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PAMANTASAN NG LUNGSOD NG MAYNILA

University of the City of Manila


College of Engineering and Technology
Department of Chemical Engineering

ChE 422 – Introduction to Biotechnology


Stoichiometry of
Microbial Growth and
Product Formation

Submitted by:

Anne Paulinne P. Redublo

2013-20358

Submitted to:

Engr. Denvert C. Pangayao

03 March 2017
PAMANTASAN NG LUNGSOD NG MAYNILA
University of the City of Manila
College of Engineering and Technology
Department of Chemical Engineering

TABLE OF CONTENTS

Stoichiometry 3

Bioreactors 3

Cell Composition and Growth Reaction 4

Elemental Balances 10

Degree of Reduction 14

Measuring Yields 15

Conversation Principles to Calculate


Full Stoichiometry of Growth 16

Stoichiometry of Microbial Growth and Product Formation 2


PAMANTASAN NG LUNGSOD NG MAYNILA
University of the City of Manila
College of Engineering and Technology
Department of Chemical Engineering

Stoichiometry
Stoichiometry is founded on the law of conservation of mass where the total mass
of the reactants equals the total mass of the products leading to the insight that the
relations among quantities of reactants and products typically form a ratio of positive
integers. This means that if the amounts of the separate reactants are known, then the
amount of the product can be calculated. Conversely, if one reactant has a known quantity
and the quantity of product can be empirically determined, then the amount of the other
reactants can also be calculated.
The term stoichiometry was first used by Jeremias Benjamin Richter in 1792
when the first volume of Richter's Stoichiometry or the Art of Measuring the Chemical
Elements was published. The term is derived from the Greek words stoicheion "element"
and metron "measure".

Introduction to Bioreactors
Biochemical engineering, also biotechnology engineering or bioprocess engineering,
is a branch of chemical engineering that mainly deals with the design and construction of
unit processes that involve biological organisms or molecules, such as bioreactors. Its
applications are in the petrochemical industry, food, pharmaceutical, biotechnology, and
water treatment industries.

BIOREACTOR
 any manufactured or engineered device or system that supports a biologically active
environment.
 a bioreactor is a vessel in which a chemical process is carried out which involves
organisms or biochemically active substances derived from such organisms. This
process can either be aerobic or anaerobic.
 commonly cylindrical
 ranging in size from litres to cubic metres
 often made of stainless steel
 may also refer to a device or system meant to grow cells or tissues in the context of
cell culture. These devices are being developed for use in tissue engineering or
biochemical engineering.

Stoichiometry of Microbial Growth and Product Formation 3


PAMANTASAN NG LUNGSOD NG MAYNILA
University of the City of Manila
College of Engineering and Technology
Department of Chemical Engineering

 On the basis of mode of operation, a bioreactor may be classified as batch, fed batch
or continuous (e.g. a continuous stirred-tank reactor model).
Organisms growing in bioreactors may be submerged in liquid medium or may be
attached to the surface of a solid medium. Submerged cultures may be suspended or
immobilized. Suspension bioreactors can use a wider variety of organisms, since special
attachment surfaces are not needed, and can operate at much larger scale than immobilized
cultures. However, in a continuously operated process the organisms will be removed from
the reactor with the effluent. Immobilization is a general term describing a wide variety of
cell or particle attachment or entrapment. It can be applied to basically all types of
biocatalysis including enzymes, cellular organelles, animal and plant cells.

Cell Composition and Growth Reaction


All living organisms, from microbes to mammals, are composed of chemical
substances from both the inorganic and organic world, that appear in roughly the same
proportions, and perform the same general tasks. Hydrogen, oxygen, nitrogen, carbon,
phosphorus, and sulfur normally make up more than 99% of the mass of living cells,
and when combined in various ways, form virtually all known organic biomolecules. They
are initially utilized in the synthesis of a small number of building blocks that are, in turn,
used in the construction of a vast array of vital macromolecules. There are four general
classes of macromolecules within living cells: nucleic acids, proteins, polysaccharides
(carbohydraytes), and lipids. These compounds, which have molecular weights ranging
from 1000 to 1000000, are created through polymerization of building blocks that have
molecular weights in the range of 50 to150. Although subtle differences do exist between
cells (e.g., erythrocyte, liver, muscle or fat cell), they all generally contain a greater variety
of proteins than any other type of macromolecule, with about 50% of the solid matter of
the cell being protein (15% on a wet weight basis).
According to Teton New Media, a premier source of medicinal studies, about 99%
of cellular molecules are water molecules, with water normally accounting for
approximately 70% of the total wet-weight of the cell. Although water is obviously
important to the vitality of all living cells, the bulk of our attention is usually focused on the
other 1% of biomolecules. Typically 70% of cell mass is water and the remaining is dry
matter. Therefore it is conventional to express cell composition on a dry basis. The
microorganism Eschericia coli is widely used in genetic engineering. Typical elements
found in Eschericia coli are given below:

