History and Evolution of Fermentation Technology

 History and Evolution of Fermentation Technology

In human history, fermentation existed as an empirical art—a domestic craft passed down through generations, driven by tradition rather than scientific understanding. Early civilizations successfully produced bread, beer, cheese, and wine by following precise, repetitive rituals. They knew that mixing grain mash with a portion of an older, successful batch of beer would initiate the reaction, but they had no concept of the microscopic life forms carrying out the transformation. Over the past two centuries, this field has undergone a dramatic transformation, shifting from an empirical domestic art into a highly engineered molecular science.  The evolution of fermentation technology is organized into five distinct chronological phases, shifting from empirical art to molecular precision.

Era 1: The Empirical Era (Pre-1800s)

For millennia, fermentation was practised purely as a domestic art form. Civilisations exploited microbial pathways without knowing that living organisms were driving the process.

• Ancient Brewing (4000–6000 BC): Sumerians, Babylonians, and Egyptians utilized wild yeasts to ferment grain mash into beer and grape sugars into wine. Fermentation was viewed as a divine or spontaneous magical event.
• The Orleans Vinegar Process (14th Century): Developed in France, this was the earliest recorded semi-continuous open commercial fermentation method. Wine was introduced into shallow wooden vats exposed to air, allowing a surface mat of wild acetic acid bacteria (Acetobacter) to convert ethanol into vinegar.

Era 2: The Physiological Foundations (1800–1900)

This era transformed fermentation from mystery into science. It was defined by intense academic conflict regarding the true nature of chemical conversions.

·       The Spontaneous Generation & Vitalist Controversy

During the mid-19th century, a fierce scientific debate erupted between two schools of thought - The Mechanistic View - Fermentation is a dead, purely chemical decay process driven by molecular tremors and The Vitalist View - Fermentation is a    physiological process driven strictly by living organisms

The Mechanistic (Chemical) View: Liebig, Berzelius, and Wohler

Liebig argued that fermentation was a form of slow, dead decay. He believed that nitrogenous plant or animal matter (like albumin or gluten), upon exposure to air, entered a state of internal instability and decomposition. This decomposing, dead material supposedly generated atomic vibrations or "tremors." When these vibrations came into contact with stable molecules like sugar, they mechanically shattered the sugar apart, forcing it to rearrange into ethanol and carbon dioxide.  While Liebig acknowledged the presence of yeast in brewing vats, he dismissed it as an accidental, lifeless byproduct of vegetation or a dead precipitate.

The Vitalist (Biological) View: Latour, Schwann, Kutzing and Pasteur

In the late 1830s, Charles Cagniard-Latour, Theodor Schwann, and Friedrich Kutzing independently used early microscopes to observe yeast. They noted that yeast cells were globular, grew by budding, and behaved like microscopic plants. However, their ideas were aggressively ridiculed by the chemical view.  In 1857, Louis Pasteur adopted the Vitalist perspective, aiming to prove that fermentation was an unyielding biological consequence of cellular life.

Pasteur’s entry into the debate was through by a practical industrial crisis. In 1856, a beet-root alcohol manufacturer in Lille, faced a major economic problem. many of his fermentation vats were mysteriously failing, producing a sour, slimy, non-alcoholic grey broth instead of ethanol. Pasteur systematically disproved Liebig's chemical assertions through a series of experiments.

Step 1: Microscopic Differentiation  - Pasteur sampled the contents of both healthy and failed industrial vats and examined them under the microscope. In the healthy vats, he observed large, plump, spherical or oval budding cells—yeast (Saccharomyces cerevisiae). These vats produced abundant ethanol.  In the sour vats, the large yeast cells were absent or dying. Instead, the broth was crowded with vastly smaller, thin, rod-shaped microorganisms (now known as lactic acid bacteria).

Pasteur deduced that fermentation was not a uniform, random decay of dead matter. Specific chemical products were produced by specific, living microorganisms. If dead chemical vibrations were responsible, the same sugar substrate should not yield completely different chemical compounds based on which microscopic shape was present in the broth.

