1.2 _Introduction to Industrial Microbiology

Industrial microbiology

• plays a crucial role in the large-scale production of valuable products such as antibiotics, enzymes, biofuels, and fermented foods.

• A branch of applied microbiology: microbes as factories of substances

Microbial diversity

is a key driver of innovation in industrial microbiology, as different microbes possess unique metabolic pathways that can be harnessed for commercial purposes.

MIcrobes

___ are used in industries such as food and beverage production, pharmaceuticals, agriculture, and environmental biotechnology.

food and beverage production, pharmaceuticals, agriculture, and environmental biotechnology.

Microbes are used in industries such as _____

fermentation processes

In the food and beverage sector, microbes are extensively used in _____ to produce products such as yogurt, cheese, bread, beer, and wine.

Lactobacillus species

play a vital role in dairy fermentation, contributing to the texture and flavor of yogurt and cheese.

Saccharomyces cerevisiae

a yeast species, is widely used in baking and alcohol fermentation, converting sugars into ethanol and carbon dioxide.

life-saving antibiotics and bioactive compounds

In pharmaceuticals, microbial diversity has led to the discovery of _____

Penicillium species

were responsible for the production of the first antibiotic, penicillin

Streptomyces species

continue to be a major source of antibiotics such as streptomycin, tetracycline, and erythromycin

Escherichia coli and Saccharomyces cerevisiae

have been genetically engineered to produce insulin, vaccines, and other biopharmaceuticals.

improve soil fertility, control plant diseases, and promote plant growth

In agriculture, microbes are used to ____

Rhizobium and Azotobacter

Nitrogen-fixing bacteria like ____enhance soil health by converting atmospheric nitrogen into bioavailable forms for plants.

Bacillus thuringiensis

Biological control agents such as ____ act as natural insecticides, reducing the need for chemical pesticides.

Pseudomonas putida and Deinococcus radiodurans

Microorganisms like _____are employed in bioremediation to clean up oil spills, heavy metal contamination, and radioactive waste

Chlorella and Spirulina,

are used for biofuel production, offering a sustainable alternative to fossil fuels.

High Metabolic Efficiency, Genetic Stability, Non-Pathogenicity and Safety, Resistance to Environmental Stressors, Ability to Utilize a Wide Range of Substrates, Secretion of High-Value Compounds, Adaptability and Scalability

Key Characteristics of Industrially Important Microbes:

High Metabolic Efficiency

Key Characteristics of Industrially Important Microbes:

• Industrial microbes must have the ability to grow rapidly and produce high yields of desired products, such as enzymes, biofuels, antibiotics, or organic acids.

• For example, Bacillus subtilis is used in enzyme production due to its ability to secrete large amounts of amylases and proteases, which are essential in detergent and food industries.

Genetic Stability

Key Characteristics of Industrially Important Microbes:

• A crucial trait for industrial microbes is _____, ensuring that the microorganism maintains its productivity over multiple generations. Genetic variations or mutations can alter the metabolic pathways, leading to inconsistencies in production. Therefore, industrial strains are often tested and optimized to minimize genetic drift.

Non-Pathogenicity and Safety

Key Characteristics of Industrially Important Microbes:

• Industrially important microbes must be safe for human use, especially in the food, pharmaceutical, and cosmetic industries. Strains used in food fermentation, such as Lactobacillus and Bifidobacterium, are generally recognized as safe (GRAS) by regulatory authorities. For pharmaceutical production, microbial strains are carefully selected and engineered to eliminate toxic byproducts.

Resistance to Environmental Stressors

Key Characteristics of Industrially Important Microbes:

• Industrial processes often require microbes to function under extreme conditions, such as high temperatures, acidic or alkaline pH, and high osmotic pressure. Thermophilic bacteria like Thermus aquaticus thrive at high temperatures and are used in the production of thermostable enzymes, such as Taq polymerase, which is essential in PCR (polymerase chain reaction) technology.

