# Bioprocess Tutorial

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3 Difficult Question with suggested answer....

### Text of Bioprocess Tutorial

KC31003 Bioprocess PrinciplesGroup 2 Group Member: TAI WEI KEONG (BK09110063) SEAH YONG UNG (BK09110144) RUSSELL ROSS SULAU (BK09110156) SCOTT BIONDI R. VALINTINUS (BK09110151) CLARICE V. BINJINOL (BK09110005) TAN YIT ZEN (BK09110015)

Question 1The growth rate of E. coli be expressed by Monod kinetics with the parameters of max = 0.935 hr1 and Ks = 0.71 g/L (Monod, 1949). Assume that the cell yield YX/S is 0.6 g dry cells per g substrate. If CX0 is 1 g/L and CS1= 10 g/L when the cells start to grow exponentially, at t0 = 0, show how ln Cx, Cx, Cs, dlnCx /dt, and dCx /dt change with respect to time.

Given data max = 0.935 hr-1 Ks = 0.71 g/L YX/S = 0.6 g CX0 = 1 g/L Cs1= 10 g/L

Rate of microbial growth is characterized by the net specific growth rateEquation (1) Equation (2)

Equation (3)

Net specific growth rate

gross specific growth rate

rate of loss of cell mass

0, cells start to grow exponentially at

Equation (4)

We assume that a single chemical species, S is a growth rate limiting. This kinetics can be described by the Monod equation.Equation (5) Equation (6)

Based on our question, we assume

Equation (7)

Equation (8)

Equation (9)

Equation (10)

SAMPLE CALCULATION Cx increased at a step size of 0.1. Cs data is calculated by using following equation:Equation (11)

The value of t is calculated from the integration with the values of Cs and Cx for each iteration made is calculated based on the previous 2 assumptions that made.

Cx1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 : 6.4 6.5 6.6 6.7 6.8 6.9

ln Cx0.0000 0.0953 0.1823 0.2624 0.3365 0.4055 0.4700 0.5306 : 1.8563 1.8718 1.8871 1.9021 1.9169 1.9315

Cs10.0000 9.8333 9.6667 9.5000 9.3333 9.1667 9.0000 8.8333 : 1.0000 0.8333 0.6667 0.5000 0.3333 0.1667

t0.0000 0.1093 0.2093 0.3016 0.3872 0.4672 0.5423 0.6131 : 3.3949 3.7076 4.1677 4.9231 6.4171 10.8661

dlnCx/dt0.8720 0.8699 0.8676 0.8651 0.8624 0.8595 0.8563 0.8529 : 0.0496 0.0332 0.0199 0.0099 0.0033 -

dCx/dt0.9149 0.9998 1.0839 1.1674 1.2500 1.3317 1.4124 1.4922 : 0.3199 0.2173 0.1324 0.0669 0.0225 -

Cx and Cs versus time12

10

8

Cx and Cs

6

Cx CS

4

2

0 0 2 4 6 time 8 10 12

lnCx versus time10.0000

1.0000 0.0000

2.0000

4.0000

6.0000

8.0000

10.0000

12.0000

ln Cx 0.1000 0.0100

Time

dlnCx/dt versus time1.0000 0.0000 1.0000 2.0000 3.0000 4.0000 5.0000 6.0000 7.0000

0.1000

dlnCx/dt 0.0100 0.0010

Time

dCx/dt versus time3

2.5

2

dCx/dt

1.5

1

0.5

0 0 1 2 3 time 4 5 6 7

Question 2Immobilized cell or enzyme technology for formation of biocatalyst has been valuable tool in many bioprocesses. Among the technologies, entrapment of biological materials within calcium alginate gel beads is the most commonly used.

a) Explain why calcium alginate has been popular as immobilization material. Suggest alternative materials suitable for immobilization.

Reasons: 1) The hydrogel formation occurs under extremely mild conditions 2) Does not depend on the formation of more permanent covalent bonds between polymer chain. 3) It is biocompatible & has good mechanical strength for many different applications.

Alternative material1) Polyacrylamide -Non-ionic -Properties of the enzymes are only minimally modified in the presence of the gel matrix. -Diffusion of the charged substrate & products is not affected.

2) Chitosan

- Obtained from alkaline hydrolysis of chitin. - It is very biocompatible -Has been used in many applications including drug delivery system. - Disadvantage: Limited solubility in water Require dilute acidic solution for dissolution.

b) Describe at least two purposes for using immobilization system in such fashion and give example on how the each purposed is serve.