Step 2: Synthetic or chemically defined media experiments - Liebig had asserted that fermentation required the presence of complex, unstable, decaying organic nitrogenous matter (like albumin) to trigger the reaction. Pasteur formulated a completely synthetic, transparent growth medium containing pure distilled water, sucrose, ammonium salts, and ash from burned yeast to provide essential inorganic trace minerals like potassium and phosphate.  He inoculated this transparent, inorganic fluid with a microscopic trace of pure yeast.  The yeast did not decay. Instead, it multiplied, consumed the sugar, and generated a healthy alcoholic fermentation.  Because there was no complex, decomposing organic matter present to provide Liebig's "atomic tremors," the fermentation could only be attributed to the metabolic activities of the growing, living yeast cells assimilating simple nutrients to build their biomass.

Step 3: Demonstrating anaerobiosis (life without air) - Liebig claimed that oxygen from the air was the indispensable trigger that initiated the chemical decay of nitrogenous substances. Pasteur disproved this by demonstrating that yeast could thrive and ferment in the absence of atmospheric oxygen.  He introduced the concept of anaerobiosis, discovering that yeast is a facultative anaerobe. In the absence of oxygen, yeast cells converted sugar into alcohol with higher efficiency. He summarised this phenomenon with his famous declaration: "Fermentation is life without air".

This has resulted in the birth of Industrial Biotechnology.  By establishing that distinct microbes produce distinct products, Pasteur taught industries that they must maintain pure cultures. To prevent "diseases" of wine and beer (souring), he introduced Pasteurization (heating fluids to 55–60°C in the absence of air) to selectively kill bacteria before inoculating with pure yeast strains.

· The Buchner Revolution (1897)

The Vitalist theory held that fermentation could only happen within an intact, living cell body. Eduard Buchner disproved this. By grinding yeast cells with quartz sand and diatomaceous earth, he prepared a completely cell-free yeast juice.  When he added this sterile juice to a concentrated sugar solution, it bubbled vigorously, producing carbon dioxide and ethanol. Buchner discovered cell-free fermentation mediated by a soluble intracellular enzyme complex (he called it as Zymase). This discovery bridged biochemistry and microbiology, proving that cells are chemical engines powered by enzymes. Buchner was awarded the Nobel Prize in Chemistry in 1907.

Era 3: True Industrial Scaling & Solvents Era (1900–1940)

The pressures of World War I forced countries to scale up fermentation from small glass bottles to massive industrial operations to produce raw materials for military weapons.

The ABE Fermentation - During World War I, the British military faced a critical shortage of acetone, an essential solvent used to manufacture cordite (smokeless gunpowder). A severe blockade cut off Britain's traditional imports of wood-derived acetone. During this time, Chaim Weizmann isolated a novel anaerobic bacterium, Clostridium acetobutylicum. He developed a massive, large-scale fermentation process that converted grain starch into Acetone, Butanol, and Ethanol (ABE) at a specific ratio ().  This  acetone yield provided exactly what the British military required.

The ABE process required the design of large, sealed fermenter tanks ( to  liters). Clostridium acetobutylicum is an obligate anaerobe—even trace exposure to atmospheric oxygen stops its growth and kills active vegetative cells. Furthermore, wild airborne bacteria (like lactic acid producers) could easily outgrow the clostridia if given the chance.  To overcome these hurdles, engineers built the world’s first dedicated, large-scale industrial fermentation facilities.  This marked the birth of modern industrial biotechnology, demonstrating that bacteria could replace traditional chemical synthesis for bulk solvent production.

Fed-Batch Cultivation of Baker’s Yeast - During the same period, the production of Baker's yeast (S. cerevisiae) faced a major biological barrier: the Crabtree Effect. When yeasts are given high concentrations of sugar, they automatically switch to fermenting it into ethanol, even if there is plenty of oxygen around. This caused waste of sugar and lowered cell biomass yields.  To bypass this, engineers invented Fed-Batch cultivation. Instead of dumping all the sugar into the tank at the start, nutrients are pumped in incrementally at low concentrations. This keeps the cells locked in pure aerobic respiration, maximizing biomass output while completely preventing the formation of toxic alcohol.

Era 4: Submerged Aerated Engineering Era (1940–1970)

The urgent demand for mass-produced penicillin during World War II completely reshaped bioprocess engineering, driving the creation of the modern bioreactor.

