BIOPROCESS ENGINEERING
LESSON 10
BASIC DESIGN AND CONSTRUCTION OF FERMENTOR AND ANCILLARIES
Introduction
Biological Treatment For Recovery
Biological treatment forms the basis of many treatment
processes as a first step in the recovery of water, while often
presenting the possibility of recovery of other materials. It is
therefore important to first address the principles of biological
treatment. This module addresses very briefly some of the
underlying microbiology as relevant to water and wastewater
treatment before embarking on some treatment principles,
growth kinetics and recovery applications.
Microbiological Principles
The science of classification, especially of living forms, is called
taxonomy. The object of taxonomy is to establish the relation-
ship between one group of organisms and another, and to be
able to differentiate between them. Until the end of the
nineteenth century, all organisms were divided into two
kingdoms: plant and animal. Then, when microscopes were
developed and biologists came to understand the structure and
physiological characteristics of microorganisms, it became
apparent that micro-organisms did not really belong to either
the plant or animal kingdom, although many microbes have
either plant or animal characteristics. In 1866, a third kingdom,
the Protista, was proposed by Ernst Haeckel to include all
microorganisms - bacteria, fungi, protozoans and algae.
Subsequent research indicated that among the Protista, two
different basic cell types could be distinguished: procaryotic and
eucaryotic.
Five-kingdom System of Classification
Higher organisms, structurally much more complicated, are
based on the eucaryotic cell, in which a membrane separates the
nucleus from the cytoplasm. This type of cell apparently
evolved more recently, a little more than a billion years ago.
Simpler eucaryotic organisms, mostly unicellular, are grouped as
the Protista and include fungus-like slime moulds, animal-like
protozoans, and some algae. Some algae are more complex
multicellular types. Algae are assigned to kingdoms based on
evolutionary relationships. The more primitive algae (actually
cyanobacteria) are placed in the Protista, and other algae are
included with the plant kingdom.
Figure 1 Five kingdom system of classification
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Plants, animals, and fungi comprise three kingdoms of more
complex eucaryotic organisms, most of which are multicellular.
The main distinguishing factor among these three kingdoms is
the nutritional mode; another important difference is cellular
differentiation.
Fungi are eucaryotic organisms that include the unicellular yeasts,
multi-cellular moulds, and macroscopic varieties such as
mushrooms. To obtain raw materials for vital functions, a
fungus absorbs dissolved organic matter through the mem-
branes of cells in rootlike structures called hyphae.
Plantae are multi-cellular eucaryotes, and they include some algae
and all mosses, ferns, conifers and flowering plants. To obtain
energy, a plant photosynthesizes, a process involving the
conversion of carbon dioxide from the air to organic molecules
for use by the cell.
Animalia are multi-cellular animals with eucaryotic cells. These
include sponges, various worms, insects, and animals with
backbones (vertebrates). Animals obtain carbon and energy by
ingesting organic matter through a mouth of some kind.
In 1978, Whittaker and Margulis proposed a revised classifica-
tion scheme, organizing the five kingdoms according to
procaryote or eucaryote as overleaf.
Superkingdom Procaryotae Superkingdom Eucaryotae
Kingdom Monera Kingdom Protista
Branch Protophyta, plantlike
protists
Branch Protomycota, funguslike
protists
Branch Protozoa, animallike
protists
Kingdom Fungi
Kingdom Plantae
Kingdom Animali
The division of all living organisms into procaryote and
eucaryote has been challenged recently by a number of biolo-
gists who claim that there is a third basic category. The
organisms belonging to this new category are bacteria, that is,
procaryotes. Now it is becoming clear that these bacteria are no
more closely related to other bacteria (called eubacteria or true
bacteria by these biologists) than they are to eucaryotic organ-
isms such as animals and plants.
The bacteria belonging to this new category look like typical
bacteria and lack a nucleus and other membrane-bounded
organelles. For years, however, it has been known that they are
unusual in a number of ways. For example, their cell walls
never contain peptidoglycan, they live in extreme conditions and
their metabolism is unusual. Their genetic make-up is
complete different from that of eubacteria. A classification
allowing the archeabacteria is shown in Figure 2.
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 The dotted arrows also explains another long held view that
BIOPROCESS ENGINEERING
chloroplasts really originate from cyanobacteria, except that they
are not free-living but really almost live independently, produc-
ing photosynthetic products for its host. Similarly,
mitochondria are really bacteria-like separate entities within the
eucariotic cell.
Figure 2 The 3 Superkingdom classification of living
organisms
Classification According To Nutritional Requirements
Organisms can also be classified according to their nutritional
requirements. This form of classification is most appropriate
to wastewater treatment as it describes the opportunities for
application of these organisms. It also describes, in general
terms, the conditions to be designed for if a certain organism is
to be put to use. Classification according to nutritional
requirements is shown in Table 1.
Microorganism metabolism (principally a process of energy
conversion) is sustained by redox reactions, providing the
ultimate source of energy. The three major classes of these
energy-yielding processes are:
• Respiration (aerobic), constituting the class of biological
oxidation processes in which molecular oxygen is the
electron acceptor;
• Respiration (anaerobic), constitutes the class of biological
oxidation processes in which inorganic compounds other
than oxygen are electron acceptors;
• Fermentation, constituting the class of energy yielding
biologica redox reactions in which organic compounds
serve as the final electron acceptors .
