BKB 3423

Bioreactor Engineering

Dr Farhan Binti Mohd Said

Condition and assessment

Attendance :100%

Assessment:-Assignments/ Mini project : 15%Quizzes : 5%Test 1 : 20%Test 2 : 20%Final Examination : 40%

Class hourTuesday 2.00 – 4.00 pm WDK 06Thursday 2.00 – 3.00 pm WDK 06

Chapters covered

Introduction to Bioprocess Principles and Growth Kinetics

Bioreactor SystemOperation Considerations for Bioreactor and

Cell ImmobilizationAeration and Mixing in Submerged CultureSterilization and Aseptic Technique in

FermentationScale up and Instrumentation of Bioreactors

Course outcome

Summarize the current issue, development and application of bioreactor

Solve the calculation problem regarding to the cell growth and kinetic in fermentation mode, explain and discuss the different operation mode of bioreactor and its related instrumentation

Design a mass, heat transfer and sterilization system in a bioreactor

References

Shuler, M.L. & Kargi, F. “Bioprocess Engineering: Basic Concepts”. Prentice Hall

Bailey, J.E. & Ollis, D.F. “Biochemical Engineering Fundamentals”. McGraw Hill

Asenjo, J.A. “Separation Process in Biotechnology. Marcel Dekker

Mini project

Form a group of 3 to 5 studentsSelect one topic:

Production of …Limit review on bioreactor/ fermentation

related topic ONLYPamphlet, assignment and presentation

Pamphlet and assignment due on week 8 (before mid term break), 1st of Nov. 2011

Presentation on week 10, 15th of Nov. 2011

Mini project (cont’d)

Size of pamphlet – a piece of A4 size, should include: Limit pages for assignment not more than 15 pages Information should covered:

Introduction Substrate, microbes Reaction pathway/ fermentation process- anaerobic/aerobic Growth condition Type of bioreactor – justify Mode of bioreactor – justify Product and the application Conclusion References

Pamphlet and assignment must stated: Subject and subject code Name of group Group members with metric number Lecturer name

BIOREACTOR ENGINEERINGBKB 3423

Introduction to Bioprocess principle & growth kinetics

Chapter 1

Dr Farhan Binti Mohd Said

Topics outline

Introduction to Bioprocess EngineerIntroduction to fermentationKinetic cell growth of batch and continuous

fermentation

Lesson outcome

Discuss types of fermentation processDescribe microbial cell growth phase in batch

fermentationPerform calculation of kinetic cell growth of

batch and continuous fermentation

1.1 Introduction to Bioprocess Engineer

Chemical engineer vs Bioprocess engineer

What is bioprocess engineering??

Include the work of biological and engineering sciences to “Bring Engineering to Life” for development of effective manufacturing processes and designing better products

Example of bioprocess:

Fermentation of sugar to alcoholProduction of Hepatitis B vaccineProduction of insulin Conversion of milk to cheeseDestruction of pollutantsProduction of artificial skin for burn therapy

Application of bioprocess engineering

Production of biofuelsDesign and operation of fermentation systemDevelopment of food processing systemDesign of instrumentation

1.2 Introduction to fermentation

Introduction to fermentation

Fermentation can be used to describe any process which involved the production of organic product by mass culture of microorganism (bacteria, yeast, fungi)

Fermentation can be carried out either as submerged (liquid medium) or solid-state (solid or semi solid medium) culture.

> 90% of industrial processes are carried out as submerged fermentation

Typical Bioprocess Stock culture

Shake flask

Seed fermenter

Raw materials

Medium formulation

Sterilization

Computer controlProduction fermenter

Air

Recovery

Purification

Products

Effluent treatment

Medium preparation

Microorganismcell preparation

Microbial growth phases

Modes of fermentation process

Batch fermentation process batch fermentation process the entire medium is removed from the fermentation tank. The tank is then thoroughly washed, cleaned and the new batch is started only thereafter.

Continuous fermentation processIn the continuous fermentation process, a part of the medium is removed at more or less regular intervals when the fermentation process is in force, and new medium is added therein. Thus, The process of fermentation continues non-stop.

Batch fermentation

batch fermentation : fermenter is filled ONCE until its full, (no more substrate added through reaction)

fed batch : fermenter is only partly filled (certain amount of substrate is added throughout the reaction)

Batch fermentation

Considered as a closed system

At t=0, sterilized nutrient solution is inoculated and incubated

During the process, NO additional fresh medium is added, except 02 (aerobic microorganism), antifoam agent, acid or alkali to control the pH

Composition of culture medium, biomass and metabolite concentration change constantly

Fed batch fermentation

Substrate is added in increment as the fermentation progress

NO growth medium is removed

Nutrient solution is added in small concentration at the beginning and these continue to be added in small doses during production phase

Continuous fermentation

An open systemFresh medium is added continuously during

fermentation and spent medium is removed simultaneously

Fermentation can last up to 1 000 h

Why do cell stop growing?

Why do cell stop growing?

Depletion of nutrientsLack of 02

Change in pHGrowth inhibition from metabolic end

product

1.3 Kinetic growth cell

Batch cultureGrowth rate of bacterial culture

X = biomass concentrationµ = specific growth rate t = time

Doubling time and the specific growth rate

The doubling time (t0) is an expression commonly used to describe the rate of cell growth.