The Penicillin Scale-Up Crisis - When Alexander Fleming discovered penicillin in 1928, it was produced in minute amounts by the mold Penicillium notatum. Early attempts to harvest it required growing the fungus on the surface of thousands of shallow bottles, which was slow and highly prone to contamination.  To solve this crisis, Howard Florey, Ernst Chain, and a team of engineers revolutionized the process between 1940 and 1945:

1. Strain Switch: They replaced Fleming's original strain with a high-yielding version of Penicillium chrysogenum.
2. The Submerged Bioreactor: Because this fungus requires massive amounts of oxygen, scientists could no longer use shallow pans. They engineered deep, enclosed steel vessels where the mold grew fully submerged throughout the liquid broth. 

3.     Penicillium is an obligate aerobe. It requires enormous amounts of dissolved oxygen to synthesize its secondary metabolites. If the mold was submerged in liquid without a constant air supply, it literally suffocated and stopped producing the drug.  To deliver oxygen to these deep tanks without introducing contaminants, engineers invented the Sparger - A perforated ring at the base of the tank that continually pumps sterile, pressurized air into the broth and

4.     The Mechanical Agitator (Impeller): High-speed internal blades that break up incoming air into tiny bubbles, ensuring maximum oxygen distribution while blending the thick fungal growth.

5.     Sterilization Protocols: Systems designed to sterilize large volumes of media using high-pressure steam, along with specialized air filters to prevent contamination over long fermentation runs.

Era 5: The Biotechnology & Molecular Era (1970–Present)

The birth of genetic engineering completely separated fermentation technology from its traditional reliance on natural, wild-type microbes.

• Recombinant DNA (rDNA) technology (1973): Herbert Boyer and Stanley Cohen successfully sliced a specific gene out of one organism and pasted it into a bacterial plasmid. This technological leap allowed scientists to turn simple microbes into highly specialised cellular factories.
• Insulin (1978/1982): Scientists successfully inserted the human insulin gene into Escherichia coli. By 1982, Humulin became the world’s first commercially approved recombinant pharmaceutical product made via fermentation, replacing insulin extracted from pig and cattle pancreases.
• Modern Metabolic Engineering: Today, industrial microbiologists do not just rely on random mutations. They use precision tools like CRISPR-Cas9 and computer-guided metabolic models to completely modify a cell's metabolic pathways. By deleting genes that make unwanted side products and overexpressing target genes, we can engineer microbes that convert cheap sugars into complex bioplastics, biofuels, monoclonal antibodies, etc with near-perfect efficiency.

Scope and Importance of Industrial Microbiology

 Scope and Importance of Industrial Microbiology

Industrial Microbiology is a specialised branch of applied microbiology that involves the screening, improvement, cultivation, and use of microorganisms for large-scale, economically driven production of commercially useful products.

While laboratory research focuses on pure biochemical pathways, industrial microbiology is profit-oriented. It operates at the intersection of microbiology, biochemistry, metabolic engineering, and biochemical engineering.

Core Characteristics of an Industrial Process

An industrial microbial process must meet strict economic and operational criteria:

• The organism must be genetically stable, non-pathogenic, and easily cultivable in mass quantities.
• Media must be cheap and readily available (e.g., molasses, corn steep liquor, whey).
• Yield must be high enough to justify the costly optimization of downstream processing (the extraction and purification of the product).

An industrial process can be divided into three operational segments:

Upstream Processing (USP): Includes producer strain selection, strain improvement, and media formulation.

Bioreactor Fermentation: The core conversion phase where the environments (pH, temperature, dissolved oxygen) are kept under strict automated control to allow growth of microbes to produce the desired product.

Downstream Processing (DSP): The multi-step operations (filtration, centrifugation, chromatography, crystallisation) needed to separate, concentrate, and purify the product from fermentation broths.

Primary Driver in Industrial Microbiology is Economic profit, yield optimisation, and scalable commercialization.  The working scale is 1,000 to >500,000 liters in giant fermenters and the aim is the production of pure, concentrated, marketable biochemicals, biomass, or metabolites.

The Scope of Industrial Microbiology

The modern scope of industrial microbiology spans several trillion-dollar sectors globally. Rather than relying on traditional synthetic chemistry—which often requires high pressures, toxic solvents, and high temperatures—industries favor microbes because they function under ambient, biocompatible conditions.

