Bioreactor System

ERT 314

Sidang 1 2011/2012

Chapter 2:Biological Systems

and Media Design

Week 2 - 3

2.1 Introduction to Biological Systems

The selection of the suitable and specific microorganism

for use in fermentation process has direct influence on

the design of the bioreactor

This chapter discusses the effect that varying the

microorganism has on the choice of bioreactor

However, the selection of bioreactor depends not only on

the organism chosen but other factors such as:

Factors which affect microbes’ growth – growth rate,

temperature, induction type, duration of growth

Factors which affect the product – chemical & physical nature

of the product, kinetics of product synthesis

Microorganisms that Have Been Suggested for

use as ‘Model’ Cells for Bioreactor Research

Microorganism Special feature of interest

Bacillus subtillis To characterize mixing and oxygen transfer evaluated

by the production of acetoin and butanediol

Beauveria tenella fungi

imperfectii

Useful for evaluation of wall growth

Chaetomium cellulolyticum Fungus, used to test more highly viscous broths

Candida utilis For studying the effects of hydrostatic pressures

Escherichia coli Use as a test organism due to its role as a producer

of many recombinant proteins

Methylomonas M15 Used for comparison of mass transport, growth, and

product quality in CSTR and column reactor

Pseudomonas fluorescens Used to test the influence of varying hydrostatic

pressure

Trichosporon cutaneum Strict aerobic yeast, model for maximal transfer

Xanthomonas campestris Bacteria used for studying the effect of viscous media

2.1.1 Growth Characteristics of Microorganism

Unicellular Microbes Multicellular Microbes Budding Microbes

Most of the bacteria – size

in the range 0.5 to 2 µm

Most of the fungi – consist

hyphae and hyphal cells

composed of cellulose and

chitin

Yeasts – unicellular

microbes, larger than

bacteria, has typical

diameter of 5µm

Distinguishing parameter is

bacteria’s growth

requirements such as

requirement of O2, C, N

and energy

Generally aerobic at

optimal temperature of

25°C and at pH 5-6

They grow in solid forming

colonies like bacteria

Reproduction – binary

fission and sporulation

Reproduction – sexual,

asexual or both

Reproduction – sexual

method

Most of them produce

polysaccharide slime layer

(Capsule), which lead to

increase the viscosity of

media

Has 3 division in fungal

kingdom – Eumycota, Slime

molds & Linchens

Common industrial fungi –

Aspergillus, Neurospora,

Penicillium, Rhizophus,

Trichoderma

Example: S. cerevisae used

in bread, beer and wine

industry, regarded as safe

to human

Morphology Structures of Bacteria

Binary Fission

Sporulation

Morphology of Fungi

Shapes of Fungi

Reproduction of Fungi

2.1.2 Influence of Microbial Characteristic

On Bioreactor Selection

Rheological Properties: Shear and Viscosity

Oxygen Demand

Growth Rate

Effect of Culture pH

Growth Temperature

Medium Composition and Preparation

Requirement of Light

Foam Production

Sterility

Safety and Regulation

Rheological Properties:

Shear and Viscosity

Shear forces act perpendicular to direction of fluid

motion and are usually and quantitated by measuring the

shear rate and shear stress

Newtonian and non-Newtonian

The main factor that influence rheological properties of

fermentation are:

Microorganism – single cell, mycellium or pellets

Metabolites – biopolymers

Substrates – starch, soyameal, cellulose

Suspension of single celled microbes – low viscosity,

similar to water

Rheological Properties:

Shear and Viscosity

Some microbes produce very viscous broth due to secretion of polymers (pullulan, hyaluronic acid, alginate, xanthan gum) during fermentation

These polymers shown pseudoplastic behaviour – the effective viscosity decreases with increasing shear rate

For most of filamentous fungi – increase the medium viscosity and behave as non-Newtonian

Eg. Penicillum and Aspergillus

During their growth, a complex dependence on cell morphology is found, which some fungi grow in pellets lead to increase of viscosity

Therefore, shear rate and shear stress should be measured to avoid cell damage – increase the agitation speed, decrease the growth and product rate for B. flavum but for Xanthamonas, increase of agitation will increase xanthan gum production (WHY?)

Morphological Changes in Microbes Due to

Increased Shear

Microbes Description of Effect

Aspergillus flavus Mycellium is short and strongly branched. Starch is produce instead of

kojic acid

Filamentous fungi Number of growing tips

Penicillum chrisogen Length of hyphae changes with stirrer speed

Penicillum Biomass growth decrease but rate of penicillin synthesis increase as

agitation decreases

Aspergillus niger Biomass growth decreases but rate of citric acid synthesis increases as

