Sunday, 6 August 2017

Introduction To Sterilization Programme In Fermentation Processes

What is sterilization?

Sterilization is technique to make anything free from organisms either by removing them or killing them.

The removal or killing all organisms from fermentation medium is the main aim of the sterilization process or else the contaminant will deteriorate the process.

Contaminant can enter into the fermenter from various points or sight like
  • Improper sterilization of media 
  • Partial sterilization of air
  • Cooling water system
  • Through out-lets, in-lets or other openings
  • Faulty inoculum procedure
  • Due to faulty process used for pretreatment of crude ingredients
  • Due to leakage in design of fermenter
Why sterilization is required?

If any foreign microorganism invades, the fermentation process then product will not be produce.

Thus, it is very necessary to sterilize the medium and other materials so that only required organisms, which we inoculate, will grow and give high yield of fermentation product. Thus, process of sterilization plays vital role in the fermentation processes.

We have discussed importance of sterilization. Following are the consequences of contamination of fermenter.
  • The medium would have to support the growth of both the production organism and the contaminant resulting in a loss of productivity
  • If the fermentation is a continuous one then the contaminant may outgrow the production organism and displace it from the fermentation
  • The foreign organism may contaminate the final product for example single-cell protein where the cells separated from the broth constitute the product
  • The contaminant may produce compounds, which make subsequent extraction of the final product difficult
  • The contaminant may degrade the desired product this is common in bacterial contamination of antibiotic fermentations where the contaminant would have to be resistant to the normal inhibitory effects of the antibiotic and degradation of the antibiotic is a common resistance mechanism for example, the degradation of beta lactam antibiotics by beta lactamase producing bacteria
  • Contamination of a bacterial fermentation with phage could result in the lysis of the culture
Contamination may be avoid in fermenter by using following precautions
  • Using a pure inoculum to start the fermentation
  • Sterilizing the medium to be employed
  • Sterilizing the fermenter vessel
  • Sterilizing everything that is used during the process
  • Maintaining aseptic conditions during the fermentation
Principles of Sterilization
Microorganisms will be removed by sterilization process which otherwise will create problem in fermentation process.
There are two main methods for the sterilization.
  • Destruction of microorganism
  • Removal of microorganism
Methods of Sterilization
Sterilization is carry out by many methods like
  • Filtration
  • Radiations
  • Ultra sonic treatment 
  • Heat treatment 
  • Chemical treatment
The extent to which this procedure adoptsdetermine by the likely probability of contamination and the nature of its consequences.
Microbes that may contaminate the fermenter vessel include bacteria, fungi, protozoa, spores, and phages.
Destruction of microbes may be achieve by either chemical or physical methods.
The common physical agent is Moist Heat
  • Moist heat is the satisfactory sterilization process in industry
  • Removal of microorganisms done by filtration
  • Various kinds of filters are available for this purpose
Filtration for the removal of microbes depends on.
  • Size of microorganism
  • Effectiveness of filter
The strictness of sterilization condition depends on.
  • Types of fermentation process
  • Time of fermentation process
  • Type and magnitude of contamination of the other article 
Some fermentations are described as 'protected' - that is, the medium may be utilized by only a very limited range of microorganisms or the growth of the process organism may result in the development of selective growth conditions such as a reduction in pH.
The brewing of beer falls into this category hop resins tend to inhibit the growth of many micro-organisms and the growth of brewing yeasts tends to decrease the pH of the medium. Thus, brewing worts are boiled but not necessarily sterilized and, the fermenters are thoroughly clean with disinfectant solution but are not necessarily sterile. Also the precautions used in the development of inoculum for brewing are far less stringent than for example in an antibiotic fermentation.
However the vast majorities of fermentations are not 'protected' and if contaminated would suffer some of the consequences previously listed.
Which article to be sterilize?
  • Sterilization is required for Media, Air, Fermenter, Feed and additives
  • Different articles are sterilize by different methods
  • Heat labile (Serum, blood or animal tissue) medium is sterilized by filter
  • Heat stable media is sterilize by steam under pressure
  • Air is mainly sterilized by filter
Depending on the volume and process sterilization of media may be.
  • Continuous sterilization process or
  • Batch sterilization process

Wednesday, 17 May 2017

Research Institutes Statewise In India

Andhra Pradesh
Arunachal Pradesh
Himachal Pradesh
Jammu & Kashmir
West Bengal

Monday, 24 April 2017

Why You Should Compost The Organic Waste ?

