactobacillus plantarum and Lact. amylovorus at ensiling on wheat silage stored at 25 and41 °C was studied under laboratory conditions. The inoculants were applied at 106 cfu g−1.Silages with no additives served as controls. Three jars per treatment were sampled on days 2, 8 and 60 after ensiling, for chemical and microbiological analyses. After the ensiling period, the silages were subjected to an aerobic stability test. The control and Lact. plantarum inoculated wheat fermented faster at 25 than at 41 °C, whereas silages inoculated with Lact. amylovorus fermented faster at 41 °C. This was apparent from the rate of pH decrease and from the contents of residual sugars and lactic acid in the final silages. The numbers of lactobacilli in the control and Lact. plantarum silages at 41 °C after 2 and 8 days of ensiling were lower than in the corresponding silages at 25 °C. For the Lact. amylovorus silage the opposite held true. The control silages at both temperatures and the Lact. plantarum silage at 41 °C were the most stable silages under aerobic exposure.
We have used isolates of Trichoderma spp. collected in South-East Asia, including Taiwan and West... more We have used isolates of Trichoderma spp. collected in South-East Asia, including Taiwan and Western Indonesia, to assess the genetic and metabolic diversity of endemic species of Trichoderma. Ninety-six strains were isolated in total, and identified at the species level by analysis of morphological and biochemical characters (Biolog system), and by sequence analysis of their internal transcribed spacer regions 1 and 2 (ITS1 and 2) of the rDNA cluster, using ex-type strains and taxonomically established isolates of Trichoderma as reference. Seventy-eight isolates were positively identified as Trichoderma harzianum/Trichoderma inhamatum (37 strains) Trichoderma virens (16 strains), Trichoderma spirale (8 strains), Trichoderma koningii (3 strains), Trichoderma atroviride (3 strains), Trichoderma asperellum (4 strains), Hypocrea jecorina (anamorph: Trichoderma reesei; 2 strains), Trichoderma viride (2 strains), Trichoderma hamatum (1 strain), and Trichoderma ghanense (1 strain). Analysis of biochemical characters revealed that T. virens, T. spirale, T. asperellum, T. koningii, H. jecorina, and T. ghanense formed clearly defined clusters, thus exhibiting speciesspecific metabolic properties. In biochemical character analysis T. atroviride and T. viride formed partially overlapping clusters, indicating that these two species may share overlapping metabolic characteristics. This behavior was even more striking with T. harzianum/T. inhamatum where genotypes defined on the basis of ITS1 and 2 sequences overlapped significantly with adjacent genotypes in the biochemical character analysis, and four strains from the same location (Bali, Indonesia) even clustered with species from section Longibrachiatum. The data indicate that the T. harzianum/T. inhamatum group represents species with high metabolic diversity and partially unique metabolic characteristics. Nineteen strains yielded three different ITS1/2 sequence types which were not alignable with any known species. They were also uniquely characterized by morphological and biochemical characters and therefore represent three new taxa of Trichoderma.
Aspergillus fumigatus is an anamorphic euascomycete mold with a ubiquitous presence worldwide. De... more Aspergillus fumigatus is an anamorphic euascomycete mold with a ubiquitous presence worldwide. Despite intensive work to understand its success as a pathogen infecting immunosuppressed patients, the population dynamics and recent evolutionary history of A. fumigatus remain understudied. We examined patterns of genetic variation at three intergenic loci for 70 natural isolates from Europe, North America, South America, Asia, Africa, and Australia. The same loci were used to analyze within-population genetic variation for 33 isolates obtained from five geographic locations. Neither data set detected evidence of population differentiation or found any association between the genetic and geographic distances among these isolates. No evidence for genetic differentiation within the two A. fumigatus mating types was detected. The genetic diversity of A. fumigatus, contrasted with that of its close teleomorphic relatives, Neosartorya fischeri and Neosartorya spinosa, is remarkably low.
... Phialides 26 verticillatae, ampulliformes, 4.5 7.0 × 3.45.4 µm. Conidia breviter cylindrac... more ... Phialides 26 verticillatae, ampulliformes, 4.5 7.0 × 3.45.4 µm. Conidia breviter cylindracea, 4.05.8 × 2.33.2 µm, vel in culturis veteribus usque ad 10.5 × 4.2 µm. Simile Trichodermati longipili, sed conidia magis lata et per sequentem ITS12 et tef1α distinguibile. ...