A. Pharmaceutical and Medical Biotechnology

Microbes are the primary source for many life-saving therapeutics:

• Antibiotics: Mass submerged production of secondary metabolites like -lactams (Penicillin from Penicillium chrysogenum), aminoglycosides (Streptomycin from Streptomyces griseus), and macrolides.
• Recombinant Therapeutics: Inserting human genes into E. coli or Saccharomyces cerevisiae to produce human insulin, interferon, and human growth hormone.
• Steroid Transformation: Using Rhizopus nigricans or Corynebacterium species to perform stereospecific hydroxylation of progesterone, drastically cutting down the chemical steps needed to manufacture anti-inflammatory cortisone and birth control pharmaceuticals.

B. Food, Beverage, and Nutritional Industries

• Fermented Beverages: Industrial-scale ethanol production using S. cerevisiae variants for the brewing, distilling, and wine making industries.
• Organic Acids: Production of citric acid by Aspergillus niger via sugar fermentation (used as an antioxidant and acidulant in beverages), and lactic acid by Lactobacillus species for food preservation.
• Amino Acids: Bulk production of L-Glutamic acid by Corynebacterium glutamicum (for MSG production) and L-Lysine (an essential animal feed supplement).

C. Enzymes

Microbial enzymes are highly efficient and biodegradable alternatives to synthetic chemical catalysts:

• Amylases and Glucoamylases: Produced by Bacillus subtilis or Aspergillus oryzae to hydrolyze starches into high-fructose corn syrups for baking and brewing.
• Proteases and Lipases: Alkaline-tolerant versions isolated from Bacillus strains used in laundry detergents to break down protein and lipid stains.
• Pectinases: Used in the commercial juice industry for clarifying fruit juices and increasing extraction yields.

D. Bioenergy, Biofuels, and Green Chemicals

As fossil fuels decline, microbes offer a sustainable alternative path for chemical energy production:

• Bioethanol: Large-scale fermentation of simple sugars and lignocellulosic biomass by yeast strains to produce fuel-grade ethanol.
• Biogas (Methane): Anaerobic digestion of organic municipal and agricultural wastes by methanogenic archaeal consortia.
• Bioplastics: Bacterial synthesis of Polyhydroxyalkanoates (PHAs) like Polyhydroxybutyrate (PHB) by Cupriavidus necator, which serves as a fully biodegradable alternative to petrochemical plastics.

E. Environmental Management & Bioremediation

Industrial processes are also deployed to mitigate anthropogenic pollution:

• Bioremediation: The application of specialized microbial strains (such as Pseudomonas putida) to degrade xenobiotics, persistent organic pollutants, and crude oil spills.
• Bioleaching: Using acidophilic chemolithotrophs like Thiobacillus ferrooxidans to extract metals (such as copper, uranium, and gold) from low-grade ores, minimizing traditional mining pollution.

 

Transport phenomena in bioprocess

 

Biomass maintenance – the focal point of the process.

The growth of biomass either as a primary product (bakers yeast) or as a cell factory producing primary or secondary metabolites is the main aim of the bioprocess

Ensuring that ideal conditions for biomass growth and maintenance are in place is the engineers responsibility.

·         Biomass often requires oxygen (aerobic growth). Oxygen is sparingly soluble in aqueous solutions. Therefore the challenge is there to provide adequate levels of oxygen at all stages of the process

·         Providing adequate oxygen transfer requires considerable kinetic energy input. This energy will eventually become heat energy. This heat has to be removed in order to maintain the optimal temperature for biomass growth (30^oC for yeast and some bacteria, 37^oC for animal cells and microbial pathogens).

·         Microbial metabolism also results in heat evolution. This heat has also to be dealt with.

The specific oxygen uptake rate increases with increase in the dissolved oxygen concentration up to a certain point (referred to as C_crit) above which no further increase in oxygen uptake rate occurs. Thus, maximum biomass production may be achieved by satisfying the organism's maximum specific oxygen demand by maintaining the dissolved oxygen concentration greater than the critical level. If the dissolved oxygen concentration falls below the critical level then the cells may be metabolically disturbed.  Oxygen is normally supplied to microbial cultures in the form of air, this being the cheapest available source.

The transfer of oxygen from air to the cell during fermentation occur in a number of steps:

(i) The transfer of oxygen from an air bubble into solution.

(ii) The transfer of the dissolved oxygen through the fermentation medium to the microbial cell.

(iii) The uptake of the dissolved oxygen by the cell.

Defining the mass transfer problem

There are eight resistances to mass transfer in this system.