Ability to Utilize a Wide Range of Substrates

Key Characteristics of Industrially Important Microbes:

• Some microbes can ferment agricultural waste, making them valuable in biofuel production. For example, Clostridium acetobutylicum can convert plant biomass into bioethanol and butanol, reducing reliance on petroleum-based fuels.

Secretion of High-Value Compounds

Key Characteristics of Industrially Important Microbes:

• Many industrial microbes naturally secrete valuable metabolites, including antibiotics, organic acids, vitamins, and enzymes. Aspergillus niger produces citric acid, a key ingredient in food and beverage industries, while Streptomyces species are prolific producers of antibiotics. Through metabolic engineering, scientists can enhance these traits to improve yield and efficiency.

Adaptability and Scalability

Key Characteristics of Industrially Important Microbes:

• Microbial strains must be capable of growth in large-scale fermentation systems while maintaining their efficiency. Some microbes require specific conditions, while others can be adapted for high-density cultivation.

• For example, Escherichia coli and Saccharomyces cerevisiae are widely used in recombinant protein production due to their ability to grow in bioreactors under controlled conditions.

Natural Sources of Industrial Microbe, Techniques for Isolation and Screening, Strain Improvement and Optimization

Sources and Isolation of Industrially Important Microbes:

Soil and Agricultural Environments

Sources and Isolation of Industrially Important Microbes:

Natural Sources of Industrial Microbes:

• One of the richest sources of industrial microbes, soil harbors bacteria, fungi, and actinomycetes capable of producing antibiotics, enzymes, and bioactive compounds. Streptomyces species, known for their antibiotic production, are commonly isolated from soil samples.

• is a rich source of antibiotic-producing actinomycetes, fungi, and bacteria.

Example microbes:

• Streptomyces spp. – Produces antibiotics (e.g., streptomycin, tetracycline).

• Bacillus spp. – Produces enzymes and biopesticides (e.g., Bacillus thuringiensis).

• Pseudomonas spp. – Used for bioremediation and plant growth promotion.

Aquatic Environments/Marine waters

Sources and Isolation of Industrially Important Microbes:

Natural Sources of Industrial Microbes:

• Marine and freshwater environments contain microbes with unique metabolic properties. Algae such as Chlorella and Spirulina are cultivated for biofuel and nutritional supplements, while extremophilic bacteria from deep-sea hydrothermal vents produce enzymes with high stability.

• have unique metabolic pathways due to their adaptation to high salinity and pressure.

Example microbes:

• Alcanivorax spp. – Used for oil spill bioremediation.

• Salinispora spp. – Produces novel antibiotics.

• Halophilic archaea – Used in enzyme production for high-salinity environments

Decomposing Organic Matter

Sources and Isolation of Industrially Important Microbes:

Natural Sources of Industrial Microbes:

• Fungi and bacteria that decompose plant material can be isolated for use in bioconversion processes, such as converting agricultural waste into bioethanol. Trichoderma reesei, for instance, is a fungus used in cellulase production for biomass degradation.

Extreme Environments (Temperature, Salinity, pH))

Sources and Isolation of Industrially Important Microbes:

Natural Sources of Industrial Microbes:

• Microbes from extreme habitats, such as hot springs, salt flats, and acidic lakes, are valuable for producing enzymes that function under harsh conditions. Thermophiles like Thermus aquaticus are used in molecular biology applications due to their heat-resistant enzymes.

• Microbes from hot springs, deep-sea vents, and acidic/alkaline lakes are used in biotechnology.

Example microbes:

• Thermus aquaticus – Produces Taq polymerase for PCR.

• Pyrococcus furiosus – Used in biofuel production under extreme conditions.

• Sulfolobus spp. – Acidophilic archaea used for bioleaching in mining

Selective Media and Culturing

Sources and Isolation of Industrially Important Microbes:

Techniques for Isolation and Screening:

• Microbes are cultivated on specific media designed to promote the growth of target species while inhibiting others. For example, antibiotic producing bacteria are screened on agar plates containing indicator strains to observe zones of inhibition.