Table 1 Classification of microorganisms according to
nutritional needs
Class Nutritional Requirements
Autotrophic The organisms depend entirely on
inorganic compounds for energy.
These are divided into:
Phototrophic Use radiant energy for growth.
Chemotrophic Use oxidation of inorganic com-
pounds as energy source. e.g. nitrifying
bacteria.
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Heterotrophic Organic compounds are required as
nutrient.
Lithotrophic Use inorganic electron donors (e.g.
hydrogen gas, ammonium ions,
hydrogen suIphate and sulphur) .
Organotrophic Require organic compounds as electron
donors.
Strictly Aerobic Cannot grow without molecular
oxygen, which is used as oxidant.
Strictly Anaerobic Use compounds other than oxygen for
chemical oxidation. Sensitive to the
presence of traces of molecular
oxygen.
Facultative Anaerobic Can grow either in the presence or
absence of oxygen. Can use nitrate or
sulfate as electron acceptor.
The presence or absence of oxygen in water influences the
products likely to be present and is of considerable importance
as it limits the type of microorganism that can be active.
Most biological systems used to treat organic waste, depend
upon heterotrophic organisms, which utilize organic carbon as
their energy source. Heterotrophic bacteria also play a role in
water treatment and distribution. Chemotrophic bacteria are
important in nitrogen removal and in corrosion processes
during water distribution.
Classification According To Identifiable Characteristics
Microbiologists like to classify organisms according to character-
istics identifiable under a microscope, according to chemical
reactions they may cause and/or their reaction to certain stains.
All of these make routine identification easier to accomplish.
Bergey’s Manual is the authoritative guide on this.
Another very important group of organisms in the field of
water and wastewater treatment are the pathogens. Pathogens
are parasitic organisms that cause disease and include viruses,
bacteria, yeasts, fungi, protozoa and even animalia, such as
worms. Many of these are water-borne or involve vectors, such
as mosquitoes, that breed in water.
Microbiological Aspects of Water Pollution Control
Biological processes are used extensively to treat municipal and
industrial wastewater. In advanced countries, the requirement
of industry to implement biological treatment (secondary
treatment) was the result of governmental regulations enacted
over 30 years ago. Prior to that time, many industries used only
sedimentation (primary treatment), which removes solids but is
rather ineffective in removing dissolved organic substances. In
the 1970s, the primary treatment facilities were supplemented
with biological systems such as activated sludge and trickling
filters. These processes have become sophisticated as a result of
advances in process control and microbiology and are capable of
removing over 90% of the dissolved organics from the primary
effluent.
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Microorganisms
Both procaryotic, or unicellular organisms that lack a true
nucleus, and eucaryotic, multicellular organisms that have a
membrane enclosed nucleus, are active in biological treatment
systems. The bacteria are procaryotes. They are typically 0.5 mm
wide and 1 mm long and exist as coccus, rod, and spiral shapes.
Fungi are filamentous eucaryotes that have rigid cell walls that,
unlike eucaryotic algae, lack chlorophyll.
Microorganisms require both a carbon and energy source for
growth. The pollutant serves as both the carbon and energy
source for heterotrophic microorganisms. Autotrophic organ-
isms use either light (photoautotrophs) or an inorganic
compound (chemautotrophs) as the energy source and CO2 as
the carbon source. Examples of the former are the algae and of
the latter are the nitrifying bacteria that convert ammonia to
nitrate:
2 NH4+ + 3 O2 è 2 NO2- + 4 H+ + 2 H2O
2 NO2-+ O2 è 2 NO3-
The bacteria converting ammonia to nitrite are usually
Nitrosomonas.
Nitrobacter further oxidizes the nitrite to nitrate.
Temperature and pH are important parameters influencing
growth rate. Each organism has a minimum, optimum, and a
maximum temperature for growth. The three temperatures are
referred to as cardinal temperatures. Psychrophilic organisms
grow at low temperatures (0-20oC), mesophiles at higher (15 -
45oC), and thermophiles at the highest temperatures (40-70oC).
Most microorganisms grow best at pH near neutrality (pH 7)
but some are acid tolerant or acidophiles. Thiobacillus
thiooxidans, a sulfur-oxidizing bacterium, is capable of growth at
a pH of 1.
In anaerobic environments byproduct formation is common.
Incomplete dechlorination of pesticides has been found to
occur both in the laboratory and in the natural environment.
However, some chemicals, such as 3-chlorobenzoic acid, are
capable of being completely mineralized to methane under
anaerobic conditions.
The effect of growth rate inhibition at substrate concentrations
exceeding a toxicity threshold is shown in Figure 2. Such
behavior has been observed with growth of bacteria on
dichloromethane and pentachlorophenol (PCP), for instance.
Figure 1 Monod Equation
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From: Davis, M.I. and Cornwell, D.A. 1985 Introduction to
BIOPROCESS ENGINEERING
Environmental Engineering. 2nd ed. McGraw-Hill Fig 5-6 p.323
Figure 2 Growth Rate Inhibition From: La Grega, MD.,
Buckingham, P.L., and Evans, J.C. 1994. Hazardous Waste
Management. McGraw-Hill, Inc. Fig 10-13 p.578
Upsets or shock loads that result in abrupt increases in pollut-
ant feed concentration commonly occur in industrial wastewater
treatment. During such episodes, the increase in pollutant
concentration in the biological treatment tank may result in
growth rate inhibition and a further increase in concentration.
