Comparative mycelial and spore yield by Trichoderma viride in batch and fed-batch cultures

The effects of cultural parameters such as carbon and nitrogen source and environmental factors including temperature and pH were investigated on spore and mycelial yield of Trichoderma viride, which has potential as a biocontrol agent against species of Fusarium in batch culture and fed-batch culture where there was limiting nutrient. The results obtained indicated that growth and sporulation of T. viride were greatly influenced by various carbon and nitrogen sources, and by environmental factors such as pH and temperature. Mannitol, wheat bran and rice bran as sole carbon sources appear to stimulate high mycelial growth and spore yield in fed-batch culture. Growth and sporulation were also favoured by NaNO₃, peptone and NH₄SO₄ as the nitrogen sources in fed-batch and batch cultures_. Maximum growth and sporulation was between pH 4.5 and 6.0. Temperatures between 30 and 37 °C were good for mycelium growth of T. viride while temperatures between 30 to 45 °C were good for sporulation. The amount of spore and mycelium produced and the time required for attainment of maximum spore yield increased with increasing carbon and nitrogen source in batch culture. The final spore yield obtained in fed-batch culture was two times higher than the apparent spore-carrying capacity of batch culture. These results show that T. viride is capable of growing and sporulating with varied nutritional and environmental conditions, and, therefore, this strain of T. viride may be useful as a biocontrol agent under diverse physiological and environmental conditions.     

Carbon Requirements of Some Nematophagous, Entomopathogenic and Mycoparasitic Hyphomycetes as Fungal Biocontrol Agents

Thirty-three carbon sources were evaluated for their effects on spore germination, hyphal growth and sporulation of 11 fungal biocontrol agents, i.e. the nematophagous fungi Paecilomyces lilacinus, Pochonia chlamydosporia, Hirsutella rhossiliensis, H. minnesotensis and Arkansas Fungus 18, the entomopathogenic fungi Lecanicillium lecanii, Beauveria bassiana and Metarhizium anisopliae, and the mycoparasitic fungus Trichoderma viride. Variations in carbon requirements were found among the fungal species or strains tested. All strains studied except for T. viride grew on most carbon sources, although B. bassiana had more fastidious requirements for spore germination. Monosaccharides and disaccharides were suitable for fungal growth. For most isolates, d-glucose, d-mannose, sucrose and trehalose were superior to pectin and soluble starch among the polysaccharides and lactic acid among the organic acids. Both ethanol and methanol could accelerate growth of most isolates but not biomass. d-mannose, d-fructose and d-xylose were excellent carbon sources for sporulation, while d-glucose, sucrose, cellobiose, trehalose, chitin, dextrin, gelatin and lactic acid were better for some isolates. Neither sorbic acid nor linoleic acid could be utilized as a single carbon source. These findings provided a better understanding of the nutritional requirements of different fungal biocontrol agents that can benefit the mass production process.     

Chemical changes in humic substances from compost due to incubation with ligno-cellulolytic microorganisms and effects on lettuce growth

 Differences in the chemical properties of the organic matter from a highly lignocellulosic compost after incubation with two ligno- and cellulolytic microorganisms were studied in this work. Inoculation with either of the two microorganisms assayed, Trichoderma viride or Bacillus sp., of soil-compost mixtures enhanced degradation processes and the degree of organic matter humification. According to the humification index, inoculation with T. viride produced the highest humification rate in all the compost-soil proportions studied (10, 20 and 30%). To evaluate the quality of the extracted humic substances according to their electrofocusing behaviours a new index was established. This index showed an increased yield of humic substances of the lowest electrophoretic mobility (highest molecular weight) in treatments inoculated with Bacillus sp., whereas inoculation with T. viride enhanced the formation of molecules of the fastest electrophoretic mobility. These results, together with the fibre analysis performed, showed that the nature of the humic substances produced after incubation appeared to depend greatly on the degradation pathway carried out by the inoculated microorganism, T. viride or Bacillus sp.. Both degradation-humification pathways beneficially affected lettuce growth, demonstrating that inoculation with any of these two microorganisms may be a useful tool to modify agronomic properties of unripe composts. Received: 2 August 1995/Received revision: 14 November 1995/Accepted: 11 December 1995     