Stoichiometry of Microbial Growth and Product Formation 4


PAMANTASAN NG LUNGSOD NG MAYNILA
University of the City of Manila
College of Engineering and Technology
Department of Chemical Engineering

ELEMENT % DRY WEIGHT


C 50
O 20
N 14
H 8
P 3
S 1
K 1
Na 1
Ca 0.5
Mg 0.5
Cl 0.5
Fe 0.2
others 0.3

Table 1. Elemental Composition of E. Coli (Strainer et. Al)

Nearly half of the dry matter in cells is carbon and the elements carbon, oxygen,
nitrogen and hydrogen total up to about 92% of the total. This observation for E. coli is also
found to be generally true for other cellular organisms.

Carbon Growth Composition Empirical


Microorganism MW
Source Rate C H N O Formula
Klibsiella
Glycerol 0.1 50.6 7.3 13.0 29.0 CH1.74O0.43N0.22 23.7
aerogenes
Aerobacter
Complex 48.7 7.3 13.9 21.1 CH1.78O0.33N0.24 22.5
aerogenes
Saccharomyces
47.0 6.5 7.5 31.0 CH1.66O0.49N0.13 23.5
cerevisiae
Sachromyces
50.3 7.4 8.8 33.5 CH1.75O0.15N0.5 23.9
cervisiae
Candida utilis Glucose 0.4 46.9 7.2 10.9 35.0 CH1.84O0.56N0.2 25.6
Candida utilis Ethanol 0.43 47.2 7.3 11.0 34.6 CH1.84O0.5N0.2 25.5

Table 2. Elemental Composition of Microogranisms (Strainer et. Al)

Stoichiometry of Microbial Growth and Product Formation 5


PAMANTASAN NG LUNGSOD NG MAYNILA
University of the City of Manila
College of Engineering and Technology
Department of Chemical Engineering

Table 2 shows that, in different microbes, the carbon content varies from 46-50%,
hydrogen from 6-7%, nitrogen 8-14%, and oxygen from 29-35%. These are small
variations and the variations appear to depend on substrate and growth conditions. For
many engineering calculations, it is reasonable to consider cell as a chemical species having
the formula
CH1.8O0.5N0.2

This engineering approximation is a good starting point for many quantitative


analyses while a more carefully formulated empirical formula based on proximate
analysis may be necessary for complete material flow analysis. The cell molecular weight
for the above cell formula is 12+1.8 + 0.5(16) +0.2 (14) = 24.6.

1. Determine the molecular formula of Aerobacter Aerogenes provided the dry cell
composition (% mass) from Table 2.

Given:
C H O N
48.7% 7.3% 21.1% 13.9%
Required: MFAerobacter Aerogenes
Solution:
Basis: 100g AA
1𝑚𝑜𝑙 1𝑚𝑜𝑙
𝑛𝑐 = 48.7𝑔 ( ) = 4.0550 𝑚𝑜𝑙 𝐶 𝑛𝑂 = 21.1𝑔 ( ) = 1.3188 𝑚𝑜𝑙 𝑂
12.01𝑔 16𝑔
1𝑚𝑜𝑙 1𝑚𝑜𝑙
𝑛𝐻 = 7.3𝑔 ( ) = 7.2277 𝑚𝑜𝑙 𝐻 𝑛𝑁 = 13.9𝑔 ( ) = 0.9921 𝑚𝑜𝑙 𝑁
1.01𝑔 14.01𝑔

Construct a molecular formula based on the computation of the amount of substance.

C4.0550H7.2277O1.3188N0.9921

Divide all subscripts by the subscript of the dominant element, ie. Carbon, therefore:

CH1.78O0.33N0.24

The molecular formula of Aerobacter Aerogenes is CH1.78O0.33N0.24

Stoichiometry of Microbial Growth and Product Formation 6


PAMANTASAN NG LUNGSOD NG MAYNILA
University of the City of Manila
College of Engineering and Technology
Department of Chemical Engineering

2. Determine the molecular formula of yeast - Saccharomyces Cerevisiae provided the dry
cell composition (%mass) from Table 2.

