BIOCHEMICAL ASPECTS OF MICROBIAL

CELLS
 Typical composition of bacterial cells consist of 50%
C, 20% O, 14% N, 8% H, 3% P, 1% S, and small amount
of Mg 2+, K+, Na+, Ca 2+, Cl – and vitamins.

 Living cells basically is a complex reactor in which

there are more than 2000 reactions happens.
NURITIONS OF MICROBIAL CELLS
 All organisms except virus contain about 80%
water.
 More or less 50% of cell dry weight is protein,
most of which are enzymes.
 Nucleic acid content varies between 10-20%,
except virus which can reach 50%.
 Fat content is about 5-15%, except in microbes
producing PHB which may reach 90%.
NUTRITION OF MICROBIAL CELLS
Nutrition needed by microbial c ells consist of:
 Macronutrients, namely elements of C, O, N, H, P,
S, Mg 2+ , K+. These elements are needed at the
concentration ≥ 10-4 M.
 Micronutrients/trace elements), namely Mo 2+, Zn
2+, Cu 2+, Mn 2+, Fe 2+, Ca 2+, Na+, vitamins, growth

hormone and metabolic precursors.

MACRONUTRIENTS
Carbon:
 Carbon compound is a main energy and carbon
sources.
 A common carbon sources in bioindustry are:
molasses (sucrose), starch (hydrolyzed into
glucose or dextrin), corn syrup, sulfite waste from
pulp and paper industry (glucose), methanol,
ethanol, methane.
CARBON
 Common carbon source s in lab scale cultivation
are glucose, sucrose, and fructose.
 In aerobic cultivation, 50% carbon substrate is
used for cellular materials, the rest 50% is used for
energy source.
 In anaerobic cultivation, less than 30% of carbon
substrate is converted into cell, the rest 70%
carbon source is converted into products (cell or
metabolite).
OXYGEN:
 More or less 20% cell dry weight is oxygen.
 Oxygen exists in all organic components and
water.
 Oxygen molecule is needed as electron acceptor in
aerobic metabolism of carbon compound.
 Oxygen gas is supplied into bioreactor through
aeration.
NITROGEN
 10-14% of cell dry weight is Nitrogen.
 Main N sources are ammonia, or ammonium salts
(NH4Cl, (NH4)2SO4, NH4NO3), protein, peptide,
amino acids, and urea.
 Nitrogen source in lab cultivation is organic nitrogen
such as yeast extract and peptone.
 Some bacteria such as Azotobacter sp and
cyanobacteria obtains nitrogen directly from the air
and convert into ammonium.
 Nitrogen is used by cell as element to develop protein
and nucleic acid.
Table 1: Some C and N sources which
are used in bioindustry
Carbon sources Nitrogen sources

Starch wastes (corn and Soybean flour

potato) Yeast extract
Molasses (cane and beet) By product of distillation
Whey Cotton seed extract
n-Alkane Dry blood
sulfite waste Corn step liquor
Household waste Fish flour
Cellulose waste Peanut flour
Nuts
HYDROGEN:
 About 8% of cell dry weight is
hydrogen.
 Hydrogen element is obtained from
carbon compound such as
carbohydrate.
 Methanogenic bacteria uses hydrogen
as energy source.
PHOSPHATE:
 About 3% of cell dry weight is Phosphor.
 Phosphor exist in nucleic cid and cell wall
of gram positive bacteria.
 P is key element in regulation of cell
metabolims.
 Main P sources are inorganic phosphate
salt, such as KH2PO4 and K2HPO4.
SULPHUR:
 About 1% of cell dry weight is sulphur.
 Sulphur exists in protein and some coenzymes.
 Main sulphur source is sulphate salt such as
(NH4)2SO4.
 Other sulphur sources are amino acids containing
Sulphur.
 Some autotrophic bacteria use S2- and S0 as
energy source.
POTASSIUM (KALIUM):
Potassium is cofactor for some
enzymes and needed in
carbohydrate metabolisms.
Main potassium sources are salts
of KH2PO4 and K2HPO4.
MAGNESIUM :
 Mg is cofactor for some enzymes , and
exists in cell wall and membrane.
 Ribosome needs Mg 2+ ion.
 Magnesium is generally supplied in the
form of salts MgSO4.7H2O or MgCl.
Table 2. Eight Macronutrients and their
roles
Element Physiological functions Requirement
(M)

