Hacettepe University

Department of Chemical Engineering

KMÜ446
BIOTECHNOLOGY
Mixed Cultures
III
Prof. Dr. Zümriye AKSU

M.L. Shuler and F. Kargi, Bioprocess Engineering: Basic Concepts, Third

Edition, Prentice Hall, 2017.
 1. INTRODUCTION
The dynamics of mixed cultures are important considerations in some commercial
fermentations. They are critical to understanding the response of many ecological systems to
stress. The use of organisms with recombinant DNA has added another dimension to our
consideration of how cells within a population interact with each other.

Many food fermentations, such as cheese manufacture, depend on multiple interacting

species. The biological treatment of waste waters relies on an undefined complex mixture of
microorganisms. The ratio of various species in the treatment process is critical; sudden
shifts in the composition of the population can lead to failure of the unit to meet its
objectives.

In all natural environments, cells exist in potentially mixed populations. Understanding how
these cells interact with one another is critical to understanding the natural cycles for the
elements (e.g., C, N, and S), the response of ecosystems to environmental challenges (for
example, acid rain), and the rate and extent of degradation of chemicals introduced into such
ecosystems.

M.L. Shuler and F. Kargi, Bioprocess Engineering: Basic Concepts, Third Edition, Prentice Hall, 2017. 2
 2. MAJOR CLASSES OF INTERACTIONS IN MIXED
CULTURES
The major interactions between two organisms in a mixed culture are competition,
neutralism, mutualism, commensalism, amensalism, and prey–predator interactions.

Competition is an indirect interaction between two populations that has negative effects on
both. In competition, each population competes for the same substrate. Two populations or
microorganisms with similar nutrient requirements usually compete for a number of
common, required nutrients when grown together.

Neutralism is an interaction where neither population is affected by the presence of the other.
That is, there is no change in the growth rate of either organism due to the presence of the
other. Neutralism is relatively rare. One example of neutralism is the growth of yogurt starter
strains of Streptococcus and Lactobacillus in a chemostat. The total counts of these two
species at a dilution rate of 0.4 h-1 are quite similar whether the populations are cultured
separately or together. Neutralism may occur in special environments where each species
consumes different limiting substrates and neither species is affected by the end products of
the other.

M.L. Shuler and F. Kargi, Bioprocess Engineering: Basic Concepts, Third Edition, Prentice Hall, 2017. 3
Mutualism and protocooperation are more common than neutralism and may involve
different mechanisms. In both cases, the presence of each population has a positive effect on
the other. For mutualism, the interaction is essential to the survival of both species. In
protocooperation, the interaction is nonessential. One mechanism is the mutual exchange of
required substances or the removal of toxic end products by each organism.

The metabolisms of partner populations must be complementary to yield a mutualistic

interaction. An example is the growth of a phenylalanine-requiring strain of Lactobacillus
and a folic-acid-requiring strain of Streptococcus in a mixed culture. Exchange of the
growth factors phenylalanine and folic acid produced by partner organisms helps each
organism to grow in a mixed culture, while separate pure cultures exhibit no growth.
Another example of mutualistic interaction exists between aerobic bacteria and
photosynthetic algae. Bacteria use oxygen and carbohydrate for growth and produce CO 2
and H2O. The algae convert CO2 to carbohydrates and liberate oxygen in the presence of
sunlight.

Note that symbiosis and mutualism are not the same. Symbiosis implies a relationship when
two organisms live together. A symbiotic relationship may be mutualistic, but it may also be
neutralistic, parasitic, commensalistic, and so on.

M.L. Shuler and F. Kargi, Bioprocess Engineering: Basic Concepts, Third Edition, Prentice Hall, 2017. 4
Commensalism is an interaction in which one population is positively affected by the
presence of the other. However, the second population is not affected by the presence of the
first population. Various mechanisms may yield a commensal interaction. Two common
mechanisms are the following:

1. The second population produces a required nutrient or growth factor for the first
population.
2. The second population removes a substance from the medium that is toxic to the first
population.

