EXPERIMENT NO. 2 BATCH FERMENTATION OF RECOMBINANT E.

COLI IN BIOREACTOR USING DIFFERENT CONTROL CONDITION OBJECTIVES To study the growth kinetics of E coli in benchtop bioreactor using different control condition BACKGROUND Escherichia coli Bacteria E. coli is classified as nonphotosynthetic and mesophiles bacteria. There are hundreds of different types of E. coli recognized by the combination of sugars and proteins displayed on the bacterial surface. E. coli bacilli have long rods without separation when grown under limiting conditions. It is enterotoxigenic secretes toxins that specifically affecting cells of intestinal mucosa, causing vomiting and diarrhea. The toxins are; Cholera-like, heat-labile toxin (LT) - adenylate cyclase activator and heatstable toxin (ST) - intestinal membrane-bound guanylate cyclase activator. The toxicity factor must be taken into consideration to ensure safety of handling and product. Growth kinetic of E. coli (doubling time) has been recognized at 40C is 0.35 hours. Meanwhile, the minimum, optimum and maximum growth temperature of E. coli are 10C, 37C, and 45C respectively. As a neutrophile, pH optimum of E. coli has been identified between 6 and 7 with the lower limit 4.4 and upper limit 9.0. There are several advantages of using E. coli. It is one of the most-studied organisms for recombinant protein synthesis and the best-studied microorganism where its genetics and physiology are far better understood than any other living organism, which greatly facilitates genetic manipulations. E. coli also do not require any growth factors: they can synthesize all essential purines, pyrimidines, amino acids and vitamins, starting with their carbon source, as part of their own intermediary metabolism. Peptone used in this project (in the media preparation) contains carbon source for that purpose. In addition, a wide range of mutations with specific characteristics and the well-defined vectors and promoters are available, which has greatly speeded up the development of an appropriate biological catalyst. Furthermore, it is easy to attain a very high volumetric productivity because E. coli has a relatively high growth rate and it can reach a very high cell concentration (>50 g dry wt/l), as well as high expression levels by selecting a combination of specific vector and promoter (about 25% to 50% more of total protein). Finally, the production cost of protein is low since E. coli can grow on simple and inexpensive media, and the operational cost of the fermentation process is also relatively low. However there are also disadvantages of using E. coli. One major problem is it does not normally secrete protein, therefore the active protein present intracellularly is limited due to the either proteolytic degradation or the formation of inclusion body and it makes the product separation and purification difficult. This problem can be solved by protein secretion. And also, production of recombinant proteins in E. coli typically, is severely curtailed under slow-growth conditions, such as during the production stage of

fed-batch cultivation, due to the intense competition between normal cell function maintenance and recombinant protein production for the limited available metabolic machinery. So, careful medium selection and controlled growth rate is important. Growth Kinetics Growth phase of microorganism consists of 4 phases; first, the lag phase in this phase cell synthesizes new components and ready to divide. Second, the exponential/log phase microorganism grows and divides at maximum rate possible. Third, the stationary phase population growth ceases and growth curve becomes horizontal. Usually attained at a population level of around 10 9 cells per ml. Reasons are nutrient limitation, limited oxygen availability and accumulation of toxic waste products. And the final phase is, death phase decline in number of viable cells, caused by depletion of nutrients and buildup of toxic wastes. There is also mathematical consideration of growth. During the exponential phase each microorganism is dividing at constant intervals. Thus, the population will double in number during a specific length of time called the generation time or doubling time. Because the population is doubling every generation the increase in population is always 2n, n is number of generations. The resulting population increases is exponential or logarithmic. Bioreactor Experimental Design The bench top bioreactor, 2-litre B-Braun fermenter, is set to operate at the optimum growth condition for E. coli. The design of experiment is shown in Figure 1 and Bioreactors parameters are set according to Table 1.

