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The growth of a microorganism is extremely dependent upon the surrounding environmental conditions. Every organism has a range of optimum conditions at which members of its species will grow ideally. Inoculum, temperature, pH and dissolved oxygen concentration (among other factors) within the media is often carefully controlled in industrial applications as deviations from the optimal conditions often have profound effects on the growth rate and activity of the organism (Shuler and Kargi, 1992).
When directing experiments involving microbial growth, the preparation and quality of the inocula are as important as any other parameter control. In Streptomyces if mycelia are used in the inoculum, many uncontrolled variables will emerge. These variables include age, stress level and growth stage of the cells, all of which obligatory should be controlled (Kieser et al., 2000). This problem gets even worse when attempting to initiate trials over a period of several months as even small variations in the population size of the inoculum can result in large changes in the level and rate of growth. Streptomyces vegetative inocula are often prepared through the addition of several colonies sometimes from many starter plates to small amounts of media. This is afterward incubated until sufficient biomass is produced, as determined through optical density measurements of the media. This method of vegetative inoculum used by Jakeman et al. (2004) is effective in obtaining biomass; nevertheless it does not provide the consistent inoculum required for comparative trials. The number of cells within the inoculum is hard to directly measure and the inconsistent manner of initially adding cells can result in cells at different stages of growth or stress level. Another method of preparing an inoculum is to harvest spores from the selected culture and store under conditions not satisfactory for germination. The cells within a spore suspension remain in a consistent state while still providing an "instant inoculum" (Kutzner and Nitsch, 1970). This technique also allows long term storage of a viable inoculum that should produce reliable results. By suspending the spores in a soft agar spore suspension, Kutzner and Nitsch (1970) have proven that a suspension of Streptomyces spores will remain viable as an inoculum over a year.
The bacterial growth behavior is greatly influenced by many factors such as environmental conditions and the physiological state of the bacteria. The temperature of the environment directly affects the activity and growth of cells; every species has an ideal temperature for growth that is influenced by its physiology (Shuler and Kargi, 1992). This optimum temperature for growth may not be the same as the temperature at which metabolites are produced most efficiently. Streptomyces venezuelae ISP 5230 are mesophilic bacteria, which means that their optimal growth temperature is between 15 and 40 Â°C. For every increase of 10 °C closer to the optimum growth temperature, the growth rate of bacteria approximately doubles (Truelstrup-Hansen et al, 2002). If the media temperature is above optimal, the rate of growth is reduced. The temperatures used for Streptomyces venezuelae growth have been cited as 30 °C Â± 2 °C (Wang and Vining, 2003; Jakeman et al., 2006). At 42 °C Streptomyces venezuelae express heat shock genes that motivate sporulation and antibiotic production (Yang et al., 2008).
Similarly to temperature, the pH (hydronium ion concentration) of the media affects the growth rate of bacteria by influencing the activity of cellular enzymes (Shuler and Kargi, 1992). To achieve the maximum growth rate level, current literature recommends growing Streptomyces venezuelae in a media with the maintenance of pH between 6.0 - 7.8 (Jakeman et al., 2006). The optimal pH levels are often different when maximizing growth rate and highest product yield (Shuler and Kargi, 1992). However, bacteria are often able to grow within a huge range of pH. Studies on the Streptomyces species by Kontro et al. (2005) have found the optimal pH ranges of growth and sporulation for this species to be between 5.5 and 11.5. The study executed by Kontro et al. (2005) has also found that the media strongly affects the acid tolerance of Streptomyces species. When grown at pH 4 in a media that contains yeast extract most species included in the studies were unable to grow, while at the pH of 7 the majority of species displayed maximum growth rate on most types of media. In order to buffer the pH of the growth medium to near neutral values, a buffer called MOPS (3-(N-morpholino) propanesulfonic acid) is normally used for this species of Streptomyces. While studying the effects of carbon sources on antibiotic production by Streptomyces venezuelae, MOPS was added to jadomycin B production media by Jakeman et al. (2006) however, no buffer was added to the Maltose yeast extract malt extract (MYM) agar that was used for the growth during the study. Glazebrook et al. (1990) studied the effect of different carbon sources on Streptomyces venezuelae sporulation in minimal LS media. In this media, enough amounts of MOPS were added to maintain the pH of the media at near neutral values for every carbon source except glucose. During growth in glucose-based LS media, a high production of oxo-acids was observed resulting in growth inhibition.
