6-Mass Balances

Introduction to Bioengineering
4. Mass Balances
Objective
i. Perform mass balances to processes with and without reaction
SUB-SECTIONS:
1 – Introduction to mass balances

2 – Mass balances with reaction
BIBLIOGRAPHY:
P.M. Doran “Bioprocess Engineering Principles“ (1997), chap. 4, pp 51 - 85
José Santos/Miguel Prazeres

4. Mass balances
Introduction to mass balances

Introduction to Mass Balances
Mass conservation principle
Mass balances result from the application of the «Mass conservation principle»
“In Nature nothing is created,

nothing is lost,
everything is transformed"
Antoine-Laurent de Lavoisier

Systems and boundaries
In order to perform a mass balance, the system under study must be clearly defined.
A system can be defined as a portion or as the totality of the process under analysis.
This portion is limited by the boundary between the system and the exterior.
system
• Systems can be open or closed.
• In open systems mass is transferred across the

boundary.
• In closed systems there is no transfer of mass

across the boundary.
boundary

Systems and boundaries

  Unit 1
 Unit 2

 
system 1
system 2 system 3

   
Unit 1 Unit 2
 
system 4

General Mass Balance Equation
system
ACCUMULATION (Acc) MASS OUT (OUT)
MASS IN (IN) PRODUCTION (P)
CONSUMPTION (C)
boundary
MASS IN + PRODUCTION = MASS OUT + CONSUMPTION + ACCUMULATION

Acc OUT
IN P
C
IN + P = OUT + C + Acc (kg/s, mol/s)
OUT
i. If no chemical/biological reaction: IN = OUT + Acc IN Acc
ii. Steady state system (Acc=0): IN = OUT OUT

IN

Q38. Water is added to a tank that already contains 100 L at a rate of 20 L/h
and removed at a rate of 5 L/h. What is the volume after 3 h?
20 L/h 5 L/h
boundary
IN = OUT + Acc => Acc = IN-OUT
ACC = 20 – 5 = 15 L/h
V = Vo + ACC = 100 + 15 L/h x 3 h = 145 L

Techniques for Mass Balance Calculations
Q39. Evaporation is used to concentrate an aqueous solution of sodium hydroxide from
5% to 29%. Perform mass balances assuming an initial flow rate of 100 kg/h of solution.
Calculations:
1 - Draw the block diagram of the process
2 - Attribute algebraic symbols to the stream variables. Take note of all the known
variables in the problem.
FM3 100% H2O, (XH2O)3 = 1

FM1 = 100 kg/h FM2
 Evaporator 
95% H2O, (XH2O)1 = 0.95 71% H2O, (XH2O)2 = 0.71

5% NaOH, (XNaOH)1 = 0.05 29% NaOH, (XNaOH)2 = 0.29

3 - Prepare a table showing all streams and components in the problem. Indicate the
units of the numerical values introduced in the table.
Kg/h   
H2O
NaOH
Total
4 - Write a number of equations equal to the number of unknown variables. These

equations can be mass balances, thermodynamic laws, empirical correlations, etc.
Unknown variables: FM2 and FM3, i.e. NV = 2

Mass balance equations: H2O and NaOH, i.e. NEQ = 2

Mass balance equations
IN + P = OUT + C + Acc (kg/s, mol/s)
FM3 100% H2O, (XH2O)3 = 1

FM1 = 100 kg/h FM2
 Evaporator 
95% H2O, (XH2O)1 = 0.95 71% H2O, (XH2O)2 = 0.71

5% NaOH, (XNaOH)1 = 0.05 29% NaOH, (XNaOH)2 = 0.29

- Total mass balance: FM1 = FM2 + FM3
- Mass balance NaOH: (XNaOH)1 FM1 = (XNaOH)2 FM2 + (XNaOH)3 FM3
5 – Select a basis for the calculations (according to the type of problem, the basis of
calculation should be expressed in moles, mass or volume).
Calculation basis: 100 kg/h in stream  (real calculation basis)
6 – Solve the equations.
100 = FM2 + FM3  FM3 = 100 - 17.2 = 82.8 kg/h
0.05 x 100 = 0.29 FM2  FM2 = 17.2 kg/h