Effects of carbon and nitrogen sources,carbon-to-nitrogen ratio,and initial pH on the growth of nematophagous fungus <Emphasis Type="Italic">Pochonia chlamydosporia</Emphasis> in liquid culture

The effects of carbon and nitrogen sources, carbon-to-nitrogen ratio (C:N) and initial pH value on the growth and sporulation of the nematophagous fungus Pochonia chlamydosporia in liquid culture were examined. Among the 21 carbon sources and 15 nitrogen compounds tested, the optimal carbon and nitrogen sources for mycelial growth were sweet potato and L-tyrosine, and for sporulation were sweet potato and casein peptone. A C:N ratio of 10:1 at pH 3.7 gave the maximum yield of conidia and a C:N ratio of 40:1 at pH 6.8 gave the maximum biomass. The initial pH value had a significant effect on mycelial growth and conidial production, with the optimal ranges being 3.5–4.5 for sporulation and 5–6 for growth. Maximum conidial production was obtained at an initial pH of 4.0 and the maximum biomass at pH 6.0. The results also showed that the final pH after 7 days cultivation was always higher than the initial value. The variability in growth and sporulation of seven strains of P. chlamydosporia in liquid culture was also compared and discussed.     

SCP production by Chaetomium cellulolyticum,a new thermotolerant cellulolytic fungus

Chaetomium cellulolyticum, a newly isolated cellulolytic fungus, showed 50–100% faster growth rates and over 80% more final biomass-protein formation than Trichoderma viride, a well-known high cellulase-producing cellulolytic organism, when cultivated on Solka-floc (a purified, predominantly amorphorous form of cellulose) or partially delignified sawdust (consisting of a mixture of hardwoods) as the sole-carbon source in the fermentation media. However, in both cases, T. viride produced much higher quantities of free cellulases at faster rates and also degraded more substrate than C. cellulolyticum. It is concluded that the synthesis mechanisms and/or the nature of the cellulase complexes of the two types of organisms are quite different such that C. cellulolyticum is more optimal for single-cell protein (SCP) production, while T. viride is more optimal for the production of extracellular cellulases. It was also found that the amino acid composition of C. cellulolyticum is generally better than that of T. viride and compares favorably with those of the FAO reference protein, alfalfa, and soya meal. In addition, preliminary feeding trials on rats have shown no adverse effects of the SCP produced by C. cellulolyticum fermentations.     

Production of laccases by Pleurotus ostreatus in submerged fermentation in co‐culture with Trichoderma viride

Aims: To evaluate the production and stability of laccases by Pleurotus ostreatus in liquid co‐cultures with Trichoderma viride as a function of infection time and agitation rate. Methods and Results: Pleurotus ostreatus cultures were infected with T. viride spores at 30 and 48 h. Maximal laccase volumetric activity was seen after 48 h (control cultures) or 72 h (co‐cultures) of cultivation time. Only the cultures infected at 30 h showed an increased laccase volumetric activity compared to control cultures. After maximal laccase volumetric activity value was reached, a sharp decrease in it was observed in control cultures. Co‐cultures exhibited a comparatively lower loss of activity. The influence of P. ostreatus and/or T. viride on the stability of laccase volumetric activity and isoenzyme pattern was evaluated. Trichoderma viride induced changes in the laccase isoenzyme pattern. Agitated cultures increased biomass growth and specific productivity threefold and sevenfold, respectively, to the static cultures. Conclusions: The laccase volumetric activity is very likely the result of the balance between biosynthesis and degradation/biotransformation rates occurring during the cultures. The individual presence of P. ostreatus or T. viride in the culture negatively affected the volumetric laccase activity. Significance and Impact of the Study: The evaluation of culture parameters that could influence Trichoderma–basidomycetes interaction and laccase production during submerged fermentation has not been reported. This study showed how laccase production in co‐cultures of P. ostreatus and T. viride was influenced by the infection time and agitation/oxygenation conditions.     

