Solution: Assignment 4

Bioreactor design and analysis – 2022

You are a biochemical engineer in a pharmaceutical company and you are asked to
design a well-mixed two-stage chemostat system as shown below in the following figure
for production of a novel secondary metabolite.

James M. Lee, Biochemical Engineering, Prentice Hall, 1992

The volume in each reactor is 0.5 m3; the flow rate of feed is 50 L/ h. It is a two stage
process, where mycelial growth occurs in the first reactor and the second reactor is used
for product synthesis. The concentration of substrate in the feed is 10 g /L.

Kinetic parameters and the yield coefficients of the organism are: YX/S (Biomass yield
with respect to substrate) = 0.5 kg/kg; KS = 1 kg/m3; μmax = 0.12 h-1; ms (maintenance
coefficient) = 0.025 kg kg-1 h-1; qp (Specific product formation rate) = 0.16 kg kg-1 h-1, YP/S
(Product yield with respect to substrate) = 0.85 kg kg -1.

Assume that product synthesis is negligible in the first reactor and growth is negligible
in the second reactor. You are asked by the manager to determine:

Given, V1 = 0.5 m3 = 500 L, V2 = 0.5 m3 = 500 L

Yx/s = 0.5 gg-1, Ks = 1 gL-1
µmax = 0.12 h-1, mS = 0.025 gg-1h-1
qp = 0.16 gg-1h-1, Y p/s = 0.85 gg-1
F = 50 Lh-1

1. Substrate concentration in the inlet feed of the second reactor

For substrate concentration,

 Steady-state mass balance equation for biomass:
𝐹𝑋0 − 𝐹𝑋1 + μ𝑋1𝑉 = 0

X0 = 0, (Assuming sterile feed)

𝐹 50 𝐿ℎ
−1
−1 µ𝑚𝑎𝑥 * 𝑆1
µ≈𝐷 = 𝑉
= 500 𝐿
= 0. 1 ℎ = 𝐾𝑆 + 𝑆1

𝐷 * 𝐾𝑠
𝑆1 = = (0.1 ℎ−1)(1 𝑔𝐿−1)
µ𝑚𝑎𝑥− 𝐷 −1 −1
(0.12 ℎ − 0.1 ℎ )

S1 = 5 gL-1

2. Cell concentration in the inlet feed of the second reactor:

Steady-state mass balance equation for substrate:

µ 𝑞𝑝
𝐹𝑆0 − 𝐹𝑆1 − ⎛ 𝑌 + 𝑥 𝑌𝑝
+ 𝑚 ⎞𝑋1𝑉1 = 0
𝑠 𝑠
⎝ 𝑠⎠

(Assuming that product synthesis is negligible in the first reactor)

𝐹𝑆0 − 𝐹𝑆1 − ( µ
𝑌𝑥
𝑠
+ 𝑚𝑠 𝑋1𝑉1 = 0 )
Rearranging the above equation

𝐹
𝑉
(𝑆0 − 𝑆1) =
1
( µ
𝑌𝑥/𝑠
+ 𝑚𝑠 𝑋1 )
𝐹
(𝑆0−𝑆1) −1 −1 −1
𝑉1 0.1 ℎ (10 𝑔𝐿 −5 𝑔𝐿 )
𝑋1 = =
( ) ( )
−1
µ 0.1 ℎ −1 −1
𝑌𝑥/𝑠
+𝑚𝑠 −1 +0.025 𝑔𝑔 ℎ
0.5 𝑔𝑔

𝑋1 = 2. 2 𝑔/𝐿

3. Substrate concentration in the exit stream from the second reactor

X2 = X1 = 2.22 g/L (only product is being formed in the second fermenter)

Steady-state mass balance equation for substrate:

𝐹𝑆1 − 𝐹𝑆2 −
( µ
𝑌𝑥/𝑠
+
𝑞𝑝
𝑌𝑝/𝑠
+ 𝑚 𝑋1𝑉2 = 0
)
𝑠

(Assuming that growth is negligible in the second reactor)

𝐹𝑆1 − 𝐹𝑆2 − ( 𝑞𝑝
𝑌𝑝/𝑠
+ 𝑚𝑠 𝑋2𝑉2 = 0 )
𝑆2 = 𝑆1 − ( 𝑞𝑝
𝑌𝑝/𝑠
+ 𝑚𝑠 𝑋2 ) 𝑉2
𝐹

( )(2. 22 𝑔𝐿 )( )
−1 −1
−1 0.16 𝑔𝑔 ℎ −1 −1 −1 500 𝐿
= (5 𝑔𝐿 ) − −1 + 0. 025 𝑔𝑔 ℎ −1
0.85 𝑔𝑔 50 𝐿ℎ