  • Home composting is a valuable tool in educating children about nature and the cycle of life. 
  • Adding organic materials to the soil improves moisture retention.
  • Healthy plants from healthy soil look better, produce better and have a much greater ability to fight off pests and diseases.  
  • Compost provides a balanced, slow–release source of nutrients that helps the soil hold nutrients long enough for plants to use them. 
  • The nutrients from compost are not washed away by rainfall. No waste!
  • Keeps a valuable resource out of the landfill.
  • Reduce civic costs for waste collection and thereby reduce fuel use
  •  Extend the life of landfills. Remember residential waste is 40% compostable materials.
  • Compost is a mild, slow release, natural fertilizer that won’t burn plants like chemical fertilizers.
  • It also improves texture and air circulation for heavier soils (like Manitoba gumbo) and helps to increase the water retention of sandy soils
  • Provides organic matter and nutrients which will improve plant growth and  lead to better yields.
  •  Organics in landfills break down anaerobically (without oxygen) to produce methane gas, a greenhouse gas 21 times more harmful than CO2.
  • Buried organics can react with metals in the landfill to produce toxic leachate, a potential source of groundwater pollution.

Saturday, 4 March 2017

Classification of Fermentation Process

Fermentation Processes can be classify into five different categories
  • Processes Producing Microbial Enzymes
  • Processes Producing Microbial Metabolites
  • Processes Producing Microbial Cells [ Biomass]  as the Product
  • Processes Producing Recombinat Products
  •  Processes Modifying Substrates [ Transformation Processes]
Processes That Produce Microbial Enzymes

  • Microbes, plants and animal are the major source of enzymes
  • Commercial production of many enzymes exploiting these sources have been achieved
  • As being produced in large quantities by the fermentation processes, microbial enzymes have the enormous economic potential
  • Microbes are more prone to change in its genetics to enhance its productivity compared to plant or animal system
  • It is possible to produce enzymes of eukaryotes into the prokaryote systems with the help of recombinant DNA technology
  • It is possible to control and improve microbial enzyme production by introducing inducers and activators in the production medium
  • It is also possible to increase the copy number of gene coding for the a specific enzyme using principles of recombinant DNA technology
Process That Produce Microbial Metabolites

