Lecture 3: Chapter 4

Stoichiometry of growth and product formation, growth stoichiometry, material balance calculations, balances on reactive process, combustion reactions, additional considerations on chemical and biological processes.

BTE 2523: Biochemical Engineering Fundamental

Balance on a Cell

Typical Inputs into Biological Systems Carbon Source (Glucose,amino acid) Nitrogen Source, NH4Cl, (NH4)2SO4, amino acids, proteins Oxygen Source, Air (O2) Hydrogen Source Phosphate Source, PO4-3

Typical Outputs in Biological Systems Biomass Products Metabolites Products and metabolites are easy to write a chemical formula

How to write a formula for cells?

Stoichiometry of Growth and Product Formation

A mass balance that involves a reaction the stoichiometry of the conversion needs to be known before the mass balance can be solved Firstly, need to show how equations for growth and product formulation are formulated

Elemental Balances and Growth

In any fermentation, biotransformation or animal cell culture all the atoms of carbon, hydrogen, oxygen, nitrogen and the other elements can be accounted for In terms of growth, the substrate provide these elements and they are modified to cells and products The simplest case is when the only extra-cellular products are CO2 and water. Under such circumstances the following generalised equations can be written: CwHxOyNz + aO2 + bHgOhNi Where cCHON + dCO2 + eH2O

CwHxOyNz is the formula of the main carbohydrate source HgOhNi is the formula of the main nitrogen source cCHON is the formula for ash free biomass

a-e are the stoichiometric coefficients based on 1 mole of substrate

An absence of such compounds as NADH and ATP from the equation since they are not exchanged with the environment. Other compounds such as minerals and vitamins often added to medium their contribution can be neglected due to such a small quantity the biomass contains only the C, H, O and N elements, since these make up over 90% of the cells, Table 1 illustrates how these elements vary amongst different species

Table 1: Typical composition of organic and inorganic components of microorganisms

Table 2: The elemental composition of E.coli, one of the most widely used organisms in the biotechnology industry.

Clearly the ash free component of E.coli cells, the components containing C, O, N and H amount to 92% of the cell dry weight. It is common to merge all the other components into the ash content for mass balance calculations

Table 3: The elemental formula for various organisms

Table 4: The elemental composition as a function of culture conditions Shows the effect of culture conditions on elemental formula of a range of microorganisms

The stoichiometry balance:

CwHxOyNz + aO2 + bHgOhNi cCHON + dCO2 + eH2O

Is not complete unless the stoichiometric coeffiecients a-e are known if the formula for the biomass is known from tables 3 and 4, these can be evaluated by solution of the sinultaneous equations given below:
C balance : w = c +d H balance : x + bg = c +2e O balance : y + 2a + bh = c +2d + e N balance : z + bi = c

However, only four equations and five unknowns so additional information is required. Various parameters can be utilized to overcome this problem. These include:
(i) Respiratory quotients RQ = d/a = Moles CO2 produced Moles O2 consumed

(ii) Yield of biomass on substrate Yxs= kg biomass = Kg substrate c (MW cells) (MW substrate)

However, a large parameters affect the yield, including medium composition, pH, temperature, electron acceptor. The yield figure would be based on the mass of biomass including the ash content and so correction for this has to be made

Product stoichiometry
If in addition to the biomass an extracellular product can be formed with a composition CjHkOlNm. The mass balance can then be rewritten: CwHxOyNz + aO2 + bHgOhNi cCHON + dCO2 + eH2O + fCjHkOlNm With the additional stoichiometric coefficient f, another unknown. The product yield, experimentally determined is utilised, defined as: YPS= g product formed g substrate consumed = f (MW product) (substrate)

the relationship does not hold of product formation is linked to growth, secondary metabolite production or biotransformation. In such cases, independent equation must be used to describe growth and product formation.