When this occurs, flow to the system must be stopped until
residual pollutant concentrations have decreased to below toxic
levels. Reseeding of the system with microorganisms may also
be necessary.
The cell yield is the mass of microorganisms formed per
nutrient, usually organics:
dX/dt = -YdS/dt
where Y is the cell yield, mg dry wt. cells/ mg pollutant and S is
the substrate (pollutant) concentration. Values of the cell yield
depend upon the specific type of organism, oxygen status of
the environment, and carbon content of the pollutant. Under
anaerobic conditions, less ATP is produced than under aerobic
conditions resulting in a lower cell yield. Aerobic growth on
compounds containing a relatively small fraction of carbon,
such as pentachlorophenol, results in the production of fewer
cells. The cell yield for PCP-degrading bacteria has been reported
to be 0.15 mg dry wt/ mg PCB. By contrast, highly carbon-
aceous materials are metabolized with the production of high
cell numbers. The growth yield on naphthalene is 0.7 mg dry
wt/mg naphthalene.
Pollutant Biodegradability
The public health threat associated with pollutants entering the
environment will be greater if they are persistent. The half-life
or time required for 50% disappearance of a chemical, is an
indicator of susceptibility of the chemical to biodegradation.
Half-lives may range from one day to several months depend-
ing on the environment and pollutant. Compounds that resist
biodegradation are refractory or recalcitrant. Examples of
recalcitrant compounds include DDT and PCBs.
The factors that influence persistence are related to the indig-
enous microbiology of the environment and chemical
structures of the pollutants. The following chemical structural
parameters are normally involved:
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 1. number of substituents Nutrient Requirements for Aerobic Growth
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2. type of substituents Hazardous waste sites are often contaminated in the subsurface
with petroleum hydrocarbons. Such contamination arises from
3. position of substituents
petroleum spills on the surface or from underground tank
4. degree of branching leaks. Effective biological treatment of the contamination
5. low solubility in water (bioremediation) requires the presence of oxygen. The oxygen
6. atomic charge difference requirements for degradation of an organic compound may be
determined if assumptions are made regarding cellular compo-
Chlorine is a common substituent, which imparts both toxicity
sition and the fraction of organic compound oxidized for
and persistence. The incorporation of chlorine into an organic
energy. Consider the oxidation of a branched alkene C7H12. The
compound increases its longevity. In the examples given below
complete oxidation reaction is:
the compound on the right is more persistent than the one on
the left. C7H12 + 10 O2 → 7 CO2 + 6H2O
If 1 mg alkene reacts, the O2 required is:
Less persistent More persistent 10(2 x 16)/[(7+12) + (12 x1)] = 3.33 mg O2/mg alkene
propionic acid 3-chloropropionic acid Oxygen will also be required for the production of biomass
monochloroacetic acid dichloroacetic acid C7H12 + 5O2 + NH3 → C5H7O2N + 2CO2 + 4 H2O
phenol mono-, di-, tri-, and pentachlo 5(2 x 16)/((7x12)+(12 x1)) = 1.67 mg O2/mg alkene
rophenol We assume that 50% of the hydrocarbon is oxidized and the
benzoic acid 3-chlorobenzoic acid remainder is converted into cell mass, thus
monochlorobiphenyl polychlorinated biphenyls 1 mg alkene requires (0.5)(3.33 mg O2) + (0.5)(1.67 mg O2)
(PCBs) = 2.5 mg O2
The type of substituent will influence pollutant residence time The nitrogen required to produce cells:
in the environment. Di-substituted benzenes with a carboxyl 0.5 mg alkene x (1 mmol cells/mmol alkene)(14 mg N/mmol cells)
or hydroxyl replaced with nitro, sulfonate, or chlorine degrade 96 mg/mmol)
more slowly. The position of the substituent also influences
half-life of the compound in the environment as illustrated = 0.073 mg N/ mg alkene
below. The cell yield is:
(0.5 mg) x (5x12+7x1+2x16+1x14) = 0.59 mg cells/mg
Less persistent More persistent (7x12 + 12x1alkene)
o,p-chlorophenol m-chlorophenol The cell formula containing P, C60H87O23N12P, is used to obtain
o,p-chlorophenoxyacetic acid m-chlorophenoxyacetic acid the phosphorus requirement:
o,p-amino,nitro,methoxy (0.59 mg cells /mg alkene)x31 mg P = 0.0133 mg P/mg alkene
benzoic acid mamino,nitro,methoxybenzoic (60x12+87+23x16+12x14+31 mg cells)
acid The yield on oxygen Yxo of the mass of microorganisms
m-nitrophenol p-nitrophenol produced per mass oxygen consumed:
p-methylaniline o-methylaniline Yx/o = 0.59/2.5 = 0.24 mg cells/mg O2
Highly branched compounds are more resistant to biodegrada- Methods of Anaerobic Biological Treatment
tion than straight chain compounds. The branched aliphatic Anaerobic processes in industrial wastewater treatment are used
acid, b-methylbutyric acid, is degraded more slowly than butyric mainly when high concentrations of organic material have to be
acid. removed or converted or for the further treatment of the
Water solubility may affect the rate at which pollutants are organic sludges removed from other biological wastewater
degraded. Many microorganisms, which grow on compounds treatment. Simple forms of anaerobic treatment, such as
of low solubility, utilize only the dissolved form of the anaerobic ponds and septic tanks, however, are used for treating
pollutant. The growth rate then becomes controlled by the rate wastewater (rather than sludge) although, even in these cases,
of dissolution of the pollutant. Crushing crystals of naphtha- the most intense anaerobic action takes place in the layer of
lene, for instance, results in an enhanced growth rate of concentrated sludge which settles to the bottom. Although the
microorganisms, due to the higher available surface area at poor level of mixing, especially in the simpler processes, makes
which naphthalene dissolves, making the naphthalene more classification a little difficult, most conventional anaerobic
available as substrate. processes are essentially suspended growth systems.