Represents the time take for the cell population to double

Growth limiting nutrient

Nutrient exhausted, the specific growth rate reduces

Monod equation

µ = specific growth rate µmax = max specific growth rateS = concentration of limiting nutrientKs = half saturation coefficient

Equation (1) can be rewrite as:

(1)

(2)

Exercise 2

Growth of an organism on a glucose based medium is described by following parameters:◦ µm = 0.065 h-1

◦ Ks = 0.05 g l-1

The specific growth rate of the organism at the following [glucose] is :-

What happens when S>> Ks?

Glucose Concentration (g/L) µ h-1

0.01

0.1

0.5

1.0

20

90

Yield factor

Measure efficiency conversion of one substrate into biomass Growth / biomass yield, Y x/s Product yield, Y p/s

Batch culture

How environmental condition effect growth kinetic?

TpHDOOsmotic pressureSalt concentrationNutrient availability

Product Formation

Product formation Primary Metabolites

Growth associated Secondary Metabolites

Non-growth associated Mixed growth associated

Primary Metabolites (growth associated product)

Produced simultaneously with growthSpecific rate product formation ∞ specific growth

rate

Product: Primary metabolites, essential for the metabolic activity & growth, Produced from growth substrate by the cell activity

E.g: Enzyme protease (Bacillus subtilis), amino acid,

alcohol (Saccharomyces cerevisiae)

Secondary Metabolites (non-growth associated product

Production occur at the end or during stationary phase

The specific rate of product formation is constant, growth rate is zero

Product: Secondary metabolite, not essential for growth, the growth conditions crucial to determine the synthesis rate of secondary metabolites

E.g: Hormones, antibiotics (penicillin)

Mixed growth associated

Production occur during slow growth and stationary phase

Eq given:

E.g: xanthan gum, lactic acid & certain secondary metabolites

Biomass and product formation

Many products of commercial importance are produced in a manner of non-growth associated

Product formation is dependent on : Growth rate Medium composition Mixing and oxygen transfer conditions Biomass formation Specific precursor

Continuous culture

Start up as batch, change to continuous as the state variables approach steady state

Why the continuous culture fail to prevail:

Why continuous process used?

Continuous culture

Start up as batch, change to continuous as the state variables approach steady state

Why the continuous culture fail to prevail: Contamination Mechanical failure Stability of producing organism

Why continuous process used? Extremely large throughput Unstable product Very low productivity – need a high throughput to

compensate

Continuous culture design

Feed Effluent

X = concentration of biomass in the fermenter and effluentS = concentration of substrate in the fermenter and effluentP = concentration of product in the fermenter and effluentX0 = concentration of biomass in the feedS0 = concentration of substrate in the feedP0 = concentration of product in the feedF = feed and effluent flow rateV = liquid volume in the fermenter

Reactors for continuous culture

Chemostat: Also known as a continuous stirred tank reactor

(CSTR) Cellular growth is typically limited by one essential

nutrient, other nutrients are in excess When operated at steady state, nutrient, product and

cell concentrations are constantPlug Flow Reactor (PFR):

Substrate and cell concentrations vary with axial position in the reactor

An ideal PFR resembles a batch reactor in which distance along the fermenter replaces incubation time

Turbidostat Cell concentration in the culture vessel is maintained

constant by monitoring OD and feed flow rate Culture volume is kept constant Suitable for microbe which able to withstand stree

environment Operates best in high dilution rate

The ideal chemostat

A material balance on the cell concentration around the chemostat yields:

F = flow rate of nutrient solution (l/h)VR = culture volume (l)X = cell concentration (g/l)µg , kd = growth and endogenous metabolism (death) rate constants, respectively (h-1)

The cell mass balance equation can be as:

Usually the feed medium is sterile, and therefore X0=0

If the death rate is negligible compared to the growth rate (kd<<µg), and if the system is at steady state (dX/dt=0), then:

D = dilution rate and D = F/ VR

D is the reciprocal of residence time

Since in a chemostat, the growth rate is limited at least one substrate, the Monod equation can be substituted into:

Grow faster rate, increase D; grow slower rate, decrease D

If D >> µm, the culture cannot reproduce quickly enough to maintain itself and is washed out

S = steady-state limiting substrate concentration (g/l)

(a) can be rearrange to:

(a)

Steady state of chemostat

Steady state of biomass

Steady state of substrate concentration

Productivity of a continuous culture

Productivity = rate of production of a component Productivity of biomass formation = mass flow rate of

biomass out of reactor

Productivity of substrate = mass flow rate of the product out of the reactor

Exercise (example 6.4)

QUIZ 1

1) What are the possible reasons that cause the microbes to enter the stationary phase.

(2 marks)

2) Describe briefly the correlation of growth of microbes with:- Production of primary metabolites- Production of secondary metabolites

(4 marks)

3) List two purposes of secondary metabolites to the microbes.

(2 marks)

4) What are the advantages and disadvantages of continuous culture? (at least 2 answers of each)

(4 marks)