MCB 402

Microorganisms of industrial importance

Industrial microbiology includes the use of microorganisms to manufacture food or industrial
products in large quantities. Numerous microorganisms are used within industrial microbiology;
these include naturally occurring organisms, laboratory selected mutants, or even genetically
modified organisms (GMOs).

The specific microorganisms employed in industrial microbiology were often isolated from the
natural environment, which involved the random screening of a large number of isolates.
Alternatively, suitable microorganisms were acquired from culture collections. Most of these
microorganisms, irrespective of their origins, were subsequently modified by conventional strain
improvement strategies, using mutagenesis or breeding programmes, to improve their properties
for industrial use.

In most cases, regulatory considerations are of major importance when choosing microorganisms
for industrial use. Fermentation industries often prefer to use established GRAS (generally
regarded as safe) microorganisms (Bacteria-Bacillus subtilis, Lactobacillus bulgaricus,
Lactococcus lactis, Leuconostoc oenos; Yeasts- Saccharomyces cerevisiae, Candida utilis,
Kluyveromyces marxianus, Kluyveromyces lactis; Filamentous fungi- Aspergillus niger,
Aspergillus oryzae, Mucor javanicus, Penicillium roqueforti), particularly for the manufacture of
food products and ingredients.
Actinomycetes is a successful group of bacteria possessing the characteristics of both bacteria
and fungi. They are spore forming gram-positive bacteria, present aerobically in nature. They are
successful industrial microorganisms linked with production of large number of important
secondary metabolite such as antibiotics, enzymes and help in degrading a wide range of
hydrocarbons, pesticides, aliphatic and aromatic compounds. Actinomycetes include the genera
Streptomyces, Micromonospora, Nocardia, Rhodococcus, Actinoplanes and
Thermoactinomyces.

Characteristics of Industrial strains

Irrespective of the origins of an industrial microorganism, it should ideally exhibit:
1. Genetic stability
2. Efficient production of the target product, whose route of biosynthesis should preferably be
well characterized
3. Limited or no need for vitamins and additional growth factors
4. Utilization of a wide range of low-cost and readily available carbon sources
5. Amenability to genetic manipulation
6. Safety, non-pathogenicity and should not produce toxic agents, unless this is the target product
7. Ready harvesting from the fermentation e.g. Yeast is easily centrifuged compared to bacteria
8. Ready breakage, if the target product is intracellular
9. Production of limited byproducts to ease subsequent purification problems.
10. Resistance to predators such as bacteriophages
11. Competitive physiological requirement/characteristics against contaminants e.g. High
temperature with low pH requirements

Microbial products of industrial interest

These include
 Microbial cells
 Enzymes
  Antibiotics, steroids, alkaloids
 Food additives
 Amino acids
 Fuels and chemicals
 Vitamins
 Polymers

