A bioreactor is a vessel in which a chemical process is carried out which involves organisms or biochemically active substances derived from such organisms. This process can either be aerobic or anaerobic; process has usually 3 major stages:
1 .Preparation of nutrient media for the cultivated microorganism and the cultivation process.
2 .The course of the microorganism reproduction process in bioreactors called also fermenters.
3 .Obtaining of the final product from the cultivated medium.
TYPES AND CLASSIFICATIONS OF BIOREACTORS
Bioreactors are generally classified into two broad groups ;
I) suspended growth bioreactor
Ii, biofilm bio reactors
I. SUSPENDED GROWTH BIOREACTORS
The reactors use microbial metabolism under aerobic, anaerobic, conditions to biosorb organic compounds and biodegrade them to harmless residuals. The microbial activity in the systems produces biomass that is removed by gravity sedimentation.
Types of SGB is 1, Batch reactors, 2, CSTR’S, 3, Plug-flow reactors etc
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1.I, THE BATCH BIOREACTOR: A typical batch reactor consists of a tank with an agitator and integral heating/cooling system. These vessels may vary in size from less than 1 liter to more than 15,000 liters. They are usually fabricated in steel, stainless steel, glass lined steel, glass or exotic alloy.
Liquids and solids are usually charged via connections in the top cover of the reactor. Vapors and gases also discharge through connections in the top. Liquids are usually discharged out of the bottom.
ADVANTAGES OF THE BATCH BIOREACTOR
1 .Easy operation and absence of mechanical pumps.
DISADVANTAGES1. Where mixing is a critical parameter, they are not the ideal solution.
i.2, THE CONTINUOUS STIRRED TANK REACTOR :{CSTRS}
The CSTR also known as vat or back mix reactor. The liquid or slurry stream is continuously introduced and liquid contents are continuously removed from the reactor. The basic characteristic of the ideal CSTR is that the concentration of the substrate and microorganisms are the same everywhere throughout the reactor .
ADVANTAGES: The rate of many chemical reactions is dependent on concentration; continuous reactors are generally able to cope with high concentrations due to their superior heat transfer capabilities
Disadvantages: consumption of more power due the presence of mechanical pumps
1.3 ,THE PLUG FLOW REACTOR
This is also referred to as a tubular reactor or a piston- flow reactor. The liquid or slurry stream continuously enters one end of the reactor and
leaves at the other end .
ADVANTAGES:1. Can run for long periods of time without maintenance .
DISADVANTAGES:1. Temperatures are hard to control and can result in undesirable temperature gradients2. Expensive to maintain.
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II. BIOFILM BIOREACTORS: In biofilm reactors most of the microorganisms are attached to a surface.Biofilm is also used regularly for wastewater treatment, and the bacteria can either absorb or break down toxic substances in the water .
The different kinds of biofilm reactors include membrane, fluidized bed, packed bed and the rotating biological contactor
2,1 .PACKED BED BIOREACTORS :The medium to which the microorganisms are attached is stationary e.g. plastic media or pea sized stones.
Commonly packed bed reactors are used for aerobic treatment of waste waters and are known as tricking filters and or biological towers.
ADVANTAGES:1. There is improved contact between the waste stream and the micro organisms.
2.2 ,Fluidized BED REACTOR; The fluidized bed reactor depends upon the attachment of particles, that are maintained in suspension by a high upward flow rate of the fluid to be treated.
The particles are often called biofilm carriers. The carriers may be sand grains, granular activated carbon, and diatomaceous earth
ADVANTAGES:1. Uniform particle mixing
2 .The ability to operate reactor in continues state.
DISADVANTAGE: 1. Increased reactor vessel size
2 .Pressure loss scenario
2.3, THE ROTATING BIOLOGICAL CONTACTOR
The RBC process involves allowing the wastewater to come in contact with a biological medium in order to remove pollutants in the wastewater before discharge of the treated wastewater to the environment.
It consists of a series of closely spaced, parallel discs mounted on a rotating shaft which is supported just above the surface of the waste water. Microorganisms grow on the surface of the discs where biological degradation of the wastewater pollutants takes place. The discs are
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submerged in waste water to about 40% of their diameter and are rotated by power supplied to the shaft. ADVANTAGES: 1.Due to high amount of aeration, waste water is degraded faster and more efficiently.
Immobilized bioreactors
Numerous types of bioreactors are currently used at laboratory or industrial scale. The bioreactor with immobilized biocatalysts is derived from the “classical” bioreactors. The more advantages offered by the use of the immobilized microorganisms, cells or enzymes.
Immobilization has been used for production of metabolites or their bioconversion.
immobilization has been used for commercial production of amino acids, e.g., E. coli cells entrapped in polyacrylamide gel for the production of L-aspartic acid.
L-alanine production using a mixture of E. coli and Pseudomonas dacunhae immobilized in K-Carrageenan edible seaweed, and organic acids
Type of Bioreactors for Immobilized
1, Immobilized Enzyme Bioreactors:Continuous flow reactors are based on immobilized enzymes. They offer the greater productivity per unit amount of enzyme, and it can be used for substrates having low solubility.
These reactors may be of the following types: packed bed reactor and fluidized bed reactor.
1.1 PACKED BED REACTORS
Packed bed reactors, also known as fixed bed reactors, are often used for catalytic processes. Packed bed reactors consist of a cylindrical shell with convex heads.
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In packed bed reactors, cells are immobilized on large particles. These particles do not move with the liquid. The Packed bed reactors are heterogeneous reaction systems. Packed bed reactors have a wide range of uses for catalytic reactions. They are also widely used in small scale commercial reactions. It`s cost-effective methods of removing ammonia from livestock wastewater.1.2 FLUIDIZED BED REACTORSFluidized bed reactors are heterogeneous catalytic reactors in which the mass of catalyst is fluidized. This allows for extensive mixing in all directions, As a result of the mixing is excellent temperature stability ,increased mass-transfer and reaction rates.Fluidized bed reactors are capable of handling large amounts of feed and catalyst. Fluidized bed reactors are commonly used in catalytic cracking processes.
2, Fibrous-Bed BioreactorIn fibrous-bed bioreactor, the cells are immobilized on the fibers in the bioreactor.
Fibers as a support matrix for cell attachment and entrapment or immobilization .it's have High specific surface area and High permeability than compared to granular materials. The fibrous-bed concept offers many advantages over traditional stirred-tank systems. Cells are completely protected from the shear of rotating impellers & sparged gas, while products are easily harvested from the cell-free medium.
The other type of fbb are rotating fibrous bed bioreactor and Spouted bed bioreactor
Fibrous bed bioreactor used for carboxylic acids and alcohols production. Rotating fibrous bed bioreactor using for viscous fermentations and Spouted bed bioreactor for solid state fermentations.
2, MBBR (Moving bed biofilmreactor)MBBR is also known as moving bed biofilm reactor. It is preferred to the wastewater treatment systems.