1: Low Level

2: High Level

F: Factor

Table 1: Design of Experiment (DOE) by Taguchi Method Table 1: Reference for designed experiment for bioreactor condition optimization Parameters Run 1 Run 2 Run 3 Run 4
F: Factor

F1 Airflow (vvm) 0.5 0.5 1 1

F2 Agitation 200 300 200 300

F3 Temp (oC) 35 37 37 35

Prior to the inoculation, the DCU Tower control unit is switched on, and the culture medium parameter such as pH, partial oxygen pressure (pO2), agitation, aeration, and temperature are set according to the predetermined optimum values. pH is calibrated by using standard solution of pH 4.0, 7.0 and 10. pO2 is calibrated by using nitrogen gas (N2). Media for fermentation is prepared without autoclave. Top plate of the bioreactor vessel is unscrewed and lifted-off. The media then is poured into the bioreactor vessel, and closed by screwing back the top plate. The bioreactor vessel then equipped with air sparger, 1M sodium hydroxide (NaOH) solution, 1M hydrochloric acid (HCl) solution,

antifoam solution, pH probe, pO2 probe, and temperature probe. The bioreactor vessel is then autoclaved. Preparation of E. coli cell culture E. coli source is taken from stock culture stored at -80C freezers. The tube is thawed, and a wire loop was used to transfer E. coli into a bijou bottle containing 10ml media. The culture is incubated for overnight at 37 C. Preparation of inoculum The first requirement for a batch scale process is sufficient inoculum culture to start full-scale process. A one tenth volume (10%) inoculum was used to achieve rapid growth without a lag phase. Using aseptic technique, 1 ml of E. coli liquid culture is pipetted into a bijou bottle containing 9 ml of media, and incubated for 5 hours at 37 C. The 10ml starter culture in the bijou bottle is then transferred into a 250 ml flask containing 90 ml media resulting 100 ml culture, and then allowed to incubate in a thermostatted rotary shaker set at 37 C and 300 rpm for 6 hours (for inoculation in bioreactor). The 100 ml inoculum is transferred aseptically into the inoculum flask. Cell Cultivation in Stirred Tank Bioreactor The inoculum flask is taken to the sterilized bioreactor vessel. By using aseptic technique, the connection tube of the inoculum flask is connected the inoculum pipeline of the bioreactor vessel. The connection tube was clamped-off, and by gravity action, the inoculum inside the inoculum flask will flow through the connection tube into the culture vessel. The inoculum vessel needs to be lifted quite high to ensure stable flow of the inoculum. Finally, after all the inoculum has been transferred into the sterilized bioreactor vessel, the connection tube was disconnected aseptically. Do fermentation with conditions as shown in Table 1. The agitation, temperature, pO2 level, and pH are monitored during the fermentation process. Sampling Sampling will be carried out by closing the airflow supply first. Then the plunger of the syringe is pushed down, which opened the outlet tubing. Hose clamp is removed from the connected tube to the culture vessel, and outlet tubing is clamped-off. The plunger of the syringe is slowly pulled to suck the sample through the sampling tube into the sampling reservoir tube. Required amount of sample is transferred into the sampling reservoir tube, and then the plunger is pushed back a little to empty the sampling tube from the culture. This action avoids residues of previous samples in the sampling tube. The hose clamp is removed from the outlet tubing and the transfer tubing from the culture vessel is clamped-off. The outlet tubing is led into a collector (bijou bottle or yellow-