Given:
C H O N
47.0% 6.5% 31.0% 7.5%
Required: MFSaccharomyces Cerevisiae
Solution:
Basis: 100g SC

1𝑚𝑜𝑙 1𝑚𝑜𝑙
𝑛𝑐 = 47𝑔 ( ) = 3.9134 𝑚𝑜𝑙 𝐶 𝑛𝑂 = 31𝑔 ( ) = 1.9375 𝑚𝑜𝑙 𝑂
12.01𝑔 16𝑔
1𝑚𝑜𝑙 1𝑚𝑜𝑙
𝑛𝐻 = 6.5𝑔 ( ) = 6.4356 𝑚𝑜𝑙 𝐻 𝑛𝑁 = 7.5𝑔 ( ) = 0.5353 𝑚𝑜𝑙 𝑁
1.01𝑔 14.01𝑔

Construct a molecular formula based on the computation of the amount of substance.

C3.9134H6.4356O1.9375N0.5353

Divide all subscripts by the subscript of the dominant element, ie. Carbon, therefore:

CH1.64O0.50N0.14

The molecular formula of Saccharomyces Cerevisiae is CH1.64O0.50N0.14

3. Determine the molecular formula of Eschericia Coli provided the dry cell composition
(%mass) from Table 2.

Given:
C H O N
50% 8% 20% 14%
Element Composition below 5% which includes S, Ca, N, etc. are neglected.
Required: MFEschericia Coli
Solution:
Basis: 100g EC
1𝑚𝑜𝑙 1𝑚𝑜𝑙
𝑛𝑐 = 50𝑔 ( ) = 4.1632 𝑚𝑜𝑙 𝐶 𝑛𝐻 = 8𝑔 ( ) = 7.9208 𝑚𝑜𝑙 𝐻
12.01𝑔 1.01𝑔

Stoichiometry of Microbial Growth and Product Formation 7


PAMANTASAN NG LUNGSOD NG MAYNILA
University of the City of Manila
College of Engineering and Technology
Department of Chemical Engineering

1𝑚𝑜𝑙 1𝑚𝑜𝑙
𝑛𝑂 = 20𝑔 ( ) = 1.25 𝑚𝑜𝑙 𝑂 𝑛𝑁 = 14𝑔 ( ) = 0.9993 𝑚𝑜𝑙 𝑁
16𝑔 14.01𝑔

Construct a molecular formula based on the computation of the amount of substance.

C4.1632H7.9208O1.25N0.9993

Divide all subscripts by the subscript of the dominant element, ie. Carbon, therefore:

CH1.90O0.30N0.24

The molecular formula of E. Coli is CH1.90O0.30N0.24

4. Determine the molecular formula of Candida Utilis, which feeds on glucose, provided the
dry cell composition (%mass) from Table 2.
Answer: CH1.83O0.56N0.20

5. Determine the molecular formula of Candida Utilis, which feeds on ethanol, provided the
dry cell composition (%mass) from Table 2.
Answer: CH1.84O0.55N0.20

6. Suppose we want to produce 10 g of cells using glucose as a carbon source. What is the
minimum amount of glucose that would be needed? Assume cell composition as
CH1.8O0.5N0.2 (MW = 24.6)

Given: Glucose is C6 H12 O6


MW of glucose = 180
Required: Minimum Amount of Glucose
Solution:
10𝑔 𝑐𝑒𝑙𝑙𝑠
𝑀𝑜𝑙𝑒𝑠 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑡𝑜 𝑏𝑒 𝑔𝑟𝑜𝑤𝑛 =
24.6𝑔/𝑚𝑜𝑙
Since glucose has 6 moles of carbon per mole of glucose,

1 10
𝑀𝑜𝑙𝑒𝑠 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑛𝑒𝑒𝑑𝑒𝑑 = 𝑥
6 24.6
Therefore,
1 10
𝑀𝑖𝑛𝑖𝑚𝑢𝑛 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑛𝑒𝑒𝑑𝑒𝑑 (𝑚𝑎𝑠𝑠) = 𝑥 𝑥 180
6 24.6
𝑀𝑖𝑛𝑖𝑚𝑢𝑛 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑛𝑒𝑒𝑑𝑒𝑑 (𝑚𝑎𝑠𝑠) = 12.2𝑔

Stoichiometry of Microbial Growth and Product Formation 8


PAMANTASAN NG LUNGSOD NG MAYNILA
University of the City of Manila
College of Engineering and Technology
Department of Chemical Engineering