FM3 = 82.8 kg/h 82.8 kg/h H2O

FM1 = 100 kg/h FM2= 17.2 kg/h
 Evaporator 
95 kg/h H2O 12.2 kg/h H2O

5 kg/h NaOH 5 kg/h NaOH
Kg/h   
H2O 95 12.2 82.8
NaOH 5 5.0 -
Total 100 17.2 82.8

1 – Draw the block diagram of the process.
2 - Attribute algebraic symbols to the stream variables. Take note of all the known
variables in the problem.
3 – Prepare a table showing all streams and components in the problem. Indicate the
units of the numerical values introduced in the table.
4 – Write a number of equations equal to the number of unknown variables. These

equations can be mass balances, thermodynamic laws, empirical correlations, etc.
5 – Select a basis for the calculations (according to the type of problem, the basis of
calculation should be expressed in moles, mass or volume).
6 – Solve the equations.

Overall mass balances
In processes with more than one UO we usually make mass balances to the individual units.
B C
A

  Unit 1
 Unit 2

 
In many situations, the mass balances to individual units can be replaced by a mass
balance that includes the full process: i.e an overall mass balance
B C
A 
   
Unit 1 Unit 2
 
An overall mass balance will include the streams entering and exiting the global process.
Intermediate streams  and  will not be a part of these balances.
Overall mass balances
Q41. Calculate the mass flow rate and mass composition of the effluent stream of the
following process.
FM4 = 40 kg/h
A-20%
B-80%

 
FM1 = 100 kg/h Unit 1 Unit 2 FM7 = ? kg/h
 
A-50%
  A-?
B-50% A-10% A-80% B-?
B-90% B-20%
FM2 = 30 kg/h FM6 = 50 kg/h (mass percentages)
(we do not need to know the composition of streams  and , i.e. there is no need to
perform individual mass balances to the two units or stream junction.

Overall mass balances FM4 = 40 kg/h
A-20%
B-80%

 
 
A-50%
  A-?
B-50% A-10% A-80% B-?
B-90% B-20%
FM2 = 30 kg/h FM6 = 50 kg/h (mass percentages)
Total, overall MB: FM1 + FM4 = FM2 + FM6 + FM7
Overall MB to component A: xA1FM1 + xA4FM4 = xA2FM2 + xA6FM6 + xA7FM7
Composition stream 7: xA7 + xB7 = 1

FM4 = 40 kg/h A-20%
Overall mass balances B-80%

 
 
A-50% 
 A-?
B-50% A-10% A-80% B-?
B-90% B-20%
FM2 = 30 kg/h FM6 = 50 kg/h
FM7 =100 + 40 – 30 - 50 = 60 kg/h
(0.5 x 100) + (0.20 x 40) = (0.10 x 30) + (0.80 x 50) + (xA7 60)  xA7 = 0.25
xB7 = 1-0.25 = 0.75
Kg/h       
A 50 3 8 40 15
B 50 27 32 10 45
Total 100 30 40 50 60

4. Mass balances
Mass balances with reaction

Mass Balances With Reaction
Acc OUT
IN P IN + P = OUT + C + Acc (kg/s, mol/s)
C
Steady state system (Acc=0):

OUT
i. If no chemical/biological reaction: IN = OUT IN
ii. With chemical/biological reaction: IN + P = OUT + C OUT

P
IN
C
We must consider the reaction stoichiometry, which relates the mass (or moles) of
elements and compounds that combine with each other (reagents) to originate new
compounds (products) through the corresponding stoichiometric coefficients.
Molecular and Atomic Mass Balances
Chemical/Biological Reaction
In problems involving reactions, one of the first tasks is to write the equation of the
chemical or biological transformation.
REAGENTS  PRODUCTS
We should take into consideration the fact that the number of atoms of each element must
be the same in both members of that equation.
Atomic species mass balance (law of conservation of atomic species):
(atoms of species X)in = (atoms of species X)out

