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
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
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
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