1. The gas film
2. The gas-liquid interface
3. The liquid film at the GL interface
4. The bulk liquid
5. The liquid film surrounding the solid
6. At the liquid-solid interface
7. In the solid phase
8. At the reaction sites in the solid phase

Reactions 1-7 are purely physical and occur in series.

Assuming that oxygen transfer to the bulk liquid phase is limited by the liquid layer at the gas-liquid interface and that the bulk liquid phase is well mixed.

Oxygen Transfer rate   

                                

Where….

 is the concentration of oxygen in the liquid layer adjacent to the gas-liquid interface at infinite time (saturation concentration)

C_L is the bulk liquid oxygen concentration

Lets call the constant of proportionality k_La

Integrating results in the following…

DETERMINATION OF KLa VALUES

The sulphite oxidation technique

The measurement of dissolved oxygen concentrations relies on the rate of conversion of a 0.5 M solution of sodium sulphite to sodium sulphate in the presence of a copper or cobalt catalyst:

Na₂SO₃ + 0.5O_z = Na₂SO₄

The rate of reaction is such that as oxygen enters solution it is immediately consumed in the oxidation of sulphite, so that the sulphite oxidation rate is equivalent to the oxygen-transfer rate.

The procedure is carried out as follows: the fermenter is batched with a 0.5 M solution of sodium sulphite containing 10^-3 M Cu₂⁺ ions and aerated and agitated at fixed rates; samples are removed at set time intervals (depending on the aeration and agitation rates) and added to excess iodine solution which reacts with the unconsumed sulphite, the level of which may be determined by a back titration with standard sodium thiosulphate solution. The volumes of the thiosulphate titrations are plotted against sample time and the oxygen transfer rate may be calculated from the slope of the graph.

Advantage is simplicity and, also, the technique involves sampling the bulk liquid in the fermenter and, therefore, moves some of the problems of conditions through the volume of the vessel.

However, the procedure is time consuming (one determination taking up hours, depending on the aeration and agitation and is mostly inaccurate.

Gassing-out techniques

Depends upon monitoring the increase in dissolved oxygen concentration of a solution during aeration and agitation. The oxygen transfer rate will decrease during the period of aeration as the solution get saturated

To monitor the increase in dissolved oxygen over an adequate range it is necessary first to decrease the oxygen level to a low value. Two methods have been employed to achieve this lowering of the dissolved oxygen concentration - the static method and the dynamic method.

THE STATIC METHOD OF GASSING OUT

In this technique the oxygen concentration of the solution is lowered by gassing the liquid out with nitrogen gas, so that the solution is 'scrubbed' free of oxygen. The deoxygenated liquid is then aerated and agitated and the increase in dissolved oxygen monitored using some form of dissolved oxygen probe.

THE DYNAMIC METHOD OF GASSING OUT

The respiratory activity of a growing culture in the fermenter is used to lower the oxygen level prior to aeration. The procedure involves stopping the supply of air to the fermentation which results in a linear decline in the dissolved oxygen concentration due to the respiration of the culture.

The dynamic gassing-out method has the advantage over the previous methods of determining the KLa during an actual fermentation and may be used to determine K L a values at different stages in the process. The technique is rapid and only requires the use of a dissolved-oxygen probe, of the membrane type.

A major limitation in the operation of the technique is the range over which the increase in dissolved oxygen concentration may be measured. It is Important allow the oxygen concentration to drop during the deoxygenation step.

The oxygen-balance technique

The KLa of a fermenter may be measured during fermentation by the oxygen balance technique which determines, the amount of oxygen transferred into solution in a set time interval. The procedure involves measuring the following parameters:

(i) The volume of the broth contained in the vessel

(ij) The volumetric air flow rates measured at the air inlet and outlet

(iii) The total pressure measured at the fermenter air inlet and outlet

(iv)The temperature of the gases at the inlet and outlet

(v) The mole fraction of oxygen measured at the inlet and outlet

The oxygen-balance technique appears to be the simplest method for the assessment of KLa and has the advantage of measuring aeration efficiency during fermentation. The sulphite oxidation and static gassing out techniques have the disadvantage of being carried out using either a salt solution or an uninoculated, sterile fermentation medium.

The effect of medium and culture rheology on KLa

Fluids may be described as Newtonian or non-newtonian depending on whether their rheology characteristics obey Newton's law of viscous flow.  A Newtonian liquid has a constant viscosity regardless of shear, so that the viscosity of a Newtonian fermentation broth will not vary with agitation rate. However, a non-Newtonian liquid does not obey Newton's law of viscous flow and does not have a constant viscosity.

