Single-cell protein

Single-cell protein (SCP) refers to edible unicellular microorganisms. The biomass or

protein extract from pure or mixed cultures of algae, yeasts, fungi or bacteria may be
used as an ingredient or a substitute for protein-rich foods, and is suitable for human
consumption or as animal feeds.

Whereas industrial agriculture is marked by a high water footprint, high land use,

biodiversity destruction, general environmental degradation and contributes to climate
change by emission of a third of all greenhouse gases, production of SCP does not
necessarily exhibit any of these serious drawbacks. As of today, SCP is commonly grown
on agricultural waste products, and as such inherits the ecological footprint and water
footprint of industrial agriculture. However, SCP may also be produced entirely
independent of agricultural waste products through autotrophic growth.[4] Thanks to
the high diversity of microbial metabolism, autotrophic SCP provides several different
modes of growth, versatile options of nutrients recycling, and a substantially increased
efficiency compared to crops.

Single-cell proteins develop when microbes ferment waste materials (including wood,

straw, cannery, and food-processing wastes, residues from alcohol production,
hydrocarbons, or human and animal excreta). The problem with extracting single-cell
proteins from the wastes is the dilution and cost. They are found in very low
concentrations, usually less than 5%. Engineers have developed ways to increase the
concentrations including centrifugation, flotation, precipitation, coagulation, and
filtration, or the use of semi-permeable membranes.

The single-cell protein must be dehydrated to approximately 10% moisture content

and/or acidified to aid in storage and prevent spoilage. The methods to increase the
concentrations to adequate levels and the de-watering process require equipment that
is expensive and not always suitable for small-scale operations. It is economically
prudent to feed the product locally and soon after it is produced.

Microorganisms
 Yeast: Saccharomyces cerevisiae, Pichia pastoris, Candida utilis, Torulopsis,
Geotrichum candidum
 Fungi (Mycoprotein): Aspergillus oryzae, Fusarium venenatum, Sclerotium
rolfsii, Polyporus, Trichoderma, Scytalidium acidophilum
 Bacteria: Rhodobacter capsulatus 
 Algae: spirulina (dietary supplement),Chlorella

Advantages

Large-scale production of microbial biomass has many advantages over the traditional
methods for producing proteins for food or feed.

1. Microorganisms have a much higher growth rate (algae: 2–6 hours, yeast: 1–3
hours, bacteria: 0.5–2 hours). This also allows to select for strains with high
yield and good nutritional composition quickly and easily compared to breeding.
2. Whereas large parts of the crop, such as stems, leaves and roots are not edible,
single-cell microorganisms can be used entirely. Whereas parts of the edible
fraction of crops contains is undigestible, many microorganisms are digestible at
a much higher fraction.
3. Microorganisms usually have a much higher protein content of 30–70% in the
dry mass than vegetables or grains. The amino acid profiles of many SCP
microorganisms often have excellent nutritional quality, comparable to a hen's
egg.
4. Some microorganisms can build vitamins and nutrients which eukaryotic
organisms such as plants cannot produce or not produce in significant amounts,
including vitamin B12.
5. Microorganisms can utilize a broad spectrum of raw materials as carbon sources
including alkanes, methanol, methane, ethanol and sugars. What was considered
"waste product" often can be reclaimed as nutrients and support growth of
edible microorganisms.
6. Like plants, autotrophic microorganisms are capable to grow on CO2. Some of
them, such as bacteria with the Wood–Ljungdahl pathway or the reductive
 TCA can fix CO2 between 2-3, up to 10 times more efficiently than plantswhen
also considering the effects of photoinhibition.
7. Some bacteria, such as several homoacetogenic clostridia are capable to
perform syngas fermentation. This means they can metabolize synthesis gas, a
gas mixture of CO, H2 and CO2 that can be made by gasification of residual
intractable biowastes such as lignocellulose.
8. Some bacteria are diazotrophic, i.e. they can fix N2 from the air and are thus
independent of chemical N-fertilizer, whose production, utilization and
degradation causes tremendous harm to the environment, deteriorates public
health, and fosters climate change.
9. Many bacteria can utilize H2 for energy supply, using enzymes
called hydrogenases. Whereas hydrogenases are normally highly O2-sensitive,
some bacteria are capable of performing O2-dependent respiration of H2. This
feature allows autotrophic bacteria to grow on CO2 without light at a fast growth
rate. Since H2 can be made efficiently by water electrolysis, in a manner of
speaking, those bacteria can be "powered by electricity".
10. Microbial biomass production is independent of seasonal and climatic variations,
and can be easily shielded from extreme weather events that are expected to
cause crop failures with the ongoing climate-change. Light-independent
microorganisms such as yeasts can continue to grow at night.
11. Cultivation of microorganisms generally has a much lower water footprint than
agricultural food production. Whereas the global average blue-green water
footprint (irrigation, surface, ground and rain water) of crops reaches about
1800 liters per kg crop due to evaporation, transpiration, drainage and runoff,
closed bioreactors producing SCP exhibits none of these causes.
12. Cultivation of microorganisms does not require fertile soil and therefore does
not compete with agriculture. Thanks to the low water requirements, SCP
cultivation can even be done in dry climates with infertile soil and may provide a
means of fail-safe food supply in arid countries.
13. Photosynthetic microorganisms can reach a higher solar-energy-conversion
efficiency than plants, because in photobioreactors supply of water, CO 2 and a
balanced light distribution can be tightly controlled.
 14. Unlike agricultural products which are processed towards a desired quality, it is
easier with microorganisms to direct production towards a desired quality.
Instead of extracting amino acids from soy beans and throwing away half of the
plant body in the process, microorganisms can be genetically modified to
overproduce or even secrete a particular amino acid. However, in order to keep
a good consumer acceptance, it is usually easier to obtain similar results by
screening for microorganisms which already have the desired trait or train them
via selective adaptation.

