Industrial Microbiology
Industrial Microbiology Introduction
Industrial microbiology is the study of the large-scale, profit-motivated production of microorganisms or their products for direct use or as inputs in manufacturing. Examples include yeast production for food, animal feed, or bread-making, and ethanol production for beverages, perfumes, or pharmaceuticals.
It is associated with the commercial exploitation of microbes for human benefit, impacting economics, the environment, and social parameters.
Although humans have made use of microbes in the production of products (e.g. alcoholic beverages and dairy products) since the beginning of civilization, they were once 'oblivious' to what role microbe even played.
Historical Context
Pasteur's Contributions
Louis Pasteur demonstrated in 1857 that alcoholic fermentation in beer and wine is a result of microbial activity, dismissing the chemical process theory.
He identified organisms that could spoil beer and wine and differentiated between aerobic and anaerobic fermentations.
He invented pasteurization, which contributed significantly to food and beverage preservation which was initially introduced to preserve wine.
His publications, such as Études sur le Vin (1866) and Études sur la Bière (1876), were catalysts for industrial fermentation progress.
Hansen and Pure Culture Techniques
Hansen developed pure culture techniques at the Carlsberg Brewery in Denmark.
In 1883, pure strain brewing was first carried out using Carlsberg Yeast No. 1 (Saccharomyces carlsbergensis, now Saccharomyces cerevisiae).
Early 20th Century Advancements
Progress was slow during this period, though there were significant advancements in large-scale sewage treatment.
Weizmann developed the acetone-butanol fermentation process (1913–15) using Clostridium acetobutylicum.
In the early 1920s, citric acid manufacturing was introduced using Aspergillus niger.
Penicillin Production
The need for penicillin during World War II accelerated fermentation technology in the 1940s.
Production shifted from small-scale surface culture to large-scale submerged fermentations, leading to media and microbial strain development.
Recent Progress
Milstein and Kohler pioneered the production of monoclonal antibodies in the early 1970s for analytical, diagnostic, therapeutic, and purification purposes.
Genetic Engineering
Genetic engineering (recombinant DNA technology) has significantly influenced fermentation processes and products over the last 20 years.
It allows for gene transfer between organisms, enabling new approaches to strain improvement.
Expression vectors can be inserted into prokaryotes like Escherichia coli or eukaryotes like yeast, depending on whether post-translational processing is needed.
Characteristics of Industrial Microbiology
Industrial microbiology is often divided into sub-disciplines such as medical microbiology, environmental microbiology, and food microbiology. These boundaries are blurred and for convenience purposes only.
Industrial vs. Medical Microbiology
Industrial and medical microbiology differ in motivation, economic value, and scale.
Motivation: Industrial microbiology aims for profit and wealth generation, whereas medical microbiology focuses on restoring patient health.
Economic Value: Microorganisms in medical microbiology have little direct economic value, whereas those in industrial microbiology or their products are highly valuable.
Scale: Industrial microbiology involves large-scale cultivation in fermentors (up to 50,000 liters or more), unlike the small scale of routine medical microbiology.
Multi-Disciplinary Teamwork
Industrial microbiologists work in teams with chemical engineers, biochemists, economists, lawyers, and marketing experts to generate profit.
Key Roles of the Microbiologist
The microbiologist has a central role in an industrial establishment. Some of these functions include:
Selecting the organism for the process.
Choosing the growth medium.
Determining optimal environmental conditions (pH, temperature, aeration, etc.).
Monitoring for contaminants during production and ensuring quality control.
Maintaining the culture collection to retain desirable properties.
Improving microorganism performance through genetic manipulation or medium reconstitution.
Obsolescence in Industrial Microbiology
Less efficient methods are replaced by better ones due to the profit-driven nature of industrial microbiology. For example, fermentation ethanol was abandoned for cheaper chemical methods using petroleum around 1930, but has since become profitable again with rising petroleum prices.
Free Communication of Procedures
Many industrial microbiology procedures are kept secret or patented by companies. This secrecy aims to maintain a competitive edge, causing textbooks to lag in describing current industrial methods.
Patents and Intellectual Property Rights
Governments establish patent laws to encourage inventors to disclose inventions and prevent exploitation without reward.
Patentability Requirements
Inventions must be new, useful, and unobvious to be patentable.