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Moving Bed Bioreactors (MBBR) biologically treat wastewater by circulating moving media in aerobic and anaerobic activated sludge environments. MBBR technology employs thousands of polyethylene biofilm carriers operating in mixed motion within an aerated wastewater treatment basin. This polyethylene biofilm carriers that protected surface area to support the growth of bacteria while achieves high-rate biodegradation within the system.Application Food and beverage plants, steel mills, oil refineries, petrochemicals, chemical plants, paper mills and any industries requiring wastewater treatment for BOD removal, nitrification and denitrification.3, Membrane bioreactor The Membrane Bio Reactor (MBR) is a biological system coupled with a membrane to enhance solid-liquid separation and filtration. The membrane is made of fibrous material. The MBR can achieve desired permeation rate at low transmembrane pressure. a membrane bioreactor can be categorized into the following groups:
1. Solid State Fermentation:In such fermentations, microbial growth and product formation occur at the surface of solid substrates. Examples of such fermentations are mushroom cultivation, mold-ripened cheeses, starter cultures, etc. More recently, this approach has been used for the production of extracellular enzymes, certain valuable chemicals, fungal toxins, and fungal spores used for biotransformation. Traditional substrates are several agricultural products, e.g., rice, wheat, maize, soybean, etc.
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The substrate provides a rich and complex source of nutrients, which may or may not need to be supplemented. Such substrates selectively support mycelial organisms, which can grow at high nutrient concentrations and produce a variety of extracellular enzymes .
According to the physical state, solid state fermentations are divided into the following two groups: (i) low moisture solids fermented without or with occasional/continuous agitation, and (ii) suspended solids fermented in packed columns through which liquid is circulated.
Solid state fermentations on large scale use stationary or rotary trays. Temperature and humidity controlled and air is circulated through the stacked solids. Less frequently, rotory drum type fermenters have been
used.
2. Submerged Fermentation:Batch Culture:Batch culture is a closed culture system, which contains limited amount of nutrient medium. After inoculation, the culture enters lag phase, during which there is increase in the size of the cells and not in their number. The culture then enters lag phase or exponential growth phase during which cells divide at a maximal rate and their generation time reaches minimum.
The increasing population of bacterial cells, after sometime, enters into a stationary-phase due to depletion of the nutrients and the accumulation of inhibitory end products in the medium. Eventually, the stationary, phase of bacterial population culminates into death-phase when the viable bacterial cells begin to die.
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This typical growth curve is only obtained in a batch culture
2, 1, Fed-Batch Culture:When a batch culture is subsequently led with fresh nutrient medium without removing the growing microbial culture, it is called fed-batch culture. e.g., production of secondary metabolites.
2,2 .Continuous Culture:This condition is obtained by growing microbes in a continuous culture that nutrients are supplied and end products are continuously removed.
3. Anaerobic Fermentation:In anaerobic fermentation, a provision for aeration is usually not needed. But in some cases, aeration may be needed initially for inoculum build-up. The air present in the headspace of the fermenter should be replaced by CO2, H2, N2 or a suitable mixture.
This is particularly important for obligate anaerobes like Clostridium. The fermentation usually liberates CO2 and H2, which are collected and used,
4. Aerobic Fermentation:The main feature of aerobic fermentation is the provision for adequate aeration; in some cases, the amount of air needed per hour is about 60-times the medium volume. Therefore, bioreactors used for aerobic fermentation have a provision for adequate supply of sterile air,
Aerobic fermenters may be either of the (i) stirred-tank type
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)ii (air-lift type
Ex: is penicillin production
Fermentation kinetics and Monods Model-:
I, Growth kinetics and Monod’s Model
These basic forms of the kinetic rate expressions Will be used in the material balance equations for batch, fed-batch and continuous
Microbial cultures in bioreactors.
Requirements for growth
The essential requirements for the growth of any microbial Culture are as follows :
I, a viable inoculum: use an inoculum as a starter
II, a carbon source
II, an energy source;
III, essential nutrients for biomass synthesis;
IV, suitable physico chemical conditions;
The specific growth rate can be expressed in two different ways:
)i (Substrate dependent and (ii) substrate independent
Monod kinetics for growth
Cell metabolism is made up of hundreds of sequential, branched and parallel biological reactions that are normally catalysed by enzymes. The Production of these enzymes themselves is an important aspect of metabolism, that growth is the result of hundreds of such enzyme-catalysed reactions .
)Fig:(
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The simplest of the expressions relating the enzyme reaction rate to the rate limiting Substrate concentration is the Michaelis–Menten expression:
1.1
If we identify an enzyme reaction of the Michaelis–Menten type with the rate controlling Step for growth (Fig) and if we assume moreover that the concentration of This rate controlling enzyme, is proportional to the viable cell concentration, while the Concentration of the substrate for the rate controlling step is proportional to the limiting Substrate concentration in the nutrient medium, then we can write an analogous expression the classical Monod equation for cell growth.
Normally the specific growth rate function µ(S) is simply abbreviated as µ, and it has the dimensions h−1. Using the definition of the volumetric and specific rates for growth, Equation (1.2)
Eq (1.2)
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We have got
) 1.3(
The relationship between the specific growth rate and limiting substrate concentration Proposed by Monod states that:
) 1.4(
Where rx is the volumetric rate of cell growth, kg cells m−3 h−1; µ max is the maximum specific growth rate, h−1; S is limiting substrate concentration, kg substrate m−3; KS is the Saturation constant, kg substrate m−3; xv is the viable cell concentration, kg cells m−3.
When the substrate concentration is not limited, that is when S >> KS numerically, KS Can be ignored in Equation (1.4) and then S cancels each other, the specific growth rate Approaches µ max and the growth rate becomes independent of S and only proportional to the cell concentration. This is the zero order asymptote of the Monod expression, that is The specific growth rate is zero order with respect to substrate concentration:
) 1.5(
When the substrate concentration is lower than the numerical value of the Monod saturation Constant, S << KS, then S can be ignored in the denominator of Equation (1.4) and the specific growth rate becomes first order with respect to the growth limiting substrate Concentration as
(1.6)
This is the first order asymptote of the Monod expression. For the substrate concentrations In between these two asymptotes, the specific rate is a function of the substrate concentration According to the Monod equation (Equation 1.4). In transition from zero order to Monod equation for the specific growth rate, we can define arbitrarily a critical substrate
Concentration, Scrit, in such as way that:
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Eq (1. 7)
Then inserting Equation (1. 7) into Equation (.1. 4), we get:
) Eq 1.8(
It should be noted that Equation (1.8) will depend on the arbitrary Definition we used for the critical substrate concentration; if we had
Defined it in Equation (1.7) as 90% Instead of 99%, then we would get Scrit = 90KS.
When the substrate concentration S is numerically equal to the saturation constant KS Then, the specific growth rate from Equation 1.4) is:
) 1.9(
For this reason, and considering the arbitrary definition of the critical substrate concentration, sometimes the Monod saturation constant, KS is called the critical substrate concentration. the critical substrate concentration is useful in the design of the medium Composition, indicating the concentration of a particular substrate when it becomes growth limiting.
Substrate accelerated death
During the course of fermentation some of the cells become nonviable, i.e. incapable of Growth and reproduction. In this case, the cells cannot maintain their physiological activities, the cell membranes and wall lose their integrity and autolysis can occur .