Disadvantages

Although SCP shows very attractive features as a nutrient for humans, however there
are some problems that deter its adoption on global basis:

 Fast growing microorganisms such as bacteria and yeast tend to have a high
concentration of nucleic acid, notably RNA. Levels must be limited in the diets
of monogastric animals to <50 g per day. Ingestion of purine compounds arising
from RNA breakdown leads to increased plasma levels of uric acid, which can
cause gout and kidney stones. Uric acid can be converted to allantoin, which is
excreted in urine. Nucleic acid removal is not necessary from animal feeds but is
from human foods. A temperature hold at 64 °C inactivates fungal proteases and
allows. However, this problem can be remediated.[20] One common method consists
in a heat treatment which kills the cells, inactivates proteases and allows
endogenous RNases to hydrolyse RNA with release of nucleotides from cell to
culture broth.
 Similar to plant cells, the cell wall of some microorganisms such as algae and
yeast contain non-digestible components, such as cellulose. The cells of some kind of
SCP should be broken up in order to liberate the cell interior and allow complete
digestion.
 Some kind of SCP exhibits unpleasant color and flavors.
 Depending on the kind of SCP and the cultivation conditions, care must be taken
to prevent and control contamination by other microorganisms because
contaminants may produce toxins such as mycotoxins or cyanotoxins. An interesting
approach to address this problem was proposed with the fungus Scytalidium
 acidophilum which grows at a pH as low as 1. This allows to hydrolyse paper wastes
to a sugar medium and creates aseptic conditions at low-cost.
 Some yeast and fungal proteins tend to be deficient in methionine.
BIOFILM

A biofilm comprises any group of microorganisms in which cells stick to each other and

often also to a surface. These adherent cells become embedded within a
slimy extracellular matrix that is composed of extracellular polymeric substances (EPS).
The cells within the biofilm produce the EPS components, which are typically
a polymeric conglomeration of extracellular polysaccharides, proteins and DNA.
Because they have three-dimensional structure and represent a community lifestyle for
microorganisms, they have been metaphorically described as "cities for microbes".

Biofilms may form on living or non-living surfaces and can be prevalent in natural,
industrial and hospital settings. The microbial cells growing in a biofilm
are physiologically distinct from planktonic cells of the same organism, which, by
contrast, are single-cells that may float or swim in a liquid medium. Biofilms can form
on the teeth of most animals as dental plaque, where they may cause tooth
decay and gum disease.

Microbes form a biofilm in response to various different factors, which may include
cellular recognition of specific or non-specific attachment sites on a surface, nutritional
cues, or in some cases, by exposure of planktonic cells to sub-inhibitory concentrations
of antibiotics. When a cell switches to the biofilm mode of growth, it undergoes
a phenotypic shift in behavior in which large suites of genes are differentially regulated.

A biofilm may also be considered a hydrogel, which is a complex polymer containing

many times its dry weight in water. Biofilms are not just bacterial slime layers but
biological systems; the bacteria organize themselves into a coordinated functional
community. Biofilms can attach to a surface such as a tooth, rock, or surface which may
include a single species or of a diverse group of microorganisms. The biofilm bacteria
are able to share nutrients and are sheltered from harmful factors in the environment,
such as desiccation, antibiotics, and a host body's immune system. A biofilm usually
begins to form when a free-swimming bacterium attaches to a surface.

Formation

The formation of a biofilm begins with the attachment of free-floating microorganisms

to a surface. It is thought that the first colonist bacteria of a biofilm adhere to the surface
initially through weak, reversible adhesion via van der Waals forces and hydrophobic
effects. If the colonists are not immediately separated from the surface, they can anchor
themselves more permanently using cell adhesion structures such as pili.

Hydrophobicity can also affect the ability of bacteria to form biofilms. Bacteria with
increased hydrophobicity have reduced repulsion between the extracellular matrix and
the bacterium. Some bacteria species are not able to attach to a surface on their own
successfully due to their limited motility but are instead able to anchor themselves to
the matrix or directly to other, earlier bacteria colonists. Non-motile bacteria cannot
recognize surfaces or aggregate together as easily as motile bacteria. During surface
colonization bacteria cells are able to communicate using quorum sensing (QS) products
such as N-acyl homoserine lactone (AHL). Once colonization has begun, the biofilm
grows through a combination of cell division and recruitment. Polysaccharide matrices
typically enclose bacterial biofilms. In addition to the polysaccharides, these matrices
may also contain material from the surrounding environment, including but not limited
to minerals, soil particles, and blood components, such as erythrocytes and fibrin. The
final stage of biofilm formation is known as dispersion, and is the stage in which the
biofilm is established and may only change in shape and size.

The development of a biofilm may allow for an aggregate cell colony (or colonies) to be
increasingly resistant to antibiotics. Cell-cell communication or quorum sensing has
been shown to be involved in the formation of biofilm in several bacterial species.

USES

Medicine

Infections associated with the biofilm growth usually are challenging to eradicate. It is
mostly due to the fact that mature biofilms display tolerance towards antibiotics and the
immune response. Biofilms often form on the inert surfaces of implanted devices such
as catheters, prosthetic cardiac valves and intrauterine devices.

The rapidly expanding worldwide industry for biomedical devices and tissue
engineering related products is already at $180 billion per year, yet this industry
continues to suffer from microbial colonization. No matter the sophistication, microbial
infections can develop on all medical devices and tissue engineering constructs. 60-70%
of nosocomial or hospital acquired infections are associated with the implantation of a
biomedical device. This leads to 2 million cases annually in the U.S., costing the
healthcare system over $5 billion in additional healthcare expenses.

Industry

Biofilms can also be harnessed for constructive purposes. For example, many sewage
treatment plants include a secondary treatment stage in which waste water passes over
biofilms grown on filters, which extract and digest organic compounds. In such biofilms,
bacteria are mainly responsible for removal of organic matter (BOD),
while protozoa and rotifers are mainly responsible for removal of suspended solids
(SS), including pathogens and other microorganisms. Slow sand filters rely on biofilm
development in the same way to filter surface water from lake, spring or river sources
for drinking purposes. What we regard as clean water is effectively a waste material to
these microcellular organisms. Biofilms can help eliminate petroleum oil from
contaminated oceans or marine systems. The oil is eliminated by the hydrocarbon-
degrading activities of microbial communities, in particular by a remarkable recently
discovered group of specialists, the so-called hydrocarbonoclastic bacteria (HCB).
Biofilms are used in microbial fuel cells (MFCs) to generate electricity from a variety of
starting materials, including complex organic waste and renewable biomass. Biofilms
are also relevant for the improvement of metal dissolution in bioleaching industry

Food industry

Biofilms have become problematic in several food industries due to the ability to form
on plants and during industrial processes. Bacteria can survive long periods of time in
water, animal manure, and soil, causing biofilm formation on plants or in the processing
equipment. The buildup of biofilms can affect the heat flow across a surface and
increase surface corrosion and frictional resistance of fluids. These can lead to a loss of
energy in a system and overall loss of products. Along with economic problems, biofilm
formation on food poses a health risk to consumers due to the ability to make the food
more resistant to disinfectants. As a result, from 1996 to 2010 the Center for Disease
Control and Prevention estimated 48 million foodborne illnesses per year. Biofilms have
been connected to about 80% of bacterial infections in the United States.