Influence of culture conditions on production and freeze-drying tolerance of Paecilomyces fumosoroseus blastospores

With the goal of developing a defined medium for the production of desiccation-tolerant blastospores of the bioinsecticidal fungus Paecilomyces fumosoroseus, we evaluated the impact of various media components such as amino acids, carbohydrates, trace metals and vitamins on hyphal growth and sporulation of P. fumosoroseus cultures and on the freeze-drying tolerance of blastospores produced under these conditions. A comparison of 13 amino acids as sole nitrogen sources showed that glutamate, aspartate, glycine and arginine supported biomass accumulations (12–16 mg ml⁻¹) and blastospore yields (6–11 × 10⁸ blastospores ml⁻¹) comparable to our standard production medium which contains casamino acids as the nitrogen source. Using glutamate as the sole nitrogen source, tests with various carbohydrates showed that P. fumosoroseus grew best on glucose (18.8 mg biomass ml⁻¹) but produced similar blastospore concentrations (7.3–11.0 × 10⁸) when grown with glucose, glycerol, fructose or sucrose. P. fumosoroseus cultures grown in media with sodium citrate or galactose as the sole carbohydrate produced lower blastospore concentrations but more-desiccation-tolerant spores. Zinc was the only trace metal tested that was required for optimal growth and sporulation. In a defined medium with glutamate as the nitrogen source, vitamins were unnecessary for P. fumosoroseus growth or sporulation. When blastospores were freeze-dried in the absence of a suspension medium, residual glucose (>2.5% w/v) was required for enhanced spore survival. Thus, a defined medium containing basal salts, glucose, glutamate and zinc can be used to produce optimal concentrations of desiccation-tolerant blastospores of P. fumosoroseus. Received 27 October 1998/ Accepted in revised form 06 May 1999     

Effect of yeast extract and succinic acid on growth and sporulation of alternaria species

Summary Alternaria solani andA. nyctanthi, these pathogens causing leafspot disease were able to metabolize a variety of nitrogen compounds when grown on different culture media. The amount of growth varied with the nitrogen source. Peptone produced the best zonation when added in definite proportion to the yeast extract medium. Ammonium compounds were found to be moderately effective for growth but poor for sporulation. The effect of adding succinic acid in media containing ammonium sources and the role of pH in the utilization of nitrite nitrogen was investigated.The fungus gave more vegetative growth on a mixture of aminoacids than in culture media in which the same amino acids were supplied singly to study the effect produced on growth and sporulation.     

Nitrogen nutrition and regulation of cephalosporin production in Streptomyces clavuligerus.

When used as sole nitrogen source, certain amino acids (e.g., proline, asparagine) supported both growth and sporulation by Streptomyces clavuligerus streaked onto solid defined medium. Ammonium supported growth but suppressed sporulation. Amino nitrogen was best for cephalosporin production in liquid defined medium, although urea was almost as useful. A comparison of amino acids showed asparagine and glutamine to be the best nitrogen sources and arginine to be almost as good. Ammonium salts supported a somewhat lower growth rate than asparagine, but antibiotic production was very poor on these inorganic nitrogen sources. Addition of ammonium to asparagine did not affect growth rate but increased mycelial mass; cephalosporin production was reduced by about 75%. Antibiotic production was more closely associated with growth in the absence of ammonium than in its presence, indicating a strong inhibitory and (or) repressive effect of NH4+ on antibiotic production. Ammonium exerted its negative effect when added at 24h or earlier, i.e. before antibiotic formation began.     

