Optimizing of Trichoderma viride cultivation in submerged state fermentation

American Journal of Applied Sciences, July, 2009 by Hayyan Ismaeil Al- Taweil, Mohammad Bin Osman, Aidil Abdul Hamid, Wan Mohtar Wan Yusoff

INTRODUCTION

In the recent years, the environmental contamination caused by excessive use of chemical pesticides increased the interest in integrated pest management, where chemical pesticides are substituted by biopesticides to control plant pests and plant diseases. Trichoderma-Based Biocontrol Agents (BCAs) possess better ability to promote plant growth and soil remediation activity compared to their counterparts (virus, bacteria, nematodes and protozoa (3), (4). Their capability to synthesize antagonistic compounds (proteins, enzymes and antibiotics) and micro-nutrients (vitamins, hormones and minerals) enhance their biocontrol activity.

Like other fungal BCAs, conidial mass of Trichoderma is the most proficient propagule, which tolerates downstream processing (e.g., air drying). Despite the advantages, mass production of Trichoderma BCAs is less prevalent, owing to high-cost raw materials like Mendel's medium, molasses, corn steep liquor and other (8), (9).

Trichoderma spp. Have gained wide acceptance as effective BCAs against several commercial phytopathogens. These antagonistic fungi are most common among fungal biocontrol agents because of their multiple BCA characteristics, namely, antagonism and plant-growth stimulation (10). Thus, mass-scale production of Trichoderma spp. would have great potential for commercial use. Micropropagules of Trichoderma spp. In the form of conidia are preferred over chlamydospores and mycelial biomass because of the viability and stability in field application. Therefore, there are several BCA products of Trichoderma spp. In the market containing conidia of Trichoderma spp. As active ingredients. Multiple BCA action renders the production of Trichoderma spp. Conidia of commercial and environmental interest. There is abundant literature on the use of conventional synthetic media like glucose, cellulose, soluble starch and molasses to produce Trichoderma spp. (5), (6). However, the cost of these raw materials for commercial production of BCAs is one of the major limitations behind the restricted use. To overcome the cost limitation, many researchers have successfully used substrates like corn fiber dry mass, sewage sludge compost and cranberry pomace. Despite the use of alternate sources, the cost of production was still high, as these raw materials need to be supplemented by othernutrients (10). Dominguesa et al. (2) found that the influence of the size binoculum and the composition of the fermentation medium on the morphology and cellulase production were studied. Different inoculums sizes were studied but the significative change in fungus morphology was observed for spore's concentration between [10.sup.5] and [10.sup.7] spores m[L.sup.-1] (i.e., [10.sup.2] and [10.sup.4] spores m[L.sup.-1] in pre-culture medium). In the medium without Tween 80, at low inoculum size, the majority of the pellets were large and well individualized, in contrast, at higher inoculation densities small flocs were obtained, with higher production of soluble protein and higher filter paper activity. It was found that the average pellet size seems to be inversely proportional to the inoculum size. Medium composition, namely Tween 80, also influences the morphology of T. reesei Rut C-30 and enzyme production. The presence of Tween 80 in fermentation medium inhibited the pellet formation of this strain (2). The optimum conditions for cellulase production were [([NH.sub.4]).sub.2][SO.sub.4], 0.5 g [L.sup.-1] as nitrogen source, pH (5.0), temperature (28[degrees]C) and incoulum size ([10.sup.8] spores m[L.sup.-1]). Tween 80 (0.1%) improved the overall cellulase production as under these optimized conditions (7).

Trichoderma viride, a mould of family Hypocreaceae, order Hypocreales and class Ascomycetes, is well known for the biological control and the production of cellulose and chitinase (1). In this study we reported the existence of biological agent strain of T. viride.

The aim of this study is to evaluate and investigate the effects of the submerged state fermentation parameters; concentrations of Carbon g [L.sup.-1]; (10, 45 and 80) Glucose. Nitrogen g [L.sup.-1]; (0.10, 0.35 and 0.60) Ammonium Sulfate. Temperature; (20, 30 and 40). PH; (4.0, 6.0 and 8.0). Rpm; to obtain optimal submerged culture conditions for bio-by production from Trichoderma, to the best of environmental conditions for submerged culture of Trichoderma viride.

The purpose of this study is to optimize the submerged culture conditions to produce the suspension by Trichoderma with respect to several operating variables in shake flask fermentation for industrial biotechnological uses as biocontrol agent on large scale.

MATERIALS AND METHODS

Isolation of producer microorganism: Fungal species was isolated from soil samples by using a selective medium (PDA). Samples were dusted over the plates and the plates were incubated at 30[ or -]1[degrees]C for four days. The microbial colonies were developed which were picked up and purified by streaking and incubated at 30[ or -]1[degrees]C for 3 days. A green colonies forming culture with was selected and identified to be Trichoderma viride (1). The culture was maintained on potato dextrose agar slants.