Growth

Laboratory Culture Conditions

The majority of Bacillus species will grow at mesophilic temperatures on commercially prepared nutrient media, although in some cases it is necessary to modify the pH or salt concentration. Obligate thermophilic species such as B. stearothermophilus that do not grow satisfactorily at 37 °C are usually grown at 60 °C. Moderately thermophilic species, such as B. coagulans, are grown between 45 °C and 50 °C. The more fastidious insect pathogens, B. larvae and B. popilliae, require the addition of thiamine for growth and are usually grown at 25 °C to 30 °C. B. stearothermo-philus requires additional calcium and iron, and B. pas-teuri requires 0.5-1% urea.

B. subtilis and many other species are able to grow in simple salts media containing ammonium or amino acids as nitrogen sources and glucose or other simple sugars as carbon sources. The most commonly used general-purpose chemically defined medium is Spizizen's minimal medium (27). The growth rate in this medium can be improved by the replacement of ammonium, a poor nitrogen source for B. subtilis, with glutamine.

B. subtilis is able to use a number of amino acids as nitrogen sources (e.g., arginine, glutamine, glutamate, as-paragine, and aspartate), and the respective catabolic pathways are induced in the presence of these compounds. Consequently, the concentration of many amino acids recommended to overcome auxotrophy are actually growth limiting. It should also be borne in mind that derivatives of the well-studied B. subtilis strain 168 often show tryp-tophan auxotrophy, and consequently complex media in which acid-hydrolyzed casein provides the main source of nitrogen need to be supplemented with this amino acid.

Although many Bacillus species sporulate readily, special media and growth protocols are required for efficient sporulation (14). Sporulation is induced in response to nutrient deprivation and occurs after the end of exponential growth. In B. subtilis, sporulation is repressed in the presence of glucose and at low cell densities. A widely used sporulation medium was described by Schaeffer and colleagues (101). In some species sporulation can also be induced by decoyinine, an analog of GTP (14).

Growth in Batch Culture. Although many species of Bacillus grow under anaerobic conditions, they are normally grown aerobically. Some strains (e.g., B. subtilis) have a tendency to lyse in the absence of an energized membrane (33). Growth is normally carried out in conical flasks in an orbital (reciprocating) incubator at >200 rpm, although most species can be grown in batch fermentors. Ideally, the culture volume should not exceed 20% of the flask volume. The media should be prewarmed as temperature shock can also induce autolysis.

Nutrients are usually supplied in excess and growth continues until a particular substrate is exhausted, the oxygen tension or pH falls to an inhibitory level, or an inhibitor accumulates to an inhibitory level. The various phases in the growth cycle have been described in detail elsewhere (102).

Growth in Continuous Culture. Bacillus species undergo marked metabolic, physiological, and morphological changes during batch culture. Many of these changes occur during transition to and during stationary phase and include morphologically distinct spores and physiologically distinct competent cells. To some extent these population variations can be reduced by growth in a chemostat under continuous culture conditions. This method allows the effect of individual growth parameters (e.g., pH, oxygen tension, growth rate, limiting substrate) on particular cellular characteristics to be identified. Chemostat cultures can be used to study cells under "steady-state" conditions, or during transitions from one steady state to another (103).

In principle any liquid culture medium that supports growth in batch culture can be used for continuous culture. However, because the pH is actively controlled during growth, it is not necessary to include components required only for buffering pH. The concentration of the limiting nutrient determines the culture density, and all other essential nutrients are supplied so that they are in excess. For aerobes, the concentration of the limiting nutrient is set so that the culture density is not so high as to impose an excessive oxygen demand. For B. subtilis this may mean limiting the culture density to about 1-2 g dry weight per liter. Complex media can be used if a defined limitation can be achieved (104). Several defined media have been described using, for example, phosphate, magnesium, potassium, glucose, and sulfate as limiting substrates.

Commercial Culture Conditions

Most of the products traditionally produced from Bacillus species, including enzymes, antibiotics, and insecticides, are synthesized during stationary phase using a tradi tional batch fermentor. However, insecticides require both a high growth yield and efficient sporulation because the ¿-endotoxin accumulates in the sporangium. The production of a-amylase and of B. thuringiensis ¿-endotoxin are given as examples of the factors that need to be taken into account during commercial Bacillus fermentations.

Most species of Bacillus grow well on commercial formulations of nutrient media, although in some cases the medium needs to be modified by adjustment of the pH or ionic strength or by the addition of specific nutrient supplements. This means that media can, to some extent, be based on cheap, readily available substrates. Despite the extensive expertise in the fermentation of Bacillus species, commercial considerations have limited the published literature on large-scale fermentations. Culture conditions and media formulations are highly geared to the particular strains and processes used and these have been selectively reviewed elsewhere (105-107).

Media components have been developed empirically over a number of years to avoid catabolite repression that prevents the synthesis of a number of the commercially important products from Bacillus species. For example, a-amylases are produced in media containing starch in various forms including corn, wheat malt, and potato that is noncatabolite repressing. When carbohydrates are used as a carbon source, phosphate is required at a concentration of about 100 mM. Peptones and amino acids are a better source of nitrogen than ammonium for B. subtilis, probably because of the efficiency with which nitrogen is assimilated into amido groups. In the case of B. apiaries, however, ammonium is the preferred nitrogen source. Glutamine is the nitrogen source that sustains the fastest growth rate. The use of amino acids as a nitrogen source represses the synthesis of proteases and the onset of sporulation, allowing the productive stationary phase to be extended.