Some industrial products and their producer microorganisms

Bacteria Yeasts and filamentous fungi
1 Traditional products
Bread, beer, wine and Mainly Saccharomyces
spirits cerevisiae
Cheeses, other dairy Lactic acid bacteria
products
Ripening of blue and Penicillium species
Camembert-type cheeses
Fermented meats and Mostly lactic acid bacteria
vegetables
Mushrooms Agaricus bisporus,
Lentinula edodes
Soy sauce Aspergillus oryzae
Zygosaccharomyces rouxii
Sufu (soya bean curd) Mucor species
Vinegar Acetobacter species
2 Amino acids
L-Glutamine Corynebacterium glutamicum
L-Lysine Brevibacterium lactofermentum
L-Tryptophan Klebsiella aerogenes
3 Enzymes
Carbohydrases
α-amylase Bacillus subtilis
β-amylase Aspergillus niger
Amyloglucosidase Aspergillus niger
glucose isomerase Streptomyces olivaceus
Invertase Kluyveromyces species
lactase (β-galactosidase) Kluyveromyces lactis
Cellulases Trichoderma viride
Lipases Candida cylindraceae
Pectinases Aspergillus wentii
Proteases
Subtilisin (alkaline) Bacillus licheniformis
Microbial rennet (acid) Rhizomucor miehei
Neutral Aspergillus oryzae
4 Fuels and chemical
feedstocks
Acetone Clostridium species
Butanol Clostridium acetobutylicum
Ethanol Zymomonas mobilis Saccharomyces cerevisiae
Glycerol Zygosaccharomyces rouxii
 Methane Methanogenic archaeans
5 Organic acids
Acetic acid Acetobacter xylinum
Citric acid Aspergillus niger;
Yarrowia lipolytica
Fumaric acid Rhizopus species
Gluconic acid Acetobacter suboxydans
Itaconic acid Aspergillus itaconicus
Kojic acid Aspergillus flavus
Lactic acid Lactobacillus delbrueckii
6 Agricultural products
Gibberellins Fusarium moniliforme
Fungicides Coniothyrium minitans
Insecticides Bacillus thuringiensis
Silage Lactic acid bacteria
7 Pharmaceuticals and
related compounds
Antibiotics
Aminoglycosides Streptomyces griseus
(streptomycin)
Macrolides Saccharapolyopora erythraea
(erythromycin)
Tetracyclines Streptomyces aureofasciens
(chlortetracycline)
β-Lactams
Penicillins Penicillium chrysogenum
Cephalosporins Acremonium chrysogenum
clavulanic acid Streptomyces clavuligerus
Peptides
Bacitracin Bacillus licheniformis
Gramicidin Bacillus brevis
8 Hormones and related
compounds
Insulin Recombinant Escherichia coli Recombinant Saccharomyces
cerevisiae
Human growth hormone Recombinant Escherichia coli Recombinant Saccharomyces
cerevisiae
Interferon Recombinant Escherichia coli Recombinant Saccharomyces
cerevisiae
Steroids Arthrobacter species Rhizopus species
Vaccines Bacillus anthracis
Clostridium tetani
Recombinant Escherichia coli
Salmonella typhi
9 Vitamins
B12 (cyanocobalamin) Pseudomonas denitrificans
b-Carotene (provitamin A) Blakeslea trispora
Ascorbic acid (vitamin C) Acetobacter suboxydans
Riboflavin Recombinant Bacillus subtilis Ashbya gossypii
 10 Polymers
Dextran Leuconostoc mesenteroides
Cellulose Acetobacter xylinum
Xanthan Xanthomonas campestris
Alginates Azotobacter vinelandii
Pullulan Aureobasidium pullulans
Gellan Sphingomonas paucimobilis
11 Single cell protein Methylococcus capsulatus Candida utilis
Methylophilus methylotrophus Fusarium venenatum
Kluyveromyces marxianus
Paecilomyces variotii
Saccharomyces cerevisiae

STRAIN IMPROVEMENT
Biotechnology industry is basically based on utilising microbes, plants and animal cells to obtain a diverse
range of products. Microorganisms generate a wide variety of products as part of their metabolism that are
of huge benefit for humans day to day use. Some of these products include antibiotics, enzymes, organic
compounds, biofuels, etc.
In nature the microbial metabolism is controlled to avoid wasteful expenditure of energy thus microbes
produce these metabolites in low concentrations; however, prerequisite for any industrial scale
biotechnological process is a microbial strain which generates high amounts of any desired industrial
product.
Strain improvement is defined as the science and technology of genetically modifying and manipulating
microbial strains to enhance/improve their potentials for numerous biotechnological applications and it
majorly involves in iteration the genetic alterations, fermentation techniques and assay. Strain
improvement is a new discipline which integrates metabolic engineering with other disciplines that
includes strain improvement such as;
 Upstreaming process which include systems biology i.e. systems of biological components, synthetic
biology and evolutionary engineering
 Midstreaming process that includes fermentation
 Downstreaming process that involves separation and purification

Purpose of Strain Improvement

 Increase the productivities
 Regulating the activity of the enzymes
 Introducing new genetic properties into the organism by Recombinant DNA technology / Genetic
engineering.