A fermentation broth consists of the liquid medium in which the organism grows, the microbial biomass and any product which is secreted by the organism. Thus, the rheology of the broth is affected by the composition of the original medium and its modification by the growing culture, the concentration and morphology of the biomass and the concentration and rheological properties of the microbial products. Therefore, it should be apparent that fermentation broths vary widely in their rheological properties and significant changes in broth rheology may occur during fermentation.

Highly viscous non-Newtonian broths of fungal and streptomycete fermentations present major difficulties in oxygen provision.

The effect of the degree of agitation

It has been demonstrated to have a profound effect on the oxygen-transfer efficiency of an agitated fermenter.

(i) Agitation increases the area available for oxygen transfer by dispersing the air in the culture fluid in the form of small bubbles.

(ij) Agitation delays the escape of air bubbles from the liquid.

(iij) Agitation prevents coalescence of air bubbles.

(iv) Agitation decreases the thickness of the liquid film at the gas-liquid interface by creating turbulence in the culture fluid.

Bioreactors: Essential Requirements

• Support high biomass concentration
• Maintain sterile conditions
• Provide adequate aeration (if aerobic)
• Agitation (homogeneous suspension, mass transfer)
• Removal of generated heat (temperature control)
• Shear conditions appropriate to biological system

Mass transfer in the bioreactor

All biochemical reactors of significance involve hetrogenous systems generally with more than one phase

To ensure effective biotransformation, interphase mass transfer must occur between these phases

Typically the transfer of oxygen from the gas phase to the liquid phase (where it is sparingly soluble) is the most difficult mass transfer problem in the reactor.

Another mass transfer problem is the removal of CO₂, a byproduct of metabolism from the fermentation broth.

Definition of mass transfer coefficients

Example: oxygen

 

Design of a fermenter

Design of a fermenter

Construction materials

The fermenter must withstand repeated steam sterilization cycles. Glass and/or stainless steel are generally used.

On laboratory scale glass is mainly used. Glass gives smooth surfaces, is non-toxic, corrosion proof and it is usually easy to examine the interior of the vessel.

Two basic designs where glass is used as the fermenter body material are

1. A glass vessel with a round or flat bottom and a top flanged carrying plate

2. A glass cylinder with stainless-steel top and bottom plates

At pilot and industrial scale, vessels are normally constructed of stainless steel or mild-steel.  The corrosion resistance of stainless steel is an advantage. The inclusion of nickel, chromium, molybdenum, tungsten, silicone, etc in steel enhances their engineering properties.

In a vessel there will be joints between glass and glass or glass and metal or metal and metal joints such as between a fermenter vessel and a detachable top or base plate. A reliable aseptic seal should be made between such joints. With glass and metal, a gasket seal, a lip seal or an O ring seal may be used. In metal to metal joints only an O ring is suitable. O ring seal ensures a good liquid- and/or gas-tight joint despite the glass or metal expanding or contracting at different rates with changes in temperature during a sterilization cycle or an incubation cycle. Nitryl or butyl rubbers are normally used for these seals as they will withstand fermentation process conditions.

Temperature control

In a fermenter there should be provision for temperature control. Heat will be generated by microbial activity and also through mechanical agitation.  If the heat generated by these two processes is not ideal for the particular manufacturing process, then we may have to add or remove heat from the system.

On a laboratory scale extra heat is generally provided by placing the fermenter in a thermostatically controlled bath, or by the use of internal heating coils or a heating jacket through which water is circulated or by a silicone heating jacket.

In larger vessels internal coils are used and cold water is circulated to reduce the temperature to achieve the correct temperature. 

Aeration and agitation

Primary purpose of aeration is to provide microorganisms with sufficient oxygen for metabolic requirements, while agitation ensure the uniform suspension of microbial cells in a homogeneous nutrient medium. Some fermenters work with mechanical agitation system while some others employ non mechanical aeration-agitation system.

The structural components of the fermenter involved in aeration and agitation are:

(a) The agitator (impeller).

(b) Stirrer glands and bearings.

(c) Baffles.

(d) The aeration system (sparger).