Production of Bacillus sphaericus larvicide on industrial peptones

Summary A range of industrial peptones in combination with other carbon and nitrogen sources were investigated in order to define a medium for production of insecticide from Bacillus sphaericus, strain 2362. Industrial protein hydrolysates with an average peptide chain length above 30 were found to be poor substrates. Fermentation of B. sphaericus on protein-based media supplemented with glycerol resulted in production of 4–5x10³ units of larvicide per ml fermentation broth, and toxicity of the larvicide up to 10⁴ units per mg. Equally high larvicide production was obtained, when peptone was substituted with 30 mM l-glutamate and 2 mM proline. Addition of 2 mM l-arginine to this medium prevented sporulation and larvicide production.     

Fusant Trichoderma HF9 with enhanced extracellular chitinase and protein content

Strain improvement was carried out to obtain higher chitinase and protein by inter-specific protoplast fusion between Trichoderma harzianum and Trichoderma viride. Fusant HF9 and parental strains of Trichoderma were compared for chitinase and protein production. 1% of glucose, sucrose and fungal cell wall (Rhizoctonia solani), were used as carbon source for cultivation of Trichoderma and fungal cell wall was the best to induce chitinase and protein. Usage of 0.5% colloidal chitin for the fungal growth under aerated conditions at pH 6.5 and 28°C led to higher chitinase and protein production. In these conditions fusant Trichoderma HF9 in comparison with parent strains had 3-, 2.5- and 1.5-fold increase of total chitinase, specific chitinase and protein, respectively. SDS-PAGE analysis revealed that it had 9 major protein bands with up-regulation compared to parent strains. Amino acid analysis showed that protein of culture filtrate of T. harzianum, T. viride and fusant Trichoderma HF9 had 8, 6 and 10 amino acids, respectively. The results obtained suggested that fusant HF9 could be an integration of T. harzianum and T. viride through protoplast fusion.     

Enhancement of cellulase production by Trichoderma viride using carbon/nitrogen double-fed-batch

Summary The production of cellulase by Trichoderma viride has been remarkably raised by means of a fed batch fermentation with the addition of cellulose as the carbon source and ammonia as nitrogen source during cultivation.     

Production of cellulase and xylanase by a wild strain of Trichoderma viride

Summary The production of cellulase and xylanase was investigated with a newly isolated strain of Trichoderma viride BT 2169. The medium composition was optimized on a shake-flask scale using the Graeco-Latin square technique. The temperature and time for optimal growth and production of the enzymes in shake cultures were optimized using a central composite design. The temperature optima for maximal production of filter paper cellulase (FPase), xylanase and -gluosidase were 32.8°, 34.7° and 31.1° C, respectively, and the optimum times for production of these enzymes were found to be 144, 158 and 170 h, respectively. The optimized culture medium and conditions (33° C) gave 0.55 unit of FPase, 188.1 units of xylanase and 3.37 units of -glucosidase per milliliter of culture filtrate at 144 h of shake culture. Among different carbon sources tested, the maximum enzyme activities were produced with sulphite pulp and all three enzymes were produced irrespective of the carbon sources used. Batch fermentation in a laboratory fermentor using 2% sulphite pulp allowed the production of 0.61 unit of FPase, 145.0 units of xylanase and 2.72 units of -glucosidase. In a fed-batch fermentation on 6% final Avicel concentration FPase and -glucosidase were 3.0 and 2.4 times higher respectively than those in batch fermentation on 2% Avicel. The pH and temperature optima as well as pH and temperature stabilities of T. viride enzymes were found to be comparable to T. reesei and some other fungal enzymes.     

