Bioreactor Bioprocessing

PRACTICAL 1 Kinetics Study Batch Fermentation of Baker’s Yeast Objective To study the growth kinetic of Baker’s yeast in batch production Introduction Batch fermentation can be considered to be a “closed system”. At the time of zero, the sterilized nutrient solution in the fermenter is inoculated with microorganism & incubation is allowed to proceed under optimal physiological conditions. In the course of the entire fermentation, nothing is added except oxygen (in a form of air), an antifoam, and acid/base to control the pH. Composition of the culture medium, biomass, and metabolite concentration change constantly as a result of the cell metabolism. The growth of a microbial culture may be divided into a number of stages and already discussed by Monod (1949) and Stanbury and Whitaker (1984). After the lag phase where there are no increase in cell number, one period of faster growth cell was occur (cell increase with time exponentially) or called log phase. After substrate was exhausted the growth ceases and this is called stationary phase. The exhausted of substrate for maintenance and the presence of toxic cause the cell death and this was called death phase. Many fermentation industries still used batch fermentation even the increase of productivity for many types of fermentation can be achieved using continuous and fed-batch fermentation (Whitaker, 1980) Yeast has been used since the beginning of human civilization. It is still a very versatile microorganism widely used in a wide range of fermentation industries. For example, the carbon dioxide released as a result of carbohydrate metabolism is used to raise dough in baking, supporting multibillion dollar alcoholic beverages industry, and single cell protein industry (animal food supplement). Methodology Microorganism The Baker’s yeast, Saccharomyces cerevisiae, was used in all experiment. This is hybrid yeast produced for commercial baker. Media Most practical industrial fermentation processes are based on complex media because of the cost and the choice of the nutrients and the ease of nutrient preparation. The well formulated defined medium was used in this experiment.

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Bioreactor Bioprocessing

Stock Culture Medium Compound Concentration (g/L) Glucose 50 Yeast extract 5 KH2PO4 2 MgCl2 1 NH4Cl2H2O 1 Technical Agar 15 pH 5.5 Pre-seed Culture and Batch Fermentation Medium Concentration (g/L) Compound Pre-seed Batch fermentation Glucose 50 100 Yeast extract 5 10 KH2PO4 2 4 MgCl2 1 2 NH4Cl2H2O 1 2 pH 5.5 5.5 Batch Fermentation Batch fermentation was carried out in fermenter with 1L culture volume. The inoculum was prepared as instructed. A single colony of pure culture from a petri dish, or a slant tube (containing solid gel) was inoculated into the medium flask (250mL) containing 100mL pre-seed medium. Medium was sterilized by autoclaving at 121°C, 15psi for 20 min prior to inoculation. The inoculated flask was constantly agitated in a temperature control flask shaker for 12-14 hours to reach exponential growth phase, or log phase. The S.cerevisiae culture was then inoculated into sterile fermenter containing 900mL batch culture medium to start the fermentation. During the fermentation, samples were withdrawn at time intervals. In normal practice for preparation of inoculums, subculturing was done repeatedly for several times to ensure that the culture is acclimated before employed in fermentation. Sample Analysis Measuring dry weight 1. 1.5mL of sample was inserted into the eppendorf tube. 2. Step (1) was repeated for another four samples from different time interval. 3. Samples were centrifuged at 4000rpm for 20 min. 4. The cell pellet was dried in the oven at 100°C for approximately 30 minutes and dry weight of the sample was calculated. Measuring pH value and optical density at 610nm 1. Spectrophotometer was reset. Wavelength was calibrated at 610nm. 2. Sample was taken and inserted into up to 2/3 of the cuvette.