In produce, microorganisms attach to the surfaces and biofilms develop internally.

During the washing process, biofilms resist sanitization and allow bacteria to spread
across the produce. This problem is also found in ready-to-eat foods, because the foods
go through limited cleaning procedures before consumption. Due to the perishability of
dairy products and limitations in cleaning procedures, resulting in the buildup of
bacteria, dairy is susceptible to biofilm formation and contamination. The bacteria can
spoil the products more readily and contaminated products pose a health risk to
consumers. One bacteria that can be found in various industries and is a major cause of
foodborne disease is Salmonella. Large amounts of salmonella contamination can be
found in the poultry processing industry as about 50% of salmonella strains can
produce biofilms on poultry farms. Salmonella increases the risk of foodborne illnesses
when the poultry products are not cleaned and cooked correctly. Salmonella is also
found in the seafood industry where biofilms form from seafood borne pathogens on
the seafood itself as well as in water. Shrimp products are commonly affected by
salmonella because of unhygienic processing and handling techniques. The preparation
practices of shrimp and other seafood products can allow for bacteria buildup on the
products. New forms of cleaning procedures are being tested in order to reduce biofilm
formation in these processes which will lead to safer and more productive food
processing industries. These new forms of cleaning procedures also have a profound
effect on the environment, often releasing toxic gases into the groundwater reservoirs.

Aquaculture

In shellfish and algae farms, biofouling species tend to block nets and cages and
ultimately outcompete the farmed species for space and food. Bacterial biofilms start
the colonization process by creating microenvironments that more favorable for
biofouling species. In the marine environment, biofilms could reduce the hydrodynamic
efficiency of ships and propellers, lead to pipeline blockage and sensor malfunction, and
increase the weight of appliances deployed in seawater. Numerous studies have shown
that biofilm can be a reservoir for potentially pathogenic bacteria in freshwater
aquaculture. As mentioned previously, biofilms can be difficult to eliminate even when
antibiotics or chemicals are used in high doses. The role that biofilm plays as reservoirs
of bacterial fish pathogens regarding has not been explored in detail but it certainly
deserves to be studied.
MICROBIAL BIOPOLYMERS

Microorganisms produce several biopolymers. Of these, intracellularly produced

polyhydroxyalkanoates (PHAs) and extracellularly produced exopolysaccharides
(EPS) are gaining importance over the other biopolymers. These naturally produced
polymers can replace plant-based or petroleum-derived polymers. There are
innumerable reports and reviews on the production of PHA and EPS by several
bacteria, fungi, actinomycetes, and algae. This chapter briefly gives an introduction to
PHA and provides recent developments in the genetic and metabolic pathways for the
synthesis of microbial EPS. Different strategies used for fermentative production and
various means of downstream processing are discussed. Possible ways to minimize
the cost of production and downstream processing are covered in this chapter.
Applications of these EPS in various fields such as agriculture, cosmetics, foods,
medical and healthcare industry, mining, oil recovery, packaging, pharmaceuticals,
printing and textile industry, wastewater treatment, etc., are presented. The potential
of these polymers indicates that these microbial cell factories can be exploited for the
better of mankind.

BIOSURFACTANTS

Biosurfactants are amphiphilic compounds produced in living surfaces, mostly on

microbial cell surfaces or excreted extracellular hydrophobic and hydrophilic moieties
that confer the ability to accumulate between fluid phases, thus reducing surface and
interfacial tension at the surface and interface respectively. Most biosurfactants are
either anionic or neutral and the hydrophilic moiety can be a carbohydrate, an amino
acid, a phosphate group, or some other compounds. The hydrophobic moiety is mostly a
long carbon chain fatty acid. These molecules reduce surface and interfacial tensions in
both aqueous solutions and hydrocarbon mixtures. This property of biosurfactant
makes them potential candidates for enhancing oil recovery (Sarkar et al. 1989).
Because of surface active property of biosurfactants, micro emulsions are created in
which micelle formations occur where hydrocarbons can solubilize in water or water in
hydrocarbons (Banat 1995). Biosurfactants enhance the emulsification of
hydrocarbons, have the potential to solubilize hydrocarbon contaminants and increase
their availability for microbial degradation. The use of chemicals for the treatment of a
hydrocarbon polluted site may contaminate the environment with their by-products,
whereas biological treatment may efficiently destroy pollutants, while being
biodegradable themselves.

Production

Quite a lot of microorganisms have been reported to produce several classes of

biosurfactants such as glycolipids, lipopeptides, phospholipids, neutral lipids or fatty
acids and polymeric biosurfactants (Cooper and Zajic 1980; Cooper 1986; Kosaric
1993). These compounds are produced during the growth of microorganisms on water
soluble and water insoluble substrates (Sheppard and Mulligan 1987; Desai et al. 1988;
Ron and Rosenberg 2001). Microorganisms utilize a variety of organic compounds as
the source of carbon and energy for their growth. When the carbon source is an
insoluble substrate like a hydrocarbon (CnHn), microorganisms facilitate their diffusion
into the cell by producing a variety of biosurfactants. Some bacteria and yeasts excrete
ionic surfactants which emulsify the CnHn substrates in the growth medium. Some
examples of this group of biosurfactants are rhamnolipids which are produced by
different Pseudomonas sp. (Burger et al. 1963; Guerra-Santos et al. 1984; Guerra-Santos
et al. 1986), or the sophorolipids which are produced by several Torulopsis sp. (Cooper
and Paddock 1983). Some other microorganisms are capable of changing the structure
of their cell wall, which they achieve by synthesizing lipopolysaccharides or nonionic
surfactants in their cell wall. Examples of this group are: Candida lipolytica and Candida
tropicalis which produce cell wall-bound lipopolysaccharides when growing on n-
alkanes (Osumi et al. 1975) and Rhodococcus erythropolis, many Mycobacterium sp.
and Arthrobacter sp. which synthesize nonionic trehalose corynomycolates
(Kretschmer et al. 1982; Ristau and Wagner 1983). There are lipopolysaccharides, such

as emulsan, synthesized by Acinetobacter sp. (Rubinowitz et al. 1982) and lipoproteins

or lipopeptides, such as surfactin and subtilisin, produced by Bacillus subtilis (Cooper et
al. 1981). Other effective biosurfactants are mycolates and corynomycolates which are
produced by Rhodococcus sp., Corynebacteria sp., Mycobacteria sp. and Nocardia sp.
(MacDonald et al. 1981; Kretshmer et al. 1982) and ornithinlipids, which are produced
by Pseudomonas rubescens, Gluconobacter cerinus, and Thiobacillus ferroxidans
(Knoche and Shively 1972; Tahara et al. 1976).
Biomining

Biomining is the process of extracting valuable metals from ores and mine tailings with
the assistance of microorganisms. It is an effective and green technology to mine metals.