An invention is patentable if it is new, results from inventive activity, and is capable of industrial application; or if it improves upon a patented invention and is industrially applicable.
New: Not part of existing body of knowledge.
Inventive: Not obviously derived from existing knowledge in method, application, combination, or industrial result.
Industrially Applicable: Can be manufactured or used in any industry, including agriculture.
Exclusions from Patentability
Patents cannot be obtained for:
Plant or animal varieties or essentially biological processes for their production, excluding microbiological processes and their products.
Inventions contrary to public order or morality.
Scientific principles and discoveries.
Legal Advice
It is advisable to consult a patent attorney due to the complexity of patent laws. The United States Code Title 35 – Patents (Revised 3 August, 2005) and the UK Patent Act 1977 are examples of patent laws.
Patent Validity
Patents are valid for 20 years in the UK and some other countries, and for 17 years in the United States.
Organizational Set-up in Industrial Microbiology
The organization varies among firms but generally includes:
A culture collection (sourced internally or from public collections and linked to a patent).
Nutrient preparation from raw materials, including saccharification for complex carbohydrates.
Inoculum preparation from lyophilized vials, with purity checks on agar plates.
Scale-up through shake flasks and pilot fermentors to production fermentors.
Extraction methods specific to the end product (organism or metabolite).
Maintenance of sterility using steam, filtration, or chemicals.
Appropriate treatment of air, water, and steam before use.
Disposal of industrial wastes.
Packaging and sales.
Microorganisms Commonly Used
Living things are classified into three groups: Archae, Bacteria, and Eukarya. Industrial microbiology primarily uses bacteria and eukarya.
Archaea
Currently, Archaea are not widely used, but this may change due to their ability to grow under extreme conditions.
Advantages of Microorganisms
Microorganisms have the following advantages over plants or animals as inputs in biotechnology:
Rapid growth: Microorganisms have shorter generation times (e.g., 15 minutes for E. coli) compared to plants and animals resulting in faster product obtention.
Small space requirement: Fermentors can be housed in smaller spaces than plants or animals.
Independence from weather: Microorganisms are not affected by weather conditions.
Resistance to diseases of plants and animals: Microbes are more so resilient to diseases that plants and animals are affected by.
Bacterial Phyla
The bacterial phyla used in industrial microbiology and biotechnology are:
Proteobacteria
Firmicutes
Actinobacteria
Proteobacteria
The Proteobacteria are Gram-negative bacteria divided into five groups, including:
Acetobacter and Gluconobacter: Acetic acid bacteria.
Zymomonas: Produces a lot of alcohol, but not yet widely utilized.
Acetic Acid Bacteria
Acetobacter (peritrichously flagellated) and Gluconobacter (polarly flagellated) have the following properties:
Incomplete oxidation of alcohol to acetic acid for vinegar production.
Gluconobacter lacks the complete citric acid cycle and cannot oxidize acetic acid.
Acetobacter has all citric acid enzymes and can oxidize acetic acid further to .
Tolerance of acid conditions (pH 5.0 or lower).
Under-oxidation of sugars:
Production of glucoronic acid from glucose, galactonic acid from galactose, and arabonic acid from arabinose.
Production of sorbose from sorbitol for ascorbic acid (Vitamin C) manufacture.
Production of pure cellulose in unshaken cultures.
Firmicutes
The Firmicutes are Gram-positive bacteria divided into:
Spore-forming (Bacillus, Clostridium)
Non-spore forming (Lactic Acid Bacteria)
Wall-less (Mycoplasmas, but no industrial organisms).
Spore-Forming Firmicutes
Bacillus spp. (aerobic) are used in enzyme production and can kill insects. For example:
Bacillus papilliae: infects and kills the larvae of the beetles in the family Scarabaeidae
Bacillus thuringiensis is used against mosquitoes.
Clostridia are mainly pathogens.
Non-Spore Forming Firmicutes
Lactic Acid Bacteria:
Includes Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Streptococcus.
Important in food, industrial, and pharmaceutical products.
Lack porphyrins and cytochromes, obtaining energy by substrate-level phosphorylation.
Grow anaerobically but are not killed by oxygen; grow with or without oxygen.
Require amino acids, vitamins, and nucleotides.
Divided into homofermentative (producing only lactic acid) and heterofermentative (producing lactic acid, ethanol, and ).