Strategies for Microbial Strain Improvement

To develop an improved strain a proper blueprint should be developed which should employ some or all
of the following strategies.

1. Basic considerations
When designing a project for strain improvement, various technical, economical, legal considerations need
to be made. Once the choice between products of interest has been made, techno-economic analysis needs
to be carried out with various candidate microbial strains. The microbial analysis should include the type
of fermentation; whether it’s aerobic or anaerobic fermentation, its cheap carbon source and whether the
fermenter used is batch or continuous or fed- batch fermenter while considering down streaming
 equipment as well. Various strategies can be used depending on the nature of the product, but whatever
strategy is used, product titre, yield and productivity should be properly estimated.

2. Selection of microbial strain

When selecting a strain, the ability of the organism to utilise carbon feedstock of choice should be
considered. There is need to make a choice between making an organism more susceptible to gene
manipulation or choose another organism that is easily engineered. Other factor like the cost and ease of
upstreaming, midstreaming and downstreaming processes should be contemplated.

3. Metabolic pathway reconstruction

In some cases, the formation of product of interest is absent in the microbe of choice, therefore, this
production pathway needs to be added to the organism by inspecting the candidate enzymes or genes
through genome and metagenome analysis. Obviously, this strategy is not required in organisms
producing the desired product. However, this strategy is of increasing significance because of our interest
in the production of products that are not natural or have inefficient production in hosts.
Heterologous pathways can be introduced into the host based on biological knowledge or genomic and
chemo-informatic analysis. Development of these pathways increases the host’s capacity to produce
multiple chemicals and drugs including antibiotics or their derivatives, certain acids etc. Once the pathway
has been constructed it can be optimised by expression optimisation or codon optimisation or
increasing/altering the enzyme specificity.
4. Increasing tolerance to product
Microbes have a proper regulated system that prevents the wasteful expenditure of energy, thus the
metabolites are produced in low amounts. Once the organism is modified to overproduce a certain product
it is absolutely necessary to expand the tolerance of that strain to the product. This strategy can be adopted
at any point in strain development. When it is done in early stages it will open up metabolic fluxes thus
increasing the tolerance.
Tolerant strain is developed by serial culturing with increasing concentration of product at each step, this
is then followed by screening of strains that survive increased concentrations. By continued repetition of
this process the tolerance of the strain can be increased.
Other strategies include removing negative regulatory pathways, rerouting fluxes to optimise precursor
availability, optimising metabolic fluxes towards product formation, optimising microbial culture
conditions, gene modification to further enhance production and scale-up fermentation of developed strain

Techniques for strain improvement

1. Mutation
Mutation is a sudden change in the genetic make-up of an organism which can be heritable from
generation to generation. Any agent capable of inducing mutation is referred to as mutagen. The
application of mutagens to induce mutation is referred to as mutagenesis. Mutagenesis is the main reason
behind genetic variation but it is not possible for a single mutagenic treatment to give us all possible kinds
of mutations required. Mutagen can be distinguished into two categories:
a. Physical mutagens such as ultraviolet, gamma and X-rays
b. Chemical mutagens such as ethyl methane sulphonate - EMS, nitrosomethyl guanidine – NTG, etc.
The type of mutation produced is determined by the kind of DNA damage induced by mutagen as well as
the influence of DNA repair pathways on this damage. For example, UV radiation gives huge percentage
of pyrimidine dimers while ionising radiation causes chromosomal distortion at a very high rate whereas
EMS and NTG cause alkylation.
Another circumstance which should be taken into account when selecting a mutagen is its specificity.
Each mutagen acts on certain parts of the genome while rarely affecting others, if at all.
 High doses of mutagen decrease the chances of survivors and can also result in undesired secondary
mutations, therefore, it is essential to do optimisation of mutagen dose. Mutation can be grouped into
three:
i. Spontaneous mutations: The naturally occurring mutations in the cell. It has low frequency of
occurrence and usually occur at about 10-10 to 10-6 per generation per gene.
ii. Classical mutagenesis: This type of mutagenesis involves the usage of physical and chemical mutagenic
agents to manipulate the genetic structure of microbes. This is done to improve the desired characteristic
of an organism.
iii. Directed mutagenesis: This mutation involves targeting a known site on the genome for the activity of
the mutagen. A prerequisite for directed mutagenesis is the knowledge of genes to mutate and the
availability of tools for directing the mutagenesis to specific genes controlling the product formation.
2. Recombination
Recombination is the process in which two genetically different strains combine to generate a hybrid that
is superior and different from either of the parents. Recombination is useful in erasing the neutral and
deleterious which arise during random mutagenesis. Mutagenesis and recombination are the dominant
techniques for strain improvement. Recombination is more successful if it is used as a complement and
not as an alternate technique.
The major advantage of this technique is that the developed strains are not considered genetically
modified microorganisms (GMMO).