The reason for choosing anaerobic over aerobic treatment
should be based on financial considerations. Anaerobic
processes usually have a higher capital cost than anaerobic
processes due to the longer retention times required, but the
cost of treatment is not significantly influenced by the organic

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loading. Aerobic processes have a higher operating cost, but
this operating cost is mainly a function of the oxygen require-
ments and sludge production, ie aerobic treatment becomes
progressively more expensive with increasing organic loading.
The total cost usually overtakes that of anaerobic treatment at a
BOD or COD of a few thousand mg/L.
Anaerobic Ponds.
Anaerobic lagoons are designed with a low surface area in
relation to volume. The combination of this ratio and the high
oxygen demand of the waste effectively prevents any part of the
lagoon becoming aerobic except for a very thin layer at the top
of the water during start up. A scum layer then forms above
the wastewater which prevents algae from growing due to the
absence of sunlight and further oxygen diffusion from the air.
The by-products of anaerobic degradation include hydrogen
sulfide and methane gas. The organic loading in anaerobic
lagoons is controlled to minimise the evolution of malodorous
gases.
Anaerobic digestion is much slower than aerobic processes.
However, in anaerobic ponds, a large portion of the colloidal
particles undergo coalescence to form the scum and sludge
which hastens the removal of the suspended BOD.
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Figure 4.3 Simple anaerobic treatment systems: (a)
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anaerobic-aerobic lagoon system, (b) septic tank, (c)
Imhoff tank
Aerobic Treatment
The main objective of conventional treatment is to remove the
dissolved and colloidal degradable organic matter that remains
after primary treatment, so that the effluent can be rendered
suitable for discharge. In many cases, reduction of the BOD to
below 20 mg/L and SS to below 30mg/L is sufficient, and
conventional secondary treatment can achieve this quality. Very
often in industrial systems the objective is to pretreat the
wastewater before sewer discharge to reduce municipal charges
or to meet disposal restrictions.
A second objective in some cases is the reduction of ammonia
toxicity and nitrification oxygen demand in the receiving water
body. This is achieved by oxidation of most of the ammonia
to nitrate during treatment (nitrification). Nitrification is
possible with aerobic biological processes if they are operated at
long retention times - hence the units must be larger than
those that would be required for oxidation of carbonaceous
matter alone.
These bacterial processes are commonly carried out on either
aerated lagoons or in activated sludge plants. The design
approach is similar, except that activated sludge plants incorpo-
rate a sludge recycle, which allows independent control of the
solids retention time or sludge age. The mean cell residence time
in aerated lagoons is therefore equal to the hydraulic detention
time. To prevent washout of active bacteria, the detention time
should not be too short - two to five days’ detention is the
general order of the size of aerated lagoons. Activated sludge
processes are commonly used industrial treatment systems and
their design requires a closer study.
Activated sludge processes are now the most widely used in
newly installed municipal treatment systems and their design
requires a closer study. Disadvantage of this approach is that
the other product, which is formed out of the organic sub-
strate, is bacterial biomass, with virtually no reuse value other
than as a fertilizer and soil conditioner as the bacterial protein is
not of particularly good quality.
Design of Activated Sludge Systems
The activated sludge process design involves details of sizing
and operation of the following main elements:
a aeration tank (reactor) - capacity and dimensions
b aeration system - oxygen requirements and oxygen transfer
system
c final sedimentation tank (clarifier)
d return activated sludge system
e excess activated sludge withdrawal system and subsequent
treatment and disposal of the waste sludge.
Since the whole process takes place in a liquid medium, the
hydraulic regime, especially in the aeration tank and the final
sedimentation tank, needs to be carefully considered. The main
system elements are discussed below.
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 Porous Diffusers
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These must be operated continuously, with an airflow less than
that which could damage the diffuser but always more than the
minimum recommended flow, so that clogging will not occur
too rapidly. This requires the provision of standby air compres-
sors, and diesel motors. Most diffused air systems require a
supply of air at a pressure of 50 to 80 kPa. Porous diffusers
have a theoretical oxygen transfer rate of about 2.8 kg/kWh.
Porous diffusers may be made of ceramic material, and they are
available in the shape of plates, domes or tubes. They may also
be made of plastic material, either fabric or moulded. Fabric
diffusers are generally lightweight and they can be readily
cleaned.
Extensive air filtering before porous diffusers is required to
prevent clogging of diffusers. Baghouse systems, sometimes 2
or 3 consecutive filters, make the pre-treatment an expensive
part of the system.
Non-porous Diffusers
These have been developed with the objectives of
a. Elimination of diffuser clogging problems
b. Reduction of equipment capital cost (loss in efficiency
compared with fine bubble porous diffusers).