An example of the first type of commensal interaction is the production of H 2S by

Desulfovibrio (through the reduction of SO2-4 ), which is used as an energy source by sulfur
bacteria.

M.L. Shuler and F. Kargi, Bioprocess Engineering: Basic Concepts, Third Edition, Prentice Hall, 2017. 5
An example of the second type of commensal interaction is the removal of lactic acid by the fungus
Geotrichium candidum, which allows the growth of Streptococcus lactis. This interaction is utilized in
cheese making using S. lactis. Lactic acid produced by S. lactis inhibits the growth of the bacteria. The
fungus metabolizes lactic acid and improves the growth conditions for S. lactis.

Amensalism is the opposite of commensalism. In amensalism, population A is negatively affected by the

presence of the other population (B). However, population B is not affected by the presence of population
A. Various amensal interaction mechanisms are possible. Two common mechanisms are the following:

1. Population B produces a toxic substance that inhibits the growth of population A.

2. Population B removes essential nutrients from the media, thus negatively affecting the growth of
population A.

Predation and parasitism are interactions in which one population benefits at the expense of the other.
These two interactions are distinguished by the relative size of organisms and the mechanisms involved.
Predation involves the ingestion of prey by the predator organism. A good example of prey–predator
interaction is the ingestion of bacteria by protozoa. This interaction is common in aerobic waste-treatment
reactors such as activated sludge units. In parasitism, the host, which is usually the larger organism, is
damaged by the parasite. The parasite benefits from utilization of nutrients from the host. A common
example of parasitism is the destruction of microorganisms by microphages. Although the physical
mechanisms in predation and parasitism differ, these two phenomena have many common features in their
conceptual and mathematical descriptions.

6
M.L. Shuler and F. Kargi, Bioprocess Engineering: Basic Concepts, Third Edition, Prentice Hall, 2017.
 3. MIXED CULTURES IN NATURE

Mixed cultures of organisms are common in natural ecological systems. Microorganisms are
involved in the natural cycles of most elements (e.g., carbon, nitrogen, oxygen, and sulfur).
Organisms living in soil and aquatic environments actively participate in carbon and nitrogen
cycles. For example, certain organisms fix atmospheric CO 2 to form carbohydrates, while
others degrade carbohydrates and release CO2 into the atmosphere. Similarly, some
organisms fix atmospheric nitrogen (N2) to form ammonium and proteins, while others
convert ammonium into nitrite and nitrate (nitrification), and others reduce nitrate into
atmospheric nitrogen (denitrification). Sulfur-oxidizing organisms convert reduced sulfur
compounds (sulfur and sulfide) into sulfate, and sulfate-reducing organisms reduce sulfate
into hydrogen sulfide.

M.L. Shuler and F. Kargi, Bioprocess Engineering: Basic Concepts, Third Edition, Prentice Hall, 2017. 7
 4. INDUSTRIAL UTILIZATION OF MIXED CULTURES

Defined mixed microbial populations are commonly used in cheese making, a good example
of using mixed cultures in food production. Cheeses of various types are produced by
inoculating pasteurized fresh milk with appropriate lactic acid organisms. The bacteria used
for lactic acid production are various species of Streptococcus and Lactobacillus in a mixed
culture. Other organisms are used to develop flavor and aroma. Among these are
Brevibacterium linens, Propionibacterium shermanii, Leuconostoc sp., and Streptococcus
diacetilactis. After inoculation of pasteurized milk, a protein-rich curd is precipitated by the
acidity of the medium, and the liquid is drained off. The precipitated curd is allowed to age
by action of bacteria or mold. Some molds used in cheese making are Penicillum
camemberti and Penicillum roqueforti.

