BT5021- Metabolic Engineering

Assignment-1: (Due 16-9-16)

1. The steady- state metabolic parameters for a marine hybridoma cell line have been determined in continuous
suspension culture over a wide range of dilution rates {Hiller et al. Biotechnology Bioengineering 38,733(1991)].
Listed below are the glucose and glutamine specific uptake rates q glu and qgluc at different dilution rates. Using these
data estimate the yield coefficients and maintenance requirement for glucose and glutamine.

Dilution Rate (days-1) 0.54 0.60 0.66 0.77 0.92 1.05 1.15 1.25

qglu 1.00 0.81 1.03 1.07 1.55 1.59 2.26 3.03

qgluc 4.20 5.44 6.69 7.21 10.4 9.81 10.6 12.7

2. Clostridium acetobutylicum anaerobically converts glucose to acetone, butanol and smaller concentrations of
butyrate , acetate etc. In a fermentation, the following products were obtained from 100 moles of glucose and 11.2
moles of NH3 as nitrogen source.

(i) By performing C, N, H, O balance, determine the cell’s elemental composition.

(ii) Determine the redox status of the fermentation using available electron balance equation.

3. A fed batch reactor is operated at quasi steady state (pseudo steady state) with a microorganism having the following
growth expression.

With µmax = 1.5 /h; Ks = 1 g/L, KI= 50 g/L. The cell yield coefficient YX/S is 0.5 g-dw/ g substrate. The reactor is
started in batch mode with an initial volume of 100 liters and substrate concentration of 100 g/L. After the substrate
is nearly exhausted, a feed is stated at the rate of 1 L/h with a substrate concentration of 100 g/L.
(a) What will be the volume of the reactor after 24 h?
(b) What will be the concentration of the substrate at 24 h?
(c) What will be the concentration and total amount of biomass when t=24 h?
(d) If qp= 0.1 g-product/(g-cells h), what will be the product concentration at t=24h?
(e) After 24, the volume of the medium is reduced back to 100 liters and the fed batch operation continued. Now
what will be the concentration of the product after 48 h?
(f) What will be the product concentration if this cycle is continued 20 times?
(g) What will be the product concentration if the process is carried out in a continuous reactor operated at steady
state with dilution rate of Dw (dilution rate at the time of withdrawal in fed batch)?

4. The metabolic pathway involved in the butyric acid bacteria is shown below. (Ref: Biotechnol Bioeng. Volume 26,
174 (1984) by Papoutsakis et al.).

From the above diagram, the production of pyruvate from glucose through the EMP pathway and the
phosphoenolpyrvate phosphotransferase system, since are in a linear pathway, can be represented by the net
reaction.

Glucose 2 pyruvate + 2 ATP + 2NADH

a) Smilary develop similar reaction schemes for the following metabolites

Pyruvate
Hydrogen production from the electron carrier ferrodoxin (Fd)
Reduction of NAD by FdH
Production of acetate from acetyl-CoA (via phosphotransacetyase and acetate kinase.
Formation of butyryl-CoA from acetyl-CoA
Formation of butyrate from butyryl –CoA (note there are two possible routs)
Butanol production from butyryl –CoA
Acetone production
Ethanol roduction
Isopropanol production from acetone

b) How many independent equations describe the metabolism and how many chemical species are involved?
c) What are the degrees of freedom?