The concentrations of divalent metal cations Mn2 + , Ca2 + , and Zn2+ are important to the final yield of a-amylase, even when they have little or no effect on the growth of the bacterium. For example, Ca2 + is required for both the enzymatic activity and structural integrity of the liquefying a-amylases such as those from B. licheniformis, B. amyloliquefaciens, and B. stearothermophilus. We have evidence that this results, at least in part, from the rate of folding of the enzyme on the trans side of the cytoplasmic membrane (62). Other metal ions, including Fe2 + , Fe3+, Na + , and Mg2 + , enhance enzyme production presumably in part as a result of their effect in increasing growth yield.

The growth kinetics in Bacillus cultures is complicated by the operation of the stringent response and sporulation, both of which are similarly induced by nutrient limitations and other negative growth factors. Commercial processes often use fully or partially (oligosporogenous) sporulation-deficient strains. The stringent response, modulated by variations in the levels of ppGpp(p), appears to be a cause of linear growth. Substrates with a low carbon to energy ratio gives rise to carbon-limited growth and consequential "energy spilling" and the production of overflow metabolites. Glucose excess chemostat cultures of B. stearother-mophilus accumulate acetate up to 34% of the carbon consumed. Under these conditions, energy efficiency is sacrificed to obtain the ATP turnover required to sustain the high growth rates. Similar metabolite overflows have been observed for B. licheniformis (48%) and B. subtilis (22%).

Catabolite repression is one of the major factors limiting the production of many commercially important products. Although mutants (e.g., ccpA) have been isolated that lack elements of the catabolite repression pathway, these often grow poorly. An alternative approach in which CRE elements are deleted or inactivated may be frustrated by their occurrence within DNA sequences encoding the protein itself. Often, the most effective method for overcoming ca-tabolite repression involves a combination of appropriate media formulations and growth regimes.

Many commercially significant products are produced after the end of exponential growth (transition phase) and during the early stages of sporulation. It is therefore essential that, when sporulation-deficient strains are used, they still retain the ability to synthesize the required product. For example, mutants deficient in Spo0A (the sporulation-specific response regulator) are unable to induce various extracellular proteases (108).

Although batch cultures are traditionally used for a-amylase production, because the main production phase is during stationary phase, Emanuilova and Toda (109) compared the production of a-amylase by B. caldolyticus in batch and continuous culture. Casitone and starch were required for optimal a-amylase production. The concentration of amylase was 10- to 20-fold higher in continuous rather than batch culture, although there was an inverse relationship between growth rate and amylase production.

Industrial fermentations are carried out at 30-40 °C and at pH 7.0 with CaCO3 as the calcium source to stabilize the enzyme. The complex medium (110,111) allows growth to 1010 cell/ml within 20 h, after which time production continues for about 6 days (111). The fermentations also produce proteases that are important for the utilization of protein substrates, but which have to be separated from the a-amylase during subsequent downstream processing.

a-Amylase production can be increased by cloning the structural gene onto a multicopy plasmid, and production, possibly up to the limitation of the native secretory pathway, is achievable (112). However, because many of the plasmids used in Bacillus species are based on staphylo-coccal plasmids (e.g., pUB110), both segregational and structural instabilities are frequently observed.

Because ¿-endotoxin production of B. thuringiensis occurs during sporulation, growth and process conditions need to be optimized to give cell densities >5 X 109 per milliliter and sporulation rates greater than 90%. Although significant strain-to-strain variations are observed, certain basic requirements can be defined: these include good oxygenation, a growth temperature between 26 and 30 °C (higher temperatures can lead to plasmid curing and consequential loss of ¿-endotoxin production [113]), and pH 6.5-7.5. The production of amylases and proteases by B. thuringiensis allows a wide range of natural raw materials to be used as growth substrates. Glucose is catabolized with the production of acetate, lactate, pyruvate, and ace-toin (114,115), and pH control is required for some strains to prevent the acidic conditions restricting growth. The production of poly-S-hydroxybutyrate during early stationary phase under carbon excess can be mistaken for ¿-endotoxin but disappears during sporulation.

Cultures are usually grown in submerged liquid cultures as large as 200 L using fermentors agitated with paddle blades and with static baffles on the side wall of the vessel (99). Occasionally vessels with impellers or airlift fermentors are used. Because the industrial media contain significant amounts of insoluble particles, sterilization is accompanied by agitation. Toward the end of the fermentation, the released spores attach to and stabilize the foam, and silicon- or polypropylene-based antifoam agents are required as foam suppressers. Antifoam agents can form aggregates that interfere with downstream processing, and consequently their use is kept to a minimum. During stationary phase, oxygen consumption is markedly reduced and, provided saturation levels do not fall below 20%, agitation can be reduced, with a concomitant reduction in foaming. At the end of fermentation, the fermentation vessel will be heavily contaminated with spores that need to be inactivated if different strains are used in the next fermentation run.

Bacillus thuringiensis will grow on glucose minimal medium only when supplemented with sources of amino acids (e.g., peptones, casamino acids) or proteins. Because B. thuringiensis produces amylases, glucose can be replaced with starch as the carbon source. Growth is stimulated by yeast extract and optimal concentrations of Fe3 + ,Co +, and Zn + , and sporulation is improved by metal ions such as Ca2+ and Mn2+ . Phosphate is added both as a source of phosphorus and as a buffer.

Preparations of B. thuringiensis are applied at a rate of about 0.5 to 2 kg per hectare. Spores can be applied directly as a pest control agent, but are often processed to improve their physical characteristics. For example, granular or bait formulations are applied to the plants or soil; in other cases, concentrates are diluted in water and applied by spraying. Stickers, spreaders, or even chemical insecticides can be added to improve dispersion and activity (99).

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