Biomining an Effective Mining Technology:

1. Low infrastructure

2. Low-labour input

3. Green Technology, low gaseous emission

4. Low energy demand

5. Low operating cost compared to other mining technologies

6. Cleaner tailings

7. Mine high/low grade ores and economically viable tailings

BIODIESEL 

It refers to a vegetable oil- or animal fat-based diesel fuel consisting of long-

chain alkyl (methyl, ethyl, or propyl) esters. Biodiesel is typically made by chemically
reacting lipids (e.g., vegetable oil, soybean oil, animal fat with an alcohol producing fatty
acid esters.

Biodiesel is meant to be used in standard diesel engines and is thus distinct from the
vegetable and waste oils used to fuel converted diesel engines. Biodiesel can be used
alone, or blended with petrodiesel in any proportions. Biodiesel blends can also be used
as heating oil.

Blends of biodiesel and conventional hydrocarbon-based diesel are products most

commonly distributed for use in the retail diesel fuel marketplace. Much of the world
uses a system known as the "B" factor to state the amount of biodiesel in any fuel mix

 100% biodiesel is referred to as B100

 20% biodiesel, 80% petrodiesel is labeled B20
 5% biodiesel, 95% petrodiesel is labeled B5
 2% biodiesel, 98% petrodiesel is labeled B2

Applications

Biodiesel can be used in pure form (B100) or may be blended with petroleum diesel at
any concentration in most injection pump diesel engines. New extreme high-pressure
(29,000 psi) common rail engines have strict factory limits of B5 or B20, depending on
manufacturer. Biodiesel has different solvent properties than petrodiesel, and will
degrade natural rubber gaskets and hoses in vehicles (mostly vehicles manufactured
before 1992), although these tend to wear out naturally and most likely will have
already been replaced with FKM, which is nonreactive to biodiesel. Biodiesel has been
known to break down deposits of residue in the fuel lines where petrodiesel has been
used. As a result, fuel filters may become clogged with particulates if a quick transition
to pure biodiesel is made. Therefore, it is recommended to change the fuel filters on
engines and heaters shortly after first switching to a biodiesel blend.
Properties

1. Biodiesel has promising lubricating properties and cetane ratings compared to

low sulfur diesel fuels.

Fuels with higher lubricity may increase the usable life of high-pressure fuel injection
equipment that relies on the fuel for its lubrication. Depending on the engine, this might
include high pressure injection pumps, pump injectors (also called unit injectors)
and fuel injectors.

2. The calorific value of biodiesel is about 37.27 MJ/kg. This is 9% lower than

regular Number 2 petrodiesel.

Variations in biodiesel energy density is more dependent on the feedstock used than the
production process. Still, these variations are less than for petrodiesel. It has been
claimed biodiesel gives better lubricity and more complete combustion thus increasing
the engine energy output and partially compensating for the higher energy density of
petrodiesel.

3. The color of biodiesel ranges from golden to dark brown, depending on the
production method.

4. It is slightly miscible with water, has a high boiling point and low vapor pressure.

5. The flash point of biodiesel exceeds 130 °C (266 °F), significantly higher than

that of petroleum diesel which may be as low as 52 °C (126 °F).

6. Biodiesel has a density of ~0.88 g/cm³, higher than petrodiesel (~0.85 g/cm³).

7. Biodiesel contains virtually no sulfur, and it is often used as an additive to Ultra-

Low Sulfur Diesel (ULSD) fuel to aid with lubrication, as the sulfur compounds in
petrodiesel provide much of the lubricity.

Production

Biodiesel is commonly produced by the transesterification of the vegetable oil or animal

fat feedstock, and other non-edible raw materials such as frying oil, etc.
Methods:

There are several methods for carrying out this transesterification reaction including
the

Common batch process,

Heterogeneous catalysts,

Supercritical processes,

Ultrasonic methods,

Microwave methods.

Chemically, transesterified biodiesel comprises a mix of mono-alkyl esters of long

chain fatty acids. The most common form uses methanol (converted to sodium
methoxide) to produce methyl esters (commonly referred to as Fatty Acid Methyl
Ester – FAME) as it is the cheapest alcohol available, though ethanol can be used to
produce an ethyl ester (commonly referred to as Fatty Acid Ethyl Ester – FAEE)
biodiesel and higher alcohols such as isopropanol and butanol have also been used.
Using alcohols of higher molecular weights improves the cold flow properties of the
resulting ester, at the cost of a less efficient transesterification reaction.
A lipid transesterification production process is used to convert the base oil to the
desired esters. Any free fatty acids (FFAs) in the base oil are either converted to
soap and removed from the process, or they are esterified (yielding more biodiesel)
using an acidic catalyst. After this processing, unlike straight vegetable oil, biodiesel has
combustion properties very similar to those of petroleum diesel, and can replace it in
most current uses.

The methanol used in most biodiesel production processes is made using fossil fuel
inputs. However, there are sources of renewable methanol made using carbon dioxide
or biomass as feedstock, making their production processes free of fossil fuels.

A by-product of the transesterification process is the production of glycerol. For every 1

tonne of biodiesel that is manufactured, 100 kg of glycerol are produced.
The supplements from plant sources are referred to as "phytopharmaceuticals." They

are produced from fresh, dried from medicaly practiced (Ayurveda) plants or parts of

plants. The active ingredients are usually not completely isolated but rather are isolated

along with other naturally occurring components of the plant. (These other components

are often believed to influence the efficacy of the active ingredient.).Sometimes the

active ingredients are concentrated, and undesirable substances such as chlorophyll,

tannins, or resins are removed. It is divided into few parts in order to know production

of dietary supplements of plant origin.

Collection and cultivation of plant materials:

Most of the plants used for dietary supplements or medicinal purposes are cultivated,

that is, grown in farms however some may be collected from the wild. The below

sections discusses both methods for obtaining botanicals or herbals.

Cultivation:

Cultivation allows producers to have more control over quality and purity than does

collecting plants from the wild. The many dedicated farm houses grow a number of

medicinal plant species have been developed to produce high yields of the desired
constituents. Some plants that are grown commercially for medicinal purposes are

propagated vegetatively. (This means that new plants are grown from cuttings of old

plants. Plants grown in this way are genetically identical to the parent plant.) Some

medicinal plants are grown from selectively bred hybrid seeds, while others are

varieties of plants that are unchanged from their natural form .