Metagenomics and Direct Sequencing

Sources and Isolation of Industrially Important Microbes:

Techniques for Isolation and Screening:

• Traditional culturing methods can only detect a fraction of microbial diversity. Metagenomic approaches allow researchers to extract and analyze microbial DNA from environmental samples, identifying new species without the need for cultivation.

High-Throughput Screening

Sources and Isolation of Industrially Important Microbes:

Techniques for Isolation and Screening:

• Automated systems enable rapid testing of microbial strains for desirable traits, such as enzyme activity or fermentation efficiency. This accelerates the discovery of high-performance industrial microbes.

Mutagenesis and Adaptive Evolution

Sources and Isolation of Industrially Important Microbes:

Strain Improvement and Optimization:

• Random mutations or exposure to selective pressures can lead to strains with improved metabolic capabilities.

Genetic Engineering

Sources and Isolation of Industrially Important Microbes:

Strain Improvement and Optimization:

• Recombinant DNA technology allows scientists to insert or modify genes to improve productivity, stability, or resistance to environmental factors.

Fermentation Process Optimization

Sources and Isolation of Industrially Important Microbes:

Strain Improvement and Optimization:

• Microbial growth conditions are optimized to maximize yield and efficiency in large-scale production.

Antibiotics o Food products o Enzymes o Proteins o Amino Acids o Vaccines o Other fine chemicals

microbes as factories of substances:

Microbial lipases

• Substrate specificity

• Versatility

  • Extreme pH

  • Temperature

  • Presence of metals and solvents

fermentation

• Industrial Microbiology is synonymous with the term ___

• Energy extraction process

• Redox metabolism w/o the presence of O2

• Alternative to aerobic respiration

  • Large MW molecules are broken down

  • Electrons are donated to others

  • -> ATP and other organic molecules

• Glucose -> Pyruvate -> Ethanol

• Industrial processes of high-potency microbes are scaled-up for commercial production.

• Have an Efficient and cheap production

End Goal of Industrial Microbiology:

• Begin with the end in mind

• Be diligent with the details

• Prepare for the unexpected

Three key principles of IM:

Yeasts

are living organisms responsible for beer and wine fermentation

Enzymes

are identified as important factors for bioconversions

golden age of antibiotics

The need for anti-infection drugs (____)

Biotechnology/Industrial microbiology

as specialty domains

Antonie van Leeuwenhoek

in 1680s --> animalcules

Lazaaro Spallanzani

--> microscopic investigation of cells such as sperm and microbial growth

Antoine Lavoisier

• in 1780s --> Substance transformations; Sugars are composed of C, H, O

• Sugarcane --> Alcohol and CO2 = HOW? Quantitative studies

Joseph Louis Gay-Lussac

in 1810s --> Maintaining grape juice in an unfermented state (boil and cover); but when 'ferment' (yeast) was added, fermentation would begin.

Theodor Schwann (1837)

Vorläufige Mittheilung, betreffend Versuche über die Weingärung und Fäulnis. Ann Phys 11(2):184 [Preliminary communication concerning experiments on wine fermentation and putrefaction]

Charles Cagniard-Latour (1838)

Mémoire sur la fermentation vineuse. Ann Chim 68:206

alcohol fermentation

Schwann (1837) and Cagniard-Latour (1838): Yeasts are 'organized' microorganisms and they are associated with ___

Louis Pasteur

• (1850s) proved that the fermentation processes common during his time were linked to the specific microorganisms present and that the observed chemical changes were based on the physiological abilities of these microorganisms.

• Pasteurization (Sterilization) --> Breeding pure microbial cultures

Robert Koch

• studied the causative agent of anthrax and invented methods to isolate and purify the bacterial causative agent of the infection.

• He found that the microbial agent cannot survive outside of the host for a long time but can persist through spore formation.