In the above example, we have assumed that all of the carbon found in substrate
(glucose) is incorporated into cell mass. This does not happen as the cell needs to
"oxidize" or respire some of the carbon to produce energy for biosynthesis and
maintenance of cellular metabolic machinery. In addition cells may produce
extracellular products that accumulate in the broth. Hence we can represent growth as:

Cells + (C source, N source, others) + O2 → More Cells + Extracellular Biomass + CO2 + H2O

The medium is the "food" for the cell. It serves as a source for all elements needed
by the cell to grow (or biosynthesis) and for product formation. The compounds carbon
dioxide and water on the product side of the reaction above result from oxidation of
glucose in the medium.
Since the cellular material contains C, N, P, S, K, Na, Ca, etc, the medium must be
formulated to supply these elements in the appropriate form.
If we neglect the "others" and assign stoichiometric coefficient for each of the
species in the above equation on the basis of one mole of glucose (C-source) consumed, we
re-write the above as:

C6H12O6 + aO2 + bNH3 → cCH1.80O0.50N0.20 + dCHXOYNZ + eCO2 + fH2O

Where-in ammonia represents the nitrogen source. We will refer to this reaction as
growth reaction.

Note that whatever nitrogen that is supplied in the medium, it is expressed as


equivalent nitrogen in the form of ammonia. Cells require nitrogen in both organic and
inorganic form. It is common to supply the inorganic nitrogen as salts of ammonium (e.g.
ammonium phosphate) while the organic nitrogen is usually supplied as amino acids or
proteinous extracts which are rich in nitrogen. In most production processes using
recombinant cells, glucose is used as the carbon source. However, in the production of low
value products, less expensive carbon sources such as molasses ($ 0.10 / lb) or corn
meal ( about $ 0.12 / lb ) are used. Compare this against glucose at $ 1.00 /lb.
The growth reaction derived above is useful in interpreting laboratory data
reported in the literature. Because the early work in cell growth were reported by
microbiologists, it is necessary for us to learn the terms used by microbiologists to describe
growth stoichiometry.

Stoichiometry of Microbial Growth and Product Formation 9


PAMANTASAN NG LUNGSOD NG MAYNILA
University of the City of Manila
College of Engineering and Technology
Department of Chemical Engineering

Elemental Balances:
During the growth of cells, substrates that provide energy and raw materials are
needed for the synthesis of additional cell mass. Generally, in a biochemical process, the cell
environment should contain elements required in order to form additional cell mass and
the free energy from the substrate consumed should exceed the free energy of cells and
metabolic products formed.
In order to obtain the amount of every element that took part in a growth process,
the cellular content of all elements should be known. These include carbon, nitrogen,
oxygen and hydrogen (C, N, O and H respectively).

One mole of biological materials is defined as the amount containing 1 gram atom of
carbon. Assumption: No extracellular products other than water and carbon dioxide are
produced.

Stoichiometry of Microbial Growth and Product Formation 10


PAMANTASAN NG LUNGSOD NG MAYNILA
University of the City of Manila
College of Engineering and Technology
Department of Chemical Engineering

We have five equations and five unknowns (a, b, c, d, e). With a measured value of
RQ, these equations can be solved to determine the stoichiometric coefficients. Elemental
balances provide no insight into the energetics of a reaction. The concept of degree of
reduction has been developed and used for proton-electron balances in bioreactions.
Degree of reduction (γ) for organic compounds is defined as the number of equivalents of
available electrons per gram atom C.
The method of obtaining a balanced growth equation is very much similar to the
equation describing chemical reactions. One needs to establish a chemical formula for dry
cell material if the elemental composition of a particular strain growing under conditions is
known, the ratios of subscripts in the empirical cell formula can be determined.

Consider an aerobic growth which the products are cells, carbon dioxide and water,
and the initial components such as carbon and nitrogen sources; CHxOy and HlOmNn
respectively the equation can be written as follows:

From the above stoichiometric equation, 4 relationships consist of the stoichiometric


coefficients a, b, c and d are:

The above equations is accompanied with an additional relationship obtained from


experimental determination, i.e. respiratory quotient (RQ):

Stoichiometry of Microbial Growth and Product Formation 11


PAMANTASAN NG LUNGSOD NG MAYNILA
University of the City of Manila
College of Engineering and Technology
Department of Chemical Engineering

Example 1: A production of single-cell protein from hexadecane is given by the following


reaction equation:

with CH1:66O0:27N0:20 represent the biomass. Given that RQ = 0.43, determine the
stoichiometric coefficients a, b, c, d and e.