The agitator or impeller

The agitator is required for mixing, for fluid and gas-phase mixing, air dispersion, oxygen transfer, heat transfer, suspension of solid particles and maintaining a uniform environment throughout the vessel contents.

Agitators are classified as disc turbines, vaned discs, open turbines of variable pitch and propellers.

The disc turbine consists of a disc with a series of rectangular vanes set in a vertical plane around the circumference.  The vaned disc has a series of rectangular vanes attached vertically to the underside. Air from the sparger hits the underside of the disc and is displaced towards the vanes where the air bubbles are broken up into smaller bubbles.

The vanes of an open turbine and the blades of a marine propeller are attached directly to a boss on the agitator shaft. In this case the air bubbles do not initially hit any surface before dispersion by the vanes or blades.

Stirrer glands and bearings

The satisfactory and aseptic sealing of the stirrer shaft assembly is difficult.  The stirrer shaft can enter the vessel from the top, side or bottom of the vessel. Top entry is most commonly used, but bottom entry may be advantageous if more space is needed on the top plate for entry ports, and also shorter shaft will be needed here which permits higher stirrer speeds without shaft whipping. But  bottom entry stirrers would be submerged

Four basic types of seal assembly used are the stuffing box (packed-gland seal), the simple bush seal, the mechanical seal and the magnetic drive.

Most modern fermenter incorporate mechanical seals instead of stuffing boxes and packed glands. Mechanical seals are more expensive, but are more durable and less likely results in contamination or leakage. Magnetic drives which are quite expensive, are being used in some animal cell culture vessels.

The stuffing box or packed-gland seal

The shaft is sealed by several layers of packing rings of asbestos or cotton yarn, pressed against the shaft by a gland follower. At high stirrer speeds the packing wears quickly and excessive pressure may be needed to ensure tightness of fit. The packing may be difficult to sterilize and it is necessary to check and replace the packing rings regularly.

The mechanical seal

The mechanical seal assembly is composed of two parts, one part is stationary in the bearing housing, the other rotates on the shaft, and the two components are pressed together by springs or expanding bellows. Steam condensate is used to lubricate and cool the seals during operation and the steam also serve as a containment barrier.

Magnetic Drives

In magnetic drive the impeller shaft does not pierce the vessel.  It consists of two magnets: one driving and one driven. The driving magnet is held in bearings in a housing on the outside of the head plate and connected to a drive shaft. The driven magnet is placed on one end of the impeller shaft, held in bearings in a housing on the inner surface of the headplate. Using magnets power will be transmitted.  This is ideal for minimizing contamination.

Baffles

Four to eight baffles are normally incorporated into fermenter to prevent vortex and to improve aeration efficiency.  These are metal strips roughly one-tenth of the vessel diameter and attached radially to the wall

Sparger

A sparger is a device for introducing air into the liquid in a fermenter. Three basic types of sparger are porous sparger, the orifice sparger (a perforated pipe) and the nozzle sparger (an open or partially closed pipe). A combined sparger-agitator are also used.

Porous sparger

The porous sparger of sintered glass, ceramics or metal, are used on a laboratory scale vessel. The bubble size produced from such spargers is 10 to 100 times larger than the pore size of the aerator.  There is a problem of the fine holes becoming blocked by growth of the microbial culture.

Orifice sparger

These are perforated pipes. In small stirred fermenters the perforated pipes are arranged below the impeller in the form of crosses or rings (ring sparger).

Nozzle sparger

Most modern mechanically stirred laboratory to industrial scale fermenter have a single open or partially closed pipe as a sparger which is positioned below the impeller.

Combined sparger-agitator

The combined sparger-agitator introduce the air via a hollow agitator shaft and emit it through holes drilled in the disc between the blades. The design gives good aeration.

Sterilization of the fermenter

The fermenter may be steam sterilized under pressure. The medium may be sterilized in the vessel or sterilized separately and subsequently added aseptically to the fermenter. Steam should be introduced through all the entry and exit points and steam should be allowed exit through the air outlet. Steam should reach all parts of the equipment and each drainage point in the pipework should be fitted with a steam trap.

Sterilization of the air supply

Sterile air will be required in very large volumes in many aerobic fermentation processes.  Filter sterilization is generally adopted.  Most fermenters are fitted with cartridge-type filters. 

Sampling

The sampling points should be working while maintaining sterility. A sterile barrier must be maintained between the fermenter contents and the exterior when the sample port is not being used and it must be sterilizable after use.

