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- Inert carrier

- Cereal (like) carrier

• Defined artificial medium

• Some influence on medium

• Semi-sterile to sterile

• Downstream easy - complicated

• Attenuation possible

• Variable quality

• Downstream complicated

• Defined artificial medium

• Downstream easy

• Can be expensive

• Risk for attenuation

• Unnatural

2.2.2 Mass-Production Equipment

2.2.2 Mass-Production Equipment

The fundamental question to be asked at the start of product development is which type of bioreactor should be used for the mass production of a pathogen. Many factors play a role and it depends on the possibilities of production of the organisms (Table 3.2), but decisive is the expected scale of the production for the coming years in relation to investment costs. A well-performed marketing study will give estimates of the expected market size, but nothing more than an estimate. Looking ahead for more than 5 years is difficult and investments should not be based on long term projections. It is wiser to choose a system that can be extended or scaled up once the market demand increases. It should also be kept in mind that registration requires detailed information on production and product and that, when scaling up, no major changes occur that would need additional registration information to be submitted and evaluated.

It is beyond the scope of this book to elaborate in detail on the various types of bioreactors and to consider which one to choose for which organism. It is a case by case study depending on the organism, the required propagule for the final product and the scale of production. For solid state fermentation, non-sterile or sterile reactors could be used, non-stirred or stirred or rotating. For LSF sterile reactors are needed. Solid state fermentation is used for many purposes, such as for production of enzymes, metabolites and for the production of (Asian) foods like soy and tofu. To some extent it is also used for the production of entomopathogenic fungi. Many different types of bioreactors have been designed. An overview of techniques and bioreactors is given by Roussos et al. (1997). The literature on solid state fermentation is vast, including on production of entomopathogenic fungi and on nematodes. A careful study of this literature is useful before a new production system is built. Scale and costs and the biology of the pathogen are decisive. It is essential to study the organism in detail in order to know its nutritional and environmental requirements and to adapt the production system to meet these requirements. Process control and management of the crucial parameters are vital. There is no standard recipe for the production method of an entomopathogenic fungus, bacterium or nematode. Each case needs its own approach, although much can be learned from other cases. Fine-tuning is always needed and must be continued over the years.

What companies use is also strongly influenced by their expertise and available equipment. Experience tells us that, particularly in the case of solid state fermentation reactors, many companies develop their own equipment, focusing on their first and main microbial organism. Koppert built their own solid state fermentation reactors, with a tray system, for production of aleyrodis and Lecanicillium muscarium. The former one needs light to sporulate which made it necessary to build "windows" in the reactor, making it more vulnerable to contamination (see Fig. 3.1). Other companies have built and even patented their equipment and technology, like Mycotech (now part of Laverlam) (1995: WO 95/10598) and Prophyta (2004: EP 1502946). Mycotech developed a packed bed reactor for the production of B. bassiana (1995: WO 95/10598); Prophyta produces a biofungicide based on conidiospores of Coniothyrium minitans (Kiewnick 2001). NPP (now part of Arysta LifeSciences Europe) used a packed bed, non-sterile reactor, designed and patented (WO 94/18306) by INRA Dijon (Durand et al. 1997), for the production of B. bassiana for corn borer control. Another reason for these in-house developed bioreactors is that solid state fermentation reactors for pathogen production are hardly available on the market, in contrast to various types of LSF reactors which are standard in the biotechnical and pharmaceutical industry. Sterility, removal of metabolic heat, maintaining water content, and transfer of gases must all be carefully managed in the production of pathogens in SSF reactors. This is similar for LSF reactors, and additional critical aspects are the shear forces. When developing or buying equipment, care should be taken that these parameters can be controlled.