A number of medicinal plants are cultivated for use by the pharmaceutical industry.

Some examples include yams, which are used in the production of steroids; foxglove,

which is used for digitalis; belladona, which is used for atropine and opium, which is

used to make morphine. The following is a list of major commercially cultivated

medicinal plants many of which are used in dietary supplements.

A number of countries commercially cultivate and export substantial quantities of

medicinal plants. These countries include China, India, Thailand, South Korea, Brazil,

Mexico, Egypt, Indonesia, Nepal, the Philippines, and Kenya. Eastern European countries

cultivate medicinal plants as well, but mostly for their own consumption. As for any

agricultural crop producers of medicinal plants must provide plants with adequate

moisture and nutrients and must control pests and diseases. Pesticides must be used

cautiously to reduce the risk of harmful residues on plants. Production of medicinal

plants is generally labor intensive. In many cases only the portions of the plant that

contain the active ingredients -not the whole plant- are used. Sometimes harvesting

involves picking leaves and flowers by hand. In the future tissue culture may be used for

producing plant material.

Collection from the Wild:

Tropical forests are the source of a number of plants used for medicinal purposes. There

are several disadvantages to collecting wild plants however This practice along with

deforestation has caused some wild plant species to become endangered. Also, when

plants are collected from the wild, there is a risk that they have been incorrectly
identified . One advantage to using wild plants however is that they are unlikely to

contain any pesticide residues.

Cleaning: After the plants are harvested or gathered, they must be cleaned. Cleaning

may involve screening, washing, peeling, or stripping leaves from stems. Any

unnecessary parts are removed prior to drying to avoid wasting time and energy.

Cleaning is often done by hand.

Drying:

In some cases botanicals are used for extraction while fresh but generally they are dried

first. The purpose of drying is to reduce the water content so that the plant can be
stored. Most plants contain 60 to 80 percent moisture when harvested and must be

dried to within 10 to 14 percent moisture before storage. Plants must be dried or

processed as soon as possible after harvest because they begin to deteriorate

immediately. Processing up to this point is generally done by the producer of the plants .

Plants can be dried naturally or by a number of artificial methods. The type of plant or

plant part being used will determine the appropriate drying technique.

Natural Drying:

A practice that has been used since ancient times is sun-drying in the field. Although this

method requires no drying equipment and uses solar energy, it requires large amounts

of space and plants can be damaged by the weather. Sometimes plants are placed by

hand on drying frames or stands, to be air-dried in barns or sheds. This method of

drying is labor-intensive and can take several weeks. The exact length of time for

adequate drying depends on temperature and humidity. The natural drying may change

the structure of the active intermediate due to photochemical reaction.

Artificial Drying:

With the use of artificial dryers, drying time can be reduced to hours or minutes and

labor can also be greatly reduced. Fans that blow unheated air (cold-air drying) can

reduce drying time to several days. Warm-air drying, which is the most widely used

method for medicinal plants, uses a counter-current flow of warm air. There are several

different types of systems for warm-air drying. One type is the plate chamber dryer,

which blows warm air across plates on which plants have been placed. This method is

useful for fragile flowers and leaves but requires large amounts of labor. Workers must

load and unload the plants from the plates manually. The capacity of these dryers is

relatively low, as well. Conveyor dryers are a commonly used type of warm-air dryer.

Fresh plants travel on a conveyor belt through a counter-current flow of warm air.
These dryers can operate continuously, require relatively little labor, and have high

throughput. However, they require a large capital investment and have high energy

requirements. The drying time required for conveyor dryers ranges from 2.5 to 6 hours

and the temperature of the drying air ranges from 40 to 80°C. Hot air dryers which use

very high temperatures (200 to 1,000°C) for very short periods (2 to 5 minutes) are not

commonly used for drying medicinal plants .

Packaging of Dried Plants:

Once drying is complete plants are packaged in preparation for shipping and further

processing. Dried herbaceous plants are generally compressed into bales weighing from

60 to 100 kg (13 to 220 pounds) which are then sewn into fabric bags or wrapped in

plastic. Materials that cannot be baled such as roots and bark are placed in sacks.

Smaller bags may be used for dense materials such as dried fruits or seeds. Very fragile

materials such as flowers are packaged in crates. Dried plant materials tend to be

hygroscopic (readily absorbing moisture) and must be stored under controlled

humidity. Highly hygroscopic materials are generally packed in plastic

Cleaning and Sorting:

When the sacks or bales arrive at the processing facility processors open the packages

and clean the dried plants to remove as many impurities as possible. Sand is removed

pneumatically and iron-containing metals are removed magnetically. Next processors

sort the plant pieces by size since different end-uses require different particle sizes. For

example finely shredded material may be used for tea bags and somewhat less finely

shredded material for loose teas or infusions while coarsely shredded material may be

sold directly to consumers or used for extraction. Particles that are already the desired

size can go directly into storage to await further processing. Particles that are too big

undergo additional grinding, cutting, shredding and sieving. Various methods are used
to reduce particle size including hammer action, pressure, friction, impact cutting and

shredding . Some plant materials are packaged and sold at this point without any

additional processing. Some proceed through an extraction process which the following

section describes.

Extraction:

Extraction is a process whereby the desired constituents of a plant are removed using a

solvent. The following section describes several methods used for preparing extracts,

including organic solvent extraction, supercritical gas extraction and steam distillation.

Organic Solvent Extraction:

Organic solvent extraction is one process for separating the desired substance from

plant material. As previously mentioned dried plants are usually used for extraction

although fresh plants are sometimes used. The plants are first ground and then

thoroughly mixed with a solvent such as hexane or toluene inside a tank. The choice of

solvent depends on several factors including the characteristics of the constituents

being extracted, cost and environmental issues. If the end product will contain trace
amounts of residual solvent a nontoxic solvent must be used. Once the solvent dissolves

the desired substances of the plant it is called "miscella." The miscella is then separated

from the plant material. There are a number of techniques for solvent extraction which

include maceration, percolation and countercurrent extraction. The following is a brief

description of each.

Maceration:

This method involves soaking and agitating the solvent and plant materials together.

The solvent is then drained off. Remaining miscella is removed from the plant material

through pressing or centrifuging. This method does not totally extract the active

ingredients from the plant materials.

