Construction and standardization of a bioreactor for the production of alkaline protease from Bacillus licheniformis (NCIM-2044)

Standardization of a Bioreactor for Industrial purpose is of prime importance. The construction of the Bioreactor needs a proper understanding of the different parts of the bioreactor and how they could be used. Hence the basic focus of this work was the Construction and the Standardization of the Bioreactor. The Production of Protease was done by altering the pH and checking the KL a level in the fermentation Process. Introduction Enzymes are biocatalysts, that catalyze (i.e. accelerate) chemical reactions [1]. In enzymatic reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, the products. Bacillus licheniformis is a Grampositive, spore-forming bacterium widely distributed as a saprophytic organism in the environment. B. licheniformis is a facultative anaerobe, which may allow it to grow in additional ecological niches. Optimal temperature for enzyme secretion is between 37°C to 41°C. Component parts of a fermentation process Regardless of the type of fermentation an established process may be divided into six basic component parts; • The formulation of media to be used in culturing the process organism during the development of the inoculum and in the production fermenter. • The sterilization of the medium, fermenters and ancillary equipments. • The production of an active, pure culture in sufficient quantity to inoculate the production vessel. • The growth of the organism in the production fermenter under optimum conditions for product formation. • The extraction of the product and its purification. • The disposal of effluents produced by the process. Bioreactor: Bioreactor (fermenter) is the heart of the fermentation process. In designing and construction of a bioreactor a number of points must be considered; Construction Materials: In fermentations with strict aseptic requirements, generally, it is important to select materials that can withstand repeated steam sterilization cycles. Two basic types of fermenters are used;va glass vessel with a round or flat bottom and a top flanged carrying plate. All vessels of this type have to sterilized by autoclaving. A glass cylinder with stainless steel top and bottom plates and these fermenters may be sterilized in situ, but 30 cm diameter is the upper size limit to safely withstand working pressures [10]. Temperature Control: Normally, in the design and construction of a fermenter there must be adequate provision for temperature control which will affect the design of the vessel body as heat will be produced by microbial activity and mechanical agitation [10]. Aeration and Agitation The type of aeration-agitation system used in a particular fermenter depends on the characteristics of the fermentation process under construction. The structural components of the fermenter involved in aeration and agitation are: • The agitator (impeller). • The aeration system (sparger). • Baffles. Volumetric Mass-Transfer Coefficient (KLa): The oxygen demand of an industrial fermentation process is normally satisfied by aerating and agitating the fermentation broth.. Rate of oxygen transfer from air bubble to the liquid phase may be described by the equation; dCL/dt = KLa(C* CL) Where, CL is the concentration of dissolved oxygen in the fermentation broth (Mmoles dm-3) t is time (hours) Construction and standardization of a bioreactor for the production of alkaline protease from Bacillus licheniformis (NCIM-2044) International Journal of Microbiology Research, ISSN: 0975-5276, Volume 1, Issue 2, 2009 33 dCL/dt is the change in oxygen concentration over a time period i.e., oxygen transfer rate (Mmoles O2 dm-3h-1) KL mass transfer coefficient (cmh-1) a is the gas/liquid interphase area per liquid volume (cm2 cm-3) C* is the saturated dissolved oxygen concentration (mmoles dm-3) [10] Determination of KLa values: The determination of the KLa of a fermenter is essential in order to establish its aeration efficiency and to quantify the effects of operating variables on the provision of oxygen. The following are the techniques used for determination of KLa; • Sulphite oxidation technique • Gassing-out technique • The Static Method of Gassing-out • The Dynamic Method of Gassing-out The major factors involved in scale-up are: • Inoculum development • Sterilization • Environmental parameters • Nutrient availability, • pH, • Temperature, • Dissolved oxygen concentration, • Shear condition, • Dissolved carbon dioxide concentration • Foam production All the above parameters are affected by aeration and agitation, either in terms of bulk mixing or the provision of oxygen. Points a, b, c and e are related to bulk mixing whilst d, e, f, & g are related to air flow and oxygen transfer. Modes of Bioreactor Operations: Bioreactors can be operated in following three ways; • Batch bioreactors • Continuous bioreactors Fed-batch bioreactors Materials and Method Organism and growth conditions: Stock cultures of Bacillus licheniformis, (NCIM 2044) obtained from N.C.L, Pune were maintained and subcultured weekly on Nutrient Agar slants. Inoculum preparation: A loopful of culture was taken out from the Nutrient Agar slant with the help of a sterile nichrome wire loop under aseptic conditions, and was used to inoculate the Alkaline Protease Producing Broth (APPB) which had already been autoclaved before inoculation. Shake flask studies: A 1.0 ml portion of the above inoculum was transferred to 8 different 250 ml side-arm flasks containing 100 ml of APPB each, with pH range varying from 7.5 to 11.0 respectively under complete aseptic conditions. Two type of centrifugation process were carried out in order to get the supernatant containing the extracellular enzyme; • In first process, the samples were centrifuged at 4000 rpm for 40 mins. • In second process, the samples were centrifuged at 16000 rpm for 40 mins at 4 C. Measurement of proteolytic activity: Proteolytic activity of the organisms, recovered from the 8 different side-arm flasks of various pH ranges was determined on the basis of their ability to produce a clear zone of hydrolysis on skim milk agar plates by agar ditch method. Diameter of the zone of clearance was measured in mm. Estimation of total extracellular soluble protein: The total extracellular soluble protein content present in the supernatant was quantitatively estimated according to the Bradford method of protein estimation [9]. • The above mentioned solutions after preparation were vortexed and mixed thoroughly. • The samples were prepared as 40 μl sample + 760 μl of distilled water + 200 μl Bradford reagent (volume was made up to 5 ml as previously mentioned). • The spectrophotometer was set at a wavelength of 595 nm. • The blanks were first added into clean glass cuvettes and the instrument was auto zeroed. • Firstly, the standard solutions were added into the cuvettes and the O.D. was recorded for all the standard solutions. • Subsequently, the samples were added into the cuvettes and the O.D. of the same was recorded. • A graph of concentration vs. O.D. was plotted and the values of x and y were calculated. • Using the values of x and y the concentration/200 μl was calculated. Quantification of Alkaline Protease: Protease activity was determined spectrophotometrically by the Anson method, with a slight modification. The samples were prepared by mixing 0.1 ml enzyme (supernatant) with 1.0 ml (0.5 % casein) and 1.9 ml 0.1 M Tris-HCl Buffer, at 37°C for 30 min, and then the reaction was arrested by the addition of 2.0 ml of 5% trichloroacetic acid (TCA). This mixture was centrifuged at 3500g for 20min, and the released amino acids (in supernatant) were measured as tyrosine by Folin-Lowry method. The further tests for FolinLowry were carried out as follows; Firstly, a stock solution of BSA was prepared at a concentration of 0.2 mg/ml, which was used for preparing the standard dilutions for the assay. The standards and the samples were prepared according to the table listed below: Substrate utilization: Substrate utilization was carried out in order to determine the uptake of glucose from the fermentation medium by the cells for biomass production. The yield factor (Y)