Units of rd are (kg dead biomass) m−3 h−1.
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Units of kd are (kg dead biomass) (kg live biomass) −1 h−1. The rate Cell death can be mathematically expressed as a first-order rate expression with respectTo viable cell concentration:
Where rd is volumetric rate of conversion to nonviable form) Kg cells m−3 h−1 ;(xv is the concentration of viable cells (kg cells
m−3); kd is the rate constant (kg cells Kg cells−1 h−1).
Equation 1 means that the rate of conversion of viable cells to the nonviable form, the death rate, is assumed to be directly proportional to the concentration of viable cells. It should be noted that it is the viable cells that are contributing to the act of dying and not the dead cells; once cells are dead, they cannot die again to contribute to the death Rate. Dead cells however, contribute to the volumetric rate of autolysis, if they lose their cell membrane integrity. Normally lysis means that an external factor such as a change in the osmotic pressure of the cells’ environment or a toxic chemical such as a detergent, Disrupts the integrity of the cell membrane. Autolysis on the other hand, implies a Self inflicted disruption of the cell membrane integrity, for example, following death or starvation. Lysis as a consequence of viral infection is somewhere in between the two. Ideally therefore, autolysis should be a function of dead cell concentration whereas lysis can involve viable cell concentration as well as the concentration of the external lysis-causing Factor. We shall not distinguish between autolysis and lysis in the treatment below for the sake of simplicity.Cell autolysis can be expressed as a first order rate expression with respect to the dead biomass concentration:
1.1
1.2
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Where rL is volumetric rate of cell lysis (kg cells m−3 h−1); XD is the concentration of dead Cells (kg cells m−3), and kL is the rate constant (kg cells kg cells−1 h−1).
Specific growth rate
The specific rate of a microbial activity is equal to the volumetric rate for that activity Divided by the cell concentration performing that activity. Specific rates are usually defined for growth, product formation and substrate uptake:
1.1
1.2
With these definitions, we use the following nomenclature for the basic microbial activities.For growth:
1.3 Units of Rx are (kg live biomass) m−3 h−1.
1.4
Units of µ are (kg live biomass) (kg live biomass) −1 h−1 or simply h−1.Here, with xv we denote the concentration of living cells as opposed to dead, and we make the distinction that growth is a biological activity performed by living cells.The Monod equation for the Correlation of specific growth rate and substrate concentration does not hold true when the intracellular substrate concentration is reduced during fast growth, even though adequate Substrate is still available in the medium. In order to handle such situations, additional Models have been developed. The logistic equation,
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which is a substrate independent Method is an alternative empirical function to the Monod equation. The logistic equation is also useful in processes where information on the limiting carbon substrate is not available, For instance, the limitation of an unidentified component in complex media. In the Logistic equation, the specific growth rate can be expressed as:
1.1
And for the cell growth:
1.2
Where, µmax is the maximum specific growth rate (h−1) and xvm is the maximum viable Biomass concentration.A few examples of other forms of growth rate expressions are given below.
Tessier model:
Moser model:
Where` λ `is a constant.
Contois model:
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Where B is a constant implying that Bx is an apparent Monod constant that is proportional to biomass concentration x.
The Stringent Response
The stringent response is a global regulatory system, which mediates major changes in gene expression in response to growth-limiting stress conditions. A decrease in nutrient availability requires a rapid metabolic adaptation of the bacterial cell. In bacteria stringent response is mediated by guanosine 5` triphosphates (pppGpp) and guanosine 5` diphosphate 3 diphosphate (ppGpp)
The ppGpp was first discovered as a molecule which accumulated upon nutrient starvation and apparently promoted the down regulation of rRNA promoters in E. coli. Since then, it was shown for various Gram-negative and Gram-positive organisms, that ppGpp, often in cooperation with co-regulatory protein DksA (DnaK suppressor protein A), severely affects bacterial transcription profiles.
Induction of the stringent response is primarily aimed at growth inhibition by repression of genes involved in active bacterial growth, such as translation and replication but also at the expression of factors involved in stress adaptation.
In many bacteria cellular ppGpp levels are controlled by the bifunctional protein Rel, which possesses both ppGpp synthetase and hydrolase functions. Meanwhile, in most γ-proteobacteria bacteria ppGpp levels are determined by proteins RelA and SpoT, First described in E. coli
The SpoT is a bifunctional protein capable of both synthesizing and degrading ppGpp. The alkaline pH values trigger a SpoT-mediated stringent response during anaerobic conditions, RelA lacks a His-Asp motif essential for hydrolase activity and therefore is only capable of ppGpp synthesis .The RelA-dependent stringent response can be simulated by the addition of serine hydroxamate.
The stringent response during oxygen-limiting conditions
The stringent response is involved in mediating bacterial adaptation to oxygen limitation, anaerobiosis and the allocation of alternative electron acceptors. It was shown that a Mycobacterium tuberculosis mutant
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incapable of synthesizing ppGpp showed decreased survival rates during long-term anaerobic survival .the expression of the rmf (ribosome modulation factor) gene regulated by the stringent response in E. coli
Ntr and Pho system,
Ntr system
The activity of Ntr system is activated in response to nitrogen starvation, when ammonia is the growth limiting substrate .the bacteria are able to turn on transcription of genes for glutamine synthetase. The system enables the bacteria e.g. :enterobacteria to assimilate very low levels of ammonia by catalyzing the assimilation of ammonia into glutamine in an ATP dependent reaction .it also activates additional operon e.g.: Nac operon .that are involved in the utilization of organic nitrogen source .this permits the cells to use alternative source of nitrogen.
Bacteria can utilize a wide range of nitrogen compounds as sole sources of cellular nitrogen. These range from simple inorganic compounds such as dinitrogen and nitrate to complex compounds including amino acids such as histidine and arginine or nucleosides such as cytidine.
The global nitrogen regulatory (Ntr) system is composed of four enzymes :
A uridylyltransferase/uridylyl-removing enzyme (UTase/UR), encoded by the glnD gene,
A small trimeric protein, PII, encoded by glnB and a two-component regulatory system composed of the histidine protein kinase NtrB and the
response regulator NtrC
Adenylylation by ATase is promoted by deuridylated PII which is produced by UR action on PII (UMP) 3under nitrogen sufficiency (high glutamine/α‐ketoglutarate ratio). Deadenylylation by ATase is promoted by PII (UMP) 3 formed by UTase action on PII under nitrogen limitation (low glutamine/α‐ketoglutarate ratio).
A regulatory gene (glnG) in E. coli and other enteric bacteria encodes nitrogen regulator I , a dimer protein . It activates transcription of glnA, encoding glutamine synthetase, under nitrogen limitation and represses it
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under conditions of excess nitrogen. NRI binds to DNA at or near the promoter of glnA and is thought to regulate production of all nitrogen‐regulated systems
Pho system
In natural environments, inorganic phosphorus is commonly the major growth‐limiting nutrient. Thus, biological systems have evolved a variety of responses to modulate their phosphorus requirement or to optimize its utilization. The Phosphate (Pho) regulon is a global regulatory mechanism involved in bacterial Pi management that was first characterized in Escherichia coli, and later in many other bacterial species. The most common members activated by the Pho regulon are: extracellular enzymes capable of obtaining Pi from organic phosphates, Pi-specific transporters, and enzymes involved in storage and saving of the nutrient. The PST Pi-specific transporter is the most conserved member of the Pho regulon in all bacteria
The Pho regulon is controlled by a two-component regulatory system which comprises an inner-membrane histidine kinase sensor protein and a cytoplasmic transcriptional response regulator. These proteins have received different names in some bacteria, such as: PhoR–PhoB in E. coli
A phosphors utilization network pho system is activated on inorganic phosphate starvation in enterobacteria, resulting in the production of a high concentration of alkaline phosphatase, so that phosphate can be obtained from organic source.