Uses of Lactic Acid Bacteria:
Desirable characteristics:
Rapid and complete fermentation of cheap raw materials.
Minimal nitrogenous substance requirements.
High yields of stereo-specific lactic acid.
Ability to grow under low pH and high temperature.
Low cell mass production and negligible byproducts.
Choice depends on carbohydrate source (e.g., Lactobacillus delbreuckii ferments sucrose, Lactobacillus helveticus ferments lactose and galactose).
Actinobacteria
Tendency to form filaments. Industrially important members include Actinomycetes and Corynebacterium.
Corynebacterium spp. are important as amino acid secretors.
Actinomycetes:
Branching filamentous hyphae resembling fungi but are bacteria.
Produce secondary metabolites, especially pharmaceuticals.
Streptomyces is well-known for antibiotics.
Primarily soil dwellers.
Eucarya: Fungi
Fungi commonly used in industrial production are classified into:
Phycomycetes (Zygomycetes): Rhizopus and Mucor for enzyme production.
Ascomycetes: Yeasts for ethanol and alcoholic beverages, Claviceps purperea for ergot alkaloids.
Fungi Imperfecti: Aspergillus (aflatoxin) and Penicillium (penicillin).
Basidiomycetes: Agaricus for edible mushrooms.
Characteristics of Microbes in Industrial Microbiology
Microorganisms must meet certain requirements for industrial production:
Ability to grow in a simple medium without requiring additional growth factors.
Rapid and vigorous growth.
Fast production of desired materials.
Absence of toxic or undesirable end products.
Reasonable genetic and physiological stability.
Suitability for product harvest (e.g., yeasts are easier to centrifuge than bacteria).
Physiological requirements that protect against competition (e.g., thermophiles).
Resistance to predators such as Bdellovibrio spp. or bacteriophages.
Low oxygen demand.
Amenability to genetic manipulation.
Industrial Media and Nutrition
An adequate medium is essential for harnessing an organism’s industrial potential. Liquid media are generally preferred.
Basic Nutrient Requirements
Industrial or laboratory microbiological media must satisfy needs for carbon, nitrogen, minerals, growth factors, and water, while lacking inhibitory materials.
Carbon: Usually from carbohydrates like glucose.
Nitrogen: Required by proteins and nucleic acids. For bacteria the average Nitrogen content is 12.5% such that to produce of bacterial cells per liter requires about N..
Minerals: Include P, S, Mg, and Fe. Trace elements include Mn, B, Zn, Cu, and Mo.
Growth factors: Vitamins, amino acids, and nucleotides.
Criteria for Raw Materials
Factors to consider in choosing raw materials:
Cost: Cheaper materials lead to more competitive selling prices. For example, lactose is more suitable than glucose in some processes (e.g. penicillin production). However due to the slower rate of lactose utilization, lactose is typically replaced with glucose.
Availability: The raw material must be readily available, and large stocks need to be maintained.
Transportation costs: Proximity of the user-industry to the production site is important.
Waste disposal: The disposal of waste must be easy and inexpensive.
Uniformity in quality: Constant raw material quality ensures the same final product quality and customer satisfaction.
Adequate chemical composition: The medium must have adequate of carbon, nitrogen, minerals and vitamins for optimum product output.
Presence of precursors: Contains precursors necessary for the synthesis of products. Precursors stimulate secondary metabolites via increasing the product amount or inducing enzyme biosynthesis.
Satisfaction of growth and production requirements of the microorganisms: There are two phases of growth in batch cultivation being the trophophase and the idiophase. Both require different nutrients or different ratios of the same nutrients.
Raw Materials Used
Corn steep liquor: A byproduct of starch manufacture rich in carbohydrates, nitrogen, vitamins, and minerals.
Pharmamedia: Made from cotton-seed embryo and rich in protein and carbohydrates, as well as various minerals.
Distillers solubles: A byproduct of alcohol distillation rich in nitrogen, minerals, and growth factors.
Soya bean meal: Has about 11% nitrogen, and 30% carbohydrate and may be used as animal feed.
It is used particularly in tetracycline and streptomycin fermentations.Molasses: A source of sugar and byproduct of the sugar industry which can be derived from sugar cane or beet.
Sulfite liquor: Aqueous waste from cellulose manufacturing, containing hexose and pentose sugars.
