Preservation techniques and

maintenance methods of industrial

microorganisms

What are microorganisms?

Microorganisms are microscopic, living, single-celled organisms such as
bacteria. Ubiquitous throughout the world, microorganisms play a vital role in
supporting and maintaining nature and life. The vast majority are
beneficial: They keep nature clean by removing toxins from water and soil,
and degrade organic matter from dead plants and animals. In the human body
they aid in digestion and help prevent invasion by harmful bacteria. Without
bacteria, life would not be possible.

Microorganisms are living organisms

Novozymes harnesses the natural capabilities of live, beneficial bacteria to
help degrade and remove organics that ordinary cleaners or products miss.
Novozymes liquid products contain live bacteria in a dormant state called
spores, while dry products may contain both dormant and nondormant (active)
bacteria. All of the bacteria used in Novozymes products are considered safe
and nonpathogenic (do not cause disease/illness in healthy individuals) and
are in the Biosafety Level 1 containment group, which means they pose a low
risk to individuals and communities.

Found in nature, refined in the lab

Microorganisms produced by Novozymes are initially isolated from the
environment (water, soil, and air). Microorganisms are selected based on their
natural abilities to degrade certain substrates. The most effective strain is then
selected, fermented, and sold to customers in dry or liquid form.

Putting microorganisms to work

The beneficial microorganisms in Novozymes products perform their work by
producing enzymes. They provide an intelligent, efficient system by detecting
the organics present in the application and only producing the enzymes
needed to break down these organics. The degradation of organics will not be
immediate since the microorganisms will need time to adapt to the new
environment and then produce enzymes. While enzymes generally carry out a
single reaction, our beneficial microorganisms degrade the organic substrates
into water and carbon dioxide.
Some beneficial microorganisms can form spores that are dormant (not
metabolically active). The spores will only become active when certain
conditions, nutrients, and organics (food sources) are available. Once the
spores become active (vegetative), the beneficial microorganisms are ready
to synthesize the appropriate enzyme to degrade the substrate. The
microorganisms will continue to work until all the organics have been
exhausted or the conditions become unfavorable. Some will go back to the
spore (dormant) state, and some will die off.

What are industrial microorganisms?

Microorganisms are used extensively to provide a vast range of products and
services. They have proved to be particularly useful because of the ease of
their mass cultivation, speed of growth, use of cheap substrates (which in
many cases are wastes) and the diversity of potential products. Their ability to
readily undergo genetic manipulation has also opened up almost limitless
further possibilities for new products and services from the fermentation
industries. Traditional fermentations were originally performed (and still are in
many cases) by a mixture of wild microorganisms emanating from the raw
materials or the local environment, e.g., some food and alcoholic beverage
fermentations. Initial attempts to improve the microorganisms involved
occurred little more than 120 years ago, when they were first isolated from
these processes as pure cultures from which the most useful strains were
then selected. Those fermentation processes developed in the first 80 years
of the 20th century have mostly used monocultures. The specific
microorganisms employed were often isolated from the natural environment
which involved the random screening of a large number of isolates.
Alternatively, suitable microorganisms were acquired from culture collections.
Most of these microorganisms, irrespective of their origins, were subsequently
modified by conventional strain improvement strategies, using mutagenesis or
breeding programmes, to improve their properties for industrial use. Several
processes developed in the past 20 years have involved recombinant
microorganisms and genetic engineering technologies have increasingly been
used to improve established industrial strains. In most cases, regulatory
considerations are of major importance when choosing microorganisms for
industrial use. Fermentation industries often prefer to use established GRAS
(Generally Regarded As Safe) microorganisms, particularly for the
manufacture of food products and ingredients. This is because requirements
for process and product approval using a new microorganism are more
stringent and associated costs are much higher. Where pathogens and some
GMMs are used as the producer organism, additional safety measures must
be taken. Special containment facilities are employed and it may be possible
to use modified (crippled) strains that cannot exist outside the fermenter
environment.

PRESERVATION TECHNIQUES OF INDUSTRIAL

MICROORGANISMS

Microbes are required for the production of fermentation products. They are
very valuable for specific product. Not all the microbes will give one product
produced efficiently by specific microbe. The isolation of a desired organism
for a fermentation process may be time consuming and very expensive
procedure and it is therefore essential that it retain the desirable
characteristics that led to its selection. In addition, the culture used for the
fermentation process should remain viable and free from contamination. Thus,
industrial cultures must be preserved and maintained in such way as to
eliminate genetic change, protect against contamination, and retain viability.

PRESERVATION
Different techniques are used for maintenance and preservation of different
organisms based on their properties. Selected method should also conserve
the properties of the organisms.
Techniques for the Preservation of microbes broadly divided into two
1. Methods where organisms are in Continuous metabolic active state
2. Methods where organisms are in Suspended metabolic state

1-Continuous metabolic active state preservation technique

In this technique, organisms preserved on nutrient medium by repeated
sub-culturing. In this technique, any organisms are stored by using general
nutrient medium. Here repeated subculturing is required due to depletion or
drying of nutrient medium. This technique includes preservation by following
methods.
1.1 Periodic transfer to fresh media
Organisms grown in general media on slant, incubated for particular period
at particular temperature depending on the characteristics of the selected
organisms, then it is stored in refrigerator. These cultures can be stored for
certain interval of time depending on the organism and its growth
conditions. After that time interval, again these organisms transferred to
new fresh medium and stored in refrigerator.