agitation decreases

E. coli Mean cellular volume increases with increased stirrer speed

B. cereus, S. epidermidis,

S. cerevisae

Mean cellular volume increases with increased stirrer speed

Clostridium

acetobutylicum

CO2, H2, butanol, acetone and ethanol production increased as

stirring rate increased up to 350 rpm, then decreased

Aureobacterium

pullulans

Change of metabolite production: dimorphic fungus: above 200 rpm,

culture changes from filamentous to yeast type and pullulan

production increase

Oxygen Demand

Anaerobic Microaerophilic Aerobic

Microbes do not require O2

for growth, O2 sensitive

Microbes require only minor

amounts O2, does not impose

serious constraints on

bioreactor design

Micros require large amount

O2 depending on the

substrate used and growth

rate

Having redox potential below

11mV – producing reducing

compound such as H2 or H2S

The higher initial oxidation

level of substrate used

(glucose vs methane), the

more ATP can be produced

from oxidation

Obligate anaerobe – will grow

at redox of - 500 mV

Concentration of dissolved O2

in water is very low, therefore

O2 must be supplied

continously

Eg. Clostridium acetobutylicum

Main application – wastewater

treatment and bioleaching

O2 supplied as gas (bubbles)

have to be broken up in order

to provide sufficient gas hold

up and kLa value

Oxygen Demand

At maximal growth rate, maximal OTR is equal to the OUR

Not to use all dissolved O2 in the medium, as it can hampered

the cell growth

Some microbes can grow either in aerobic or anaerobic

conditions such S. cerevisae and E. coli

Therefore, it is important to control the condition so that the

cell’s metabolism cannot change dramatically as O2

concentration change

S. cerevisae – produce biomass in aerobic, ethanol in anaerobic

E. coli – produce less cell weight per g carbon source consumed

O2 or oxygen-enriched air is generally limited to 1000L

bioreactor scale, due to the cost of oxygen (SOLUTION?)

Growth Rate

Bacteria Yeast Fungi

Max Doubling time – in

the range of 20 -120 min

Max Doubling time – in

the range of 1 – 4 hours

Max Doubling time – in

the range of 4 – 24 hours

Fermentation period –

in the range of 12 – 24

hours

Fermentation period –

in the range of 12 – 24

hours

Fermentation period –

in the range of 24 - 48

hours, plus another few

days of product synthesis

Growth Rate

At high growth rate,

pH control and addition of medium component (glucose) is

needed to ensure the high cell density and product yield

high output energy is produced, thus, require high energy input

for cooling

At low growth rate,

requirement for cooling are not so important, just maintaining

the fermentation at 37°C (need energy input for heating)

culture more susceptible to microbial contamination (WHY?)

Effect of Culture pH

Most microbes grow in pH 5.5 – 8.8

Optimal fungi growth at pH 5 – 7

Optimal yeast growth at pH 4 – 5

Some acidophilic microbes (Thiobacillus ferroxidans) can

grow at optimum pH of 1.5 and can survive at pH 7,

produce highly corrosive condition and by-product of

H2S – (concern in fabricating bioreactor from steel)

But, B. macerans can grow above pH 9

All organisms reduce pH during growth

Effect of pH on Cell Growth

Growth Temperature

Growth Temperature

Growth Temperature

Psychrophilic microbes will be used to an increasing extent in the future (WHY?)

Thermophilic microbes known as causative agent of contamination in industry, but have several advantages such as

Cooling is easy for fermentation (WHY?)

Easy to collect volatile product

Fermentation can be done without excessive concern of sterile condition

Cell growth is relatively fast and large turnover result in fermenter

Produce thermostable proteins (protease) – household detergent, amylase – beer industry

Medium Composition and Preparation

The main factors in the medium which affect growth rate are the sources of carbon and nitrogen, as well as vitamins, or any limiting elements i.e K or PO4, and minor minerals

Types of substrate are also important i.e autotrophic or photoheterotroph microbes tap the carbon source from CO2 and light respectively

Mineral salts (Cu, Zn, Fe, Co, Mn, Mo) are essentials but some are inhibitory when they present in high concentration

In industrial setting, the cost of medium can reach up to 70% (high) of the cost of the fermentation process (SOLUTION?)

Medium might be viscous or contain particulate matter may affect the methods of medium transportation, filtration and sterilization

Requirement of Light

Special groups of bacteria – cyanobacteria and anoxygenic

photothropic bacteria are chemolitrothrophic can

assimilate CO2 by photosynthesis

Growth of these microbes require both CO2 and

presence of light

The growth of these microbes has profound impact on

the reactor design i.e photobioreactor

Foam Production

Production of foam is common phenomenon in

fermentation

It rises from the flow of air through liquid fermentation

and form small bubbles which fill up the headspace of

fermenter

Breaking the foam is done by with antifoam agents or

mechanical foam breakers

Sterility

Selective conditions are those which enable the growth of only

limited number of microbial species, including pH, temperature

and medium composition

In term of sterility, 3 main ways in which a fermentation run

may be approached:

There are no selective conditions, fermentation has to perform

under strict aseptic conditions

Partly selective conditions exist, other organisms will proliferate

Very selective conditions exist, so there is hardly any chance of

contaminating microbes

The longer the duration of the fermentation, more stringent

demand on the bioreactor design for aseptic conditions

Safety and Regulation

Microbes must defined according to their pathogenicity

Low level, GRAS – generally regarded as safe, most common use

P1 – nonpathogenic microbes

P2 and P3 – often required for the growth of microbes for production

of vaccines

P4 – extremely pathogenic