The growth of microbial culture can be divided into major four phases :
  • Lag Phase
  • Log Phase
  • Stationary Phase
  • Death Phase
Lag Phase
  • Once the inoculation of the cells into fresh medium is done, the bacterial population remains temporarily unchanged
  • There is no cell division during this phase
  • The cells grow in volume and mass by synthesizing the population remains temporarily unchanged etc
  • Metabolic activity is at high rate
  • This period is known as the period of adaptation
  • There are various factors that affects the this phase are size of inoculum, time required to recover shock in the transfer, time required for synthesizing essential coenzymes and other factors
  • Time required for synthesis of necessary new enzymes to metabolize the substrates present in the medium
Phase of Exponential Growth
  • This period is also known as the phase of exponential growth
  • During this period, the growth rate of the cells gradually increases
  • The cells grow at a constant, maximum rate 
  • Cells are growing in geometric progression dividing by binary fission
  • The incubation conditions and composition of the growth medium control the rate of cell division
Stationary Phase
  • During this phase growth cease
  • In a batch culture (in test tube or EM flask) , exponential growth cannot be continued forever
  • Various factors like exhaustion of available nutrients, accumulation of inhibitory, metabolites or end products and lack of biological space limit the growth during this phase
  • During this phase the number of dividing cells equals the number of dyeing cells
  • This is not a quiescence period like lag
  • This is the phase during which bacteria produce secondary metabolites, such as antibiotics
Death Phase
  • This phase is the reverse of the log phase
  • The viable cell population declines exponentially during this phase
Base on the various products produced, the phases of bacterial growth can be categorized into two phases. These are
  • The Trophophase
  • The Idiophase
The Trophophase
  • Metabolites which are essential to the growth of the cells like amino acids, nucleotides, proteins, nucleic acids, lipids, carbohydrates are produced during the log phase of the growth
  • The products (metabolites) produced during this phase (log phase) are known as primary metabolites and the phase in which they are produced(equivalent to the log, or exponential phase) is referred to as the trophophase
  • The primary metabolites are also known as central metabolites
  • Several primary metabolites are of economic importance and can be produced in large quantity by fermentation process
  • The synthesis of primary metabolites by wild-type micro-organisms aims to meet the requirements of the organism
  • The industrial production these metabolites can be achieve by providing appropriate cultural conditions to the wild-type organism to increase and improve the productivity of these compounds
  • Productivity can also be improve by modifying interested genes by the help of recombinant DNA technology
Following are few economically important primary metabolites which can be produced at large scale
  • Ethanol : Active Ingredient in alcoholic beverages, Used as a motor-car fuel when blended with petroleum
  • Citric Acid : Various uses in food industry
  • Glutamic Acid : Flavour Enhancer
  • Lysine : Feed Supplement
  • Nucleotides : Flavour Enhancer
  • Phenylalanine :  Precursor of aspartame, Sweetner
  • Polysaccharides : Application  in food industry, Enhanced oil recovery
  • Vitamins : Feed Supplements
  • During the stationary phases several microbial cultures produce certain compounds (these compunds are not produced during the “trophophase” and which do not appear to have any obvious function in cell metabolism). These compounds are called the secondary compounds of metabolism. The phase during which these compounds are produced (equivalent to the stationary phase) as the “idiophase”
  • The secondary metabolism is also known as “special metabolism”
  • The products of secondary metabolism are not absolutely required for the survival of the organisms
  • All microorganisms do not undergo secondary metabolism. It is common amongst the filamentous bacteria and fungi and the spore forming bacteria
  • The taxonomic distribution of secondary metabolism is different from that of primary metabolism
  • The physiological role of secondary metabolism and hence secondary metabolites in the producer cells has been the subject of considerable debate
  • The large scale production of secondary metabolites focus on the importance of these metabolites on organisms other than those that produce them
  • Secondary metabolites play an important physiological role several ways. Many secondary metabolites possess antimicrobial activity, some acts as specific enzyme inhibitors and growth promoters and many have pharmacological properties
  • Thus, due to a huge economic potential, the industrial production of these metabolites have formed the basis of a number of fermentation processes
  • As the wild-type microorganisms produce very low concentrations of secondary metabolites, the large scale synthesis can be controlled by induction, catabolite repression and feed-back systems
  • Following is the outline of inter-relationships between primary and secondary metabolism and their respective products

Processes That Produce Microbial Cells [ or Biomass ] as The Product
The commercial microbial biomass production can be divided into two major processes :
  • The production of yeast  to be used in the banking industry
  • The production of microbial cells which can be used as human and/or animal food [single cell protein]
Recombinant Products
  • Recombinant DNA molecules are also known as chimeric DNA, as they consist genes (DNA) of two different species
  • The nucleotide sequences used in the construction of recombinant DNA (rDNA) molecules can be from any species. For instance, plant or human DNA may be combined with bacterial DNA, or human DNA may be joined with fungal DNA
  • Genes from higher organisms can be inserted into microbial cells in such a way that the recipients are capable of synthesizing 'foreign' proteins
  • The advancement in the application of rDNA technology has made possible to produce a range of recombinant products by the fermentation process
  • A wide range of microbial cells have been used as hosts for such systems including Escherichia coli, Saccharomyces cerevisiae and filamentous fungi
  • Recombinant DNA is widely used in research, agriculture, medicine and biotechnology
  • Several products that result from the use of rDNA technology are found in almost every pharmacy, medical testing laboratory, doctor’s as well as and veterinarian’s office, and biological research laboratory
Following are the recombinant products that produced by genetically engineered organism 
  • Human Growth Hormone [rHGH]
  • Biosynthetic Himan Insulin [BHI]
  • Envelope Protein  of the Hepatitis B Virus
  • Follicle Stimulating Hormone[FSH]
  • Blood Clotting Factor III
  • Erythroprotein [EPO]
  • Granulocyte Colony -Stimulating Factor[G-CSF]
  • Alpha-Galactoside
  • Alpha-L-iduronidase
  • N-acetylgalactosamine-4-sulfatase
  • Dornasealfa
  • Tissue Plasminogen Activator [TPA]
  • Glucocerebrosidase
  • Interferon [IF]
  • Insulin like Growth Factor I[IGF-I]
  • Bovine Somatotropin[bST]
  • Bovine Chymosine
 Process Modifying Substrates [ Transformation Process ]
  • Many microbial cells may be exploited to convert a compound into a structurally related, financially more valuable compounds
  • As microbes can behave as catalysts with high positional specificity and stereo-specificity, microbial processes are more specific than purely chemical ones
  • These microbial processes enable the removal, addition and/or modification of various functional groups at predefined specific sites on a complex molecule without the use of chemical protection
  • The reactions which may be catalyzed include Dehydrogenation, Oxidation, Hydroxylation, Dehydration and Condensation, Decarboxylation, Amination, Deamination and Isomerization
  • As microbial processes can be operated at a relatively low temperatures and pressures have the additional advantage over chemical processes which require high temperatures, more pressures and presence of heavy-metal catalysts-a potential environmental pollutant
  • Production of vinegar is the most well-established microbial transformation process (conversion of ethanol to acetic acid)
  • Many transformation processes have been rationalized by immobilizing either the whole cells, or the isolated enzymes on an inert support which catalyze the reactions
  • The immobilized cells or enzymes may be reused many times