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Bioreactor Bioprocessing

3. Cuvette was placed on the sample holder and the cover was closed. 4. Machine was started. Absorbance value was determined. 5. pH value was determined by using electronic pH detector. 6. Step 1-5 was repeated for another four samples from different time interval. Specific Growth Rate µ = 1/x (dx/dt) Results Cell biomass concentration against time
Cell Biomass Concentration, X vs Time

18.000 16.000 14.000 12.000
X (g/L)

III

IV

II I

10.000 8.000 6.000 4.000 2.000 0.000

1

2

3
Time (day)

4

5

I II III IV Specific Growth Rate t (day) 1 2 3 4 5 X (g/L) 0.6667 6.7333 12.7333 15.5333 12.5333 1/X 1.4999 0.1485 0.0785 0.0644 0.0798

Legend Lag phase Log phase Stationary phase Death phase

dx 0 6.0666 12.0666 14.8666 11.8666

dt 1 2 3 4 5

µ 0 0.0490 0.3157 0.2394 0.1894

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Bioreactor Bioprocessing

X, cell biomass concentration: [(dry cell (g) + dry filter paper (g)) – (dry filter paper (g))] x 1000 1.5(ml) t (day) 1 2 3 4 5 Dry cell + filter paper 0.8507g 0.8598g 0.8688g 0.8700g 0.8685g Filter paper Difference 0.0010g 0.0101g 0.0191g 0.0203g 0.0188g Difference X 1000 1 10.1 19.1 20.3 18.8 Divide with 1.5 (ml) ≈ X 0.6667 6.7333 12.7333 15.5333 12.5333

0.8497g

µ, specific growth rate: dx/dt = µX µ = 1/X (dx/dt) t (day) 1 2 3 4 5 1/X 1.4999 0.1485 0.0785 0.0644 0.0798 dx 0 6.0666 12.0666 14.8666 11.8666 dt 1 2 3 4 5 Calculation (see formula) (1.4999)(0/1) (0.1485)(6.0666/2) (0.0785)(12.0666/3) (0.0644)(14.8666/4) (0.0798)(11.8666/5) µ 0 0.0490 0.3157 0.2394 0.1894

Value of pH of culture and optical density at 610nm t (day) 1 2 3 4 5 pH value 6.30 6.23 6.21 6.19 6.18 Optical Density (A) 0.045 0.093 0.106 0.146 0.158

Discussion Spectrophotometer and dry weight analysis  Spectrophotometer is an instrument which measures the amount of light of a specified wavelength which passes through a medium. According to Beer's law, the amount of light absorbed by a medium is proportional to the concentration of the absorbing material or solute present.  Monitoring cell growth using spectrophotometer (based on absorbance value) has some advantages: it is vary rapid means of monitoring growth with very minimum disruption to the culture.  However, this method does not directly indicate the cell concentration in the culture (especially when different medium type, condition, or cell strain was analyzed). In this experiment, error of spectrophotometer reading can result from cell shape and medium colour. Spectrophotometer does not able to distinguish between the cell and large particles of debris and viable cells or not.

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Bioreactor Bioprocessing

Dry weight measurements of biomass are independent of cell size and directly proportional to the absorbance (absorbance is a measure of cell mass rather than cell number).  Biomass concentration is one of the most critically needed measurements in fermentation studies. It is also one of the most difficult and unreliable ones. Dry/wet weight methods fail completely if the broth contains other insoluble particulate matter, which is often the case in a practical fermentor. These methods cannot distinguish the viable cells from the dead ones, too.

Specific Growth Rate µ represent the intrinsic rate of growth of the organism under the condition used (temperature, culture medium, etc). If µ is zero, then the number of organism is not decreasing and increasing. If µ is more than zero, then the number of organism will increase exponentially and if µ is less than zero, then the number of organism will decrease. pH value Value of pH becomes more acidic as growth proceeds as a result of metabolic by-product. Conclusion Through this experiment, I become aware and understood about growth kinetic of Baker’s yeast in batch production. References Robb, F.T. 1995. Archaea: A Laboratory Manual. CHSL Press, New York. 1037p. Panikov, N.S. 1995. Microbial Growth Kinetics. Chapman & Hall, London. 378p. Trun, N.J. and Trempy, J.E. 2003. Fundamental Bacterial Genetics. Blackwell Publishing, Oxford. 287p Mendes-Ferreira, A., Mendes-Faia, A., and Lea, C. 2004. Growth and Fermentation Patterns of Saccharomyces cerevisiae Under Different Ammonium Concentrations and its Implications in Winemaking Industry. Journal of Applied Microbiology. 97(7): 540545. http://www.engr.umd.edu/~nsw/ench485/lab9c.htm (accessed on 150209)

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