In this review the state of the art of lignocellulose bioconversion by solid substrate fermentati... more In this review the state of the art of lignocellulose bioconversion by solid substrate fermentation (SSF) is presented. The most important lignocellulolytic fungi and their properties are described, and their application in novel solid state bioreactors with on-line process control is discussed. The most important bioconversion products, biofuels, enzymes, animal feeds, biofertilizers, biopesticides, biopromoters, secondary metabolites, and the economy of their production by SSF is discussed. The use of SSF in the pulp and paper industry and in integrated crop management is illustrated.
Xylanase production by seven fungal strains was investigated using concentrated spent sulphite li... more Xylanase production by seven fungal strains was investigated using concentrated spent sulphite liquor (SSLc), xylan and D-xylose as carbon substrates. An SSLcbased medium induced xylanase production at varying levels in all of these strains, with Aspergillus oryzae NRRL 3485 and Aspergillus phoenicis ATCC 13157 yielding activities of 164 and 146 U ml −1 , respectively; these values were higher than those obtained on xylan or D-xylose with the same fungal strains. The highest xylanase activity of 322 U ml −1 was obtained with Aspergillus foetidus ATCC 14916 on xylan. Electrophoretic and zymogram analysis indicated three xylanases from A. oryzae with molecular weights of approximately 32, 22 and 19 kDa, whereas A. phoenicis produced two xylanases with molecular weights of about 25 and 21 kDa. Crude xylanase preparations from these A. oryzae and A. phoenicis strains exhibited optimal activities at pH 6.5 and 5.0 and at 65 and 55°C, respectively. The A. oryzae xylanolytic activity was stable at 50°C over the pH range 4.5-10. The crude xylanase preparations from these A. oryzae and A. phoenicis strains had negligible cellulase activity, and their application in the biobleaching of hardwood pulp reduced chlorine dioxide consumption by 20-30% without sacrificing brightness.
ABSTRACT Traditional ensiling of plant material by anaerobic lactic acid fermentation was combine... more ABSTRACT Traditional ensiling of plant material by anaerobic lactic acid fermentation was combined with enzymatic hydrolysis (ENLAC for short) with cell wall degrading enzymes (hemicellulases, cellulases, and pectinases) to increase fiber digestibility or to increase the recovery of cell content from plants. Such findings were made using 0.015% (w/w, wet basis) Phylacell® enzyme preparation by ENLAC of corn and corn-sorghum mixtures, but not of forage grasses. Addition to alfalfa of a mixture of cell wall degrading enzymes, such as NOVO Viscozyme® together with NOVO Celluclast® each at 0.2–1.0% (w/w, wet basis), resulted in more rapid ensiling and improvement of rumen digestibility of silage by 20%. After 20 d of ensiling at 25 °C when the same enzymes were added to alfalfa at the 1.0% level, protein recovery by pressing increased by 35%, β-carotene recovery by 80%, and chlorophyll/xanthophyll recovery by 30%. ENLAC with the same enzymes also increased the recovery of sclareol from muscatel sage by 400%.
Selection of suitable strains for biotechnological purposes is frequently a random process suppor... more Selection of suitable strains for biotechnological purposes is frequently a random process supported by high-throughput methods. Using chitinase production by Hypocrea lixii/Trichoderma harzianum as a model, we tested whether fungal strains with superior enzyme formation may be diagnosed by DNA bar codes. We analyzed sequences of two phylogenetic marker loci, internal transcribed spacer 1 (ITS1) and ITS2 of the rRNA-encoding gene cluster and the large intron of the elongation factor 1-alpha gene, tef1, from 50 isolates of H. lixii/T. harzianum, which were also tested to determine their ability to produce chitinases in solid-state fermentation (SSF). Statistically supported superior chitinase production was obtained for strains carrying one of the observed ITS1 and ITS2 and tef1 alleles corresponding to an allele of T. harzianum type strain CBS 226.95. A tef1-based DNA bar code tool, TrichoCHIT, for rapid identification of these strains was developed.
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Papers by George Szakacs