Q42. Consider the conversion of glucose (C6H12O6) to succinic acid (C4H6O4)

described by the equation below. Calculate the mass of glucose required to produce 100
g of succinic acid.
𝐶6 𝐻12𝑂6 + 𝐶𝑂2 → 𝐶4 𝐻6 𝑂4 + 𝐻2 𝑂
SOLUTION
The number of atomic species is not the same in both sides – we must determine the
stoichiometric coefficients by performing a mass balance to the atomic species involved.
1 𝐶6𝐻12 𝑂6 + 𝑎 𝐶𝑂2 → 𝑏 𝐶4 𝐻6 𝑂4 + 𝑐 𝐻2 𝑂
we choose a coefficient of 1 for glucose as a

reference. We could have used any other
number or molecule as the reference. José Santos/Miguel Prazeres
We have 3 unknowns (a, b and c) and we can make 3 atomic balances (C, H and O)
Carbon: 6 + a = 4b a = 6/7
Hydrogen: 12 = 6b + 2c b = 12/7
Oxygen: 6 + 2a = 4b + c c = 6/7
6 12 6
𝐶6 𝐻12𝑂6 + 𝐶𝑂2 → 𝐶4 𝐻6 𝑂4 + 𝐻2 𝑂
7 7 7
7𝐶6 𝐻12 𝑂6 + 6𝐶𝑂2 → 12𝐶4𝐻6 𝑂4 + 6𝐻2 𝑂
100 𝑔
𝑛𝑠.𝑎. = ≅ 0.85 𝑚𝑜𝑙
4 × 12 + 6 × 1 + 4 × 16 𝑔 𝑚𝑜𝑙 −1
7 𝑚𝑜𝑙 × 0.85 𝑚𝑜𝑙

𝑚𝑔𝑙𝑢𝑐𝑜𝑠𝑒 = × 6 × 12 + 12 × 1 + 6 × 16 𝑔 𝑚𝑜𝑙 −1 ≅ 89.3 𝑔
12 𝑚𝑜𝑙
Q43. Write the equation that describes the reaction between glucose (C6H12O6) and
oxygen (O2), which originates carbon dioxide (CO2) and water (H2O).
SOLUTION
C6H12O6 + O2  CO2 + H2O
The number of atomic species is not the same in both sides – we must determine the
stoichiometric coefficients by performing a mass balance to the atomic species involved.
1 C6H12O6 + a O2  b CO2 + c H2O
We have 3 unknowns (a, b and c) and we can make 3 balances (C, H and O)
Carbon: 6=b b=6
Hydrogen: 12 = 2c c=6 C6H12O6 + 6O2  6CO2 + 6H2O
Oxygen: 6 + 2a = 2b + c a=6
Biological reactions
Carbon source Biomass

(CaHbOcNd) (CHaObNg)
O2 CO2 ; H2O
aerobic process Cell
Nitrogen source Product (extracellular)

(NeOfHg) (ChHiOjNk)
Carbon source + Nitrogen source + Oxygen  Biomass + Product + Carbon dioxide + Water
CaHbOcNd + m NeOfHg + n O2  p CHaObNg + q ChHiOjNk + r CO2 + s H2O
If, for example, the carbon source is glucose, then: a = 6 ; b = 12 ; c = 6 and d = 0

Culture Media Carbon source Biomass
O2 CO2 ; H2O
Cell
Carbon sources: Glucose (C6H12O6)

Nitrogen source Product
Glycerol (C3H8O3)
Lactose (C12H22O11)
Starch (C6H10O5)n
Nitrogen sources Ammonia (NH3)
Ammonium chloride (NH4Cl)
Ammonium sulphate ((NH4)2SO4)

Complex Culture Media
Organic sources of nitrogen, carbon, vitamins and energy
Yeast extract
Obtained from yeast cells (Saccharomyces sp.): a by-product of the beer industry
Typical composition (%):

Humidity: 27
Protein : 44
Carbohydrates: 6 Chemical formula: CH1.91O0.56N0.23
NaCl: 10
Ashes (except NaCl): 13
Vitamins: few mg

Complex Culture Media
Corn-steep liquor: CH1.91O0.21N0.08 (water from corn maceration)
Typical composition (%):

Water: 50
Protein : 29
Carbohydrate: 6
Fat: 1
Ashes: 9
Other: 5
Tryptone: CH1.80O0.50N0.20 (from enzymatic hydrolysis of casein)
Bacto Peptone: CH1.80O0.52N0.21 (from enzymatic hydrolysis of fresh meat)