Many types of jet, valued orifice and turbulence disc are now
available. A system which uses slotted pipes with a shallow
immersion of about 0.9 m and a large volume of air at low
pressure has been developed. Under these conditions, compres-
sors can be replaced by blowers and the holes in the pipes are so
large that they seldom block. Power efficiency is in the range of
1.0 to 2.0 kg/kWh.
Vertical Shaft Mechanical Aerators
These have a rotor at the surface of the water, where the oxygen
transfer takes place through surface turbulence, splashing and air
bubble entrainment. The oxygen transfer capacity depends on
the design of the equipment, the depth of immersion, the
shape and size of the aeration tank, the energy input per unit
volume of tank and the rate of rotation; some of these factors
are interrelated. The oxygen transfer rates under average
operation conditions are of the order of 2 to 3 kg/kWh.
Horizontal Shaft Surface Aerators
Various types of brush and cage rotor have been designed.
They provide not only aeration but also a horizontal pumping
effect to maintain circulation and mixing. They may be used in
tanks of moderate depth to provide a rolling circulation, but
the most common use is in oxidation ditches and for supple-
menting oxygen supply in maturation ponds or oxidation
ponds.
A typical cage rotor of 700 mm diameter could be operated at a
speed of 75 rev / min, to give oxygen transfer of 2.9 to 3.5 kg/
kWh, depending on depth of immersion. These rotors may be
mounted on floats that slide in guide-boxes at each end.
U-tube Aeration
In U-tube aeration, the partial pressure of the oxygen can be
doubled or trebled by passing the water, with diffused bubbles
entrained, through a U-tube with its lower portion 10 to 20
meters or more below the hydraulic gradeline. This increases
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the partial pressure 2 or 3x and hence the instantaneous
saturation concentration, and speeds up the transfer of oxygen.
For ease of construction, the downward tube may be inside the
upward flow tube. Further increase of partial pressure of
oxygen can be obtained by using pure oxygen.
Turbine Aerators
These are submerged turbines with air released from spargers
immediately below them. They have been used where the
required oxygen input per unit of volume is greater than can be
obtained with diffused air. Their oxygen transfer capacity is in
the range of 1.5 to 2.0 kg/ kWh. There are also submerged self-
aspirating aerators that draw the air through a hollow drive
shaft (Kerag aerators).
Oxygen Enriched Systems
Molecular oxygen can be used instead of air to increase greatly
the partial pressure of the oxygen. If the atmosphere in contact
with the sewage under treatment is oxygen, the partial pressure
of the oxygen is 5x as large as in air and creates a much greater
driving force to transfer it into the liquid. In this way, the
saturation concentration at atmospheric pressure changes from
8 - 10 mg/L up to 40 or 50 mg/L. This makes it practicable to
maintain higher concentrations of DO in the liquor.
Where ‘pure’ oxygen is used for wastewater treatment, it has to
be obtained from such a source as cylinders, bulk tankers or
reticulated oxygen from suppliers, a cryogenic oxygen distillation
plant at the works, or enriching air with ‘molecular sieves’.
Protein Production from Organic Wastes
A major problem facing the world, in particular the developing
countries, is the explosive rate of population growth. The
world population now is annually increasing by approximately
94 million, and could well exceed 10 billion by 2050 if left
uncontrolled. Conventional agriculture may be unable to
supply sufficient food, in particular protein, to satisfy these
demands. The Food and Agriculture Organization (FAO)
predicts a widening of the protein gap between developed and
developing countries. Currently, at least 25% of the world’s
population suffers from hunger and malnutrition, a dispropor-
tionate number living in the developing countries.
In view of the insufficient world food supply and the high
protein content of microbial biomass, the use of biomass
produced in fermenters would be an ideal supplement for
animal feed. This would release plant proteins for human
consumption and increase the animal protein supply into the
human food chain. Development of methods to produce
microbial biomass protein (MBP) has become an important field of
study. MBP has a great nutritional value because of its high
protein, vitamin, and lipid content and the general presence of a
complete array of all essential amino acids.
There are many advantages in biotechnological processing for
MBP production. Microorganisms produce protein much more
efficiently than any farm animal could, as indicated in Table 1
with the major advantages being:
• Microorganisms can grow at remarkably high rates under
optimum conditions; some microbes can double their
mass every 0.5 to 1 hour;
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• Microorganisms are more easily modified genetically than
plants and animals, and more amenable to larger scale
screening programs to select for high growth rate and
improve nutrient content;
• Microorganisms have a relatively high protein content with
a good nutritional value;
• Microorganisms can be grown, independent of climate
conditions, in vast numbers, in relatively small continuous
fermentation processes, using relatively small land areas;
• Microorganisms can grow on a wide range of raw materials,
in particular low value wastes, while some can also use
plant derived cellulose.
Table 1 Time required to double the mass of various
organisms
Organism Time
Bacteria and yeasts 20-120 minutes
Moulds and algae 2-6 hours
Grass and some plants 1-2 weeks
Chickens 2-4 weeks
Pigs 4-6 weeks
Cattle (young) 1-2 months
Humans (young) 3-6 months
Many developed and developing countries, particularly Asian
nations, have long recognized the nutritional value of some
traditional foods produced by fermentation. During the last
four decades, there has been a growing interest in the use of
microbes for food production, in particular for feeding animals
such as poultry. It has been argued that the use of MBP
derived from low value waste materials for animal feed would
remove some protein-rich vegetable foods from animal diets to
become available for human consumption. MBP can serve as a
replacement for traditional protein supplements such as fish-
and soy meal.