C Constituents of organic sel. and energy source >10-2

Constituents of protein, nucleic acid, coenzyme 10-3
N
-
H Constituents of organic cell and water
-
O Constituents of cell and water. Needed in aerobic
respiration.
S Constituents of proteins and some coenzyme.. 10-4
P Constituents of nucleic acid, phospholipids, 10-4--10-3
nucleotide, and some coenzyme.
K Inorganic cation in cell and cofactor some 10-4--10-3
enzymes.
Cofactor for some enzymes and chlorophyll ,
Mg exists in cell wall and membrane. 10-4--10-3
MICRONUTRIENTS
 Lack of micronutrients (trace elements) will
prolong lag phase, and decreases specific growth
rate and product yield.
 There are 3 categories of trace elements:
 1. Trace elements which are very much needed,
namely Fe, Zn,and Mn. All are important
cofactors.
 2. Trace elements which are needed for specific
products, such as Cu, Co, Mo, Ca, Na, Cl, Ni,and
Se.
MICRONUTRIENTS
 3. Trace elements which are seldom
needed such as B, Al, Si, Cr, V, Sn, Be, F,
Ti, Ga, Ge, Br, Zr, W, Li, I. If needed, it
must be at concentration < 10-6 M and
toxic at concentration ≥ 10-4 M.
MICRONUTRIENTS
 Some ions such as Mg2+,Fe3+ and PO43-, may
make sediment in medium. Therefore it
needs chelating agent to dissolve them.
Chelating agent has ligand ( such as
carboxyl, amine, mercapto) to bind metal
ion and to form complex compound which
is dissolved in water. Citric acid, EDTA,
polyphosphate, histidine, tyrosine, and
cystein are chelating agent which are
commonly used. Chelating agent is needed
at low concentration (1 mM).
GROWTH FACTOR
 Growth factor stimulates growth and
synthesis of metabolite.
 Vitamin, amino acid and hormone are
growth factor.
 Vitamin B1 (thiamine), B2 (riboflavin), B6
(pyridoxine), B12 (cyanocobalamine),
biotin, folic acid, lipoic acid, p-amino
benzoid acid, and vitamin K, function as
coenzyme.
GROWTH FACTOR
 Vitamins are needed at concentrations
10-6 – 10-12 M.
 Some auxotrophic microbes need
certain amino acid 10-6 - 10-13 M.
 Insulin hormone is general hormone
for animal cell, while cytokinin is
growth hormone for plants/crops.
CULTIVATION MEDIA
 Defined Media ==> containing certain
amount of pure chemicals with certain
compositions. Examples: glucose,
(NH4)2SO4, KH2PO4, MgCl2.
 Cultivation results are controllable and
reproducible.
CULTIVATION MEDIA
 Complex media ==> containing natural
compounds whose composition is not
known exactly. Generally also contains
growth factor, vitamin, hormone, and trace
elements so that produce more cells.
Example : yeast extract, peptone, molasses,
corn step liquor.
Typical example of composition in
defined media and complex media:
Constituents Purpose Conc (g/L)
Group A: Source of
Glucose C and energy 30
KH2PO4 K,P 1.5
MgSO4.7H2O Mg and S 0.6
CaCl2 Ca 0.05
Fe2(SO4)3 Fe 15 x 10-4
ZnSO4.7H2O Zn 6 x 10-4
CuSO4.5H2O Cu 6 x 10-4
MnSO4.H2O Mn 6 x 10-4
Group B: Source of
(NH4)2HPO4 N 6
(NH4)H2PO4 N 5
Group C: 4
C6H5Na3O7.2H2O Chelating agent
Group D:
Na2HPO4 Buffer 20