In E. coli, over 30 genes are part of the phosphate regulon (Pho regulon) and are transcriptionally activated by phosphorylated PhoB. PhoR promotes the phosphorylation of PhoB under limiting phosphate conditions and dephosphorylation of PhoB in excess phosphate. PhoR and PhoB are thus a two‐component signal transduction system.
Nucleases and phosphatase are usually repressed by phosphate in fungi. Phosphate also suppresses the production of riboflavin by Eremothecium ashbyii
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Growth limiting substrate
The essential requirements for the growth of any microbial Culture are as follows:• A viable inoculum;• A carbon source;• An energy source;• Essential nutrients for biomass synthesis;• Suitable physicochemical conditions
The growth-limiting nutrient is the one in low proportion to the others. It will be exhausted first.
There are many ingredients that must be present in living cells. Some of these are: C - carbon, S -sulfur, N - nitrogen, etc. It is more convenient to deal with compounds that supply these elements.
The carbohydrate component glucose is the growth limitation for carbon. Although, some carbon may be derived from another element such as nitrogen. The carbon and nitrogen are the growth-limiting nutrient.
The growth limiting is to reduce concentrations of the various nutrients systematically one at a time. If this produces little or no effect, the nutrient that is being probably in excess. If there is a definite effect, the nutrient is very likely the "growth-limiting nutrient".
The form of Eq (4-5) can be used to describe dependence of µ on more than one limiting nutrient. In many practical applications availability of oxygen for respiration often limits growth. When both substrate, S, and dissolved oxygen concentration, CDO, are both limiting growth, specific growth rate can be mathematically described as
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Let consider growth under conditions of substrate limitations in a batch bioreactor. Incorporating the substrate limited condition, bioreactor material balance equation, may write,
In order to integrate the above, one of the variables, S, needs to be replaced in terms of X. The yield relationship, can be integrated as
This simplifies to
Where subscript, 0 refers to initial concentration. Substituting for S from Eq(4-8) in Eq(4-7) and integrating gives,
For analyzing batch systems, use the above to calculate cell concentration and then calculate substrate concentration using Eq(4-8).
The growth rate is ruled by the limiting substrate concentration (sugar)
Maintenance energy,
The microorganisms need energy for growth, intracellular/ extracellular product formation, and for maintenance functions such as transportation
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of cellular materials, osmotic regulation and defense against O2 stress. The kinetic approach to microbial dynamics requires the incorporation of maintenance energy expenditure with biomass growth under various types of bioprocesses such as primary/secondary metabolite production, bioremediation, bioaccumulation and biosorption.
During the fermentation process, substrates are utilized by microbial cells for biomass production, product formation, and for maintenance expenditure. The exponential growth of bacterial cell biomass is expressed as:
in which µ is the specific growth rate (h-1) according to Monod:
Where µmax is the maximum specific growth rate (h-1), Ks is the substrate saturation constant (g/L), and S is the substrate concentration (g/L).
During fermentative production of P(3HB), the initial microbial concentration (inoculum) and substrate are considered as reactants Thus, the microbial growth dynamics are determined by both the microbial specific growth rate and the substrate consumption rate.
The rate of substrate consumption, analogous to the biomass growth rate as a function of biomass concentration is given by:
Where qS is the specific substrate consumption rate and can be expressed in terms of the true growth yield and maintenance coefficients as:
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where YX /S is maximum growth yield coefficient (g/g) and ms is the maintenance energy coefficient (h-1).Upon substitution of Eq. (2) into Eq.(4),
When the product formation (P(3HB)) is related to carbon source consumption:
Upon substitution of Eq. (4) into Eq. (6)
where YP/S is the maximum P(3HB) yield coefficient (g/g).
In addition, the P(3HB) production by bacterial cells is described as:
Where qp(3HB) is the product formation rate, and K1 and K2 represent the growth and non-growth associated product constant, respectively.
The total maintenance energy expenditure can be divided into two components; one is a constant which is required throughout the cultivation process and the other component is growth dependent. The second component of maintenance energy depends on the specific growth rate (µ).
The maintenance is considered to be the consumption phenomenon, corresponding to energy wastage, which is increased under unfavorable environmental conditions. In general, a low maintenance value (ms) is
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observed at high specific growth rate that favors biomass growth and
metabolite production.
Ms =ms1 +ms2 (10)
Where, ms is maintenance energy expenditure, ms1 is the constant and ms2 is the growth-dependent component of the maintenance coefficient.
Where ` k is a positive quantity that depends on the substrate-microorganism system.
The maintenance energy consumption also depends on some physical factors such as temperature and salt concentration in the medium.
Upon substituting Eq. (11) into Eq. (4)
where k (1-µ µmax )is growth dependent and ms1 is the constant component of maintenance energy.
The percent of the total substrate (carbon source) consumption used for cell energy maintenance varies with the specific growth rate of microorganism and calculated as
Where ms (h-1) is total maintenance expenditure (ms1 +ms2) and qs is the specific substrate consumption rate (g/g).
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Growth yield and product formation
Specific rates are usually defined for growth, product formation and substrate uptake:
With these definitions, we use the following nomenclature for the basic microbial Activities:
For growth:
Units of rx are (kg live biomass) m−3 h−1.
Units of m are (kg live biomass) (kg live biomass)−1 h−1 or simply h−1.
For product formation
Units of rP are (kg product) m−3 h−1
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Units of qp are (kg product) (kg live biomass) −1 h−1.Here, we assume that product formation is performed by the living cells, and hence live biomass concentration is used in the definition of the specific product formation rate. In some rare cases, product formation may be due to dead cells, for example, if the product Is formed as a result of dead cells autolysing. For substrate uptake:
Units of rS are (kg substrate) m−3 h−1.
Units of qS are (kg substrate) (kg live biomass) −1 h−1.The cells themselves are the most important products of fermentation or biological activity. The energy required to drive the cell processes is the chemical energy of ATP or similar substances.ATP and other energy currency compounds in the cell in most cases is provided either aerobically or anaerobically.
Figure 1.1is a schematic description of anaerobic and Aerobic breakdown of carbon-and-energy substrates, and concomitant production of energy in the form of ATP and formation of various products.)