Thursday, 23 February 2017

The Developement of Inocula for Mycelial Processes


The majority of the industrial fermentation processes carried out using mycelial (filamentous) organisms like fungi and Streptomycetes

Vegetative fungi and spores of fungi used as inoculum.

The majority of industrially important fungi and Streptomycetes are capable of asexual sporulation so it is common practice to use a spore suspension as inoculum during an inoculum development programme.

A major advantage of a spore inoculum is that it contains far more 'propagules' than a vegetative culture.

A major advantage of a spore inoculum is that it contains far more 'propagules' than a vegetative culture.
  • Spores development [Sporulation] on solidified media
  • Spores development [Sporulation] on solid media
  • Spores development [Sporulation ]  on submerged culture media
First, we will see production of spores and then use of spore as inoculum.

Sporulation on Solidified Media

Most fungi and Streptomycetes will sporulate on suitable agar media but a large surface area must be employ to produce sufficient spores.
Roll bottle technique given by Parker (1950) for the production of spores of Penicillium chrysogenum on solid media.
In this technique three hundred cubic centimeters medium containing three percent agar sterilized in one cubic decimeter cylindrical bottles, which then, cooled to forty-five degree and rotated on a roller mill so that the agar set as a cylindrical shell inside the bottle.
These bottles inoculated with a spore suspension from a sub-master slope and incubated at twenty-four degree for six to seven days.

Sporulation on Solid Medium

Many filamentous organisms will sporulate freely on the surface of cereal grains from which the spores harvested.
Substrates such as barley, hard wheat bran, ground maize, and rice are all suitable for the sporulation of a wide range of fungi.
The sporulation of a given fungus affected by the amount of water added to the cereal before sterilization and the relative humidity of the atmosphere, which should be as high as possible during sporulation.
Fungi can produce relatively large number of spores on wheat bran or barley bran compared to solidified media like Nutrient agar and Sabouraud agar at particular temperature and humidity.
Humidity is very important for the growth of fungi and production of spores about ninety to ninety-eight percent of humidity is required.
Sporulation in Submerged Culture
Many fungi will sporulate in submerged culture provided a suitable medium is employed and suitable condition provided.
This technique is more convenient than the use of solid or solidified media because it is easier to operate aseptically and it may apply on a large scale.
The technique first adopted by Foster et al. (1945). He induced submerged sporulation in Penicillium notatum by including two point five percent calcium chloride in a defined nitrate-sucrose medium.
Medium components and other conditions favor the sporulation of fungi in submerged culture. According to Rhodes et al. (1957) the conditions necessary for the submerged sporulation of the griseofulvin-producing fungus Penicillium patulu, and the nitrogen level had to be limited to between point zero five and point one percent weight by volume and that good aeration had to maintain.
Most Actinomycetes do not sporulate in submerged culture due to this limitation they are more suitably cultivated using solid or solidified media for the production of spore inocula.
The Use of the Spore Inoculum
For the production of product at large scale, spore itself or vegetative cells developed from the spores used depending on the organism’s fermentation quantity and processes.
Some fermentation process can proceed with both either spore or vegetative cell produced from the spore.
In the clavulanic acid process the spore inoculum used to, inoculate the final seed stage. In the chlortetracycline process, a vegetative stage of fungi is use for the fermentation process.
Direct spore inoculation would avoid the cost of installation and operation of the seed tanks whereas the use of germinated spores would reduce the fermentation time of the final stage thus allowing a greater number of fermentations to carry out per year.
However, labor costs for the production of the vegetative inoculum could be almost as high as for the final fermentation although some of these costs may recover.
Inoculum Development for Vegetative Fungi
Some fungi will not produce asexual spores and therefore such process must use an inoculum of vegetative mycelium.
Gibberella fujikuroi is a fungus used for the commercial production of gibberellin. Cultures grow on long potato dextrose agar slants for one week at twenty-four degree.
Growth from three slants scraped off and transferred to a nine cubic decimeter. This medium aerated for seventy-five hours, at twenty-eight degree before transfer to a hundred cubic decimeter seed fermenter containing the same medium.
The major problem in using vegetative mycelium as initial seed is the difficulty of obtaining a uniform standard inoculum.
The procedure may improve by fragmenting the mycelium in a homogenizer such as a Waring blender prior to use as inoculum.