KH2PO4
Buffer 20
Complex media which are used for
penicillin production
Materials Purpose Amount
(percent from
total)
Glucose or molasses Source of C and 10%
(continuously fed) energy

Source of N 1-5%
Corn Step Liquor
Phenyl acetic acid
(continuously fed) Precursor 0.5-0.8%

Animal or plant fat Antifoam

0.5%
Acid and/or base To control pH at the
range of 6.5-7.5 As needed
CONSIDERATIONS
1. Cost and availability: ideally, materials should
be inexpensive, and of consistent quality and
year round availability
2.Ease of handling in solid or liquid forms,
along with associated transport and storage
costs, e.g. requirements for temperature
control.
3.Sterilization requirements and any potential
denaturation problems.
CONSIDERATIONS
4. Formulation, mixing, complexing and viscosity
characteristics that may influence agitation,
aeration and foaming during fermentation and
downstream processing stages.
5. The concentration of target product attained, its
rate of formation and yield per gram of substrate
utilized
6. The levels and range of impurities, and the
potential for generating further undesired
products during the process
7. Overall health and safety implications
What is the Role of Media in Fermentation
 In most industrial fermentation processes there
are several stages where media are required:
several inoculum (starter culture), propagation
steps, pilot-scale fermentations and the main
production fermentation
 The technical objectives of inoculum propagation
(The preparation of a population of m/o’s from a
dormant stock culture to an active state of growth
that is suitable for inoculation in the final
production stage) and the main fermentation are
often very different  differences in their media
formulations
you need to be well aware of the needs of
m/o’s

Depending on the purpose:

Different Levels of Downstream Processing
What is the Role of Media in Fermentation
 Where biomass or primary metabolites are the
target product, the objective is to provide a production
medium that allows optimal growth of the m/o
 Secondary metabolite production is not growth
related. Consequently, media are designed to provide
an initial period of cell growth, followed by conditions
optimized for secondary metabolite production. At
this point the supply of one or more nutrients (carbon,
phosphorus or nitrogen source) may be limited and
rapid growth ceases
Problems Frequently Encountered
 Compounds that are rapidly metabolize may
repress product formation. To overcome
this, intermittent or continuous addition of
fresh medium may-be carried out to
maintain a relatively-low concentration that
is not repressive
Problems Frequently Encountered
 Certain media nutrients or environmental
conditions may affect the physiology,
biochemistry, and morphology of the
microorganism. In some yeasts the single
cells may develop into pseudo-mycelium or
flocculate, and filamentous fungi may form
pellets. This is not desirable as it affects the
product yield
 The media adopted also depend on the
scale of the fermentation.

 For small-scale laboratory fermentations

pure chemical are often used in well
defined media
 However, this is not possible for most
industrial-scale fermentation processes,
simply due to cost, as media components
may account for up to 60-80% of process
expenditure
 Industrial-scale fermentations primarily use
cost-effective complex substrates, where
many C and N sources are almost
undefinable. Most are derived from natural
plant and animal materials, often
byproducts of other industries, with varied
and variable composition
Carbon Sources
Factors influencing the choice of C-
source
The rate at which C-sources
metabolized
Price & availability
Media sterilization
Carbon Sources
Carbohydrates: Starch from cereal, grains and maize;
malt from barley; sucrose from sugar cane; impure
form: cane molasses, corn steep liquor, whey from
dairy industry

Oil & Fats: Fatty acid contents (also antifoaming

properties))