Suitable Substrates For Microbial Biomass Protein
Production
One of the most important factors for MBP production is the
choice of substrate. If MBP is to be used to also treat wastes,
the substrates should be suitable to sustain growth. We could
grow autotrophs such as algae on wastewaters containing plant
nutrients (such as from fertilizer wastewater) or heterotrophs
on organic rich wastewaters. The latter could easily be classified
into synthetic and renewable feedstocks and waste products
from these.
Chemical Feedstocks
Materials with a high commercial value such as energy sources or
derivatives of these chemicals, e.g. gas oil, methanol, ethanol,
methane and n-alkane, have attracted wide interest in the early
stages. Many scientists have questioned the use of such
compounds with a high energy potential for food production,
including MBP, as health aspects. A few decade ago, many of
the large-scale processes in operation were forced to close down
because of health regulations and the high costs of materials
used.
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n-Alkane
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Alkanes, in particular those of intermediate size (C10 - C20) are
the most rapidly metabolised hydrocarbons and were chosen as
the substrate for the MBP projects of the 1960s. Alkanes can be
catabolised by many yeasts and by some fungi (for instance,
fungi of the orders Mucorales and Moniliales) as well as some
bacteria (partially through cooxidation). The following yeast
species have been intensively studied for MBP production:
Candida tropicalis, C. oleophia, and Saccharomycopsis lipolytica (Fukui
and Tanaka, 1981).
Methane
In some parts of the world, there is an excess of methane, the
chief compound of natural gas, making this a desirable energy
source for MBP production. Methane can be obtained as a very
pure gas. However, in contrast to higher hydrocarbons,
methane cannot be liquefied commercially, making long-distance
transport difficult and expensive. Considerable security
measures must also be taken when handling methane, due to
the risk of explosion. Methane as an MBP source has been
extensively researched but is now considered to present too
many technical difficulties to warrant exploitation. No methane
based MBP production process has been developed on a
commercial scale, to date.
Methanol
Methanol was, for some time, the most important substrate for
MBP production and extensive research on methanol utilizing
organisms was carried out. Methanol, as a carbon source for
MBP, has many inherent advantages over n-paraffins, methane
gas and even carbohydrates; composition is independent of
seasonal fluctuations. There are no possible sources of toxicity
in methanol, it dissolves easily in the aqueous phase in all
concentrations and no residue of carbon source remains in the
harvested biomass fermentation for the commercial production
of MBP. The “ICI Pressure Cycle Fermenter”, a combination
of air lift and loop reactor, was installed in a continuous culture
system with a capacity of 50,000 - 70,000 tons/y. Operation
began in 1980, but proved to be uneconomical at the present
high methanol prices and production has ceased (Hiotzman,
1986).
Renewable, Biological Feedstocks
Cellulose and lignocellulose
Cellulose is the most widely occurring organic material in nature,
and the principal source of biomass and therefore of a
renewable resource; it is a complex of three classes of polymer,
which consists of repeating glucose units largely in crystalline
fibers. This material, however, is generally difficult to hydrolyze
by microorganisms. At the present time, cellulose from natural
sources and waste wood is still an attractive starting material for
MBP production as well as a potential source for the fermenta-
tion of ethanol. Although there is an abundance of cellulose
on earth, cellulose is usually mixed with substances such as
lignin, hemicellulose, starch, protein, and salts. Therefore, the
cellulose source must be pretreated physically and chemically in
order to break down the cellulose into fermentable sugars.
Pretreatment may either be enzymatic (cellulose) or chemical
(acid) hydrolysis. Production of MBP directly on cellulose
31
 material has been very rare. There are some major difficulties
BIOPROCESS ENGINEERING
and disadvantages associated with direct use of cellulose:
• Low solubility of the raw material dictates use of very
dilute culture media;
• Low utilization rates lead to low growth rates, low
productivity, and low dilution rates in continuous
cultivation;
• high concentration of solids in the culture medium
requires high energy input for mixing and reduces the
relative protein content in the biomass.
Starch and Starch Wastes
Starch is a glucose polymer and is one of the most widely
available plant polysaccharides. In the biosphere, carbohydrate
matter, including starch and cellulose, exceeds the combined
amount of all other organic compounds. Starches are manufac-
tured from corn (maize), wheat and potato. The proportions
of these ingredients not only vary greatly between raw materials,
but also between the different grades of raw materials. A typical
analysis of three starch materials is shown in Table 2 (McNicol
et al., 1972).
Table 2 Typical analysis of selected starches containing
raw materials
Composition(%) Corn Wheat Potato
Starch 60.0 55.3 15.0
Protein 9.0 13.2 2.0
Fat and other 15.0 18.7 4.0
Moisture 16.0 12.0 79.0
Wastes from Renewable Feedstock Processes
The definition of “waste material” is changing continuously
since more and more re-use is replacing simple disposal. By-
products that have generally been considered as waste are now
in routine use. A suitable definition should take into consider-
ation alternative economical utilization, local pollution control
regulations and legislation, and the cost of waste treatment. A
major factor in the cost of MBP production is the raw materials
and the efficiency of their conversion into MBP production.
