Introduction

Out of the twenty naturally occurring amino acids, L -lysine (C₆H₁₄N₂O₂; MW 146.19) is the one of the nine essential amino acid. It's major commercially form is L-lysine –HCL (L - lysine monohydrochloride (Liebl et al., 1991). L- Lysine is commonly produced in a stable and non-hygroscopic hydro chlorinated form of purity higher than 98.5% and moisture content less than 1% (Fechter et al., 1997). It is mainly used as a feed additive in the animal feed industry, mixed with various common live stock such as cereals which do not contain sufficient levels of L-lysine for the live stock's nutritional requirement especially for single stomach animals like broilers, poultry, and swine (Zelder, et al., 2005). (Ishii et al., 1997), and as supplement for humans, improving the feed quality by increasing the absorption of other amino acids. (Georgen et al., 1982). As a fine chemical it is used in human medicine, in cosmetics and in the pharmaceutical industry, particularly as ingredients of infusion solution for pharmaceutical application (Zelder et al., 2005) and as precursor for industrial chemicals. Further more, a production method for industrially producing an optically active lysine derivative useful as a pharmaceutical intermediate is described in Nakazawa et al., 2006. Several hundred thousand tones of L- lysine (800,000 tones/year) are presumably produced annually world wide, almost exclusively using bacterial fermentations. US6984512and WO2005/059139 (Zelder et al., 2005), (Liaw et al., 2006) refer to an annual L-lysine production of approximately 250,000tonnes, instead. Optimal oxygen transfer is perhaps the most difficult task to accomplish. Oxygen is poorly soluble in water -and even less in fermentation broths- and is relatively scarce in air (20.8%). Oxygen transfer is usually helped by agitation, which is also needed to mix nutrients and to keep the fermentation homogeneous. There is however limits to the speed of agitation, due both to high power consumption (that's proportional to the cube of the speed) and the damage to organisms due to excessive tip speed. No significant information has been found in patent literature regarding the effect of air saturation and a little is known regarding the real effect of oxygen on L-lysine fermentation. L-lysine fermentation is an aerobic process [Shimazaki et al., 1983, Tosaka et al., 1981, Asakura etal. 1999] demanding large amounts of oxygen and strongly influenced by the air saturation in bioreactor. Lactic acid is formed as a byproduct under anaerobic conditions, which is reconsumed after the establishment of aerobic conditions. Aerobic conditions are maintained by aseptically adding to the culture oxygen containing gaseous mixtures, e.g. atmospheric air or pure oxygen [Kreutzer et al., 2001, Bathe et al., 2004]. Cultivation of L-lysine producing microorganisms is carried out with shaking of shake flasks (250-300 rpm) or by the aeration (0.5-1.5 vvm) of stirring bioreactors. US5268293 and US6984512 [Oh, J. W., et al., 1993, Liaw et al., 2006] describes bench scale fermentations operating at 2.1vvm aeration rate of atmospheric air and changing agitation speed during the fermentation between 600 (at 0 h) and 900 rpm (at 19 h). Enormous effects of air saturation (100% air saturation corresponds to saturation at 1 vvm aeration rate at 30 ° C and 600 rpm agitation rate) on continuous L-lysine production by B. lactofermentum have been found in chemo stat process development experiments. Many important fermentation processes have been found to depend critically on the availability of the DO in the aqueous phase in contact with the organisms. However, the choice of a proper DOT during the whole process is important to stimulate the product formation without wasting the energy source. Sugar consumption is one of the factors that govern the fermentation process. But it is closely related to other experimental conditions, for example, if the cell oxygen demand is not satisfied, the nutrient consumption will be definitely affected.

The critical DOC and its limitation

The effect of the DOC to the specific microbial oxygen uptake rate can be described by the Michaelis–Menten o r Monod type of expression.

Figure 1

Where, qo is the specific oxygen uptake rate; qm is its maximum value of qo; CL is the DOC in the solution; and Ko is a saturation constant, the DOC when qo=qm/2. The critical DOC denoted by Ccrit is defined as the DOC value at which qo becomes 99% of its maximum value. Above Ccrit, no further increase in oxygen uptake rate occurs and thus the growth rate becomes independent of the DOC. At high cell concentrations, the rate of oxygen consumption may exceed the rate of oxygen supply, leading to oxygen limitations. When oxygen is the rate-limiting factor, specific growth rate varies with DOC according to saturation kinetics. Below a critical Concentration, growth or respiration approaches a first order rate dependence on the DOC. According to Shuler, the critical DOC is about 5% to 10% of the saturated DOC for bacteria and yeast, and is about 10% to 50% of the saturated DOC for mould culture. The specific oxygen uptake rate can be larger in the exponential (growth) phase than that in any other phases, since the cell has the most vigorous activity. When the process goes in to the stationary period, the overall specific growth rate decreases, and so does qo. When glucose is finished, no oxygen is required for glucose oxidation, while oxygen is still needed for maintenance and the excreting of product, although the oxygen demand is relatively small. It may be recognized that equation can only be suitable for the log-phase. In fact, the Monod equation for the specific growth rate cannot explain phases other than the log-phase. It has been declared that the specific oxygen uptake rate is directly proportional to the specific growth rates [ Stanury et al., 1995] or may vary with time [Omstead et al., 1987]. Although linear relationship is not applicable to our case, it does imply that qo will change during different stages, even with a DOT value higher than the critical level. In other words, the oxygen demand for bacteria varies with the different growth phases.

Material and Methods

Figure 2

Fermentation Procedure

Variation of Dissolved Oxygen Tension

Results and Discussions

Figure 3

Table 1: Lysine Productions at Different Dissolved Oxygen Tensions

Variation of dissolved oxygen tension is in the range of 5%, 10% and 20%. From this highest amount of lysine formation is taking place at 20% (52.7 g/lit). The results are show in table 2 and figure 2. .

Specifications:

pH ----7.5
Rpm ---- 300
Temperature ---- 30 °C

Figure 4

Figure 1: Variation of Dissolved Oxygen Tension on Lysine Production in Stirred Tank Bioreactor

Specifications:

pH ----7.5
Rpm ---- 300
Temperature ---- 30 °C

By variation of different parameters in stirred tank bioreactor the high amount of lysine production is takes place as follows by varying, rpm, temperature, inoculum composition. By variation of Dissolved Oxygen tension at 20% (52.7g/lit) high amount of is lysine obtained.

Correspondence to

Dr.Dowlathabad Muralidhara Rao M.Sc.,M.Phil., Ph.D. Asst.Professor Bioprocess Laboratory Department of Biotechnology SriKrishnadevaraya University Anatapur -515003 A.P. INDIA http://www.sku-biotech.org http://www.skuniversity.org