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In aerobic cells, ATP is generated by the oxidation of substrate (usually a carbohydrate, the carbon and energy Source) by molecular oxygen to CO2 and water (oxidative phosphorylation) In anaerobic Cells ATP (and other energy currency compounds) is generated by the degradation of Substrate to simpler products such as ethanol, lactic acid, CO2 and water, etc., which are excreted by the cell (substrate level phosphorylation)Other extracellular products include such compounds as:• Exoenzymes (for breaking down substrates that cannot pass through the cell wall);• Polysaccharides (for cell aggregation, avoidance of desiccation, binding metal ions, etc.);
•Special metabolites (e.g., antibiotics).
There are substances produced in situations where the carbon substrate is in excess and other substrates, such as nitrogen or magnesium, are limiting. They include possible ‘Energy storage’ compounds such as glycogen or lipids, etc., which are stored within the Cell, or similar polysaccharides, etc., excreted by the cell. These products are consideredBy some to act as an ‘energy sink’; excess ATP is produced so as to use the limiting Substrate more efficiently, and the formation of energy storage products then dissipates the chemical energy of the excess.The products of biological activity can be classified based on their production kinetics As follows:Growth-associated
No growth associated
Mixed kinetics
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In these equations, α` and β are constants then is called the Luedeking–Piret Expression for product formation. Using the Monod equation for cell growth, it expands to:
Process optimization: factors of optimization
The optimization process is to determine the optimum value of fermentation production efficiency that leads to the maximum yield of product output. The optimization of fermentation process that are:
OPEN AND CLOSE ENDED SYSTEMS FOR PROCESS OPTIMIZATION
In close-ended system, a fixed number and type of component parameters are analyzed for optimization. Ex; Factorial Design, One-factor-at-a-time. Biological Mimicry.
In open-ended system any number and type of components/parameters are analyzed for optimization of fermentation process. ex. Borrowing Component Replacing,
Factorial Design
In this method, level of factors/parameters are independently varied, each factor at two or more levels. Typical factors are microbial strain, medium components, temperature, humidity, initial pH and inoculums volume .
Biological MimicryBiological mimicry is a close-ended system for fermentation process optimization. This method is useful for optimization of various components of fermentation media and based on concept that cell grow well in a medium that contains every things it needs in right proportion (mass balance strategy). The medium is optimized based on elemental composition of microorganisms and growth yield.
One-factor-at-a-timeOne-factor-at-a-time is a close-ended system for fermentation process
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optimization. This method can be applied for optimization of medium components as well as for process condition and it is based on the classical method of changing one independent variable while fixing all other at a certain level
Component ReplacingThis is an open-ended system for process optimization and only used to compare the component of one type in a fermentation medium . In this method, one of component of the medium was replaced by a new one at same incorporation level. However this method does not consider the components interactions. But this method can useful for screening different carbon, nitrogen and other source for improving the medium utilization
Genetic AlgorithmsIn recent years non-statistical optimization techniques such as genetic algorithms are used in fermentation technology. this technique can be used to optimize fermentation process without need of statistical designs and empirical models and based on the principle of mutation
individual .
These individuals strive for survivals. After some number of generations only the best individual hopefully represents the optimum solution .
Other factors are
pH
The pH of a culture medium will change with the metabolic product of microorganisms. Their optimum pH range for growth is around pH 6.0. the most bacteria are inhibited at pH values below 3.5..the acid and
base using for the controlling the pH .
Temperature
The Most fermenters operate around 30–36 °C, but certain fermentation may require control of the temperature in a range of 0.5 °C To maintain r temperature within the limited range, the system may require regulation of heating and cooling by the control system.
Dissolved oxygen
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The dissolved oxygen content of the fermentation has affecting cell growth and product formation. Control of DO can achieved by increasing the airflow rate or rpm of impeller
Foam
Foaming of the fermentation is a nuisance, reflects on the mass transfer process and must be prevented, for many reasons. The problems related to foaming are obvious if they are due to gas sparing. The problems are the loss of broth, clogging of the exhaust gas system And possible contamination. Antifoam agents are mineral oil, Vegetable oil and certain alcohol
Aeration and Agitation
The main function of aeration is to supply enough oxygen to the microbes in submerge culture technique for proper metabolism, while agitation provides proper mixing of the nutrient so that each and every organisms get proper nutrients.
RHEOLOGY OF FERMENTATION FLUID
Rheology is the science of flow and deformation of matter and describes the inter relation between force, deformation and time.
Fluid rheology is used to describe the consistency substance of different products, normally by the two components viscosity and elasticity. By viscosity is usually meant resistance to flow or thickness and by elasticity usually stickiness or structure.
Rheology is an important and relevant parameter of understanding and controlling the fermentation process.
The use of rheology part could contribute significantly in understanding the changes that occur during the fermentation process.
Rheological data could be used in trying to optimize the conditions towards optimum fermentation process.
The use of rheology data such as the type of non Newtonian fluid could indicate the right opportunity to change the mixing regime and save energy
The fermentation fluid is a very complex soup or solution. Fundamentally, the fermentation broth is the sea of nutrients in which the microorganisms grow, reproduce and 'swim'. The fermentation broths supply the microorganisms with all the nutrients the microorganisms need to grow and produce the various fermentation products.
The fermentation broth too acts as the medium for various physical, biochemical and physical reactions to take place. The fermentation broth will be implicated in all the mass and heat transfers that occur within the fermentor, and it will be the medium that holds the fermentation products formed.
The nature and composition of the fermentation broth temporally and spatially will affect the efficiency of the fermentation process. The interactions between the fermentation broth and the various components are complex and affect both directions
At any time the composition of the fermentation broth is complex consisting of:
1 Raw substrates2 Fermentation products
3 Microorganisms and its derivative components4 Chemical additives added to the fermenters
5 Gases such as oxygen and other metabolic gases
All three main phases; solid, liquid and gases are present in the fermentation broth and their possible interactions
RHEOLOGICAL PROPERTIES OF FERMENTATION BROTH----------------------------------------------------------------One of the most important singular properties of the fermentation broth will be its rheological or viscosity characteristics.
Fermentation broth as a thick gooey sticky mixture that is thick and viscous compounded by rising bubbles of gas exploding at the broth surface.
The viscous nature or the rheological properties will affect the mixing regimes of the fermentor. Viscosity is not a simple but a complex
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phenomenon that is always changing and responding to various parameters.
The viscousness of the fermentation broth is caused by the interactions of the various components in the fermentation broth. Such interactions in the viscosity of the broth could occur at the level of the ions and molecules which involve the various ionic forces or it could involve at the macro level such as between the various biopolymers tangling and sliding with each other. The overall result will be that the fermentation broth will be viscous.
Most of the viscosity in the fermentation fluid is caused by the various hydrogen and other ionic bonding. The Sugar in solution is sticky, but dry sugar is not sticky. The stickiness or viscousness of the sugar in solution is caused by hydrogen bonding which develop between the sugar molecules and water. During the interactions of sugar and water the hydrogens in the water molecules and the hydrogen in the sugar molecules have an attraction for each other. Thus it is the hydrogen bonding that makes the sugar sticky.