Waste materials should normally be recycled back into the
ecosystem, e.g. straw, bagasse, citric acid, olive and date wastes,
whey, molasses, animal manures and sewage. The amount of
these wastes can be very high in specific locations and may
contribute a significant level of pollution to water courses.
Thus, the utilization of such materials in MBP production
serve two functions - reduction in pollution and production of
edible protein.
For MBP production to be economical during waste effluent
treatment, certain requirements must be met:
1) Utilization of all the available nutrients in the treated effluent
The primary goal is BOD and COD reduction, and a
suitable microorganism with high density growth and
productivity rate must therefore be selected.
2) Sterilization of the waste should be avoided Decontamination,
if required, should be carried out using low-cost methods.
Methods applying high inoculum, cell recycling, low pH
values, and low concentrations of carbon source can be
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32
used for achieving a high disappearance rate of nutrients
from the growth system.
3) Inexpensive preparation of the end product (commercial biomass
protein) Recovery should avoid the use of expensive
techniques such as centrifugation. Sedimentation,
flocculation/flotation, and simple filtration, are usually
useful in the first step of product recovery.
4) Reduction in cooling or heating costs A microorganism that
grows at a similar temperature as the treated waste is of
great advantage.
Whey
Whey is a by-product of cheese-making and contains both
protein and lactose. The waste from large scale cheese plants can
become a serious environmental problem if it is discharged
without treatment. Utilization in the production of MBP was
the subject of extensive study (Moulin & Galzy, 1984). One
original solution concerned yeast production from crude sweet
whey using Candida kefyr LY 496 in batch or continuous culture
processes (Hitzman, 1986). Many cheese companies in Europe
have successfully used Kluyveromyces fragilis as the organism to
produce MBP. This process, however, has to compete with
fractionation of the whey as its component lactose and protein
(Pellon and Sinseky, 1983). The process also depends on large
scale plants giving steady effluent as a source of raw material.
The effluent is generally dilute and high transportation costs
may be involved in the collection of the whey. This situation
has been one of the major reasons why whey is still regarded as
an unattractive substrate for MBP production (Moulin and
Galzy, 1984).
Spent Sulfite Liquor
Spent sulfite liquor (SSL) is an effluent from pulp and paper
mills, a sugar-containing waste product. These effluents have
been identified as a possible source for the production of MBP.
Romantschuk (1975) cultivated Candida utiliz for MBP produc-
tion using SSL as the substrate. A reduction in sulfite
processing and its replacement by the sulfate process has tended
to reduce the available effluent and closed some USA facilities
for MBP production (Vasey and Powell, 1984). Pretorius et al.
(1993) investigated a continuous system of selective cultivation
of the thermotolerant Aspergillus sp. on SSL for MBP produc-
tion.
Selection of Suitable Microorganisms for Microbial
Biomass Protein Production
Most MBP processes are designed to take advantage of an
available, readily degradable substrate using a microorganism.
The choice of the MBP microorganism is usually limited to that
particular process, and a change in substrate often necessitates a
change in the type of microorganisms used. Several factors are
involved in the choice of a microorganism for the MBP
production. Hamphrey (1974) summarized five criteria for
microorganism selection: safety, use, digestibility, growth rate,
and maintenance.
Apart from substrate cost, operating expenses, and capital
investment for process equipment, other important factors in
MBP production are the protein content and the quality of the
MBP. The chemical composition of bacteria, yeast, fungi and
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algae vary, depending upon the genera and growth condition. not maintained during the large scale MBP production. The

BIOPROCESS ENGINEERING
The gross chemical composition of various microorganisms is potential contamination of algae cultures grown in sewage
presented in Table 3 (Oura, 1983). Although the protein oxidation ponds by enteric pathogenic bacteria and viruses,
content can be varied by growth conditions, genetic manipula- therefore, must be given serious consideration (Cooper, 1962).
tion can also be employed to alter the amino acid spectrum.
Fungi
Table 3 Protein and Nucleic Acid Content of Various Filamentous fungi play an important role in the food industry,
Microorganisms for example in the addition of flavor to certain mould cheeses
and acting as a major protein source as food additives and
Microorganism Protein (%) Nucleic Acid ( % ) extenders. These fungi are also used in the production of
Yeast 45 - 55 6 - 10 Oriental foods and to improve the protein contents of animal
Algae 47 - 63 4-6
feeds. Wainwright (1992) summarized various fungi that are
Bacteria 50 - 83 10 - 16
directly used in food processing, shown in Table 4.
Fungi 31 - 55 2.5 - 6
Cultivating fungi as a high protein food is especially attractive.
Protein quality and quantity are the goals of MBP production, The major advantages of fungal cultures are summarized as the
although the microorganisms also contain carbohydrates, fats, following:
vitamins and minerals. The most important factor in microor- • Fungal cells contain reasonably high levels of protein;
ganism selection is safety. The organism must be known to be
non-pathogenic and must not produce toxins. • Fungi contain less nucleic acid than yeasts and bacteria;

Various groups of microorganisms, including bacteria, yeasts, • The filamentous nature of fungal mycelia facilitates
algae and fungi, have been considered for use as a source of recovery of fungal MBP from fermentation broths and its
protein, the dried cells of these microorganisms being referred physical properties make it acceptable for human
to as MBP. consumption;
• Food produced from fungi is traditionally eaten in many
Bacteria
parts of the world.