Percolation:

With this method the plant material is moistened with solvent and allowed to swell

before being placed in one of a series of percolation chambers. The material is

repeatedly rinsed with solvent until all the active ingredient has been removed. Solvent

is reused until it is saturated. New solvent is used on plant material that is almost

completely exhausted and then re-used on subsequently less exhausted batches. This

method is more effective at removing active ingredients than the maceration technique.

Countercurrent extraction:

This is a highly effective process whereby solvent flows in the opposite direction to

plant material. Unlike maceration and percolation which are batch processes this

method is continuous. Screw extractors and carousel extractors are two types of

equipment used for countercurrent extraction.

Purification and Concentration of Miscella:

Miscella that has been separated from the plant material generally contains some

unwanted substances such as tannins, pigments, microbial contaminants or residual

solvent. Methods such as decanting, filtration, sedimentation, centrifuging, heating,

adsorption, precipitation and ion exchange are used to separate impurities from the

miscella. Sometimes the miscella resulting from solvent extraction is used as the final

dosage form. This is known as a "fluid extract". The miscella is sometimes concentrated

in order to increase the proportion of the desired substance. This is done through

evaporation or vaporization. Solvent is generally recovered and reused . The degree of

concentration depends on the desired end product. Equipment for concentrating the

miscella may include descending film, thin layer or plate concentrators. Any method

used to concentrate the miscella must avoid excessive heat because the active

compounds may be subject to degradation.

Sometimes extracts are dried completely using vacuum freeze dryers, cabinet vacuum

dryers, continuously operating drum or belt dryers, microwave ovens, or atomizers. The

technique for drying depends on the stability of the product and the amount of moisture

that must be removed. The resulting powdered extract is less subject to microbial

contamination than are liquid extracts.

Extraction with Supercritical Gases:

This is a method for extracting active ingredients using gases. The plant material is

placed in a vessel that is filled with a gas under controlled temperature and high

pressure. The gas dissolves the active ingredients within the plant material then passes

into a separating chamber where both pressure and temperature are lower. The extract

precipitates out and is removed through a valve at the bottom of the chamber. The gas is

then reused. Gases suitable for supercritical extraction include carbon dioxide, nitrogen,

methane, ethane, ethylene, nitrous oxide, sulfur dioxide, propane, propylene, ammonia,

and sulfur hexafluoride. An advantage of supercritical extraction is that it can take place

at low temperature thus preserving the quality of temperature-sensitive components .

Steam Distillation:
Steam distillation is another method for extracting active ingredients from medicinal

plants. The plant material is loaded onto perforated plates inside a cylindrical tank or

still, and steam is injected from below. The steam dissolves the desired substances in

the plant, then enters a condenser where it is condensed back into a liquid. This

condensate then passes into a flask where the extract either rises to the top or settles to

the bottom and is separated from the water. Distillation is complete when there is no

more extract present in the condensate. The water may be reused and the extract is

purified through centrifuging and filtering. Other Minor Extraction Methods. Other

minor methods for making extracts include cold pressing and the enfleurage process.
Cold Pressing:

Cold pressing is a process used to extract essential oils from citrus plants through

pressing . The enfleurage process is the same as the technique used to make perfume

from flowers purified fats are used to extract essential oils from plant parts. Plant

material is spread onto sheets of purified fat, which dissolve the essential oils.

Sometimes practitioners of herbal medicine prepare extracts for immediate use. These

include aqueous extracts known as decoctions, infusions or macerations. Plant material

is mixed, agitated, and soaked in water to dissolve the active ingredients. Controlling

microbial contamination can be difficult in aqueous extracts. Oily drug extracts, also

called "medicinal oils," may be prepared by soaking or macerating the plant material in

an oil such as almond, peanut, olive, poppy seed, apricot kernel, or peach kernel oil.

Vinegar is sometimes used to extract active ingredients as well. Plant materials are

soaked in acetic acid and the vinegar is consumed as the final dosage form.

Controlling the Quality of Extracts:

Once an extract has been produced by one of the methods mentioned above producers

can use a number of tests to evaluate the quality and purity of their product. First they

may examine the physical characteristics of the extract. This may include evaluating its
appearance, pH, solubility, total solids content, ash content, and in the case of dried

extracts, particle size. Next, they may analyze the components of the extract to be

certain it contains the appropriate quantities of desired ingredients. Chromatography

(including thin layer, column, high pressure liquid, and gas chromatography) may be

used for this. Finally, they may test the extract for impurities such as residual solvents,

herbicides and pesticides and for microbial contamination. Some extracts are labeled

and sold as standardized extracts. According to industry sources the desired

constituents in standardized extracts are measured and are listed as a percentage of the

total weight of the extract. For example echinacosides are the desired compounds

present in echinacea extract. A capsule containing 250 mg of echinacea extract

standardized to 4 percent would contain 10 mg of echinacosides. In some cases the

desired constituent is a known active ingredient. In cases where the active ingredient

has not been identified, another "marker " compound or substance that is known to be

present in the plant may be measured for the purpose of standardization.

Spectrophotometric testing and high pressure liquid chromatography may be used to

measure standardized constituents.

Pesticides (Applied During the Cultivation of Herbs)

A large number of Chinese herbs are collected wild and therefore are not subjected to

any pesticides. Some cultivated plants do not require the use of pesticides because they

have natural resistance to pathogenic organisms and insects. However some Chinese

herbs are grown with pesticides. Most herb growers are sensitive to the issue of

pesticide use and take adequate precautions to avoid contamination of the harvested

materials . Many herb cultivators in China cannot afford to purchase modern chemical

fertilizers and pesticides and rely instead on natural materials and careful cultivation

techniques. Regulations have been published in China requiring cultivators to follow

certain practices that minimize pesticide use and residues. Still some of the specific

restrictions on pesticide use that are imposed in America are not present in China. This

means that some of the pesticides that are used in China on herbs are not permitted in
the U.S. for those crops. A particular problem has been noted with ginseng cultivation.

Fungicide materials are used at times because the ginseng plant is highly susceptible to

fungal rot especially during its early growth period. Care is taken to avoid applying any

chemicals close to time of harvest so that natural processes (e.g., heavy rains) have time

to eliminate most of the fungicides prior to harvesting.Nonetheless fungicides and

pesticides have been detected in some samples of ginseng and notoginseng (tien-chi

ginseng). 

Fumigants (Applied after the Harvest of Herbs)

Many people apparently believe that Chinese herbs are fumigated at the ports when

they arrive in America. The majority of Western herbs, just like all Chinese herbs are

imported they frequently come from South or Central America and Eastern Europe and
are subject to the same rules and regulations as Chinese herb these Western herbs

might also be fumigated in their home country during storage. Fortunately the high

quality Chinese herbs destined for foreign markets such as the U.S. and Europe are

items that are in high demand and thus have a high turn-over with less likelihood of

needing any such long-term storage.