Culture Media
Typical composition
Chemically defined media
Example 1: Growth of filamentous fungi for the production of alkaline proteases
Glucose: 20 g/L Magnesium sulphate: 1 g/L

Glycerol: 10 g/L Sodium chloride: 1 g/L
Ammonium sulphate: 5 g/L Vitamin B12 : 10 mg/L
Urea: 2 g/L Water
Complex media:
Example 2: Growth of E. coli, for the production of plasmid DNA
LB media: 25 g/L Luria-Bertani broth Tryptone: 10 g/L

Kanamycin: 30 mg/L Yeast extract: 5 g/L
Water NaCl: 10 g/L

Cell composition
O2 CO2 ; H2O
Cell
Nitrogen source Product
Escherichia coli

Cell composition
Q44. Determine the empirical chemical formula and the “molar” mass of Escherichia coli
using the mass composition given in the table below. Consider only those elements whose
mass is > 1% (i.e. determine a, b , g and d in the formula CHaObNgPd).
Element Dry weight (%) Element Dry weight (%)

Carbon 50 Sodium 1.0
Oxygen 20 Calcium 0.50
Nitrogen 14 Magnesium 0.50
Hydrogen 8.0 Chlorine 0.50
Phosphorous 3.0 Iron 0.20
Sulfur 1.0 Other 0.30
Potassium 1.0

Cell composition
SOLUTION
Calculation basis: 100 g of cells:
Elements M (g/mol) Mass (g) Moles
C 12 50 4.17
Empirical chemical formula
H 1 8.0 8.00
O 16 20 1.25
C Ha Ob Ng Pd
N 14 14 1.00
P 31 3.0 0.10
8.00 1.25
a  1.92 b  0.30
4.17 4.17
C H1.92 O0.30 N0.24 P0.02
1.00 0.10
g  0.24 d  0.02
4.17 4.17
“Molar” mass
M = (1 x 12) + (1.92 x 1) + (0.30 x 16) + (0.24 x 14) + (0.02 x 31) = 22.7 g/mol

Cell composition
Reduced formulas of several microorganisms

Cell composition
Reduced formulas of the major macromolecules in yeast

Cell composition
Q45. A bacteria (CH1.90O0.30N0.14) is produced aerobically using glucose (C6H12O6) as the

carbon source and ammonia (NH3) as the nitrogen source. We want to obtain 24.8 g/L of
biomass in a 24 hour-long batch fermentation. Assuming that the molar respiratory quocient
(RQ =moles CO2 formed/moles O2 consumed) is equal to 1.2, determine:
a) a molecular equation to describe the production of biomass.
O2
Cell
Nitrogen source CO2 ; H2O

Cell composition
SOLUTION
a) The production equation can be written as:
C6H12O6 + a NH3 + b O2  c C H1.90 O0.30 N0.14 + d CO2 + e H2O
In order to determine the 5 unknown stoichiometric coefficients (a, b, c, d, e) we must use 5

equations (4 atomic species balances + RQ = 1.2):
 C: 6=c+d a = 0.362
 N: a = 0.14 c b = 2.845
 H: 12 + 3 a = 1.90 c + 2 e c = 2.586
 O: 6 + 2 b = 0.30 c + 2 d + e d = 3.414
 RQ: d/b = 1.2 e = 4.086
C6H12O6 + 0.362 NH3 + 2.845 O2  2.586 C H1.92 O0.30 N0.14 + 3.414 CO2 + 4.086 H2O

Reaction yields and OUR
Biomass Yield
(defined relatively to the carbon source (i.e. the substrate))
mass of cells (biomass) produced

YX/S 
mass of substrate consumed
Product Yield
(defined relatively to the carbon source (substrate))
with;
mass of extracellular product formed X – biomass
YP/S 
mass of substrate consumed S – substrate
P - product
Oxygen Uptake Rate (OUR)
OUR 
oxygen consumed
kg/s, mol/s)
time

Cell composition, reaction yields and OUR
Q45. A bacteria (CH1.90O0.30N0.14) is produced aerobically using glucose (C6H12O6) as the

carbon source and ammonia (NH3) as the nitrogen source. We want to obtain 24.8 g/L of
biomass in a 24 hour-long batch fermentation. Assuming that the molar respiratory quocient
(RQ =moles CO2 formed/moles O2 consumed) is equal to 1.2, determine:
a) a molecular equation to describe the production of biomass. 
b) the biomass yield.
c) the OUR in mmoles O2 L-1 h-1.