Lactic acid bacteria are also used in whiskey manufacture. Lactobacillus added to the yeast
reduces pH and, therefore, the chance of contamination. Lactobacillus also contributes to the
flavor and aroma of whiskey. A favorable interaction between yeast and lactic acid bacteria
exists in ginger-beer fermentation.

M.L. Shuler and F. Kargi, Bioprocess Engineering: Basic Concepts, Third Edition, Prentice Hall, 2017. 8
The utilization of undefined mixed microbial cultures in waste-treatment processes is typical
and unavoidable. Waste-water treatment constitutes one of the largest-scale uses of
bioprocesses. Mixed cultures are also utilized in the anaerobic digestion of waste materials.
Cellulase producers, acid formers, and methane producers are typical organisms involved in
the anaerobic digestion of cellulosic wastes. However, attempts to encourage the growth of a
particular species on waste materials have been made.

Corn and pea wastes are also treated by a mixed culture of Trichoderma viride and
Geotrichium sp. T. viride produces cellulase to break down cellulose into reduced sugar
molecules, and Geotrichium produces amylases to break down starch into reduced sugar
molecules. Both organisms utilize reduced sugar molecules for growth.

A mixed culture of Candida lipolytica and Candida tropicalis has been grown on
hydrocarbons, n-paraffins, or gas oil for single-cell protein (SCP) production purposes in
both laboratory and pilot-scale operations. The utilization of a mixed culture of yeasts was
proved to yield better product quality as compared to pure yeast strains.

M.L. Shuler and F. Kargi, Bioprocess Engineering: Basic Concepts, Third Edition, Prentice Hall, 2017. 9
Gaseous hydrocarbon substrates like methane can be utilized by certain bacteria to produce
SCP. Several experimental studies have shown that mixed cultures of methane utilizing
organisms grow faster than pure cultures.

Certain methane-utilizing species of Pseudomonas oxidize methane to methanol. However,

Pseudomonas is inhibited by the end product, methanol. Inclusion of a methanol utilizing
bacteria such as Hyphomicrobium into the growth medium eliminates the problem of
methanol inhibition. This relationship is mutualistic in the sense that Pseudomonas supplies
carbon source (CH3OH) for Hyphomicrobium, and Hyphomicrobium removes the growth
inhibitor (methanol) of Pseudomonas.

M.L. Shuler and F. Kargi, Bioprocess Engineering: Basic Concepts, Third Edition, Prentice Hall, 2017. 10
 5. BIOLOGICAL WASTE TREATMENT: AN EXAMPLE
OF THE INDUSTRIAL UTILIZATION OF MIXED CULTURES
5.1. Overview

Waste materials generated in a society can be classified in three major categories:

1. Industrial wastes are produced by various industries, and waste characteristics vary greatly from one
industry to another. Industrial wastes usually contain hydrocarbons, carbohydrates, alcohols, lipids, and
aromatic organics. Industrial wastes are rich in carbon compounds and usually deficient in nitrogen (high
C/N ratio); therefore, the biological treatment of industrial wastes usually requires supplemental addition
of nitrogen compounds and other nutrients. The presence of potentially toxic compounds must be
carefully considered in devising a treatment strategy.

2. Domestic wastes are treated by municipalities and derive from humans and their daily activities. They
include ground garbage, laundry water, excrement, and often some industrial wastes that have been
sewered into the municipal system. Domestic waste varies significantly with time in terms of flow and
composition due to the periodic nature of human activity (e.g., flow decreases at night when most people
sleep).

3. Agricultural wastes are produced by farm animals (e.g., manure) and include waste plants, such as
straws. Agricultural wastes are usually carbon rich because of high cellulosic material content, although
some wastes, such as poultry manure, are high in nitrogen.

M.L. Shuler and F. Kargi, Bioprocess Engineering: Basic Concepts, Third Edition, Prentice Hall, 2017. 11
Each of these waste materials has its own characteristics, and treatment methods vary depending
on these characteristics. Three major waste treatment methods are the following:

1. Physical treatment includes screening, flocculation, sedimentation, filtration, and flotation,

which are usually used for the removal of insoluble materials.