Example: Stoichiometric coefficients for cell growth

Electron balances: Degree of reduction

Available electron refers to the number of electrons available for transfer oxygen on combustion of a substance to CO2, H2O and nitrogen-containing compounds The number of available electrons found in organic material is calculated from the valence of the various elements: 4 for C, 1 for H, -2 for O, 5 for P and 6 for S. The number of available electrons for N depends on the reference state: -3 if ammonia, 0 for molecular N2 and 5 for nitrate The degree of reduction, can be defined as the number of equivalents of available electrons in that quantity of material containing 1g atom carbon. For substrate CwHxOyNz, the number of available electrons is (4w + x 2y 3z). the degree of reduction for the substrate, s = (4w + x 2y 3z)/w degree of reduction for CO2, H2O and NH3 is zero

 Electron available for transfer to oxygen are conserved during metabolism. in a balanced growth equation, number of available electrons is conserved by virtue of the fact that the amounts of each chemical element are conserved. Applying this principle to this equation: CwHxOyNz + aO2 + bHgOhNi cCHON + dCO2 + eH2O With ammonia as nitrogen source, the available-electron balance is: wS + 4a = cb Where s and b are the degrees of reduction of substrate and biomass, respectively

Theoretical Oxygen Demand

Oxygen is often limiting substrate in aerobic fermentations and the demand is represented by the stoichiometric coefficient a in the stoichiometric equations In relation to degrees of reduction, the oxygen requirements is related to the electrons available for transfer to oxygen and oxygen demand can be derived from an electron balance if product synthesis occurs as in the equation: CwHxOyNz + aO2 + bHgOhNi cCHON + dCO2 + eH2O + fCjHkOlNm

the electron balance is : ws 4a = cB fjP (this equation can be used to calculate oxygen demand) Where P is the degrees of reduction of the product

Maximum Possible Yield

For the example given above the electron balances:
ws 4a = cB fjP

can be written as a fractional allocation:

substrate

4a c B fj P w S w S w S
biomass

product

the expression can be used to calculate the upper bounds of the yields. If is defined as the fraction of available electrons in the substrate transferred to biomass:

c B w s

in the absence of product formation, all available electrons would be used for biomass synthesis and B =1. under such conditions, the maximum values of the stoichiometric coefficients c is:

cm ax

w S

 Cmax can be converted to a biomass yield with mass units using the yield expression given. Therefore, even if the stoichiometry of growth is unknown, we can calculate an upper limit for biomass yield from the molecular formulae for substrate and product. if the composition of the cells is unknown B, can be taken as 4.2 corresponding to an average biomass formula CH1.8O0.5N0.2.

similarly the maximum product yield in the absence of biomass synthesis given by:

f m ax

w S j P

Problem:
The chemical reaction equation for the aerobic growth of a microorganism on glucose and ammonia is as following:
aC6H12O6 + bO2 + cNH3 dC6H10O3N3 + eCO2 + fH2O

using the information given below, determine the total amount (in kg) of glucose, ammonia and oxygen that are required to achieve a biomass concentration of 20 kg (dry cell mass)/m 3 in a 15 m3 fermenter can be calculated.
Biomass contains 6.4% (w/w) ash, Atomic weight: H = 1, C = 12, N = 14, O = 16,

Example 4.8 Product yield and oxygen demand

The chemical reaction equation for respiration of glucose is: C6H1206+602 6C02+6H20. Candida utilis cells convert glucose to CO2 and H20 during growth. The cell composition is CH1.8400.55N0.2 plus 5% ash. Yield of biomass from substrate is 0.5 g/g . Ammonia is used as nitrogen source. a) What is the oxygen demand with growth compared to that without? b) C. utilis is also able to grow with ethanol as substrate, producing cells of the same composition as above. On a mass basis, how does the maximum possible biomass yield from ethanol compare with the maximum possible yield from glucose?