Purposes1)Cells last longer -Ordinary cells (only 1-2days) but immobilized cells can last for 30days. How? a) cells suspended into solution of sodium alginate. b)Added to a calcium chloride solution. c)Packed into column & a solution to be converted is fed into the top of the column d)Allowed to flow through the bed of beads contain immobilized biocatalyst in the cells. e)Conversion takes place & product comes out t the bottom. -Example: immobilized yeast cells

2. Retain the protein while allowing penetration of substrate -Example: calcium alginate -Widely used due to the mild condition -Result in minimum denaturation of the biocatalyst. -Can be formed in extremely mild conditions which ensure that enzyme activity yields over 80% can be achieved. -Entrapment in insoluble calcium alginate gel:rapid,nontoxic,inexpensive,& versatile methods.

c) Describe at least three methods for large-scale production of calcium alginate beads and state the pros and cons of each method.

Method IPreparation of Calcium Alginate Microgel Beads in an Electrodispersion Reactor Using an Internal Source of Calcium Carbonate Nanoparticles

Pulsed electric fields are utilized to atomize aqueous mixtures of sodium alginate and CaCO3 nanoparticles (dispersed phase) from a nozzle into an immiscible, insulating second liquid (continuous phase) containing a soluble organic acid.

Alginate molecules are stabilized by the electrostatic force in a gel The coalescence of the emulsified drops containing sodium alginate and CaCl2 is capable of producing micrometer-sized calcium alginate (Ca-alginate) gel beads. The scale-up potential of this process is almost unlimited. The technique is suitable for biological or food applications. The characteristic highly porous structure of Ca-alginate particles

Disadvantages The random process of droplet coagulation vary widely in size and shape This processing involves harsh conditions that can denature biological compounds. The difficulty in using dispersion/external gelation techniques with ionic polysaccharide is that the calcium source (CaC12) is insoluble in the oil phase.

Method IIProduction of calcium alginate beads by using Sound Wave Induced Vibration Method

A new process for the immobilization of calcium alginate, which employs a vibration of membrane induced by sound waves from a horn-type speaker, is developed to produce uniformly-sized beads of predictable diameter. Frequency of sound wave and production rate are varied to find the optimal control ranges for the bead production. The controllable bead size range is 1.50-3.50 mm The experimental production rates are 0-12 dm3 h-1 by a single nozzle.

Advantages Alternative to conventional continuous bead production methods Low apparatus cost More easier to set up compare to other methods Better control of bead size and shape

Disadvantages Speed of sound varies with temperature Difficult to accurately measure the exact arrival time of the wave front at different sensors

Method IIIProduction of alginate beads by emulsification/internal gelation

Alginate microspheres were produced by emulsification/internal gelation of alginate sol dispersed within vegetable oil. Gelification was initiated within the alginate solution by a reduction in pH (7.5 to 6.5), releasing calcium from an insoluble complex. Smooth, spherical beads with the narrowest size dispersion were obtained when using low-guluronic-acid and low-viscosity alginate and a carbonate complex as the calcium vector. Diameters ranging from 50 gm to 1000 tm were obtained with standard deviations ranging from 35% to 45% of the mean.

Advantages The resultant broad size distribution. Producing small-diameter alginate beads (200-1000 pm), potentially on a large scale (m3/h). Enhanced rates of bioconversion and to avoid bead rupture resulting from the accumulated fermentation gas. Ionic polysaccharide is that the calcium source. By controlling the conditions under which the water-in-oil dispersion is produced, the bead size can be controlled from an few micrometers to millimetres in diameter.

Disadvantages Its cost is high in processing. Emulsion is unstable. The emulsion tends to separate into a watery layer and an oily layer.

d) Describe one industrial application for each immobilized cell and enzyme system with the aid of PID diagram.

Application of immobilized cell in Beer brewing industry Main advantages of using immobilised cells for production of beer are: 1- Enhanced fermentation productivity due to higher biomass densities 2- Improved cell stability 3- Easier implementation of continuous operation 4- Improved operational control and flexibility 5- Facilitated cell recovery and reuse 6- Simplified downstream processing.

(Source: Shreve, R. N., & T.Austin, G. (1984). Shreve's Chemical Process Industries. New York: McGraw-Hill.)

Immobilisation cell system is applied in the fermenter. Kirin 3 stages bioreactor system.

(Source: Kirin's Three-Stage Bioreactor. (n.d.). Retrieved May 10, 2012, from SpringerImages: http://www.springerimages.com/Images/Chemistry/110.1007_s11947-010-0435-0-10)

Application of Immobilized Cell in Bioreactor Enzyme-immobilized membrane bioreactors (EMBR) is a combination of product separation process with enzymatic catalysis in continuous processes. The selective membrane are use to separate the enzymes from the reaction products. EMBR have been widely used in the field of fine chemicals synthesis.

(Source: Fang, Y., Huang, X.-j., Chen, P.-C., & Xu, Z.-K. (2011). Polymer materials for enzyme immobilization and their application in bioreactors.)

e)Describe the problems that envisaged when using immobilized c

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