4 Material Balances
2.90 gmol biomass lgmolethanol ] 126.78gbiomass
Yxs,max --
gmol ethanol 46 gethanol " 1 gmolbiomass
Yxs,max = 1.69 g biomass
g ethanol
Therefore, on a mass basis, the maximum possible amount of biomass produced per gram ethanol consumed is roughly twice
that per gram glucose consumed. This result is consistent with the data listed in Table 4.4.
Example 4.8 illustrates two important points. First, the chem-
ical reaction equation for conversion of substrate without
growth is a poor approximation of overall stoichiometry when
cell growth occurs. When estimating yields and oxygen
requirements for any process involving cell growth, the full
stoichiometric equation including biomass should be used.
Second, the chemical nature or oxidation state of the substrate
has a major influence on product and biomass yield through
the number of available electrons.
4.7 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 openand closedsystems, and batch, semi-
batch, fed-batch and continuous processes;,
(iii) understand the difference between steady state and equi-
librium;
(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 macroscop-
ic analysis of cell growth and product formation.
Problems
4.1 Cell concentration using membranes
A battery of cylindrical hollow-fibre membranes is operated at
steady state to concentrate a bacterial suspension from a fer-
menter. 350 kg min-1 fermenter broth is pumped through a
stack of hollow-fibre membranes as shown in Figure 4P1.1.
The broth contains 1% bacteria; the rest may be considered
water. Buffer solution enters the annular space around the
membrane tubes at a flow rate of 80 kg min-1; because broth
in the membrane tubes is under pressure, water is forced across
8~.
the membrane into the buffer. Cells in the broth are too large
to pass through the membrane and pass out of the tubes as a
concentrate.
Figure 4P 1.1 Hollow-fibre membrane for concentration of
cells.
Fermenlalion
brolh
Buffer
solution ~ ll~w-fihre
lllClllbrallC
The aim of the membrane system is to produce a cell suspen-
sion containing 6% biomass.
(a) What is the flow rate from the annular space?
(b) What is the flow rate of cell suspension from the mem-
brane tubes?
Assume that the cells are not active, i.e. they do not grow.
Assume further that the membrane does not allow any mol-
ecules other than water to pass from annulus to inner cylinder,
or vice versa.
4.2 Membrane reactor
A battery of cylindrical membranes similar to that shown in
Figure 4Pl.1 is used for an extractive bioconversion.
Extractive bioconversion means that fermentation and extrac-
tion of product occur at the same time.
Yeast cells are immobilised within the membrane walls. A
10% glucose in water solution is passed through the annular
space at a rate of 40 kg h - 1. An organic solvent, such as 2-ethyl-
1,3-hexanediol, enters the inner tube at a rate of 40 kg h-1.
4 Material. Balances 83

Because the membrane is constructed of a polymer which dehydrated egg product leaving the enzyme reactor is 0.2%.
repels organic solvents, the hexanediol cannot penetrate the Determine:
membrane and the yeast is relatively unaffected by its toxicity.
(a) which is the limiting substrate;
On the other hand, because glucose and water are virtually
(b) the percentage excess substrate;
insoluble in 2-ethyl-1,3-hexanediol, these compounds do not
(c) the composition of the reactor off-gas; and
enter the inner tube to an appreciable extent. Once immobil-
(d) the composition of the final egg product.
ised in the membrane, the yeast cannot reproduce but convert
glucose to ethanol according to the equation:
4.5 Azeotropic distillation
C6H1206 -~ 2 C 2 H 6 0 4- 2 C O 2.
Absolute or 100% ethanol is produced from a mixture of 95%
ethanol and 5% water using the Keyes distillation process. A
Ethanol is soluble in 2-ethyl-l,3-hexanediol; it diffuses into
third component, benzene, is added to lower the volatility of
the inner tube and is carried out of the system. CO 2 gas exits
the alcohol. Under these conditions, the overhead product is a
from the membrane unit through an escape valve. In the aque-
constant-boiling mixture of 18.5% ethanol, 7.4% H 2 0 and
ous stream leaving the annular space, the concentration of
74.1% benzene. The process is outlined in Figure 4P5.1.
unconverted glucose is 0.2% and the concentration of ethanol
is 0.5%. If the system operates at steady state:
Figure 4P5.1 Flowsheet for Keyes distillation process.
(a) What is the concentration of ethanol in the hexanediol
stream leaving the reactor?