SOLUTION
b) Starting from the molecular equation:
C6H12O6 + 0.362 NH3 + 2.845 O2  2.586 C H1.92 O0.30 N0.14 + 3.414 CO2 + 4.086 H2O
Molar masses:
Mbiomass = 1 x 12 + 1.92 x 1 + 0.30 x 16 + 0.14 x 14 = 20.7 g/mol
Mglucose = 180.2 g/mol
Biomass yield:
2.586 x 20.7
YX/S   0.297 g biomass/g substrate
1 x 180.2

SOLUTION
c) The OUR is the velocity of oxygen consumption:
“Moles” of biomass formed = 24.8 g L-1/ 20.7 g mol-1 = 1.20 moles/L
Moles of O2 consumed =
=1.20 moles biomass L-1 x (2.845 moles O2 / 2.586 moles biomass) =
= 1.32 moles O2 L-1 = 1.32 x 103 mmoles O2 L-1
OUR = 1.32 x 103 mmoles O2 L-1/ 24 h = 55.0 mmoles O2 L-1 h-1

Limiting reagent, conversion and excess
Two reagents intervening in a given reaction are in stoichimetric proportion if the ratio
between the number of moles of those reagents is equal to the ratio between the
corresponding stoichiometric coefficients.
Ex: A + 2B  C 35 mol A + 70 mol B  stoichiometric proportion
In industrial reactions, reagents are rarely used in stoichiometric proportions. It is thus

convenient to define:
The LIMITING REAGENT: reagent which is present in the reactor/bioreactor in the

lowest stoichiomeric amount (i.e. the reagent which disapears first if the reaction is
complete).
REAGENTS IN EXCESS: reagents that are present in amounts larger than what is
required stoichiometrically to guarantee the total conversion of the limiting reagent.

Limiting reactant, conversion and excess
EXCESS (%E)
N - EN
%E  x 100
EN
N – moles of the reagent in stoichiometric excess.

EN –moles of reagent in stoichiometric excess required for the total conversion of the limiting reagent.
Ex: A + 2B  C  
Reactor
A – 35 moles
B – 100 moles
B in excess, A limiting
100 - 70
%EB  x 100  43%
70

In many reactions, the limiting reagent is not totally converted. Thus we can define the
percentage of conversion of reagent, X(%) into products as:
R E - S
%X  x 100  x 100
E E
R – moles of reagent that is converted

E – moles of reagent that enters the reactor.
S – moles of reagent that exits the reactor.
Ex: A + 2B  C  
Reactor
A – 35 moles A – 5 moles
B – 100 moles
30 35 - 5
%XA  x 100  x 100  85.7%
35 35

Limiting reagent, conversion and excess
Example (assume complete reaction in all cases): 𝑁 − 𝐸𝑁

%𝐸 = × 100
𝐸𝑁
𝑅
A + B  C stoichiometric proportion %𝑋 = × 100
 𝐸
EA=0%, EB=0%, XA=100%, XB=100%
A + B  C A in excess, B limiting

EA=100%, EB=0%, XA=50%, XB=100%
A + B  C B in excess, A limiting

EA=0%, EB=100%, XA=100%, XB=50%

Q46. 100 moles of A react with 80 moles of B according to the following reaction:
1
A  B  2C
2
Assuming a conversion of 80% of A, determine:
a) The limiting and excess reagents.

b) The percentage of excess.
c) The composition of the stream exiting the reactor.
 
Calculation basis: Reactor
180 moles in 1 A
A – 100 moles
B
B – 80 moles
C

a) The limiting and excess reagents.