Hyrdocarbons and their derivatives: n-alkanes for

production of organic acids, aminoacids, vitamins
CARBON SOURCE EXAMPLE
 Molasses, a byproduct of sugar production, is one
of the cheapest sources of carbohydrate. Besides a
large amount of sugar, molasses contains
nitrogenous substances, vitamins, and trace
elements. However, the composition of molasses
varies depending on the raw material used for
sugar production
CARBON SOURCE EXAMPLE
 Malt extract, an aqueous extract of malted barley,
is an excellent substrate for many fungi, yeasts, and
actinomycetes. Dry malt extract consists of about
90-92% carbohydrates, and is composed of
hexoses (glucose, fructose), disaccrides (maltose,
sucrose), trisaccharides (maltotriose), and
dextrins. Nitrogenous substances present in malt
extract include proteins, peptides, amino acid,
purines, pyrimidines, and vitamins
Nitrogen Sources

Factors influencing choice of nitrogen sources

 Individual nitrogen sources influence the
metabolic regulations (eg: A. Nidulans, ammonia
regulates the production of proteases)
 Type, concentration (NH4Cl-NH4OH, strong pH
changes)
 Complex nitrogen sources favors antibiotic
production
 Downstream effects
Nitrogen Sources

Common ones
• Soybean meal (brings gradual breakdown &
prevents the accumulation of ammonia ion),
• ammonia,
• ammonium salts or
• nitrates
Minerals

Require minerals for growth and

metabolism
Mg, P, K, S, Ca etc. Need to be added as
distinct components
Cu, Fe, Mn, Zn, Mo etc critical for
secondary metabolite production
Water
Mineral content & demineralization
Precursors and Metabolic
Regulators
 Regulating production rather than
promoting growth

 Precursors: PAA (phenylacetic acid) for

Pen G; Cl for chlortetracycline
Precursors and Metabolic
Regulators
Inhibitors: For accumulating a metabolic intermediate
eg: product: Glycerol, inhibitor: Nabisulphite, effect:
acetaldehyde production repressed, m/o:
S.cerevisiae
product: tetracycline, inhibitor: bromide, effect:
chlortetracyline formation repressed, m/o:
Streptomyces aureofaciens
Inducers:Majority of enzymes are inducible.
foreign protein restrict cell growth, induction at
certain concentrations
Oxygen Requirements

Medium influence oxygen availability

Fast metabolism: Oxygen limitation
Rheology: Viscosity & its subsequent
behaviour with aeration & agitation
Antifoams: Surface active agents
reducing oxygen transfer rate
Oxygen Requirements
 Oxygen is usually a limiting nutrient due to its low
solubility in culture media
 While blending, uniformity is essential for oxygen
distribution in the bioreactor, bubble size
distribution is the most important factor for
governing mass transfer
 When bioreactors are scaled up from laboratory to
production size, their design must meet both
oxygen distribution and oxygen mass transfer
requirements
Oxygen Requirements

 Important parameter in pO2 control is

mixer's rotational speed n: nmin and
nmax. It means that, when controlling
pO2, n will vary only within this range.
These limits are determined in
connection with eliminating of
different undesirable phenomena:
Oxygen Requirements
 nmin choice is determined:
 to secure the minimal partly turbulent mixing level;
 by the guaranteed bubble dispersion;

 by the prevented sedimentation.

 nmax choice is determined by:

 setting in of the intensive foaming regime;

 irreversible mechanical damages of cells;

 liquid surface fluctuation and evaporation.

 Foaming
 Removal of cells from culture
 Physical changes
 Reduction in working volume
 Lower mass & heat ratios
 Invalid process data
 Decrease in sterility
3 ways to solve:
Defined media, antifoams or mechanical foam breakers

Ideal antifoam
 Should disperse easily and have fast action on foam
 Should be active at low concentrations
 Should be long acting in preventing new foam formation
 Should not be metabolized by m/o
 Should be nontoxic to m/o
 Should not cause any problem in the extraction step
 Should be cheap
 Should be sterilazable

Examples: Silicones, sulphonates, esters, fatty acids,

alcohols