(b) What is the mass flow rate of CO2? 74. 1% benzene
. ~ 18.5%ethanol
7.4% water
4.3 Ethanol distillation
Liquid from a brewery fermenter can be considered to contain
10% ethanol and 90% water. 50 000 kg h-1 of this fermenta-
95% ethanol ~ 1 1 m , , -
tion product are pumped to a distillation column on the 5% water
Distillation
factory site. Under current operating conditions a distillate of
Benzene ~ B I ~ - tower
45% ethanol and 55% water is produced from the top of the
column at a rate one-tenth that of the feed.
(a) What is the composition of the waste 'bottoms' from the
still?
(b) What is the rate of alcohol loss in the bottoms?
L ~ 100% ethanol
4.4 Removal of glucose from dried egg
The enzyme, glucose oxidase, is used commercially to remove
Use the following data to calculate the volume of benzene
glucose from dehydrated egg to improve colour, flavour and
which should be fed to the still in order to produce 250 litres
shelf-life. The reaction is:
absolute ethanol: p (100% alcohol) = 0.785 g cm-3; p (ben-
zene) = 0.872 g cm -3.
C6H120 6 + 0 2 + H 2 0 --9 C6H120 7 + H 2 0 2.
(glucose) (gluconic acid)
4.6 Culture of plant roots
A continuous-flow reactor is set up using immobilised-enzyme
beads which are retained inside the vessel. Dehydrated egg Plant roots produce valuable chemicals in vitro. "A batch cul-
slurry containing 2% glucose, 20% water and the remainder ture of Atropa belladonna roots at 25~ is established in an
unreactive egg solids, is available at a rate of 3 000 kg h - 1. Air air-driven reactor as shown in Figure 4P6.1. Because roots
is pumped through the reactor contents so that 18 kg oxygen cannot be removed during operation of the reactor, it is
are delivered per hour. The desired glucose level in the proposed to monitor growth using mass balances.
4 Material Balances
Figure 4P6.1 Reactor for culture ofplant roots.
Off-gas
Nulrien!
medium
,T
Air-driven
Roots reaclor
kir
Drained liquid
1425 g nutrient medium containing 3% glucose and
1.75% NH 3 is fed into the reactor; the remainder of the medi-
um can be considered water. Air at 25~ and 1 atm pressure is
sparged into the fermenter at a rate of 22 cm 3 min- I. During a
10-day culture period, 47 litres 0 2 and 15 litres CO) are col-
lected in the off-gas. After 10 days, 1110 g liquid containing
0.063% glucose and 1.7% dissolved NH 3 is drained from the
vessel. The ratio of fresh weight to dry weight for roots is
known to be 14:1.
(a) What dry mass of roots is produced in 10 days?
(b) Write the reaction equation for growth, indicating the
approximate chemical formula for the roots, CHaOflN6.
(c) What is the limiting substrate?
(d) What is the yield of roots from glucose?
4 . 7 O x y g e n r e q u i r e m e n t for g r o w t h o n
glycerol
Klebsiella aerogenesis produced from glycerol in aerobic cul-
ture with ammonia as nitrogen source. The biomass contains
8% ash, 0.40 g biomass is produced for each g glycerol con-
sumed, and no major metabolic products are formed. What is
the oxygen requirement for this culture in mass terms?
84
4.8 P r o d u c t y i e l d in a n a e r o b i c d i g e s t i o n
Anaerobic digestion of volatile acids by methane bacteria is
represented by the equation:
CH3COOH + NH 3 --9 biomass + CO 2 + H20 + CH 4.
(acetic acid) (methane)
The composition of methane bacteria is approximated by the
empirical formula CH1.4Oo.40N0.20. For each kg acetic acid
consumed, 0.67 kg CO 2 is evolved. How does the yield of
methane under these conditions compare with the maximum
possible yield?
4.9 Stoichiometry of single-cell protein
synthesis
(a) Cellulomonas bacteria used as single-cell protein for
human or animal food are produced from glucose under
anaerobic conditions. All carbon in the substrate is
converted into biomass; ammonia is used as nitrogen
source. The molecular formula for the biomass is
CHI.5600.s4No.16; the cells also contain 5% ash. How
does the yield ofbiomass from substrate in mass and molar
terms compare with the maximum possible biomass yield?
(b) Another system for manufacture of single-cell protein is
Methylophilus methylotrophus. This organism is produced
aerobically from methanol with ammonia as nitrogen
source. The molecular formula for the biomass is
CH 1.6800.36N0.22; these cells contain 6% ash.
(i) How does the maximum yield of biomass compare
with (a) above? What is the main reason for the differ-
ence?
(ii) If the actual yield of biomass from methanol is 42%
the thermodynamic maximum, what is the oxygen
demand?