−1 −1
𝑆2 = 0. 27 𝑔𝐿 ≈ 0. 3 𝑔𝐿

4. Overall substrate conversion in this process

(𝑆0−𝑆2)
𝑂𝑣𝑒𝑟𝑎𝑙𝑙 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 = 𝑆0
* 100

(10−0.3)
= 10
* 100 = 97 %

5. Final concentration of the product obtained in this process

Product mass balance on second reactor

𝐹𝑃1 − 𝐹𝑃2 + 𝑞𝑝𝑋2𝑉2 = 0

As P1 = 0 (Assuming that product synthesis is negligible in the first reactor)

𝑞𝑝𝑋2𝑉2 −1 −1
𝑃2 = = (
0.16 𝑔𝑔 ℎ ) (2.22 𝑔𝐿−1) (500𝐿)
𝐹 −1
(50 𝐿ℎ )

−1
𝑃2 = 3. 5 𝑔 𝐿

Consider a system of a chemostat with cell recycle, as shown in the figure below.
 Michael L. Shuler and Fikret Kargi, Bioprocess Engineering: Basic Concepts, Prentice
Hall, 1992

The feed flow rate and culture volumes are F = 100 L/h and V = 1000 L, respectively.
The system is operated under glucose limitation. The sterile inlet feed to the reactor
contains 10 g L-1 glucose. The microbial growth kinetics follows a Monod model with
𝜇max = 0.4 h-1, KS = 1.5 g L-1 and the yield with respect to substrate (YX/S) as 0.5 g
biomass/g substrate consumed. A recycle stream of flow rate 10 L h-1 has been used in
this reactor system with a biomass concentration four times as large as that of the exit
stream from the reactor. Assume that the system is at steady state with negligible
endogenous metabolism (kd = 0).

Given values, CSTR of Volume ‘V’ = 1000 L

µmax = 0.4 h-1 KS = 1.5 gL-1
YX/S = 0.5 gg-1 S0 = 10 gL-1
F = 100 Lh-1
Biomass concentration factor = C = 4

6. Recycle ratio (α) of the system

𝐹𝑙𝑜𝑤𝑟𝑎𝑡𝑒 𝑖𝑛 𝑟𝑒𝑐𝑦𝑐𝑙𝑒 𝑠𝑡𝑟𝑒𝑎𝑚, 𝐹𝑟 10 𝐿ℎ

−1
α= 𝐹𝑙𝑜𝑤𝑟𝑎𝑡𝑒 𝑡𝑜 𝑡ℎ𝑒 𝑣𝑒𝑠𝑠𝑒𝑙, 𝐹
= −1 = 0. 1
100 𝐿ℎ

7.
Calculating net specific growth rate for this system
𝐹 100 −1
𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒, 𝐷 = 𝑉
= 1000
= 0. 1 ℎ
 −1
𝑛𝑒𝑡 𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑔𝑟𝑜𝑤𝑡ℎ 𝑟𝑎𝑡𝑒, μ = 𝐷 (1 + α − α𝐶) = 0. 1 ℎ (1 + 0. 1 − (0. 1 * 4))
−1
= 0. 07 ℎ

8. Concentration of substrate inside the reactor:

𝐾𝑠 * 𝐷(1+ α− α𝐶)
At steady state, Substrate concentration, 𝑆 = µ𝑚𝑎𝑥−𝐷(1+ α− α𝐶)

= (1.5 𝑔𝐿−1) *(0.1 ℎ−1)(1+ 0.1−(0.1*4))

(0.4 ℎ−1) − (0.1 ℎ−1) (1+ 0.1−(0.1*4))
−1
= 0. 318 𝑔𝐿

9. Concentration of cells inside the reactor:

(
𝑌𝑥/𝑠 * 𝑆0−𝑆𝑓 ) −1 −1 −1
= (
0.5 𝑔𝑔 ) (10 𝑔𝐿 – 0.318 𝑔𝐿 )
𝐶𝑒𝑙𝑙 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛, 𝑋 = (1+ α− α𝐶) (0.7)

−1
= 6. 92 𝑔𝐿

10. Concentration of biomass in the recycle stream:

Concentration of biomass in the recycle stream = CX1 = 4 * 6.92 = 27.68 gL-1 h-1