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Fermentation Kinetics of Yeast Growth and Production

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1 Fermentation Kinetics of Yeast Growth and Production

2 Introduction Fermentation can be defined as an energy yielding process where yeast converts organic molecules (such as sugar) into energy, carbon dioxide or/and ethanol depending on the respiration pathway. Yeast can respire in anaerobically and aerobically. However, yeast gets more energy from aerobic respiration, but in the absence of oxygen it can continue to respire anaerobically, though it does not get as much energy from the substrate. Yeast produces ethanol when it respires anaerobically and ultimately the ethanol will kill the yeast (find out why is yeast continue to produce ethanol even the last is an inhibitor). C6H1206 → 2 CH3CH2OH + 2 CO2 + 2 ATP C6H O2 → 6CO2 + 6H2O APT

3 When the feed substrate to the reactor is not monosaccharide e. g
When the feed substrate to the reactor is not monosaccharide e.g. sucrose (C12H22O11), yeast enzyme cause glycosidic bond to break in a process called hydrolysis

4 Industrial and Commercial Applications
Food Industry ~ Beer ~ Bread ~ Cheese ~ Wine ~ Yogurt Pharmaceutical Industry ~ Insulin ~ Vaccine Adjuvants Energy ~ Fuel Ethanol

5 Objective To find the kinetics of the system by using
Nonlinear Regression (guess for ks and μm) The Sum of the Least Squares and the Lineweaver-Burk Plot methods in order to determine the parameters µm and ks To determine the yield coefficient and to project min. and max. amount yeast cell mass, carbon dioxide and ethanol produced

6 Experimental Set Up Apparatus pH probe D-oxygen probe Mixer
Temperature sensor Bioreactor YSI 2700 Biochemistry Analyzer pH meter Sampling device

7 Experimental: Procedure
Using Biochemistry Analyzer and Spectrophotometer to measure and make calibration curves for sugar and yeast cell concentrations Reactant initial concentration dextrose/or sucrose 25 g/L yeast 3 g/L volume reactant solution 2 L

8 Initial conditions & assumptions
2 L of solution 50 g sugar pH around 5.0 Temperature around 28-30°C Assumptions the bioreactor content is well mixed and has a constant medium volume at a certain initial conditions Temperature is constant pH maintained at optimal pH of 3.00 All reactants or nutrients present in excess except for sugar substrate.

9 Theory In ideal fermentation process in which the growing cells are consuming the substrate (sugars), and producing more cells according to the following scheme. rsx = rate of substrate consumption rx = rate of cell growth s = substrate concentration x = cell concentration P = ethanol concentration (in anaerobic case) rsx rx Cells (x) P Cells (x)

10 Theory The plot showing the trends for yeast cell growth over time
x Biomass The plot showing the trends for yeast cell growth over time

11 Theory continue Yeast Growth occurs in 4 stages
Lag phase, yeast mature and acclimate to environment (no growth occurs) The exponential growth section, the rate of reaction follows first order kinetics During the deceleration phase, a large number of parameters, each with saturation effects, have an effect on the kinetics of yeast growth (such as substrate and waste concentrations) The growth rate is ruled by the limiting substrate concentration (sugar) The final equation, often referred to as the Monod equation, looks very similar to the Michaelis-Menten equation. Stationary phase, no growth occurs due to high waste concentration or compleate substrate consuming ks = the Monod constant (g/L) μm = a maximum specific growth reaction rate (min-1)

12 Lineweaver-Burk Rearrangement

13 Nonlinear Regression Define Model
Solve for Rpredicted (dx/dt) (calculate dx/dt from the polynomial equation fitted to the curve x(t) Make initial guess for ks and μm (µm is the max. specific growth rate can be achieved when S >> ks ks is saturation constant or the value of limiting substrate conc. S at which µs equal to the half of µm Minimize Σ(R-Rpredicted)2 using solver function in Excel by varying ks and μm

14 Yield Coefficient Determination
Ratio of cell or Ethanol concentration to substrate concentration. Knowing Yx/s will give you an idea for how much additional yeast cell mass, on average, is produced for a given amount of sugar substrate consumed. As well allowed you to calculate a lower bound on the experimental stoichiometric coefficient, γ, and therefore to calculate ranges for ethanol and CO2 production. (Yeast Cell) + C6H12O6­ → γ (CO2 + CH3CH2OH) + (Yeast Cells)

15 Error in Lineweaver-Burk Parameters
Error in ks and μm relative to error in slope and y-intercept of linear fit Random Error in y values: STDEV of slope: STDEV of y-intercept:

16 Lower Bound on γ (stoichiometric coefficient)
Assume all yeast generated is attributable only to sugar complete consumption Conservation of mass requires that the remaining product be equimolar amounts CO2 and ethanol (Yeast Cell) + C6H12O6 → ϒ (CO2 + CH3CH2OH) + (Yeast Cells) Where, theoretically, ϒ = 2.

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