3. Recombinant DNA technology

Recombination DNA technology is a new technique for strain improvement. Recombination DNA
technology technique involves cloning of genes and expression of cloned gene into an expression vector
and its product formation. Recombination DNA technique can be used to improve titres of primary
metabolites such as amino acids and extracellular enzymes.
4. Precision engineering technology (Integrated strain improvement)
Precision engineering technology is a newly developed strain improvement technique which involves the
integration of classical metabolic engineering and screening methods with profiling technologies which
give a more clear understanding of genetics and physiology of metabolite production. Precision
engineering is a good framework of old and new techniques and is already having a huge impact on
industrial biotechnology as it takes into consideration the negative effects of old strain improvement
technique on the industrial microbes such as; formation of undesired products or slow growth, substrate
specificity and weak stress tolerance.

Microbial Deterioration of Pulpwood and Paper

Wood is made of cellulose, hemicellulose, lignin and extractives.

Cellulose forms the skeleton of plant cell walls and has the most desired properties for paper making. It
consists of a long straight chains of glucose molecule.

Hemicellulose consists of short branched chains of glucose and other sugar molecules. They are soluble
in water and thus are often removed during the pulping process.

Lignin is a three-dimensional phenolic polymer network. It holds the cellulose fibres together and make
them rigid. They are often selectively removed during chemical pulping and bleaching process without
significantly degrading the cellulose fibre.
 Extractives include hormones, resin and fatty acids alongside other substances that help the tree to grow
and resist diseases and pests. Extractives account for 3% of soft wood and about 5% of hardwood.

Classification of wood
There are two classes of wood

1. Hard wood: The fibres of this class of wood gives a sheet of paper its smooth surface and opacity.
Examples include Acacia, Aspen, Birch, Eucalyptus, Maple, Pacific Albus and Rubber tree
2. Soft wood: The fibres of this class of wood are generally used to provide strength to sheet of paper.
Examples include Pine and Spruce
Characteristics of soft wood and hard wood

Properties Softwood Hardwood

1 Cellulose content 42% 45%
2 Lignin content 28% 20%
3 Extractives 3% 5%
4 Fibre content 2-6mm 0.6-1.5mm
5 Coarseness 15-35mg/100mm 5-10mg/100mm

