Most of culture experiments in biotechnology had been studied in shake-flasks (Büchs, 2001). ). A thorough study is required to study the most effective and optimum condition for the enzyme productivity. Enzymes in general facilitates chemical reactions for either combination or separation of things. The enzymes are always in constant random motion which then bind with other specific molecules that complement its shape known as substrates. The motion of the enzyme determine the duration for complete reaction between the enzyme and substrates. However, increasing of velocity may increase collision between molecules and thus damage the specific shape of either the enzyme or substrate, preventing a proper interaction between the two. Therefore, all factors that may influence the production of tetrathionate hydrolase are studied to ensure optimum condition for its production are achieved.
2.3.1 Media Components
2.3.1.1 The Influence of Phosphate concentration
Phosphate is required for common bacterial function (Beck et al., 1964). Cells grown with phosphate limitation present a filamentous morphology due to a lack of cell division (Seeger et al., 1993). Phosphate starvation studies show changes in the degree of synthesis of more than 25 proteins, where some of the protein are synthesized under starvation conditions (Seeger et al., 1992; 1993).
According to Harahuc et al., (1999), sulfur-grown cells showed inhibition at high phosphate concentrations, similar to sulfate or chloride. Sulfur oxidation increased to twice the control level in the presence of 50 mM phosphate by growing cells (Harahuc et al., 1999). Harahuc et al. (1999) also reported that at 50 mM, potassium phosphate stimulated growth on sulfur as well as sulfur oxidation. The stimulatory effect of phosphate was confirmed with Thiobacillus ferrooxidans adapted on sulfur; 75 mM potassium phosphate increased growth on sulfur by 20% and sulfur oxidation by 30%
2.3.1.2 The Influence of Sulfate concentration
As Thiobacillus ferrooxidans is known for its ability to oxidize iron and sulfur, the concentration of sulfate present in the growth substrate plays important role in determining the rate of sulfur or iron that will be oxidized. A study showed that when the cells were grown on ferrous iron resulted in low levels of sulfur oxidizing activities compared to the cells grown in sulfur (Harahuc et al., 2000). Therefore, finding the optimum concentration of sulfate present on the media used may helps to increase the oxidation of sulfur rate during cultivation process.
Sulfate involved in transfer of electrons from the iron sulfur group to the copper-containing protein called rusticyanin. The rusticyanin reduction by ferrous ion required sulfur involvement was reported by Blake and Shute (1994). A study showed that usage of 0.2? of sulfate had very small effect to both iron and sulfur oxidation. However, above this concentration sulfur oxidation showed a very significant drop in activity that may be resulted from the changes in the osmotic pressure. The high osmotic pressure not only inhibit the sulfur oxidation in sulfur-grown cells but also in iron-grown cells (Harahuc et al., 2000).
2.3.1.3 The Influence Ammonium Concentration
Inorganic nutrients required by the bacteria include nitrogen (as ammonium), phosphorus (phosphate), sulfur (sulfate), and trace metals. Ammonium was shown to be a limiting nutrient for iron oxidation in cultures of Thiobacillus ferrooxidans. At high concentration the nitrogen-contained compound would be inhibitor and at low concentration it can helps to stimulated the iron oxidation in ammonium limited cultures. In some ammonium-unlimited media, the iron oxidation rates were enhanced which was believed to be contributed by the role of these compound in providing the trace nutrients for the bacteria growth (Tuovinen et al., 1979).
Ammonium sulfate is required for both activity and stability of tetrathionate enzyme (de Jong et al., 1997). Ammonium levels of 0.2 mM have been reported to be sufficient to satisfy the nitrogen requirement of Thiobacillus ferrooxidans (Tuovinen, 1971). Exactly how much nitrogen needs to be present in a growth medium will be dependent on the quantity of cell growth to be supported. Iron oxidation using cell suspensions was completely inhibited by sodium nitrate concentrations of 1 to 94 mM (Lazaroff, 1977). Sulfur oxidation has higher resistant, showing little to no inhibition up to 0.1 M. Increasing of nitrate concentrations, however, resulted in a significant drop in sulfur oxidation (Harahuc, 1999).
2.3.1.4 The Influence of Potassium Concentration
Harahuc (1999) reported in their iron oxidation experiments that were carried out at pH 2.3 and 1.8 showed a stronger inhibition by the potassium salts compared to sulfur oxidation while using the same Thiobacillus ferrooxidans strain. Increasing the concentration of potassium over the levels present in the basal salts of medium 9K had no effect on growth. However, increasing the concentration of dibasic potassium phosphate to 2.75 g/l (or more) resulted in complete inhibition of growth (Silverman, 1958). At 50 mM, potassium phosphate stimulated growth on sulfur as well as sulfur oxidation. The stimulatory effect of phosphate was confirmed with Thiobacillus ferrooxidans adapted on sulfur; 75 mM potassium phosphate increased growth on sulfur by 20% and sulfur oxidation by 30% (Harahuc, 1999).
2.3.1.5 The Influence of Magnesium Concentration
Beck (1964) reported that magnesium sulfate or ammonium sulfate or both, had no effect on the rate of activity or on the phosphate. Based on the batch experimentation it appeared that though Mg2+ ions were essential for the bio-oxidation of ferrous ions using Thiobacillus ferrooxidans, the dose of magnesium sulfate required for an efficient oxidation process to take place was as low as 3 mg/l (as Mg2+) (Harahuc, 1999). Similarly, the magnesium ion (Mg2+) is also one of the requirements for Thiobacillus ferrooxidans during bio-oxidation of ferrous to ferric ions (Harahuc, 1999).
2.3.1.6 The Influence of Chloride Concentration
Chloride ion is known by its role as an osmolyte in osmo-adaptation. It is important for growth as it is responsible to modulate gene expression and enzyme activity involved in osmo-adaptation. The sensitivity to high osmotic pressure was higher for sulfur oxidation process than iron oxidation, which made the need of chloride ion in the media. However, there in not many studies that explained on other biological functions of chloride in bacteria [26].
At the same time, chloride ion is a known inhibitor of cell growth and ferrous iron oxidation but the sensitivity to chloride is much higher for iron oxidation than sulfur oxidation [27]. In sulfur oxidation, the inhibition of the process by chloride occurs at much higher concentration compared to iron oxidation. Increased in chloride concentration decrease specific activity of the enzyme and thus decreases the enzyme activity. Therefore, the chloride concentration should be high enough to control the cell volume and fluid balance, but low enough to avoid inhibition of cell growth.