Combustion Reactions
Combustion the rapid reaction of a fuel with oxygen that produces CO2, H2O and frequently CO and SO2 the significance of these reactions lies in the tremendous quantities of heat they release used to produce steam to drive the turbines that generate most of the worlds electrical power most of the fuel used in power plant combustion furnaces either coal (carbon some hydrogen and sulfur and various noncombustible materials), fuels, oil, gaseous fuel (such as natural gas, primarily methane) or liquefied petroleum gas usually propane and/or butane: a combustion reaction in which CO is formed from a hydrocarbon referred to as partial combustion or incomplete combustion of hydrocarbon example
C + O2 C3H8 + 5O2 C3H8 + 7/2O2 CS2 + 3O2 CO2 Complete combustion of carbon 3CO2 + 4H2O Complete combustion of propane 3CO + 4H2O partial combustion of propane CO2 + 2SO2 Complete combustion of carbon disulfide

Air has the following molar composition

N2 78.03% O2 20.99% Ar 0.94% CO2 0.03% N2 He, Ne, Kr, Xe 78.03% 100.00%

Average MW = 29.0

Composition on a wet basis denote the component mole fractions of a gas that contains water composition on a dry basis denotes the component mole fractions of the same gas without the water the product gas that leaves a combustion chamber referred to as the stack gas or flue gas when the flow rate of a gas in a stack is measured, it is the total flow rate of the gas including water

Example : Composition on wet and dry bases

1. Wet basis Dry basis A stack gas contains 60 mole% N2, 15% CO2, 10% O2 and the balance H2O. Calculate the molar composition of the gas on a dry basis

2. Dry basis Wet basis An Orsat analysis (a technique for stack gas analysis) yield the following dry basis composition:
N2 65% CO2 14% CO 11% O2 10% A humidity measurement shows that the mole fraction of H2O in the stack gas is 0.07. Calculate the stack gas composition on a wet basis

Theoretical and Excess Air

If two reactants participate in a reaction and one is considerably more expensive than the other, the usual practice is to feed the less expensive reactant in excess of the valuable one this has the effect of increasing the conversion of the valuable reactant at the expense of the cost of the excess reactant and adding pumping cost the extreme case of an inexpensive reactant is air which is free theoretical oxygen: the moles (batch) or molar flow rate (continuous) of O2 needed for complete combustion of all of the fuel fed to the reactor, assuming that all carbon in the fuel is oxidized to CO2 and all hydrogen is oxidized to H2O theoretical air: the quantity of air that contains the theoretical oxygen excess air: the amount by which the air fed to the reactor exceeds the theoretical air (moles air)fed (moles air) theoretical Percent excess air : X 100%
(moles air) theoretical

Example: if 50% excess air is supplied,

(moles air) fed = 1.5 (moles air) theoretical

Example : Theoretical and excess air

One hundred mol per hour of butane (C4H10) and 5000 mol per hour of air are fed into a combustion reactor. Calculate the percent excess air.

Material Balances on Combustion Reactors

The procedure for writing and solving material balances for a combustion reactor is essentially same as that for any other reactive system there are some additional points: When you draw and label the flowchart, remember to include the N2 at both the inlet and outlet of the reactor, and unreacted fuel and O2 as well as combustion products (CO2, H2O, CO at the outlet If there is any percent excess air, the O2 actually fed may be calculated by multiplying the theoretical O2 determined from the fuel feed rate and composition and the reaction stoichiometery for complete combustion by (1 + fractional excess air)

Example : Combustion of Ethane

Ethane (C2H6) is burned with 50% excess air. The percentage conversion of the ethane is 90% of the ethane burned, 25% reacts to form CO and the balance for form CO2. Calculate the composition of the flue gas and the ratio of water to dry flue gas

Summary of Chapter 4
At the end of Chapter 4 you should: (i) understand the terms: system, surroundings, boundary and process in thermodynamics (ii) be able to identify open and clossed systems, and batch, semi-batch, fed-batch and continuous processes (in) understand the difference between steady state and equilibrium (iv) be able to write appropriate equations for conservation of mass for processes with and without reaction; (v) be able to solve simple mass-balance problems with and without reaction; and (vi) be able to apply stoichiometric principles for macroscopic analysis of cell growth and product formation.