Oxygenation and oxygen transfer kinetics
Oxygenation: Oxygen is one of the fundamental requirements in any aerobic fermentation. The process of treating a bioreactor with oxygen that lead to supporting or inhibition of different types of fermentation processes. The microorganisms can only use oxygen not directly from air but only in the form of dissolved oxygen. The oxygen demand of an industrial fermentation process is normally satisfied by bubbling air through the liquid, spraying the liquid into the air, Increasing stirrer speed or mixing, increasing volume of flow and increasing the air pressure. These parametric controls have unavoidably resulted in building very large and costly fermentorsThe oxygen used by the microorganisms used in the fermentation process comes from two main sources:
1 Oxygen incorporated in the organic substrate which are used directly in
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the metabolism of biosynthesis of new biomass2 Oxygen from the air which is pumped through the fermentation broth and used by the microorganisms in their respiration activities as their terminal electron acceptors However, the productivity of many fermentation is limited by oxygen availability. The maximum production achieved by maintaining the dissolved oxygen concentration. The DO level must be maintained greater than the critical level. If the dissolved oxygen concentration were to fall below the critical level then the cells may be metabolically disturbed.
PROBLEM OF MICROBIAL OXYGEN TOXICITY-----------------------------------------------------Most accept that oxygen is toxic to anaerobic microorganisms. Exposure to oxygen could kill obligate anaerobic microorganisms because their physiology is not equipped with enzymes that can make the toxic oxygen harmless. Aerobic microorganisms on the other hand have these enzymes to neutralize the toxic oxygen molecules. Therefore oxygen can be toxic to aerobic microorganisms depending on the situation of operation.The microbes are killed by the oxygen because they are not adapted to the
operating conditions of oxygenation .
Oxygen transfer kinetics.Oxygen from the gas phase transfers to the bulk liquid through the gas–liquid interface. This interface is created when air is bubbled through the bulk liquid using compressors and a sparger.
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Fig is a schematic representation of the conditions at the gas–liquid interface. The rate of the mass transfer from gas to liquid phase using the two film theories. On either side of the gas–liquid interface is a boundary layer or
Film of relatively Stagnant fluid Molecules of oxygen must move from the bulk gas through the stagnant gas film, go into solution (become dissolved) at the interface, move through the stagnant liquid film, eventually reaching the bulk liquid. Mass transfer will only occur If there is a driving force for mass transfer in the form of a concentration difference.
The concentration of oxygen on the liquid side of the interface is greater than that in the bulk liquid, providing a concentration driving force to transfer oxygen molecules into the liquid. At steady state, these two rates of mass transfer must beEqual:
Where kG and kL is the individual, gas and liquid, respectively, mass transfer coefficients with units of (µ)h−1.
At the interface, the oxygen concentrations on the gas and liquid side are in equilibrium described by Henry’s Law, which is a thermodynamic relationship:
The Equation cgi=hcli in terms of a concentration we can Measure's *g is defined as the fictitious bulk liquid concentration of the dissolved gas (in this Case dissolved oxygen), which would be in equilibrium with the bulk gas concentration, Cg. We can then write
The difference between C*g and CL provides the driving force for mass transfer. The mass Transfer rate (expressed as a flux, i.e. rate per unit
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area) can be described in terms of this Driving force and an overall mass transfer coefficient KL as:
The overall mass transfer coefficient KL is the inverse of the sum of the individual resistances, kL for the liquid fi lm and kG for the gas film
It turns out that of these, 1/kL is the largest, i.e. essentially all of the resistance to mass Transfer of oxygen from gas to liquid phase is on the liquid-film side. So KL can be replaced by kL:
Overall oxygen transfer rate per unit volume =flux) interfacial area/ liquid volume
Then
And
where a is the specific interfacial area giving:
Henry’s law is often written as:
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Where —PO is the partial pressure of oxygen in the gas phase (N m−2); H′ is the Henry’s Law constant for this case [(N m−2)/(kg oxygen m−3)] C*g is the hypothetical saturation Value of the dissolved oxygen in the liquid at the gas–liquid interface (kg O2 m−3). For an ideal gas that is not pure and has more than one component (e.g., air), the partial Pressure—P O is:
Where P is the total pressure of the gas and y is the mole fraction of oxygen in the gas phase. From this point on, shall use Co instead of CL to indicate the dissolved oxygen concentration in the liquid phase.The volumetric rate of mass transfer of oxygen from the gas to liquid phase, for constant a volume V of the bulk liquid, can be expressedMathematically as follows:
Where Na = volumetric mass transfer rate (kg O2 m−3 h−1 or mgO2/Lh); kLa = volumetric Oxygen transfer coefficient (h−1); C*g = hypothetical saturation value of the dissolved Oxygen in the liquid at the gas–liquid interface (kg O2 m−3); CO = dissolved oxygen concentration In the bulk liquid (kg O2 m−3) OTR = oxygen transfer rate (kg O2 m−3 h−1 or mg O2/Lh).The value of kLa obviously depends on the value of interfacial area, a, the thickness Of boundary layer and the resistance to the diffusion of the oxygen through the boundary Layer. These depend in a complex way on the hydrodynamics of the bioreactor and the Physical properties of the medium and its constituents.
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Chemostat and Turbidostat Cultures
Chemostats are continuous cultures grown in fermentors with control systems, which are widely used for research in microbial physiology. Parameters, like temperature, pH, and concentration of dissolved gases in the growth medium, can be monitored and controlled .
Fresh medium is supplied to the culture at a constant flow rate (F), and removed from the fermenters at the same rate, thus maintaining a constant culture volume (V). At steady state, the growth rate of the culture equals to the dilution rate, D = F/V, and the biomass concentration and other free parameters stabilize at levels depending on the dynamics of the fermentation.
Hence, chemostats allow continuous exponential growth to occur for many generations under constant physicochemical conditions. Crucially, chemostat culture allows the experimenter to control the growth rate (flux) at any value below a maximum value termed the maximum specific growth rate (max). This contrasts with batch cultures, where biomass concentration and the environmental conditions (in terms of pH, nutrient concentration etc.) change significantly during the (limited) time course of the fermentation, and the experimenter has no control over growth rate (flux).
The factor that determines the growth rate of a cell population in a chemostat is dilution rate, i.e. the rate of supply of the limiting nutrient. In a chemostat operating at a low dilution rate, the limiting nutrient is present at very low concentrations at the steady state. Therefore, most of the nutrient is converted into cells and the biomass concentration is high (close to the cell concentration of an equivalent batch culture at late exponential phase). As the dilution rate is increased, the availability of the limiting nutrient increases; however, the rate of removal of cells from the growth vessel is also higher and so the biomass concentration falls
Turbidostats,,
Turbidostats are continuous cultures in which the cells, rather than the experimenter, control the growth rate. A turbidostat employs a positive feedback control system that senses, and responds to, the biomass concentration in the fermentor. If the culture is growing faster than the
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rate imposed by the current dilution rate, then the biomass concentration will rise as a result of the positive difference between the biomass production rate and the rate of removal of cells from the culture vessel. In such circumstances, a control loop is activated that increases the dilution rate by increasing the speed at which a pump introduces fresh medium into the culture. This continues until the biomass concentration is decreased to some set point. Contrariwise, if the culture is growing slower than the current dilution rate, the biomass concentration will fall to below the set point, and the control loop acts to decrease the rate at which fresh medium is pumped into the growth vessel. Hence the turbidostat equilibrates at a dilution rate equal to the actual growth rate of the culture and the cell concentration stays constant. As our set point is equivalent to the biomass concentration of a mid-exponential phase batch culture, the turbidostat equilibrates at the maximum specific growth rate of the yeast strain used. However, if the culture is a pool of heterozygous deletion mutants of S. cerevisiae, and if the different mutants in the pool can have different maximum growth rates, those mutants that can achieve a max
greater than the population average will increase in the population over time, while those with a max less than population average will decrease in the population. Thus turbidostats, just like chemostats, represent a sensitive way of identifying haploinsufficient and haploproficient phenotypes, with the difference that (in a turbidostat) haploproficient mutants must be capable of growing at a rate greater than the previously recorded max. The turbidostat is commonly used for the selection of antibiotic resistant mutants andthe degradation of toxic wastes, where nutrient limitation is not desirable
UNIT-II:- Downstream Processing and scale up.