• The founding of modern microbiology is accredited to both ___(1843–1910), who demonstrated that infectious diseases such as anthrax, typhus, and cholera were caused by bacterial pathogens

spore formation

Robert Koch found that the microbial agent cannot survive outside of the host for a long time but can persist through ___

bacterial pathogens

The founding of modern microbiology is accredited to both Robert Koch (1843–1910), who demonstrated that infectious diseases such as anthrax, typhus, and cholera were caused by __

anthrax, typhus, and cholera

The founding of modern microbiology is accredited to both Robert Koch (1843–1910), who demonstrated that infectious diseases such as ___ were caused by bacterial pathogens

Edward Buchner

• Buchner funnel

• Introduced the Concepts of enzymes

• Set-up 1: cell-free supernatant -> ongoing fermentation that leads to bioconversion

• Set-up 2: with cell supernatant

• Discovered cell-free fermentation

Alexander Fleming

(1928) discovered penicillin that led to the introduction of antibiotics that greatly reduced the number of deaths from infection.

Howard Florey, Ernst Chain, and William Dunn

worked on the purification and further chemical studies on penicillin

Norman Heatley

worked on extracting penicillin from laboratory filtrate

Edward Abraham

used alumina column chromatography to purify penicillin

Florey

1940s – showed that mice can be protected from Streptococcal infection

Steroids o Amino acids o Organic acids o Carbohydrates o Vitamins o Solvents o Enzymes

Products with growing market relevance:

Elmer Gade

1958 – established Journal of Microbiological and Biochemical Engineering (first Industrial microbiology Journal)

UCL (UK)

1960s – MSc in Biochemical Engineering

1970s

– Technical University of Berlin

alcoholic, lactic acid, acetic acid, and alkali fermentation

There are mainly four main types of fermentation processes:

Alcohol fermentation

main types of fermentation processes:

• is the process in which ethanol is produced by fermentation by yeasts which are the predominant organisms (e.g., wines and beers)

• high amount of sugars

Lactic acid fermentation

main types of fermentation processes:

• is mainly work of lactic acid bacteria (LAB) and occurs chiefly in cereals and milk products (e.g., fermented milks, cereals, fruits, and vegetables).

Acetic acid fermentation

main types of fermentation processes:

• is work of Acetobacter species. Acetobacter aceti converts alcohol to acetic acid in the presence of excess oxygen.

Alkali fermentation

main types of fermentation processes:

• takes place during the fermentation of either fresh poultry eggs, fish, seeds, or any protein rich raw materials and this type of fermentation is popularly used as condiments

condiments

Alkali fermentation takes place during the fermentation of either fresh poultry eggs, fish, seeds, or any protein rich raw materials and this type of fermentation is popularly used as _____

Homofermentative pathway

yield almost exclusively lactic acid

Heterofermentative pathway

produce lactic acid, ethanol/acetic acid, and CO2

Enzymes and Cell Cultures

Key Elements of Industrial Fermentation:

Enzymes

Key Elements of Industrial Fermentation:

• bio-catalysts, lowers energy activation, speeds up process of reaction

Bio-catalysts

Enzymes as ____ lowers energy activation, speeds up process of reaction

Class 1 - Oxidoreductases

International classification of enzymes:

• Transfer of electrons (hydride ions or H atoms)

Class 2 - Transferases

International classification of enzymes:

• Group transfer reaction

Class 3 - Hydrolases

International classification of enzymes:

• Hydrolysis reactions (transfer or functional groups to water)

Class 4 - Lyases

International classification of enzymes:

• Cleavage of C-C, C-O, C-N, or other bonds by elimination, leaving double bonds or rings, or addition of groups of double bonds

Class 5 - Isomerases

International classification of enzymes:

• Transfer of groups within molecules to yield isomeric forms

Class 6 - Ligases

International classification of enzymes:

• Formation of C-C, C-S, C-O, and C-N bonds by condensation reactions coupled to cleavage of ATP or similar cofactor

preserve product of processes

Enzymes are inhibited to ___

Competitive

Inhibition of Enzyme Activity:

• The inhibitor adsorbs at the substrate binding site. In this case, two types of complexes are formed: enzyme inhibitor (EI) and enzyme substrate (ES); complex EI has no enzyme activity

Noncompetitive

Inhibition of Enzyme Activity:

• The enzyme-inhibitor-substrate (EIS) complex is unable to dissociate to give a product of reaction. In this case, inhibitor binds to E or to the ES complex. The binding of the inhibitor to the enzyme reduces its activity but does not affect the binding of substrate

• inhibitor binds in allosteric site

Uncompetitive

Inhibition of Enzyme Activity:

• The inhibitor binds only to the ES complex; it does not interfere with the binding of substrate to the active site but prevents the dissociation of the ES complex.

CELL CULTURES

• The cell population is a heterogeneous collection of single cells: every cell is different from the others depending on its actual internal composition and with respect to the particular life function that it is developing (i.e., growth, reproduction) or its age.

• To grow, reproduce themselves, and survive, the cells require the consumption of nutrients (substrate) taken from the environment. Usually such action is followed by the release of an end product of the cell’s metabolic process. Increasing their biomass, cells change the medium properties:

  • Temperature

  • Composition

  • Rheological properties

Rate limiting factor/nutrient

• decides if the bioprocess becomes successful

• A single component becomes the ____ and with respect to just this compound we consider the effects of medium concentration on cell growth kinetics.

“fixed”

To study the complexity of a living system, several parameters are imposed as ____ to detect one by one the effects of different agents.

Growth

is formulated so that all components but one are present at sufficiently high concentrations that could be considered not to change in quantity.

BIOREACTOR

An isothermal (commonly) process unit where biochemical reactions occur.

batch stirred tank reactor (BSTR)

BIOREACTOR:

• is a closed system;

• there are no mass flows entering or coming out from it, but it can eventually exchange heat with the environment.

• An ideal ____has the property of perfect fluid mixing. It represents the typical case of a cell culture

fed-BSTR

BIOREACTOR:

• is a semi-open system;

• mass can enter but not exit the reactor.

• It is an enhancement of the closed batch process in which the substrate is added in increments as the fermentation progresses.

• An ideal ____ has the property of perfect fluid mixing.

• can add an incremental amount of substrate

continuous stirred tank reactor (CSTR)

BIOREACTOR:

• is an open system.

• An ideal CSTR has the property of perfect fluid mixing. A classical CSTR bioreactor is individualized in the chemostat configuration, in which a sterile nutrient solution is added to the bioreactor continuously and an equivalent amount of converted nutrient solution with microorganisms is simultaneously taken out of the system.

• has an inlet and outlet, does not stop the process (continuous)

Chemostat

INDUSTRIAL FERMENTATION SET-UP:

• Originally introduced in the 1950s as a method to culture a bacterial population at a reduced growth rate for an indefinite period and is the most widely used approach to establish steady-state culture for various applications.

• Synonymous to CSTR

Aerobic reactors

INDUSTRIAL FERMENTATION SET-UP:

• Uses oxygen as the terminal electron acceptor

• Able to sustain a large number of important reactions that use a number of different electron donors

• Used in wastewater treatment and relies on the presence of oxygen to break down organic matter

CHEMOSTAT REACTOR

• A continuous fermentation system in which feeding of fresh medium to the fermentor and withdrawing fermentation broth are performed at the same rate

• There is perfect mixing in the reactor:

  • the ideal CSTR situation.

  • Only one nutrient constitutes the limiting substrate.

  • The death of cells caused by the mechanical agitation of the impeller is neglected.

  • The feed stream is sterile.

  • The reactor is isotherm.

Obligate anaerobes

These are prokaryotic microorganisms (Clostridium sp., the soil bacteria Actinomyces, and methanogenic bacteria) for which molecular oxygen is a toxic substance.

Obligate aerobes

These are organisms that require molecular oxygen as the terminal oxidizing agent to fulfill their energy needs.