The Effect of the Inoculum on the Morphology of Filamentous Organisms in Submerged Culture
When filamentous fungi grown in submerged culture they can grow as 'pellet' form consisting of compact discrete masses of hyphae or as the filamentous form in which the hyphae form a homogeneous suspension dispersed through the medium.
When they form pellet they will not be able to grow properly due to nutrients and oxygen-limiting conditions inside the pellet while if they form filamentous forms than their distribution may not be proper in production medium to their filamentous growth.
The information available on the morphology of Actinomycetes in submerged culture is very limited compared with that on fungi.

Tuesday, 14 February 2017

Biotoilet : Common Solution for Human Sewage Treatment

Biotoilet is  simplified anaerobic digestion system of human sewage. Instead of Common Septic tank, Anaerobic digestion tank connected with toilet drain line to digest the human sewage solid waste. In the absence of Oxygen, Microorganism breakdown the human solid waste into biogas and compostable manure fertiliser liquid which is utilize for gardening purpose.

Main Objective of Biotoilet :
  • No energy dependence system for sewage treatment.
  • Affordability in rural area & construction site.
  • Minimum water uses for cleaning & Sewage Treatment.
  • Allow weather system.
  • Allow toilet cleaning agents for better hygiene. 

Process & Technologies :
Biological process takes place in four stage 1] Hydrolysis : Large polymer converted into simple monomers 2] Acidogenesis :Simple monomers are converted into volatile fatty acids 3]Acetogenesis & 4] Methanogenesis : Volatile fatty acids are converted into methane & carbon dioxide. 

Salient Future of Biotoilet :
  • Does not require any septic tank, sewage tank connectivity.
  • Disposes human waste in a 100% ECO friendly manner.
  • Existing out of order septic tank can be replace by bio-digester in existing public & toilets.
  • Maintenance free biological process.

Phytorid Technology For Sewage Treatment & Domestic Waste Water Treatment

Phytorid Technology Developed By National Environment Engineering Research Institute(NEERI). Application of Phytorid Technology is to treat sewage and waste water treatment for domestic and industrial Sector.
Phytorid Technology

Phytorid is a Scientifically developed systematic treatment methodology for wastewater.
  •  Phytorid combines physical, chemical and biological process.
  • Works on gravity
  • No electric power requirement
  • Scalable technology
  • Easy to maintain
  • Adds to aesthetic
  • Cost effective
Salient future of Phytorid Technology :
  • Treatment efficiencies for the removal of faecal coliforms, BOD, COD, nutrients are up to 95%, which is greater than the traditional chemical methods.
  • It is a very cost effective technology when compared with the traditional wastewater treatment methods
  • Since it utilizes natural vegetation and rhizosphere microorganisms, it is eco-friendly method of treating sewage.
  • An important factor to be considered is the aesthetic improvement that is provided by this methodology.
  • No mosquitoes and odour nuisance
  • The treated water can be used for enhancement of environmental architecture such as roadside fountains.
  • The quality of treated water is comparable to irrigation standards