Fermentation Mass Production

There are potentially three fermentation systems that may be used for mass production of mycoherbicide agents: submerged liquid culture, solid substrate fermentation, and two-phase system (Auld 1993a). At the industrial level, liquid fermentation is the most common method for economical production of microbial inoculum. Two commercial myco-herbicides, Collego® and Devine®, are manufactured this way in the United States (Churchill 1982; Stowell 1991). By understanding the importance of nutritional and environmental factors on induction of fungal sporulation, spore yield, and bioherbicidal efficacy, rational approaches can be taken to develop the most efficient liquid production procedures for mycoherbicide agents. Jackson and Bothast (1990) reported that carbon concentrations and C/N ratios are the key to sporulation of the mycoherbicide agent C. truncatum. Carbon concentrations ranging from 0.4 to 1.5% gave highest spore yields while higher concentrations (2-4%) inhibited sporulation. Similarly, a C/N ratio at 15:1 was more favorable for sporulation than 40:1 or 5:1. In a further study, it was revealed that nutritional aspects impacted not only spore yield, but also spore efficacy of C. truncatum in controlling hemp sesbania (Jackson et al. 1996). Morin et al. (1990b) also observed that sporulation of Phomopsis convolvulus was completely inhibited when the C/N ratio was reduced from 1:1 to 1:5 using modified Richards medium, but the effect on spore efficacy was not reported. By understanding these impacts, fermentation procedures can be fine-tuned to maximize the production and potency of mycoherbicide agents. One of the drawbacks with liquid fermentation is that down-stream processing can be more complicated and costly. Large centrifuges are normally required to spin off spores and often a large number of spores can be imbedded in the mycelial biomass (Auld 1993a). Following recovery from the fermentor, it is usually necessary to dry the spores for long-term storage but retaining spore viability during the drying process may not be easy. Generally, spores should be dried rapidly and gently, but the drying conditions will vary with each organism. Drying methods frequently used include freeze, air, spray, or fluid-bed drying, or a combination of these methods (Churchill 1982). Solid substrate fermentation is used less commonly in commercial production of microbes except for the mushroom spawn industry. Often defined nutrients are added with liquids or solid materials such as vermiculite or paper pellets (Auld 1993a). Various cereal grains have been used to produce fungal inoculum and it is relatively easy to quantify and disperse the inoculum on these solid substrates (Boyette et al. 1991). In some places nutritive solid substances such as nutshells or straw may be available locally at low cost. Higher labor costs, difficulties in maintaining sterility, lack of control of cultural conditions, and recovery of spores from the substrate are inherent problems with solid substrate fermentation (Churchill 1982). Pfirter et al. (1999) evaluated a variety of solid substrates and found that Stagonospora convolvuli, for control of field bindweed, sporulated the best on cous-cous (cracked hard wheat) followed by maize semolina, yielding 5 x 108 spores/g substrate and 3 x 108 spores/g, respectively. Morin et al. (1990b) also reported the production of 7 x 108 conidia/g with P. convolvulus using pot barley grain as a solid substrate. They also compared liquid and solid fermentation methods and found that conidia produced using both systems were morphologically similar and there were no differences in pathogenicity. Particle size, moisture content, and temperature appear critical for successful solid substrate production. A mycoharvester developed at CABI Bioscience (www.dropdata.net/mycoharvester) appears to be a simple device for collecting spores of the mycoinsecticide fungus, Metarhizium anisopliae, produced on rice grains. This device has also been attempted to reduce inoculum impurity of the mycoherbicide Pyricularia setariae (Gary Peng, unpublished data). By reducing the proportion of large particles in the inoculum, the mycoherbicide can be applied at high spore concentrations and low carrier volumes using common spray equipment (Peng et al. 2001). A two-phase system produces mycelium in deep tank fermentation followed by sporulation in shallow open trays. This method may be particularly useful for fungal agents that cannot be manipulated to sporulate in submerged culture, but this system is labor-intensive and expensive, and additional handling of the material may lead to contamination of the final product (Rosskopf et al. 1999). Liquid fermentation produces a large amount of biomass efficiently and sporulation in a "dry phase" may circumnavigate costly down-stream processing issues. Walker (1980) used A. macrospora as a model system to first produce fungal mycelium in a liquid, then homogenize and mix it with vermiculite, followed by thinly spreading the homogenate on a solid surface. Using a similar system, Walker and Riley (1982) successfully produced spores of A. cassiae, a mycoherbicide agent for control of sicklepod. One area of fermentation that can often be overlooked is the feasibility of scale-up from shake flask laboratory volumes to production-plant level. Optimization of fermentation conditions and media components can be readily achieved in the laboratory while pilot-plant scale fermentation can help to effectively verify these selected parameters (Kwanmin 1989). Some sophisticated pilot-scale devices with consistent designs to large-scale facilities are now available. These units are particularly useful for scale-up studies and provide a wide range of pH, agitation speeds, impeller designs, aeration rates, choices of regulated incoming gases, variations in baffling and background pressures, and temperatures (Churchill 1982). However, conditions required to reach optimal yield, costs, and efficiencies in production scale fermentation can be more difficult to achieve. Some biological factors that need to be considered are culture stability, number of generations, and mutation rate while chemical factors include pH, water quality, and fermentation medium quality. Physical factors that should be evaluated include aeration, agitation, pressure, temperature, and medium sterilization. Kwanmin (1989) indicated that factors that may not have been important at a smaller scale could have significant impact on the operation and design of fermentation processes at the production plant level.

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