4.10 Ethanol production by yeast and bacteria
Both Saccharomyces cerevisiae yeast and Zymomonas mobilis
bacteria produce ethanol from glucose under anaerobic condi-
tions without external electron acceptors. The biomass yield
from glucose is 0.11 g g-1 for yeast and 0.05 g g-I for Z.
mobilis. In both cases the nitrogen source is NH 3. Both cell
compositions are represented by the formula CH 1.800.5N0.2.
(a) What is the yield of ethanol from glucose in both cases?
(b) How do the yields calculated in (a) compare with the
thermodynamic maximum?
4 Material Balances
4.11 D e t e c t i n g u n k n o w n products
Yeast growing in continuous culture produce 0.37 g biomass
per g glucose consumed; about 0.88 g 0 2 is consumed per g
cells formed. The nitrogen source is ammonia, and the bio-
mass composition is CH1.7900.56N0.17. Are other products
also synthesised?
4.12 Medium formulation
Pseudomonas 5401 is to be used for production of single-cell
protein for animal feed. The substrate is fuel oil. The composi-
tion of Pseudomonas 5401 is CH 1.8300.55N0.25. If the final cell
concentration is 25 g 1-1, what minimum concentration of
(NH4)2SO 4 must be provided in the medium if(NH4)2SO 4 is
the sole nitrogen source?
4.13 O x y g e n d e m a n d for p r o d u c t i o n o f
recombinant protein
Production of recombinant protein by a genetically-
engineered strain of Escherichia coli is proportional to cell
growth. Ammonia is used as nitrogen source for aerobic respi-
ration of glucose. The recombinant protein has an overall
formula CHI.5500.31N0.25. The yield ofbiomass from glucose
is measured at 0.48 g g-1; the yield of recombinant protein
from glucose is about 20% that for cells.
(a) How much ammonia is required?
(b) What is the oxygen demand?
(c) If the biomass yield remains at 0.48 g g- l, how much dif-
ferent are the ammonia and oxygen requirements for
wild-type E. coliunable to synthesise recombinant protein?
4 . 1 4 Effect o f g r o w t h on o x y g e n d e m a n d
The chemical reaction equation for conversion of ethanol
(C2H60) to acetic acid (C2H402) is:
C2H60 + 02 --~ C2H402 + H20.
Acetic acid is produced from ethanol during growth of
Acetobacter aceti, which has the composition CH 1.800.5N0.2.
Biomass yield from substrate is 0.14 g g- 1; product yield from
substrate is 0.92 g g-1. Ammonia is used as nitrogen source.
How does growth in this culture affect oxygen demand for
acetic acid production?
85
References
1. Felder, R.M. and R.W. Rousseau (1978) Elementary
Principles of Chemical Processes, Chapter 5, John Wiley,
New York.
2. Himmelblau, D.M. (1974) Basic Principles and
Calculations in ChemicalEngineering, 3rd edn, Chapter 2,
Prentice-Hall, New Jersey.
3. Whitwell, J.C. and R.K. Toner (1969) Conservation of
Mass and Energ7, Chapter 4, Blaisdell, Waltham,
Massachusetts.
4. Cordier, J.-L., B.M. Butsch, B. Birou and U. yon Stockar
(1987) The relationship between elemental composition
and heat of combustion of microbial biomass. Appl.
Microbiol. Biotechnol. 25,305-312.
5. Roels,J.A. (1983) EnergeticsandKinetics in Biotechnolog7,
Chapter 3, Elsevier Biomedical Press, Amsterdam.
6. Atkinson, B. and F. Mavituna (1991) Biochemical
Engineering and Biotechnolog7 Handbook, 2nd edn,
Chapter 4, Macmillan, Basingstoke.
Suggestions for Further Reading
Process Mass Balances (see also refs 1-3)
Hougen, O.A., K.M. Watson and R.A. Ragatz (1954)
Chemical Process Principles: Material and Energ7 Balances,
2nd edn, Chapter 7, John Wiley, New York.
Shaheen, E.I. (1975) Basic Practice of Chemical Engineering,
Chapter 4, Houghton Mifflin, Boston.
Metabolic S t o i c h i o m e t r y (see also ref5)
Erickson, L.E., I.G. Minkevich and V.K. Eroshin (1978)
Application of mass and energy balance regularities in fer-
mentation. Biotechnol. Bioeng. 20, 1595-1621.
Heijnen, J.J. and J.A. Roels (1981) A macroscopic model
describing yield and maintenance relationships in aerobic
fermentation processes. Biotechnol. Bioeng. 23,739-763.
Nagai, S. (1979) Mass and energy balances for microbial
growth kinetics. Adv. Biochem. Eng. 11, 49-83.
Roels, J.A. (1980) Application of macroscopic principles to
microbial metabolism. Biotechnol. Bioeng. 22,
2457-2514.