Feed ports

Additions of nutrients and acid/alkali to small fermenters are normally made via silicone tubes which are autoclaved separately. It can be aseptically connected to fermenter and pumped by a peristaltic pump. The ports which are used intermittently can be sterilized in situ with steam.

Sensor probes

Double '0' ring seals are used to provide an aseptic seal for glass electrodes in stainless steel housings.

Foam control

In any fermentation it is important to minimize foaming. When excessive foaming, the filters become wet and result in contamination. Also there may be loss of all or part of the contents of the fermenter. Antifoams may be used or Foam breakers are used.  Foam breakers break down foam by an impact mechanism by a rotating mechanism inside the fermenter.

Valves and steam traps

Valves attached are used for controlling the flow of liquids and gases. The valves may be:

1. Simple ON/OFF valves which are either fully open or fully closed.

2. Valves which provide coarse control of flow rates.

3. Valves which may be adjusted very precisely so that flow rates may be accurately controlled.

4. Safety valves which are constructed in such a way that liquids or gases will flow in only one direction.

The valves may open and close by

(a) raising or lowering the blocking unit with a screw thread (rising stem)

(b) a drilled sphere or plug or a disc rotating in between two bearings

(c) a rubber diaphragm or tube which is pinched.

Gate valves

In this valve, a sliding disc is moved in or out of the flow path by turning the stem of the valve. It is suitable for general purposes on a steam or a water line for use as fully open or fully closed.  It is not suitable for aseptic conditions.

Globe valves

In this valve a horizontal disc or plug is raised or lowered in its seating to control the rate of flow.  It is used for regulating the flow of water or steam.  It is not suitable for aseptic operation.

Piston valves

These are similar to a globe valve except that the flow is controlled by a piston passing between two packing rings. This is very efficient under aseptic operation. But there may be blockage problems with mycelial cultures.

Needle valves

The needle valve is similar to the globe valve, except that the disc is replaced by a tapered plug or needle fitting into a tapered valve seat. The valve can be used to give fine control of steam or liquid flow.  It is not generally used for aseptic applications.

Plug valves

In this valve there is a parallel or tapered plug sitting in a housing through which an orifice has been been machined. When the plug is turned through 90° the valve is fully open and the flow path is determined by the cross-sectional area of orifice. It can provide good flow control.

Ball valves

This valve has been developed from the plug valve. The valve element is a stainless-steel ball through which an orifice is machined. The ball is sealed inside a pipeline. The valve is suitable for aseptic operation, can handle mycelial broths and can be operated under high temperatures and pressures.

Butterfly valves

The butterfly valve consists of a disc which rotates about a shaft in a housing. The disc closes against a seal to stop the flow of liquid. It is not suitable for aseptic operation.

Pinch valves

In the pinch valve a flexible sleeve is closed by a pair of pinch bars or some other mechanism which can be operated by compressed air remotely or automatically. The flow rate can be controlled. The valve is suitable for aseptic operation and suitable with mycelial cultures.

Diaphragm valve

Like the pinch valve, the diaphragm valve makes use of a flexible closure.  This valve is very suitable for aseptic operation provided that the diaphragm is of a material which will withstand

repeated sterilization. The valve can be used for ON/OFF, flow regulation, and for steam.

The most suitable valve

Among these group of valves, globe and butterfly valves are most commonly used for ON/OFF applications, gate valves for crude flow control, needle valves for accurate flow control and ball, pinch or diaphragm valves for all sterile uses.

Check valves

The purpose of the check valve is to prevent accidental reversal of flow of liquid or gas in a pipe due to breakdown in some part of the equipment. There are three basic types of valve: swing check, lift check and combined stop and check.

Pressure-reduction valves

Pressure-reduction valves are incorporated into pipelines when it is necessary to reduce from a higher to a lower pressure.

Pressure-retaining valves

A pressure-retaining valve will maintain pressure in the pipeline upstream.

Safety valves

Safety valves must be incorporated into every air steam line and vessel which is subject to pressure

ensure that the pressure will never exceed the upper limit recommended by the manufacturer.

Steam traps

In all steam lines it is essential to remove any steam condensate which accumulates in the piping to ensure optimum process conditions. This is achieved by steam traps, which will collect and remove any condensate at appropriate points in steam lines automatically.