2. Chemical treatment includes chemical oxidations (chlorination, ozonation) and chemical

precipitation using CaCl2, FeCl3, Ca(OH)2, or Al2(SO4)3.

3. Biological treatment includes the aerobic and anaerobic treatment of waste water by a mixed
culture of microorganisms.

Certain characteristics of waste water need to be known before treatment. Among them are (1)
physical characteristics, such as color, odor, pH, temperature, and solids contents (suspended and
dissolved solids), and (2) chemical characteristics, such as organic and inorganic compounds.
Major carbon compounds in a typical industrial waste are carbohydrates, lipids–oils,
hydrocarbons, and proteins. Other compounds, such as phenols, surfactants, herbicides,
pesticides, and aromatic compounds, are usually in relatively (small concentrations <1 g/l) but are
difficult to degrade by biological means. Among in (NH+4 organic compounds present in waste
water are nitrogenous compounds), sulfur compounds (SO2-4 , S2-, S0, S2-3), phosphorus
compounds (PO3-4 , HPO2-4, H2PO1-4), heavy metals (Ni2+, Pb2+, Cd2+, Fe2+, Cu2+, Zn2+, Hg2+), and
dissolved gases, such as H2S, NH3, and CH4.

12
M.L. Shuler and F. Kargi, Bioprocess Engineering: Basic Concepts, Third Edition, Prentice Hall, 2017.
The carbon content (strength) of a waste-water sample can be expressed in several ways:
biological oxygen demand (BOD), chemical oxygen demand (COD), and total organic
carbon (TOC). Normally, a 5-day BOD value is reported. The BOD5 is the amount of
dissolved oxygen (DO) consumed when a waste-water sample is seeded with active bacteria
and incubated at 20°C for 5 days. Since the amount of oxygen consumed is
stoichiometrically related primarily to the organic content of waste water, BOD is a measure
of the strength of waste water. This stoichiometric coefficient is not always known, since the
composition of the organics is usually unknown. Also, some nitrogen-containing or inorganic
compounds will exert an oxygen demand. If the only organic compound is glucose, oxygen
consumption can be easily related to the carbon content of waste water under aerobic
conditions.

According to the stoichiometry of this reaction 1.07 g of oxygen is required for the oxidation
of 1 g of glucose. Samples of waste water need to be properly diluted to obtain an accurate
BOD5 measurement, seeded with active bacteria, and incubated at 20°C for 5 days along
with an unseeded blank. BOD5 is calculated using the following equation:

M.L. Shuler and F. Kargi, Bioprocess Engineering: Basic Concepts, Third Edition, Prentice Hall, 2017. 13
A typical waste-treatment operation employing biological treatment includes the
following steps:

1. Primary treatment includes the removal of coarse solids and suspended matter
(screening, sedimentation, filtration) and conditioning of the waste-water stream by pH
adjustment and nutrient additions (e.g., PO3-4 , NH+4).

2. Secondary treatment is the major step in biological treatment; it includes biological

oxidation or anaerobic treatment of soluble and insoluble organic compounds. Organic
compounds are oxidized to CO2 and H2O by organisms under aerobic conditions.
Unoxidized organic compounds and solids from aerobic treatment (e.g., cell wall material,
lipids–fats) are decomposed to a mixture of CH4, CO2, and H2S under anaerobic conditions.
A sludge of undecomposed material must be purged from either system.

3. Tertiary treatment includes the removal of the remaining inorganic compounds

(phosphate, sulfate, ammonium) and other refractory organic compounds by one or more
physical separation methods, such as carbon adsorption, deep-bed filtration, and in some
cases membrane-based techniques, such as reverse osmosis or electrodialysis.

M.L. Shuler and F. Kargi, Bioprocess Engineering: Basic Concepts, Third Edition, Prentice Hall, 2017. 14
15
THANK YOU 