Sulfur

It was reported by Frontier Herbs Company that several Chinese herbs have relatively

high levels of sulfur. This is the result of a processing method whereby herbs are spread

on screens underneath which is some heated sulfur. The sulfur fumes waft through the

herb material and leave some residue (which is intentional). These sulfur residues are

sometimes referred to as sulfites bringing images of sulfiting agents sprinkled on

restaurant lettuce or added to finished wines. However the sulfur compounds resulting

from this method of preserving the herb quality are not known to cause reactions in

sulfite-sensitive individuals (sulfur is one of the most prevalent elements in the human

body and is essential to all life). Treatment with sulfur is mostly carried out on those

herbs that are moist (e.g., ophiopogon) or those that discolor significantly over time

(e.g., atractylodes). Some importers specifically obtain herbs that have not been sulfur
treated and will mention that in their literature. There may not be any health problems

that can be associated with the sulfur processing as carried out in China but those who

are concerned now have a choice at least for crude herb materials.

Irradiation (after Import)

Today many of the herbs and spices sold in grocery stores are treated by ionizing

radiation as a means of sterilization. Crude Chinese herbs are not subjected to this

procedure with one exception certain animal materials, mainly deer antlers are

irradiated under direction of the U.S. Department of Agriculture to assure that

organisms that cause disease in animals are not carried into this country (the same

requirements exist in other countries). This type of irradiation does not leave
radioactive contaminants. Some manufacturers of finished products (e.g., extract

powders or granules) may utilize gamma irradiation as a means of reducing bacteria

counts on the finished products this procedure does not result in any radioactive

contamination.

Sterilizing Gases (Applied after Powdering)

Some Western herb companies routinely "sterilize" their herbs before putting them into

capsules (the treatment substantially reduces the bacteria count to a level deemed

acceptable). The treatment involves putting the herbs into an airtight chamber,

introducing a gas, such as ethylene oxide, heating the chamber to about 180 degrees

Fahrenheit for several hours and then evacuating the gas and allowing the herbs to de-

gas for another twenty-four hours. The same procedure is applied to Chinese herbs that

are distributed by these companies. When herbs are so treated there may be a small

amount of ethylene-derived residue which arises mainly from interaction of the

sterilizing gas with water in the herb materials. There is no evidence that such

treatment of the herbs is necessary for any health purpose (see below). The crude

Chinese herbs used for making most Chinese herb formulas for health professionals are

not treated by this method.

Bacteria, Mold and Yeast

Herbs are generally free of harmful bacteria, but they do contain naturally occurring

microorganisms..

The bacteria that can cause food poisoning in relative small amounts Salmonella is not

found in Chinese herb formulas for example in one series of tests no Salmonella

contamination was discovered in more than 25 random samples. Recent testing of

oyster shells similarly showed freedom from this bacteria (oyster meat is frequently a

source of it). Salmonella is found in certain animals such as lizards but the gecko lizard

used in making certain Chinese herb formulas is baked to destroy any of this organism

that might be present. E. coli an indicator of animal fecal contamination is rarely found

in Chinese herbs and in those rare cases the counts have been very low. Total coliform

counts (which include several harmless organisms) in the Chinese herb tablets are
generally less than 500 per gram; counts below 1,000 are considered low for natural

materials.

Viruses (that May Be Present in Animal Products)

Assays have not been carried out to determine the presence of viruses in animal

materials from China. A concern was raised by some American practitioners about the

possibility of human viruses in placental material from China. The processing of human

placenta before it is shipped to the West includes boiling followed by baking and the

material that arrives is very dry making it highly unlikely to contain any organisms that

might have been originally present. The materials are further heated and dried by the

grinding process used for making powders (for pills) alternatively it is sterilized by

boiling when the material is used in making a decoction. Small amounts of any residual

viable virus would be very unlikely to cause disease when consumed orally. Nonetheless

the FDA has recently restricted use of human placenta because of their general rules-

regarding potential contamination-for use of human substances in medicine.

Heavy Metals

Reports that Chinese herb products were contaminated by heavy metals emerged in the

late 1990s and included an extensive testing of patent remedies by the California Health

Department Food and Drug Branch published in 1998. Two metals detected in several
products mercury and arsenic are the result of intentional addition to herb formulas

following the belief that these compounds improve the effects of the formulas. The

primary additives are cinnabar (contains mercury) and realgar (contains arsenic).

Western manufacturers of Chinese herb formulas never add these compounds so

mercury and arsenic are not present in amounts higher than normally found in plants

(below 3 parts per million). Lead contamination of Chinese herb formulas may occur

either from intentional addition of lead compounds (rare, but sometimes done in Hong

Kong) or by unintentional contamination (environmental contamination of the herb

materials or factory contamination). Chinese herb formulas manufactured in the U.S.

and other Western countries never have added lead compounds. The level of lead found

in the imported herb materials used for manufacturing formulas is generally quite low
almost always meeting the World Health Organization standard of not more than 3

parts per million.

Western Drugs in Patent Herbs

Western drugs are present in some patent formulas made in China and this is not

always indicated on the label. A well-known case is a variety of Yin Chiao tablet

("superior quality-sugar coated") from Tianjin which includes an analgesic and an

antihistamine (it also has caffeine added). Many practitioners and consumers are not

familiar with the ingredient labeling of herbs and drug ingredients and therefore may

not realize that a drug is present in a Chinese product sold in a Chinese herb shop.

Although it is illegal to import such materials for sale in the U.S. they have found their

way into several Chinatown shops. Typical drug additives were antipyretics (e.g.,

aspirin and acetaminophen), antihistamines and antibiotics. The products were not

labeled to indicate that they contain drugs. 

Additives in Manufacturing

Many people prescribe Chinese herbs that have been processed to some extent beyond

the minimal processing to produce "crude" pharmacy materials. Chinese pills may be

made with honey or other binders as well as have a coating of vegetable oil. Most cough

syrups and herb extracts in liquid form (in vials) are made with sugar, honey or both.
Tablets are made with flow agents, binders and coatings. Sugar-coated tablets and

capsules made in China may have synthetic colors as aids to identification. Capsulated

herbs contain flow agents and the capsule is made from animal gelatin (vegetarian

capsules are rarely used and are made of the same materials used to coat tablets). Dried

decoctions are often produced with a starch carrier (such as potato starch) or from the

powdered herb dregs left over from the extraction procedure. For products made in the

U.S., most manufacturers provide a list of items that are not used in the product which

consumers may be concerned about (e.g., corn, soy, wheat, animal materials, etc.). Some

manufacturers provide disclosure of all additives used in manufacturing. Typically these

involve various cellulose materials (fillers, disintegration aids, binders, coatings),

magnesium stearate (flow agent), and various types of gums (binding agents). In most
cases all the additions to the basic herb material constitute less than 10% of the weight

of the finished product (with the exception of products comprised of isolated active

components, which may have a larger proportion of filler to control the dosage

amount). 