100 80
1
A  B  2C 80 – 50 = 30 moles of B left when all A is consumed
2
Limiting reagent: A
100 50 Excess reagent: B
b) The percentage of excess.
N - EN N = 80 moles of B available
%E  x 100
EN EN = 50 moles of B required to fully convert A
80 - 50
%E  x 100  60%
50

c) The composition of the stream exiting the reactor.
R R
%X  x 100 80  x 100  R  80 moles
A E 100
moles limiting reagent (A) converted, R = 80 moles

moles limiting reagent (A) exiting the reactor, S = 20 moles
1
A  B  2C N. moles in 2 Composition of 2
2
80 40 160 A: 100 – 80 = 20 moles A: 9.1%

B: 80 – 40 = 40 moles B: 18.2%
C: 160 moles C: 72.7%

6-Mass Balances

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6-Mass Balances

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Introduction to Bioengineering

1 – Introduction to mass balances

P.M. Doran “Bioprocess Engineering Principles“ (1997), chap. 4, pp 51 - 85

José Santos/Miguel Prazeres

Introduction to mass balances

“In Nature nothing is created,

José Santos/Miguel Prazeres

• In open systems mass is transferred across the

• In closed systems there is no transfer of mass

José Santos/Miguel Prazeres

José Santos/Miguel Prazeres

ACCUMULATION (Acc) MASS OUT (OUT)

MASS IN (IN) PRODUCTION (P)

MASS IN + PRODUCTION = MASS OUT + CONSUMPTION + ACCUMULATION

José Santos/Miguel Prazeres

IN + P = OUT + C + Acc (kg/s, mol/s)

ii. Steady state system (Acc=0): IN = OUT OUT

José Santos/Miguel Prazeres

IN = OUT + Acc => Acc = IN-OUT

V = Vo + ACC = 100 + 15 L/h x 3 h = 145 L

José Santos/Miguel Prazeres

FM3 100% H2O, (XH2O)3 = 1

95% H2O, (XH2O)1 = 0.95 71% H2O, (XH2O)2 = 0.71

José Santos/Miguel Prazeres

4 - Write a number of equations equal to the number of unknown variables. These

Unknown variables: FM2 and FM3, i.e. NV = 2

José Santos/Miguel Prazeres

Mass balance equations

IN + P = OUT + C + Acc (kg/s, mol/s)

FM3 100% H2O, (XH2O)3 = 1

95% H2O, (XH2O)1 = 0.95 71% H2O, (XH2O)2 = 0.71

José Santos/Miguel Prazeres

- Total mass balance: FM1 = FM2 + FM3

- Mass balance NaOH: (XNaOH)1 FM1 = (XNaOH)2 FM2 + (XNaOH)3 FM3

Calculation basis: 100 kg/h in stream  (real calculation basis)

6 – Solve the equations.

100 = FM2 + FM3  FM3 = 100 - 17.2 = 82.8 kg/h

0.05 x 100 = 0.29 FM2  FM2 = 17.2 kg/h

José Santos/Miguel Prazeres

FM3 = 82.8 kg/h 82.8 kg/h H2O

95 kg/h H2O 12.2 kg/h H2O

Total 100 17.2 82.8

José Santos/Miguel Prazeres

1 – Draw the block diagram of the process.

4 – Write a number of equations equal to the number of unknown variables. These

6 – Solve the equations.

José Santos/Miguel Prazeres

FM2 = 30 kg/h FM6 = 50 kg/h (mass percentages)

José Santos/Miguel Prazeres

FM2 = 30 kg/h FM6 = 50 kg/h (mass percentages)

Total, overall MB: FM1 + FM4 = FM2 + FM6 + FM7

Overall MB to component A: xA1FM1 + xA4FM4 = xA2FM2 + xA6FM6 + xA7FM7

Composition stream 7: xA7 + xB7 = 1

José Santos/Miguel Prazeres

FM7 =100 + 40 – 30 - 50 = 60 kg/h

xB7 = 1-0.25 = 0.75

José Santos/Miguel Prazeres

Mass balances with reaction

Steady state system (Acc=0):

ii. With chemical/biological reaction: IN + P = OUT + C OUT

Atomic species mass balance (law of conservation of atomic species):

(atoms of species X)in = (atoms of species X)out

José Santos/Miguel Prazeres