The Downstream Processing
The Downstream Processing took place after making the product in a fermenter.
The product can be the micro-organism, or a metabolite, but the fermenter may contain up to 95% water and much effort has to be put in
concentrating the product .
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There are many problems in downstream processing (DSP). Removal of water, recovery of the product and purify the product. Even so, the pharmaceutical products need up to 99.999% purity is required.
The various stages of processing that occur after the completion of the fermentation or bioconversion stage,
1 .Cell disruption (dealing with an intracellular product),
2 .Clarification (separation of the cells and cell debris from the liquid) ,
3 .Concentration of the product stream ,
4 .Purification (often in multiple steps),
5 .Product formulation (giving the product a suitable form).
Cell disruption
The products are released by cells into the fermentation fluid to allow direct recovery. Cell walls can be disrupted in several ways. These
methods form two main groups: mechanical and non- mechanical .
Non- mechanical
1 ,drying: freeze drying, vacuum drying
2 ,Osmotic shock: a change of ionic strength of the solution causing the cells to swell and burst.
3 ,Temperature shock
4 ,Chemo-lysis :addition of surface active chemicals, solvents, antibiotics or enzymes to degrade the cell walls.
Mechanical cell disruption
Mechanical methods are used for large applications such as Ultrasonic disrupters, Homogenizers and Bead mills.
I, Ultrasonic disrupters: Ultrasonic disrupters are frequently used in the laboratory but too expensive on a plant scale.
II, Bead mills: Bead mills consist of a cylindrical vessel containing rotating discs and beads. The cells in the process fluid are disrupted by
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shear forces between the beads. The process is accompanied by heat production and cooling is required.
Homogenizers: The homogenizer is a simple piece of equipment and it can only process large volumes, has a high wear rate,
2 . Clarification
The Clarification yields, a clear liquid containing the dissolved product. Two major techniques are available: centrifugation and filtration
2.1 , centrifugation
The suspension is put into two identical tubes that are rotated. The separation is apparent after 10 minutes of centrifugation at 3000 rpm. This separation method can be used on plant scale. In larger centrifuges the residence time is lower. In plant operations, two types of centrifuges are frequently used: tubular and disc stack
2.1:1 the tubular centrifuge
A tubular centrifuge can be seen as a sedimentation vessel turned on its side and with an increased acceleration force.
2.1.2 The disc stack centrifuge .
Disc stack centrifuges contain conical plates (discs) at a short distance from each other. That have a large area and increased acceleration force.
2.2 filtration
Its separate particles from suspensions, the particles are retained by a filter medium, which is a porous fabric on the surface of the medium. The particles form bridges over the pores and create a filter cake. . The liquid is forced through the filter by a pressure difference .
In practice two modes of filtration are used:
Batch filtration with plate filters and Continuous filtration with rotating drum vacuum filters.
2.2.1 Batch filtration with plate filters
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The plate filters have a large number of hollow frames. These are covered with the filter medium. The liquid flows from outside to inside and the solids are retained on the filter medium. After a certain time the space between the frames is filled with filter cake and the pressure drop of the equipment increases. The frames are disassembled and the cake removed. This can be done automatically.
2.2.2 Rotating drum vacuum filters
The rotating vacuum drum filter is a large drum that rotates around a horizontal axis. The filter medium covers the outside of the drum. The drum rotates slowly through a trough containing the feed. The vacuum in the drum sucks in the liquid, leaving the solids as a cake on the outside. Only the lower part of the drum is submerged in the feed, the rest of the surface can be used to wash and dry the filter cake. At the end of the cycle, the cake is scraped off the drum by a knife and collected for further processing.
3 , Concentration after Clarification ,
This usually means removing water. Concentration can also purify the product stream and sometimes this is enough to obtain the desired end-product purity. Three types are evaporation, precipitation and ultra filtration
3.1 … Evaporation
The oldest and simplest method that to evaporate water or solvent from the mixture. Biological products tend not to be very stable, so temperature and exposure time should be kept low. The temperature can be kept low by working under reduced pressure.
3.2 … Precipitation
Precipitation can be used in several parts in the process. Early in the process it can remove water or salt. The precipitation can also be performed in a fractionating manner (the precipitates obtained contain different fractions of the different products). The solubility is mostly decreased by adding salts, typically (NH4)2SO4, or organic solvents such as acetone or ethanol.
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3.3 , Ultrafiltration .
Two types are frequently used that are Ultra filtration membranes (UF), Microfiltration membranes (MF)
3.3.1 Mf: MF membranes have pores of 0.2--0.5 µm diameter and thus retain cells.
3.3.2Uf: Ultrafiltration membranes have pores of approximately 10 nm diameter and thus retain proteins, but not water and salts. A UF membrane can be used in two ways: to concentrate or to wash out salts from the product (di filtration).
4 , purification
In the downstream processing of bio-products two major groups of purification processes can be distinguished: those for bulk products such as citric acid, enzymes and penicillin. Important techniques can be identified such as liquid-liquid extraction and crystallization.
4.1 … Liquid liquid extraction
These systems are composed of an aqueous mixture of a salt and a polymer or two polymers. In certain concentration ranges these separate into two phases. The dense bottom phase contains most of the salt, whereas the top phase mainly contains the polymer. . For factors govern the extraction: Mass balances (overall, phase, local) Phase and reaction equilibrium (K) Mass transfer and reaction rates (k) and Hydrodynamics
4.2 … Crystallization
When a solution is super-saturated the solvent contains more product than at equilibrium and as a result the product crystallizes. The crystals are separated by filtration or centrifugation. This yields a fairly pure product. Other processes are Gel filtration (separation on size), Adsorption (hydrophobic interaction, separation on polarity), Ion exchange (separation on charge) and Affinity chromatography (separation on shape: some molecules perfectly fit on the surface).
Storage and packaging methods downstream processing
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Packaging is the science, art and technology of enclosing or protecting products for distribution, storage, sale, and use. It refers to the process of design, evaluation, and production of packages. Fermented foods often have a low oxidation–reduction potential, helping to inhibit the growth of aerobic spoilage organisms
The Storage is bigger issues that have used many traditional methods for the storage. The cold storage and refrigerator is widely used techniques for storage. However, both these techniques are too much expensive and power consuming. While, vacuum sealing is the best solution for storage hence it’s a green packaging technique. Oxygen content and redox potential are important factors for viability of bacteria in fermented foods during storage.