Recovery of the propagules from the mass production system is an intrinsic part of the process. In most cases propagules need to be separated form the medium and this process should not negatively influence the quality of the spores, dauer juveniles, etc. The percentage recovery should, of course, be as great as possible. Harvesting often means a sudden change in conditions and care should be taken to avoid potential damage to propagules by osmotic shock, low or high temperatures, desiccation, shear forces or high pressure. Harvesting methods depend on the production system whether from submerged or solid fermentation. Downstream equipment is usually not a standard type of equipment available to the biocontrol industry and needs to be developed in-house or bought from other industries. Typical apparatuses are centrifuges, separators, sieving and milling equipment, and machinery to dry spores or medium/carrier with spores or mycelium, such as a spray-dryer, a fluidized-bed dryer, a freeze-dryer or a simpler forced-air blower. This equipment is needed to concentrate progagules from the production phase, and in the formulation process. Biocontrol companies have to investigate which equipment is suitable for their processes and develop the appropriate protocols. This is a case by case study. Advantages and disadvantages of various production systems is given in Table 3.4. Mass production is a multi-disciplinary process. On an industry scale both advanced biological and technical knowledge and skills are necessary.

Fig. 3.1 Mass-production equipment (left: 1,000 l fermentor for liquid state fermentation; right: 1.3 m3 bioreactor for solid state fermentation)

2.2.3 Inoculum Stability

The first aspect in producing a microorganism is storing the strain in a way that it keeps its relevant characteristics, mainly its virulence. It is well known that repeated sub-culturing could lead to a reduced virulence or loss of other properties, such as reduced sporulation (Butt et al. 2006) and a decrease in production of cuticle-degrading enzymes (Nahar et al. 2008). Further there is a risk of contamination. Every production batch should lead to yielding propagules of the same quality and obviously the inoculum for the production is the basis for this. Loss of virulence can often be restored by passing it over the target host again, but in a mass-production system this is cumbersome and costly. The solution is to make a master stock from which many sub-cultures are made, also called working seed stock that can be stored for a long period at the appropriate conditions. This is often at low temperatures, deep-frozen or by cryo-preservation. A strain should also be properly identified and deposited in a public collection culture as a back up. This is also a registration requirement. Every production batch can now be started using one of the stored samples. Depending on the organism, a new master stock needs to be made every 2-3 years. A newly made master stock needs to be rigorously checked on identity, on virulence via a bio-assay and on microbial contaminants. Re-identification of the culture by a specialized laboratory further ensures the identity and that the quality of the stock material is still maintained. This makes the whole procedure of keeping stable inoculum relatively easy and cheap. Further, the number of culturing steps in producing inoculum for scaling up to the final mass production should be kept as low as possible to minimize the risk of any loss of characteristics. Any new batch needs to start with inoculum from the original master stock.

Table 3.4 Advantages and disadvantages of various mass-production methods (SSF = solid state fermentation; LSF = liquid state fermentation)

Method

Advantage

Disadvantage

In vivo

• Only economic method for baculovirus production

• Exceptionally used for bacteria (B. popilliae)

• No high technical expertise required

• Low developmental costs

• Easy to scale up when labour costs are low

• Labour-intensive

• High risk for contamination

• No effect of economy of scale

• Host and baculovirus production must be kept separate

• Little influence on "medium"

In vitro

SSF: bioreactors

• Can be used for many fungi and bacteria

• Medium to high start-up costs

• Technical expertise required

• Sterile technology

• Control of parameters possible

• Start-up costs medium to high

• Labour requirements medium

• Effect of economy of scale limited

• Production facility of medium technology required

• Limited influence on medium composition

• Limited homogeneity

In vitro SSF: bags

• Low technical expertise required

• Cheap to scale up to mid-range level

• Low quality facility will suffice

• Labour-intensive

• Risk for contamination, particularly in drying steps

• Labour-intensive downstream methods

• No effect of economy of scale

In vitro LSF

• Versatile; fermentors can be used for fungi, bacteria and EPNs (viruses in cell cultures)

• Sterile technology

• Homogeneous process

• Large effect of economy of scale

• Labour requirement low

• Good ability to control conditions

• Medium optimization possible to high degree

• Great technical expertise required

• High quality equipment and facility needed

• Fungi may not produce conidia

• Large scientific input required, particularly in virus/cell line production

Modified after Lisansky (1993)

Modified after Lisansky (1993)

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