Various species of bacteria can utilize a wide range of carbon
and energy sources, including sugars, starch, cellulose (either in Table 4 Direct food uses of fungi
pure form or as agricultural or forest product waste) hydrocar- Application Species
bons and petrochemicals. Rapid growth rates and high protein Edible macrofungi
contents make bacteria prospective candidates in the MBP Common edible mushroom Agaricus bisporus
production (Bhattacharjee, 1970). The generation time of Shii - take Lentinus edodes
bacteria is only 20 to 30 minutes compared with 2 to 3 hours Chinese or straw mushroom Volvariella volvacea
for yeast and 16 hours or more for algae, and fungi (Litchfield, Winter mushroom Flammulina velutipes
1979). Semi - batch and continuous MBP processes using Oyster mushroom Pleurous sp.
bacteria are suitable for industrial waste recovery. The conver- Truffle Tuber melanosporum
sion efficiency of substrates into MBP by bacteria is very high Cheeses
Roquefort, Stilton, ‘blue’ Penicillium roquefortii
(0.8 - 1.2 g /g substrate) (Litchfield, 1979).
Camembert, Brie, soft Penicillium camembertii
Yeasts ripened cheese
Yeasts can be utilize many substrates for MBP production. Oriental food fermentation
These substrates include n-alkanes, methanol, ethanol, diesel Ang-kak Monascus purpurea
oil, gas oil, brewery waste, sulfite waste liquor, starch, anaerobic Hamanatto Aspergillus oryzae
digester supernatant, molasses, cheese whey and domestic Miso Aspergillus oryzae /
A.sojae
sewage. The species of yeasts most commonly used as MBP
Ontjom Neurospora intermedia
include Candida, Hansenula, Kluyveromyces, Rhodotorula and
Shoyu ( soy sauce ) Aspergillus oryzae /
Torulopsis, and particularly Saccharomyces cerevisiae. Because of A.sojae
high MBP yields, wide utilization of substrates, high quality of Tempeh Rhizopus oligosporus
protein, easier and inexpensive harvesting, yeasts have become R. arrhizus, R. oryzae
the most favourable characteristics for use as a major MBP food.
Yeast Culture
Algae
Since yeasts can grow rapidly on a wide range of substrates
Algae are photosynthetic microorganisms and thus require
including waste products, they have the potential to provide an
either sunlight or artificial illumination. They can be grown
extremely versatile protein. A large number of amylolytic yeast
either photosynthetically or heterotrophically. Autotrophic
species have been screened for MBP production from starch
growth involves in using carbon dioxide, whereas heterotrophic
materials to obtain strains that can convert starch directly in a
growth occurs in the dark with organic carbon and energy
monoculture or improve the performance of Sacchromycopsis
source. Since illumination is the limiting factor in photosyn-
fibuligera in a symbiotic system. About 90 species of a total 400
thetic algal growth, outdoor cultivation is restricted to the use
yeast species currently recognised are capable of utilizing starch
of shallow pond site between latitude 35ºN and 35ºS
as a sole source of carbon and energy (Robyt, 1984).
(Litchfield, 1979). Aseptic conditions with algae in ponds are

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 Table 2-5 Production of MBP by yeast cultivation from
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starch materials
a strain substrate process µ T yield
species
−1 o (g/g
(h ) ( C)
)
C. utiliz NCYC 707 potato waste batch 0.50 30 0.55
NRRL1048 soluble starch batch 0.14 23 0.61
C. tropicalis CBS6948 cassava continuous 0.26 35 0.42
corn continuous 0.10 32 0.74
L. kononenkoae IGC4052 soluble starch batch 0.12 25 0.58
ICG4052B soluble starch continuous 0.04 28 0.48
S. cerevisiae BRG 530 soluble starch batch 25 0.50
Sa. fibuligera corn starch batch 30 0.54
NRRL Y76 soluble starch batch 0.35 30 0.50
NRRL1062 potato continuous 0.35 32 0.84
IMAT3812 peeled potato batch 28 0.40
ATCC2607 soluble starch batch 32 0.51
Sc. occidentalis IGC 2829 soluble starch batch 0.21 30 0.59
UCD54-83 soluble starch batch 0.25 30 0.62
IMAT2196 potato peels batch 28 0.54
CBS 2863 cassava flour batch 0.23 28 0.44
Sp. holsaticus FRI Y-5 soluble starch batch 0.14 23 0.43

Abbreviations for genera:

C. = Candida; L. = Lipomyces; S. = Saccharomyces;
Sa. = Saccharomycopsi; Sc. = Schwanniomyces;
Sp. = Sporobolomyces.
Fungal Culture
Because of the properties of easy harvesting, low nucleic acid
content and acceptability as traditional food, filamentous fungi
have become more and more attractive in MBP production and
biotechnological waste treatment processes.
Mixed Cultures
In many practical fermentation processes, the cheapest substrate
is always used, and this will not be of the highest purity and
will often be a mixture of several materials in large quantities.
It would be useful, for example, a mixed culture can be found
that not only degrades cellulose but also starches and sugars.
Conclusion
Experimental results indicated that the design of wastewater
treatment aimed at microbial biomass protein production
should be different from the design of conventional biological
wastewater treatment systems. Conventional systems are
typically designed to achieve a certain effluent quality, but the
major design criterion for systems using yeast cultures should
be the prevention of bacterial contamination.
Review Question
• What are the different suitable substrates for microbial
biomass protein production?

Notes:

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