capped test tube). Sample is transferred into the collector by slowly pushing the plunger of the syringe. Sample is driven through the rising tubing of the sample and the outlet tubing into the collector. Flame and ethanol 70% is used during sample collecting for aseptic purpose. Sample of 10 ml will be withdrawn every 60 minutes a bijou bottle during fermentation for measuring the optical density/absorbance (OD, A600), TCN ad CDW, substrate (glucose), and protein concentration. For protein and glucose concentration assay, 5ml samples is withdrawn into a yellow-capped test tube, and centrifuged at 5000 rpm before undergo analysis. Optical density (OD), Total Cell Number and Cell Dry Weight Analysis Please refer to Experiment 1. Substrate analysis (Glucose) Innova YSI 2700 Select biochemistry analyzer with YSI 2710 Turntable is used for direct analysis of glucose (dextrose) concentration. The enzyme Glucose Oxidase is immobilized in the YSI Dextrose Membrane (YSI 2365): Glucose (Dextrose) + O2 H2O2 + D-Glucono--lactone Glucose-oxidase 1ml of sample is transferred into an Eppendorf tube, and centrifuged for 5 minutes at 10,000 rpm. Then, the sample was transferred into a cuvette, put onto the turntable, and analyzed. Protein concentration assay The supernatant obtained from the cell lysis is used to determine the protein content. Seven hundred and eighty micro liter phosphate buffer (100mM) and 20l supernatant is pipetted into Eppendorf tubes. Then, 200l Biorad Protein Assay reagent is added carefully, as it is highly viscous and toxic. The mixtures are shaken and left for a minimum time of 5 minutes and maximum 1 hour, to allow the reaction. The content of the Eppendorf tubes then transferred into cuvettes. The spectrophotometer is set to a wavelength of 595nm and calibrated using a blank, which consist of 800l phosphate buffer and 200l Biorad Protein Assay. Standard curve was prepared earlier by using Bovine Serum Albumin (BSA), which replaces the supernatant. The standard curve then was used to determine the concentration of the protein from the OD value measured.

Growth kinetic study max, maximum specific growth rates are determined after the specific growth rate became constant. The OD600 values of the cultures are determined every one hour until 8 hour. The natural logarithms of these OD600 values were plotted against time and the

slope of the resulting linear regression line was taken as max. The values obtained for max were highly reproducible. Using Michelis-Menten kinetics; for batch growth culture, specific growth rate , is equal to maximum specific growth rate max Ks is known as the saturation constant or half-velocity constant and is equal to the concentration of the rate limiting substrate. In other words, the Monod model implies that Ks can be calculated from measured max and residual glucose concentrations when max/2, Ks been determined. True yield such as YX/S, and YP/S are often difficult to evaluate. Although true yields are essentially stoichiometric coefficients, the stoichiometry of biomass production and product formation is only known for relatively simple fermentations. However, theoretical yields can be related to observed yields such as YX/S and YP/S, which more easily to determine by plotting graph. The true yields can only determined by expression from theoretical yields.

ANALYSIS Parameters for Run 1-4 Time (min) 60 120 180 240 300 360 420 480 RPM pO2 pH OD Glucose Protein Enzyme

Question: 1) Plot the graph and compare for other Runs 2) Which condition to get the highest , td and yield coefficient (YX/S and YP/S)

REFERENCES kesson, M. 1998. A Probing Strategy for Substrate Feeding in Escherichia coli Cultivations. http://www.control.lth.se/~akesson/pub/lic_www.pdf Buckland, B.C. 2001. The Application of Computer Control To improve Fermentation Processes. Daniel R. Omstead (edit.). Computer Control of Fermentation Processes Florida.: CRC Press,Inc. Fieschko, J., and Ritch, T. 1986. Production of human alpha consensus interferon in recombinant Escherichia coli, Chemical Engineering Communications, 45, pp. 229-240. Fordyce, A.P., Rawlings, J.B., and Edgar, T.F. 1998. Control Strategies For Fermentation Processes. Daniel R. Omstead (edit.). Computer Control of Fermentation Processes. Florida: CRC Press,Inc. Lee, S.Y. 1996. High cell-density culture of Escherichia coli, Trends in Biotechnology, 14, pp. 98-105. Minihane, B. J., and Brown, D. E. 1986. Fed-batch culture technology. Biotechnol. Adv. 4:207-218. Prescott, L.M., Harley, J. P., and Klein, D.A. 1999. Microbiology, Fourth Edition. United States of America:McGraw Hill. Shuler, M.L., and Kargi, F. 2002. Bioprocess Engineering Basic Concepts 2nd Edition, USA: Prentice Hall. Stanbury, P.F., Whitaker, A., and Hall, S.J. 2003. Principles of Fermentation Technology, Second Edition. U.K :Butterwort Heiemann.