Pulpwood is a timber cut primarily to be a source of wood fiber for producing paper, cardboard, or other
fiber products. Trees of any size can be used for pulpwood, but trees in the range of 5 to 9 inches
D.B.H. (diameter at breast height) are normally used.
Pulping is a process that extracts fibrous material, cellulose, from wood or other raw material as a
prelude to papermaking. Pulping can be done mechanically or chemically.
Chemical pulping process involves the application of heat and chemicals (Aqueous NaOH and NaS for
dissolution of lignin) on wood chips in a pressure cooker known as a digester. The wood fiber is
separated into cellulose fibers, lignin (the wood glue that holds the tree together) and other substances
such as sugars. After about 2-4hours, the mixture (pulp, pulping chemicals and wood waste) is
discharged from the digester. The pulp is washed to separate it from the chemical and the wood waste
(black liquor).
Efficient pulp washing is very important because it ensures the maximum recovery of the pulping
chemicals and it minimises the amount of organic waste carried out with the pulp into the bleaching
process. Poorly washed pulps require higher bleaching chemical doses thus increasing the cost and the
amount of organic waste discharges in the bleached plant effluent.
Pulp is a clean, wood-based, renewable and biodegradable raw material. It can be used to produce
paper, tissue, board and specialty paper – making them truly sustainable bioproducts.

Biological/Microbial Deterioration of Pulpwood, Paper-Pulp and Paper

Microbial degradation of these materials involves the activities of fungi and a few bacteria.
Temperature and moisture together with an appropriate availability of oxygen play an important role in
growing the fungi to deteriorate pulp-wood. Deterioration of pulp-wood by Basidiomycetous fungi
brings about two classes of rot on the basis of the constituent of the wood been attacked.
a. White rots: White rotten patches on the pulp-wood surface characterizes the degradation of brownish
lignin in the wood leaving behind a white spongy cellulosic mass called white rot in the wood.
b. Brown rots: Brown rotten patches on the pulpwood results from a preferential microbial deterioration of
the cellulose leaving behind a brown pinky mass predominantly of lignin called brown rot.
Deterioration of stored pulp-wood surface by some ascomycetous and deuteromycetous fungi brings
about a degradation known as soft rots.
Paper-Pulp, the product of pulpwood is also subjected to microbial attack and deterioration because its
major component-cellulose is susceptible to degradation by a host of fungi and bacterial especially those
microbes that are capable of producing cellulose degrading enzymes called cellulase. Although, those
paper-pulps which are prepared by chemical treatments generally possess less nutrients for
microorganisms and hence are less susceptible to microbial attack than the physically (mechanically)
prepared paper-pulps.
Microbial degradation of the paper-pulp may be encountered in the form of “paper-pulp slime” spots on
the finished paper sheet. Paper-pulp slime is produced by the deposition of microorganisms. Bacteria,
yeasts, moulds, algae, and protozoa have been implicated and isolated from pulp slimes.
Bacteria, particularly capsulated bacilli such as Enterobacter aerogenes and Bacillus spp. represent the
most important group of pulp slime producers. Also, bacterium Alcaligenes viscosus var. dissimilis has
been obtained from pink pulp slime.
Species of Mucor (Penicillium, Trichoderma, Fusarium) and yeasts (Torula, Rhodotorula) are the fungi
that have been isolated from pulp slimes in various paper-making industries.

Finished paper which is prepared by the refinement and fabrication of paper-pulp is also attacked by
microorganisms. Various fungi (Penicillium spp., Aspergillus spp., Chaetomium, etc.) and bacteria are
the main attackers as cellulose, the main constituent of the paper, is susceptible to them causing black,
brown or yellow discoloration and spotting on the paper. The other constituents of the paper- Glue or
casein, also serve as substrate for certain microorganisms.

These staining or decolouration of the paper-sheet is caused as a result of the microorganisms producing
certain chemicals during their metabolism. Growth of cellulolytic microorganisms may result in either
weakening of fibres, perforations and/or even complete destruction of the finished paper.

Two distinct forms of fungal decay have been identified in paper:

i. Soft rot: This is associated with the degradation of the three structural wood components particularly
hemicellulose and cellulose. Soft rot is frequently associated with wood in contact with excessive
moisture condition. E.g. of fungi responsible is Chaetomium

ii. Brown rot: This is associated with the degradation caused by a diffuse polymerization of cellulose in
wood resulting in significant strength losses during the decay stages. E.g. of fungi responsible include
Poria incrassate, Merulius lachrymans, Piptoporus betulinus, Coniophora spp etc