The dissolved oxygen available to bacterial cells within the media can be a limiting factor if the rate of consumption exceeds the rate of supply (Schuler, 2000). There is a critical oxygen concentration exclusive to each bacteria species, above which the growth rate is independent of the media dissolved oxygen concentration. This value is usually 5 - 10 % of the maximum saturation level; however the maximum saturation is influenced through the existence of dissolved salts, organics, and the media's temperature (Shuler and Kargi, 1992). The production of secondary metabolites can also be influenced through dissolved oxygen concentration. During antibiotic production Streptomyces clavuligerus, increased oxygen saturation above the critical dissolved oxygen concentration which resulted in additional product and biomass yield (Yegneswaran et al., 1991) up to the maximum saturation limit of oxygen in the media.
The substrate used in the media is an important factor that can affect growth rate of the bacteria as well as its characteristics. For instance, Kontro et al. (2005) found that Streptomyces species changed its behavior as acidophiles, neutrophiles or alkalophiles, depending on the growth media. Most species would grow optimally at pH that is near to neutral or slightly alkaline conditions on a media that included yeast extract (Kontro et al., 2005). Streptomyces venezuelae is grown using many carbon sources including glycerol, glucose, lactose, mannose, maltotriose, maltose starch and more. The effect of these carbon sources on growth and secondary metabolite production is dependent upon many factors such as pH (Kontro et al., 2005). For example, studies on the sporulation of Streptomyces venezuelae in liquid media by Glazebrook et al., (1990) found that while maltose supported the growth and sporulation of Streptomyces venezuelae, glucose significantly inhibited sporulation. Specifically, when grown in minimal LS media using glucose as the main carbon source, the pH of the media dropped rapidly with the production of oxo-acids by the bacteria. The increased acidity likely contributed to growth inhibition; however the glucose was reported to also have an inhibitory effect on sporulation that was un-related to the increased acidity of the media. As the jadomycin family of antibiotics is linked to the sporulation pathway (Wang and Vining, 2003), it is visible that the media has a huge effect on the growth and antibiotic production by Streptomyces venezuelae. Work by Jakeman et al. (2006) found that the carbon source supplied in to the media also has an influence on the antibiotic production by Streptomyces venezuelae. The effect of several sugars on jadomycin B production was studied and important differences in antibiotic production between sugars were found, where glucose was found to be optimal.
The bacterial behavior is greatly influenced by the environmental temperature; however the metabolism and activity of bacteria also affects media temperature. Naturally about 50 - 60 % of energy produced during the metabolism of nutrients within a system is transformed into heat energy (Shuler and Kargi, 1992). The metabolism of sugar is a great source of thermal energy that must be taken into consideration in bioreactor design (Ben-Hassan et al., 1992). Streptomyces venezuelae aerobically metabolize sugars for energy and the production of metabolites. In industry, the amount of substrate used by the microorganism for growth and product synthesis determines the economic viability of the process. The heat energy produced can be determined through the utilization of a heat balance model (Ghaly et al., 1992). Once the heat of mixing is determined, the energy generated by Streptomyces venezuelae can be determined. The work by Ghaly et al. (1992) on the heat generated through mixing and metabolism during lactose fermentation showed that the temperature of media increased from 22 to 32 Â°C with bacterial growth (Figure 3.9). This increase in temperature was due to the heat generated by the yeast (7.4 JÂ·s-1) as well as the heat of mixing (1.01 JÂ·s-1). The amount of heat generated by the organisms in a bioreactor can be estimated by modeling the heat flux over the boundary of the enclosed system. This mathematical model or "heat balance" balances the thermal energy generated within a system with the energy stored and lost from the system (Ben-Hassan et al., 1992). To develop a heat balance using this method, a trial must first be executed on the system through normal stable state operation to evaluate the heat generation during operation of the bioreactor. A heat balance can be performed on a bioreactor to determine the amount of thermal energy generated within the system during normal operation due to mixing. To model the system under different operating circumstances the reactor can be run at several agitation speeds (e.g. 200, 400 and 600 rpm) until a state of thermal balance is reached. At thermal balance the amount of energy generated within the bioreactor is equal to the amount of energy lost; this state of stability can be used to eliminate unknown parameters within the heat balance model.