Other Substrates
Alcohols, acetic acid, methane, and fractions of crude petroleum.
Growth Factors
Materials not synthesized by the organism, functioning as enzyme cofactors.
Water
VItal for fermentation, cooling, washing, and cleaning. Requires regular analysis for minerals, color, and pH.
Potential Sources of Carbohydrates
Cassava, sweet potato, yams, cocoyam, millets, rice, and sorghum.
Protein Sources
Peanut meal, blood meal, and fish meal.
Primary and Secondary Metabolites
Products of industrial microorganisms are divided into primary and secondary metabolites.
Primary Metabolites
Produced during active cell growth (trophophase) and essential for survival. Industrial applications include ethanol, citric acid, glutamic acid, lysine, and vitamins.
Secondary Metabolites
Produced after active growth (idiophase) and not essential for survival. Examples include antibiotics. Secondary metabolites are typically absent e.g. E.coli, Salmonella, Shigella. Secondary metabolism is promoted by growth rates slow down and where nutrients are limited.
Industrial Microorganism (Screening, Isolation, Preservation and Improvement)
Initial Steps
Begin with literature, web, and patent searches.
Contact culture collections and regulatory offices for information on useful microbial cultures.
Culture Collections
Provide a rich source of microorganisms. Functions include maintaining the existing collection, collecting new strains, and providing pure, authenticated culture samples.
Isolation from the Environment
Strategies include:
Shotgun approach: Collecting samples from diverse environments and screening for desirable traits.
Objective approach: Sampling from specific sites likely to contain organisms with desired characteristics.
Prior to selection, an initial step is often to kill or repress the proliferation of common organisms and encourage the growth of rare ones.
Enrichment cultures encourage the growth of desired-trait organisms.
Culture Preservation
Involves maintaining pure and stable cultures to prevent contamination, denaturation, and death. Important for preserving productivity after screening and genetic manipulation.
Techniques for Culture Preservation
Storage at reduced temperature:
Agar slants/stabs: Cultures are streaked over solidified nutrient medium and kept in refrigerator at . Lasts for 2-4 months.
Storage at sub-zero temperature: Suspended in glycerol (-20°C, -80°C) or liquid nitrogen (-193°C). Lasts for 6-8 months.
Storage of glass beads: Cultures are suspended with cryoprotectant solution and glass beads. A few beads are removed as needed. Prevents cell death from the freeze thaw process.
Storage in dehydrated forms:
Dried cultures: Commonly used for spore forming bacteria. In this process, strains of Actinomyces have been recovered even after 20 years of storage.
Lyophilization (freeze-drying): Cultures are suspended in a buffer or skimmed milk and are frozen, removing water via sublimation and sealing under vacuum.
Strain Improvement by Genetic Manipulations
Improve productivity through:
Mutation
Protoplast fusion
Gene cloning
Protoplast Fusion
Cell walls from single cells are removed to combine traits and produce hybridizable results. Hybrid cells which exhibit the highest frequency fusion with reproducable are then plated on a selective nutrient medium to select for those cells with the right traits.
Gene Cloning
Gene of interest is cloned such that production of the bio-products is increased. Not only is the concern gene copy number increased, but expression is enhanced via replacing weak gene promoter with a strong promoter.
Industrial Fermentation Process
Fermentors and Fermentation Process
A fermenter is a vessel in which a particular microbe is grown under controlled conditions to produce a desired byproduct or biomass. During fermentation, parameters are tightened for optimum yield and production of by-products.
Features of Fermenter
Non-corrosive cylindrical steel vessel capable of withstanding high temperatures and pressure.
Inlets for gases, media components, acids/alkalis.
Outlets for gases and sample collection.
Ports for sensors (pH, temperature, dissolved oxygen).
Provision for cooling fermentation broth.
Agitator for uniform mixing.
Types of Fermentors
Classified based on design:
Stirred tank bioreactor
Bubble column bioreactor
Airlift bioreactor
Fluidized bed bioreactor
Packed bed bioreactor
Types of Fermentation Processes
Liquid (submerged, cells are suspended in medium)
Solid state (cells adsorbed to solid, nutrient-rich material)
Most fermentations are of the submerged type because they save space engineering design.
Submerged Cultivation
Guarantees a controlled environment for efficient, high-quality product production.
