Box 31 Production of Lecanicillium muscarium in SSF and LSF

In 1989 Koppert started to mass-produce Verticillium lecanii strain Ve6 (renamed as Lecanicillium muscarium) for Mycotal, in SSF home-built bioreactors. In the past, Tate & Lyle produced it in LSF as they did with the V lecanii strain Ve2 (renamed as L. longisporum) for Vertalec (Quinlan 1988). Tate & Lyle's biopesticide department became independent as Microbial Resources Ltd in 1984. In 1986 all registrations and technical know-how of Microbial Resources were acquired by Novo Industria AS of Denmark (S. Lisansky, personal communication). In 1988 Koppert bought the IP rights of these products and started to produce them. Koppert choose for SSF in order to improve the products and their efficacy. Tate & Lyle produced Mycotal in LSF, the main reason for this was the scale-up possibility which was considered too difficult with SSF technology. LSF was a "second choice" based on spore quality: conidiospores have a better shelf-life and field persistence; on the other hand economy of scale is much better with LSF (S. Lisansky, personal communication). Koppert wanted to produce conid-iospores in order to have a better shelf-life and better efficacy and persistence in the field. At the same time, there was SSF production equipment available as well as experience with the production of Aschersonia aleyrodis. The biore-actors were designed with a tray-system. The fermentors had a surface of 28 m2 (52 trays of x 0.55 m2) and each held 100 kg dry medium. Koppert built 12 fermentors in 1990 and had drying and sieving-milling equipment built. The fermentors were equipped with an inoculation system of ultrasonic nozzles on each tray, and with internal cooling systems. Pre-conditioned air can be lead through the reactors in order to keep the RH high and for supply of oxygen and transfer of CO2 and metabolic heat. Everything was built to keep the system sterile. A large autoclave was designed and built in which one reactor, equipped with wheels, could be sterilized, including the medium. Investment for the reactors was around C120,000 in 1990. A production run (including medium preparation, autoclaving and downstream processing) takes 10 days and mean yields are about 8.10E14 spores/reactor. Annual full capacity is about 25 runs with 10 reactors per run, giving 2.10E17 spores (Approximately 20,000 kg Mycotal (10E13 spores/kg)).

In 2005 our group started to investigate again the LSF technology for Mycotal. The production of blastospores reaches 2.10E10 spores/ml in a 1201 fermentor, and scaling up to 1,000 l fermentors gave a similar yield per ml. A complete production run takes 7 days. To have the same capacity on an annual basis, a LSF fermentor capacity of 1,0001 only needs 10 runs (2.10E16 spores/fermentor). This would only occupy a quarter of its annual capacity. A new set of SSF bioreactors, including autoclave and downstream equipment, would cost around €750,000 in 2007, a 1,000 l fermentor around €300,000, including all equipment. This clearly shows the economy of scale, the difference is about a factor 10: 2.5 times the equipment price, and 4 times the capacity. If variable costs are considered, SSF demands about 10 times more labour to produce 20,000 kg Mycotal. This example shows that the costs for a yearly supply of Mycotal for SSF are much higher than for LSF when investment and running costs are considered (see Table 3.5). Thus, LSF has a considerable advantage over SSF in terms of economy of scale. The decision on how to produce a pathogen, however, is also influenced by quality of spores (see Feng et al. 2002), shelf-life, efficacy, production of metabolites, contaminants, and registration issues. A final decision on whether to produce by SSF or LSF can only be made based on all aspects.

Table 3.5 Parameters of the production of L. muscarium spores in SSF and LSF (SSF = solid state fermentation; LSF = liquid state fermentation)

Production parameters

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