1.2 Overlaying culture with mineral oil

Organisms are grown on agar slant then they are covered with sterile
mineral oil to a depth of 1 cm. above the tip of the surface. This method is
simple; one can remove some organisms in aseptic condition with the help
of sterile wire loop and still preserving the initial culture. Some species
preserved satisfactorily for 15 20 years by this method.
1.3 Storage in sterile soil
This method is widely used for preserving spore forming bacteria and
fungi. In this method, organisms will remain in dormant stage in sterile soil.
Soil sterilized then spore suspension added to it aseptically, this mixture
dried at room temperature and stored in refrigerator. Viability of organisms
found around 70 80 years.
1.4 Saline suspension
Normal Saline used to provide proper osmotic pressure to organisms
otherwise high salt concentration is inhibitory for organisms. Organisms
kept in screw cap bottles in normal saline, stored at room temperature,
wherever required transfer made on agar slats, and incubated

2MethodswhereorganismsareinSuspendedmetabolicstate
Organisms preserved in suspended metabolic state by either drying or
storing at low temperature. Microbes are dried or kept at low temperature
carefully so that their revival is possible.
2.1 Drying in vacuum
In this technique, organisms dried over chemical instead of air dry. Cells
passed over CaCl2 in a vacuum and then stored in refrigerator. Organisms
survive for longer period.
2.2 Lyophilization
Lyophilization is vacuum sublimation technique. Cells grown in nutritive
media and then this culture distributed in small vials. These vials culture then
immersed in a mixture of dry ice and alcohol at -78oC. These vials
immediately connected to a high-vacuum line, and when they are completely
dried, each vial sealed under vacuum. This is most effective and widely used
technique due to long time survival, less opportunity for changes in
characteristics of organisms and small storage area. Organisms can survive
for period of 20 years or more.
2.3 Use of Liquid nitrogen
Microorganisms grown in nutritive media and then this culture frozen with
Cryoprotective agents like Glycerol and Dimethyl Sulfoxide. Frozen culture

kept in liquid Nitrogen refrigerator. Organisms can remain alive for longer
period.
2.4 Storage in silica gel
Both bacteria and yeast stored by this method. By this technique,
organisms can survive for 1 2 years. Finely Powdered Heat sterilized
Silica powder mixed with thick suspension of cell at low temperature.

MAINTENANCE OF INDUSTRIAL
MICROORGANISMS
The great increase in number and size of industrial fermentations has
accentuated the value of maintaining collections of microorganisms,
especially of production strains, assay organisms and related species.
Considerable work has been devoted to finding methods of maintaining
cultures in a vigorous and stable condition. At present, the chief methods of
preservation of industrially important microorganisms are by means of soil
culture, agar slants, mineral oil overlay, and lyophilization. Each method has a
place in any collection of microorganisms and all are in use in the collection of
the Northern Utilization Research Branch, Agricultural Research Service, U.
S. Department of Agriculture, at Peoria. The NURB Culture Collection is
organized in three parts. The first is devoted to bacteria with emphasis on the
genera Pseudomonas, Leuconostoc, Bacillus, and Streptomyces. These
cultures are designated by the prefix "B" with the culture numbers, and total
over 2800 strains. The second part is concerned with yeasts and yeast-like,
organisms. These cultures, numbering over 2200, are designated by the "Y"
preceding their specific number. The last portion of the collection consists of
filamentous fungi with emphasis on species of Penicillium, Aspergillus, and
the order Mucoraks, though many additional fungi of economic importance are
included. This mold collection numbers over 2400 isolates and can be
recognized by the fact that no letter precedes the culture number. No list or
catalogue of our cultures has been published. The primary reason for this is
that we do not wish to enter into competition with other collections. In addition
to the permanent cultures, the Collection has lyophil preparations of
approximately 10,000 strains of microorganisms in pure culture which are not
intended to be permanent accessions. However, because of the long viability
of most lyophilized microorganisms, nearly all are still living and represent a
reservoir of readily available material. They have not been incorporated into
the collection because their usefulness has not been shown, or else they are
known to duplicate cultures already in the collection. The lyophil apparatus
(figure 1) and the procedure used is essentially the same for all three groups
of microorganisms (molds, yeasts and bacteria). A description of the
apparatus and an explanation of the procedure were given by Wickerham and
Flickinger (1946). It will not be amiss to emphasize two special advantages of
the NURB process. The first of these is the relative inexpensiveness of the

lyophil apparatus. The main item of expense is the vacuum pump. It, however,
is of a type generally found in microbiological laboratories. The rest of the
equipment consists of items which either are available in all laboratories or
may be constructed fairly cheaply. The second advantageous feature is the
speed of the operation. We can routinely lyophilize 30 cultures in triplicate in a
day (3 runs with our setup). An experienced operator can increase production
to 40 cultures in triplicate (4 runs) on any day that he is free of other duties.