Forms of Submerged / Liquid Fermentation
Batch Fermentation
Known viable cells are inoculated inside of sterilized medium already containing nutrients. Additional nutrients are not added once this process has already started
Batch cultivation can be used for the production of alcoholic beverages, organic acids and amino acids used as flavor enhancers.
Fed Batch Fermentation
Nutrients are gradually and aseptically added over time. This can increase bio-product turnout. Typical food fed-batch fermentations are large-scale production of baker’s Yeast and pure ethanol iii) Continuous fermentation Continuous culture represents an open system. This type of fermentation causes increase amounts of bio-product.
###### Continuous Fermentation
Nutrients are continuously added and culture birth removed such that both are equal and there is no overall volume change in solution. Conditions are predetermined such that to flow rate of incoming nutrient solution promotes constant microbe state (aka. the steady state of growth).
The fermenter can be maintained to be the chemostat or the turbidostat.
The steady state of growth can also be maintained by constantly monitoring the turbidity of the culture medium by use of turbidometer.The typical food fed-batch fermentations are large-scale production of baker’s Yeast and pure ethanol iii) Continuous fermentation Continuous culture represents an open system
Solid Substrate Fermentation
Microorganisms are cultured on the surface of water insoluble substrate and there is a low level of free water. Different substrates and microorganisms are used. In the Western countries, certain food products, industrial enzymes and limited to feed supplements are applied.
Advantages of SSF
Concentrated medium is used; smaller reactor volume; lower capital investment costs; water from the process has a low effluent; agricultural wastes are used as substrates.
Control, By Filtering
Remove smaller bacterial based agents with the use of membrane filter.
Industrial Applications of SSF
Production of mold, ripening cheeses, vegetable fermentation or silage and mushroom cultivation. Mushroom cultivation is considered a special single-cell protein system utilizing agricultural and forestry wastes.
Sterilization
Sterilization of Culture Media and Fermenter
To achieve maximum bio-product output, sterlization needs to be performed because it achieves maximal product turnover and destroys spores.
Batch fermentation media is sterlized by subjecting the medium to for 15 min or an equal time temperature combination. In continuous fermentation case, the meduim is sterlized by subjecting the medium to for 15 - 120 seconds depending on what type of medium it si.
Another type of sterlization involves using a filtration technique, commonly used for heat labile products.
To remove bacterial based pathogens, a membrame cartridge filter is applied.
Control of chemical and physical conditions during incubation
Agitation
Mixing of solid, liquid and gas phases to distribute nutrients and gases. Gas is mixed to ensure nutrient availability and a consistent temperture. Agitation can be applied via stirrer shaft or passing high pressure air through fermenters.
Heat Transfer
Uniform heat transfer is important for proper sterilization. Temperature should be constantly monitored. To control temperature, both heating and cooling are applied by by use of internal coils.
Control and Monitoring
Parameters are controlled using electrodes and acid or alkali addition. Parameters can be measured on or offline i.e. measured using computers and chemicals.
Downstream Processing
Downstream processing (DSP) is the extraction and purification of a biological product from the fermentation broth. In general the stages are the following,
Cell harvesting
Lyses/Breakage of cells
Concentration
Purification
Formulation
Cell harvesting
Solids are separated from the liquid phase such that separation is achieved via flotation, flocculation, filtration and centrifugation. Foam fermentation from flotation is promoted by collector substances such as fatty acids. Flocculation is accelerated by the introduction of salts, hydrocolloids. Rotary drum vacuum filters can also be utilized to seperate out yeast. Based on differential densities, liquid are filtered from solids by using centrifugation.
Lyses/Breakage of cells
Cells are broken by physical or chemical means to release intracellular products,
Physical methods
Ultrasonication, osmotic shock etc are applied.
Chemical Methods
Hydroxides, organic solutions or detergents are applied.
Concentration
n Water is evaporated by applying heat to the supernatant to concentrate materials such that water gets evaporated and product can be retrieved.
2 Liquid-Liquid Extraction:
Used to concentrates (via transfering ) the product from one liquid or another. There are two flavors of this one being the extraction of low molec weight materials, and an extraction of high molecular eight products such as proteins.
Membrane filtration can be applied which is a semi peremeable technique.
Precipitation
Polymers are applied to precipitate out compounds and proteins
Chromatography
Applied to purify different mixtures of compounds.