Production of poly-3-hydroxybutyrate by Bacillus sp. JMa5 cultivated in molasses media

A strain of Bacillus sp. coded JMa5 was isolated from molasses contaminated soil. The strain was able to grow at a temperature as high as 45°C and in 250 g/l molasses although the optimal growth temperature was 35–37°C. Cell density reached 30 g/l 8 h after inoculation in a batch culture with an initial concentration of 210 g/l molasses. Under fed-batch conditions, the cells grew to a dry weight of 70 g/l after 30 h of fermentation. The strain accumulated 25–35%, (w/w) polyhydroxybutyrate (PHB) during fermentation. PHB accumulation was a growth-associated process. Factors that normally promote PHB production include high ratios of carbon to nitrogen, and carbon to phosphorus in growth media. Low dissolved oxygen supply resulted in sporulation, which reduced PHB contents and dry weights of the cells. It seems that sporulation induced by reduced supply of nutrients is the reason that PHB content is generally low in the Bacillus strain.     

Characterization of ethanol fermentation waste and its application to lactic acid production by Lactobacillus paracasei

In this study, an ethanol fermentation waste (EFW) was characterized for use as an alternative to yeast extract for bulk fermentation processes. EFW generated from a commercial plant in which ethanol is produced from cassava/rice/wheat/barley starch mixtures using Saccharomyces cerevisiae was used for lactic acid production by Lactobacillus paracasei. The effects of temperature, pH, and duration on the autolysis of an ethanol fermentation broth (EFB) were also investigated. The distilled EFW (DEFW) contained significant amounts of soluble proteins (2.91 g/l), nitrogen (0.47 g/l), and amino acids (24.1 mg/l). The autolysis of the EFB under optimum conditions released twice as much amino acids than in the DEFW. Batch fermentation in the DEFW increased the final lactic acid concentration, overall lactic acid productivity, and lactic acid yield on glucose by 17, 41, and 14 %, respectively, in comparison with those from comparable fermentation in a lactobacillus growth medium (LGM) that contained 2 g/l yeast extract. Furthermore, the overall lactic acid productivity in the autolyzed then distilled EFW (ADEFW) was 80 and 27 % higher than in the LGM and DEFW, respectively.     

Assessment of real-time PCR for quantification of Legionella spp. in spa water

Aim: To identify media and environmental conditions suitable for rapid mycelial growth and sporulation of Diplocarpon mali. Methods and Results: Liquid shake cultures were used to evaluate effects of media and environmental conditions on mycelial growth and conidial production of D. mali. Carrot sucrose broth (CSB), potato and carrot dextrose broth (PCDB) and potato and carrot sucrose broth (PCSB) were most favourable for rapid mycelial growth. PCDB, PCSB, PCB (potato and carrot broth) and carrot dextrose broth (CDB) were favourable for conidial production. All carbon sources tested and peptone favoured for mycelial growth. Carbon and nitrogen sources tested did not significantly stimulate conidial production. The optimum temperature for mycelial growth and conidial production was 25°C. No mycelial growth occurred at 5 or 30°C, but D. mali survived at these temperatures. Active mycelial growth occurred at pH 5–7, and pH 5–8 was favourable for sporulation. Conclusions: PCDB and PCSB incubated at 25°C for 14 day are recommended for mycelial growth and conidial production of D. mali. Significance and Impact of the Study: The information generated in this study will facilitate mycological and pathological research on D. mali and Marssonina leaf blotch of apple caused by D. mali.     

Cellulase and protein production from mixed cultures of Trichoderma viride and a yeast

Fermentations with mixed cultures of the cellulolytic fungus Trichoderma viride and the yeast Saccharomyces cerevisiae or Candida utiliswere examined. The fermentations were carried out in an aerated 5 liter fermentor with NaOH treated barley straw as the cellulose source (2–4%). Yeast was inoculated 24–32 hr after the fungus and the growth of the two organisms was followed through the production of CO₂ and cell protein. In comparison with fermentations with T. viridealone, the production time for maximum yields of cellulases and cell protein was reduced by several days, depending on the straw concentrations. The protein content of the growth product was 21–22% and the amino acid composition of the product resembled that of T. viride alone.     