Incorrect Herb Material Provided

Another type of "contamination" is receiving an incorrect herb material thus the one

that is not actually desired contaminates the finished product. In the case of Chinese

medicine herb substitution is a common practice and whether or not an herb is

"correct" or not may depend on certain expectations. When ordering Chinese herbs

there is a possibility that the material obtained will not be the one requested and if the

recipient is not familiar with the appearance of the proper material then a wrong item

may be used. Labels on some packages of imported Chinese patents are sometimes

deceptive. In some formulas aconite is labeled as cyperus and in a number of products

"ginseng" appears in the name of the product but not among the ingredients (it is

substituted by codonopsis) a recent label for Wuchi Paifeng Wan does not show that

black chicken is present even though it is a major ingredient. Items said to contain

musk, ox gallstone, rhino horn, pearl or other expensive items may contain various

substitutes in the case of musk the substitute may be a synthetic chemical (muscone).
None of the Chinese patents tested by the U.S. Fish and Wildlife Department showed

evidence of containing the endangered animal species such as rhino horn or tiger bone

that were listed on their labels

PENICILLIN PRODUCTION:

Penicillin was the first naturally occurring antibiotic discovered. It is

obtained in a number of forms from Penicillium moulds. Penicillin is not a
single compound but a group of closely related compounds, all with the
same basic ring-like structure (a β-lactam) derived from two amino acids
(valine and cysteine) via a tripeptide intermediate. The third amino acid of
this tripeptide is replaced by an acyl group (R) and the nature of this acyl
group produces specific properties on different types of penicillin.

There are two different types of penicillin.

Biosynthetic penicillin is natural penicillin that is harvested from the mould

itself through fermentation.

Semi-synthetic penicillin includes semi synthetic derivatives of penicillin -

like Ampicillin, Penicillin V, Carbenicillin, Oxacillin, Methicillin, etc. These
compounds consist of the basic Penicillin structure, but have been
purposefully modified chemically by removing the acyl group to leave 6-
aminopenicillanic acid and then adding acyl groups that produce new
properties.

These modern semi-synthetic penicillins have various specific properties

such as resistance to stomach acids so that they can be taken orally, a
degree of resistance to penicillinase (or β-lactamase) (a penicillin-
destroying enzyme produced by some bacteria) and an extended range of
activity against some Gram-negative bacteria. Penicillin G is the most
widely used form and the same one we get in a hypodermic form.

PENICILLIN G

Penicillin G is not stable in the presence of acid (acid-labile). Since our

stomach has a lot of hydrochloric acid in it (pH2.0), if we were to ingest
penicillin G, the compound would be destroyed in our stomach before it
could be absorbed into the bloodstream, and would therefore not be any
good to us as a treatment for infection somewhere in our body. It is for this
reason that penicillin G must be taken by intramuscular injection - to get
the compound in our bloodstream, which is not acidic at all. Many of the
semi-synthetic penicillins can be taken orally.

Penicillium chrysogenum  that produce antibiotics, enzymes or

othersecondary metabolites frequently require precursors like
purine/pyrimidine bases or organic acids to produce said metabolites.
Primary metabolism is the metabolism of energy production for the cell
and for its own biosynthesis. Typically, in aerobic organisms (Penicillium
chrysogenum) it involves the conversion of sugars such as glucose to
pyruvic acid2 and the production of energy via the TCA cycle. Secondary
metabolism regards the production of metabolites that are not used in
energy production for example penicillin from Penicillium chrysogenum. In
this case the metabolite is being utilized as a defence mechanism against
other microorganisms in the environment. In essence Penicillium
chrysogenum can kill off the competition to allow itself to propagate
efficiently. It should be noted that these secondary metabolites are only
produced in times of stress when resources are low and the organism must
produce these compounds to kill off its competitors to allow it to survive.

MEDIA FORMULATION:

Lactose: 1%

Calcium Carbonate: 1%

Cornsteep Liquor: 8.5%

Glucose: 1%

Phenyl acetic acid: 0.5g

Sodium hydrogen phosphate: 0.4%

Antifoaming Agent: Vegetable oil

FERMENTATION

To begin the fermentation process, a number of these spores will be

introduced into a small (normally 250-500ml) conical flask where it will be
incubated for several days. At this stage, explosive growth is the most
desired parameter and as such the medium in the flask will contain high
amounts of easily utilisable carbon and nitrogen sources, such as starch
and corn-steep liquor. At this stage, the spores will begin to revive and
form vegetative cells. Temperature is normally maintained at 23-280C and
pH at ~6.5, although there may be some changes made to facilitate
optimum growth. The flask will often have baffles in it and be on a shaking
apparatus to improve oxygen diffusion in the flask.

Once the overall conditions for growth have been established and there is a
viable vegetative culture active inside the flask, it will be transferred to a 1
or 2 litre bench-top reactor. This reactor will be fitted with a number of
instruments to allow the culture to be better observed than it was in the
shake flask. Typical parameters observed include pH, temperature, and
stirrer speed and dissolved oxygen concentration. This allows tweaking of
the process to occur and difficulties to be examined. For example, there
may not be enough oxygen getting to the culture and hence it will be
oxygen starved.  At this point, the cells should be showing filamentous
morphology, as this is preferred for penicillin production. As before, cell
growth is priority at this stage. At this stage, growth will continue as before,
however, there are often sudden changes or loss in performance. This can
be due to changes in the morphology of the culture (Penicillium
chrysogenum is a filamentous fungi and hence pseudoplastic) that may or
may not be correctable.
At this stage the medium being added to the reactor will change. Carbon
and nitrogen will be added sparingly alongside precursor molecules for
penicillin fed-batch style. Another note is that the presence of penicillin in
the reactor is itself inhibitory to the production of penicillin. Therefore, we
must have an efficient method for the removal of this product and to
maintain constant volume in the reactor. Other systems, such as cooling
water supply, must also be considered. If all goes well we should have
penicillin ready for downstream processing. From here it can be refined
and packaged for marketing and distribution to a global market