SOLUTION:

RQ = d/a = 0.43

C : 16 = c + d
H : 34 + 3(0.20c) = 1.66c +2e
34 = 1.06c + 2e
O : 2(d/0.43) = 0.27c + 2d + e
0.27c – 2.6157d + e

Thus,

Complete Stoichiometric Equation:

Stoichiometry of Microbial Growth and Product Formation 12


PAMANTASAN NG LUNGSOD NG MAYNILA
University of the City of Manila
College of Engineering and Technology
Department of Chemical Engineering

Example 2: Complete the stoichiometric equation for the production of ethanol by


fermentation using Saccharomyces cerevisiae using glucose as a feed. The respiratory
quotient is 0.70.

Given:
C6H12O6 + aO2+ bNH3 → cCH1.64O0.50N0.14 + dCO2 + eH2O
Respiratory Quotient: d/b = 0.70

Required: Complete Stoichiometric Equation


Solution:
EQ. 1 : C Bal.: 6 = c + d
EQ. 2 : H Bal: 12 + 3b = 1.64c + 2e
EQ.3 : O Bal: 6 + 2a = 0.50c + 2d + e
EQ. 4 : N Bal: b = 0.14c
EQ. 5 : Resp. Quotient: d = 0.70a

Using systems of linear equations:


Equation 3 & 5:

Equation 2 &4:

Equation 1, 6 & 7:

By 3 equations and 3 unknowns, we will have:

c = 6.88 d = -0.88 e =1.80 a = -1.26 b =0.96

Stoichiometry of Microbial Growth and Product Formation 13


PAMANTASAN NG LUNGSOD NG MAYNILA
University of the City of Manila
College of Engineering and Technology
Department of Chemical Engineering

Hence the stoichiometric equation for microbial growth is:

The stoichiometric equation for product formation is:

Therefore the overall biochemical reaction is:

Degree of Reduction (γ)


 The number of equivalents of available electrons per gram atom C
 The degree of reduction of any element in a compound is Equal to the valence of this
element.
 The degrees of reduction for some key elements are : C=4, H=1, N=-3, O=-2, P=5, S=6

Example on how to calculate degree of reduction:

A high degree of reduction indicates a low degree of oxidation.

Stoichiometry of Microbial Growth and Product Formation 14


PAMANTASAN NG LUNGSOD NG MAYNILA
University of the City of Manila
College of Engineering and Technology
Department of Chemical Engineering

Consider the aerobic production of a single extracellular product:

The degree of reduction of substrate, biomass and product are:

Note that CO2, H2O and NH3 the degree of reduction is zero

Measuring Yields
It is stressed that stoichiometric yield coefficient are ratios of
conversion rates (rx is given as C•mol X m3 reactor per h; r1 in mol i m3 per h).

These rates are calculated from measurements in experiments which


may be either batch, continuous or fed batch cultures, using correct mass
balances. The most frequently measured growth stoichiometric coefficient is
the biomass yield on substrate (or electron donor) Y sx (or Y px)' In a constant
volume batch culture (0 indicating time=0), Y sx will be:

Stoichiometry of Microbial Growth and Product Formation 15


PAMANTASAN NG LUNGSOD NG MAYNILA
University of the City of Manila
College of Engineering and Technology
Department of Chemical Engineering

Conservation Principles to Calculate the


Full Stoichiometry of Growth
Example: Use of conservation principles in calculation of all stoichiometric coefficients.

An aerobic micro-organism grows on oxalate using NH4 as N-source. The following


overall reaction equation can be written based on 1 C-mol biomass being produced with a
biomass yield on oxalate of Ydx= 1/5.815 C-mol biomass per mol oxalate:

-5.815 C2O4 + aNH4 + bH4+ + cH20 + dO2 + eHCO3 + 1CH1.8O0.5N0.2

There are five unknown stoichiometric coefficients for which five conservation
constraints can be formulated. C-conservation H-conservation O-conservation N-
conservation and Charge-conservation

Solving gives the full stoichiometry.

-5.815 C2O4 + O.2NH4 + O.8H - 1.857O2 -5.42H2O + 1CH1.8O0.5N0.2 + 10.63HCO3

Thus we see that Yax = 1/1.857 C-mol X / mol O2. Also Ycx =1/10.53 C-mol X/ mol HCO3

Stoichiometry of Microbial Growth and Product Formation 16

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