Evaluation of cellulases produced from four fungi cultured on furfural residues and microcrystalline cellulose

Four fungal strains—Trichoderma viride, Aspergillus niger, Trichoderma koningii, and Trichoderma reesei—were selected for cellulase production using furfural residues and microcrystalline cellulose (MCC) as the substrates. The filter paper activity (FPA) of the supernatant from each fungus was measured, and the performance of the enzymes from different fungal strains was compared. Moreover, the individual activities of the three components of the cellulase system, i.e., β-glucosidase, endoglucanase, and exoglucanase were evaluated. T. koningii showed the highest activity (27.81 FPU/ml) on furfural residues, while T. viride showed an activity of 21.61 FPU/ml on MCC. The FPA of the crude enzyme supernatant from T. koningii was 30% higher on furfural residues than on MCC. T. koningii and T. viride exhibited high stability and productivity and were chosen for cellulases production. The crystallinity index (CrI) of the furfural residues varied after digested by the fungi. The results indicated differences in the functioning of the cellulase system from each fungus. In the case of T. koningii, T. reesei and T. viride, furfural residues supported a better environment for cellulase production than MCC. Moreover, the CrI of the furfural residues decreased, indicating that this material was largely digested by the fungi. Thus, our results suggest that it may be possible to use the cellulases produced from these fungi for the simultaneous saccharification and fermentation of lignocellulosic materials in ethanol production.     

Life‐cycle kinetic model for endospore‐forming bacteria,including germination and sporulation

We develop a mechanistic life‐cycle model for endospore‐forming bacteria (EFB) and test the model with experiments with a Bacillus mixed culture. The model integrates and quantifies how sporulation and germination are triggered by depletion or presence of a limiting substrate, while both substrates affect the rate of vegetative growth by a multiplicative model. Kinetic experiments show the accumulation of small spherical spores after the triggering substrate is depleted, substantially more rapid decay during sporulation than for normal decay of vegetative cells, and a higher specific substrate utilization rate for the germinating cells than that for growth of vegetative cells. Model simulations capture all of these experimental trends. According to model predictions, when a batch reactor is started, seeding with EFB spores instead of active EFB delays the onset of rapid chemical oxygen demand (COD) utilization and biomass growth, but the end points are the same. Simulated results with low aeration intensity show that germination can consume some substrate without dissolved oxygen (DO) depletion. Biotechnol. Bioeng. 2009; 104: 1012–1024. © 2009 Wiley Periodicals, Inc.     

Bio-production of compost with low pH and high soluble phosphorus from sugar cane bagasse enriched with rock phosphate

Summary Aspergillus niger and Trichoderma viride strains were used together as a fungal activator in the presence or absence of farmyard manure (FM) for composting of bagasse enriched with rock phosphate. Quality of the composts produced was compared with that obtained from non-inoculated bagasse. The composts were evaluated as organic phosphatic fertilizers, for broad bean plants. The results showed that composting of bagasse without microbial inoculation or FM addition was not complete after 105 days of fermentation. An excellent decomposition in a relatively short time however was obtained with the use of A. niger and T. viride as inoculant agents with or without FM. The inoculation with A. niger + T. viridewith or without FM, also represented the most suitable conditions for phosphate solubilization. Acidic conditions (pH 4–5) at the end of the experiment were obtained in all piles receiving Aspergillus niger and there was a correlation between the amounts of soluble phosphorus and the reduction in pH values in the compost piles. There were no phosphate-dissolving fungi present in any composted piles except those treated with Aspergillus niger and Trichoderma viride. The number of phosphate-dissolving bacteria increased only in the treatments that were treated with FM. The non-fertilized sandy soil and the non-inoculated bagasse compost did not provide broad bean plants with phosphorus while the composts produced by inoculation with A. niger + T. viride provided the plants with the highest amounts of phosphorus.