Good Manufacturing Practice (GMP):GMP is Relates to the manufacturing, processing, and storing of materials that assure the products are safe for human consumption PACKAGING TYPES:Primary secondary and tertiary packaging Primary packaging: Glass has been widely used as this packaging
material. Ex: Ampoules, Vials, Strip package and Blister packaging :
Secondary packaging: this is outside the primary packaging ex: paper and carton.
Tertiary packaging: this is used for bulk handling , warehouse storage and transport shipping. The most common form is a palletized unit load that packs tightly into containers.
Methods of packaging
Active packaging, intelligent packaging, and Modified atmospheric packaging,
1 ,active packaging
The package headspace to enhance the performance of the package system” Active packaging includes Oxygen scavengers and antimicrobial packing.
Oxygen scavengers
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Oxygen scavengers are the most commercially important subcategory of active packaging in recent times. Oxygen scavengers can help to maintain food product quality by decreasing food metabolism, reducing oxidative rancidity, inhibiting undesirable oxidation of labile pigments and vitamins, controlling enzymatic discoloration, and inhibiting the growth of aerobic microorganisms . Oxygen scavenger technology is particularly useful in hot and humid climate, which is conducive to mold spoilage of products.e.x. Polyethylene terephthalate (PET) bottles.
Antimicrobial packaging
It prevents surface growth of pathogenic microorganisms in food, by use of antimicrobial agents where large portion of spoilage and contamination occurs. Two major functions of microbial inhibition are microbial-cidal and microbial-static effects. Chemical antimicrobial agents are the most common substances used in the industry. Ex: Benzoic acid, sorbic acid, FILMS:– LDPE, LLDPE, PET, polyolefin, EVA 6. Some plant extracts such as grapefruit seed, cinnamon, horse radish, and clove have been added to packaging systems to demonstrate effective antimicrobial activity against spoilage and pathogenic bacteria.
Intelligent packaging
Packaging that contains an external or internal indicator to provide information about the history of the package and the quality of the
product .
3 , Modified atmosphere packaging :( MAP)
Modified atmosphere is the practice of modifying the composition of the internal atmosphere of a package (commonly food packages, drugs, etc In modified atmospheric packaging the air in the package is replaced with a different gas or gas mixture. As a consequence of change in the gas atmosphere, the shelf life of the product is increased significantly the modified atmosphere slows down the degradation process. The three main gases used in MAP are oxygen, carbon dioxide, and nitrogen.
Other packaging
1 , Vacuum sealer packaging
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This is the best alternative for food storage and packaging. The vacuum sealer removes the air from the bag before sealed. Thus, it has reduced the chances of contamination of outer atmospheric factors. The shelf life of the food is increasing five times than the ordinary packaging methods. 2, Vacuum Pouche s
Also known as food vacuum pouches or barrier pouches, provide a barrier against atmosphere and moisture.
Storage 1,All materials and products shall be stored off the floor and with sufficient space between the material and the walls to allow inspection and pest control activities to be carried out.
2, the storage area shall be designed to allow maintenance and cleaning, prevent contamination and Minimize deterioration. 3, Design bulk storage areas to prevent or restrict traffic by nonessential personnel.
4 ,Provide interior storage areas with a ventilation system to give three air changes per hour
Scale up; scale down, criteria involved in scale up.
The basic idea behind scale-up is to preserve the quality of the separation achieved at small scale. A typical scale-up from laboratory to pilot plant is on the order of 50- to 100-fold. And that increase is frequently followed by another 10- to 50-fold scale-up from pilot plant to final commercial manufacturing scale.
Many parameters affect the success of scaleup, including the resin stability (physical and chemical), the product, the equipment (flow distributor, fraction collector, packing quality, system design), and the operating conditions (product loading, gradient slope). Although isocratic gradient elution are the most common modes of operation. Frontal chromatography has always played an important role at large scale, especially in the chemical industry.It could require establishing an efficient process in a larger volume vessel optimized for cultivation in larger volumes, e.g. a stirred tank reactor.
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The exercise in scaling up involved a number of programmed steps that has to be established to predict the behaviour of the final large scale production fermentor. The exercise carried out during scale up include: 1 Inoculum development2 Sterilization establishing the correct sterilization cycle at larger loads3 Environmental parameters such as nutrient availability, pH, temperature, Dissolved oxygen, Dissolved carbon dioxide.4 Shear conditions, foam production.Most of fermentation-based on same basic production blocks, And all commercial-scale bioprocess facilities can be roughly divided into two sections:
1. The upstream biosynthesis operation, where the desired end product is made, typically involves highly proprietary methods and calls for rigorous sterility requirements.
2. The downstream portion employs a site-specific mix of widely used chemical-engineering unit operations to extract and purify the target product, and appropriately dispose of all waste streams.Factors complicate the internal geometry Each of multiple fermentation vessels required by a commercial scale facility. These include introducing the fermentation broth, sterile air (both to maintain the required dissolved oxygen levels and provide air lift for low-shearing mixing inside the vessel) and sterilized nutrients (such as vitamins, amino and fatty acids, minerals and even antibiotics that ensure the health and maximize the productivity of the microorganisms). When air lift in the vessel can't provide sufficient mixing, the fermenter may be equipped with low-shear agitation devices. Agitation and Aeration provides two related functions in an aerobic fermentation process: (i) to provide mixing, and (ii) to supply oxygen.Fermentation vessels must be designed to ensure adequate heat-removal capabilities that to handle heat produced by the metabolic processes .if promotes cooling as needed that to maintain the narrow temperature range that can be tolerated by the bioengineered organisms. Sufficient safeguards must also be in place to guard against contamination and cell mutation. These include double-block and bleed valves, and steam-in-place (SIP)/clean-in-place (CIP) systems.
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Commercial-scale fermentation vessels must be equipped with a variety of advanced instruments, sensors and transmitters to monitor everything such as pressure level and temperature inside the fermenter to pH, dissolved oxygen and nutrient levels within the fermentation broth. In some cases, the number of in-line monitoring devices needed can be reduced by using external lab sampling or indirect relationships between key operating parameters.Production and recovery of productDuring fermentation, the desired product ends up in the fermentation broth. At the end of each batch fermentation, the microorganisms are destroyed, the product is separated and purified and the dead cell bodies, unreactive carbohydrate feedstock or nutrients and byproducts are removed.Successful scale-upThe identification and genetic engineering of a suitable organism, followed by careful piloting certainly is crucial to success with bio-based manufacturing. Often the case, a scale-up strategy that combines integrated teamwork with engineering can minimize efforts, costly rework and delays., and help today's manufacturing routes based on renewable feedstock's to achieve their full commercial-scale potential on time and on budget.
Scale down process
Creation and qualification of scale-down models are essential for performing several critical activities that support process validation and
commercial manufacturing .
The goal when scaling down is to create a small-scale or lab-scale system that mimics the performance of its large-scale (pilot or manufacturing).
Fermentation processes often involve several scales of operation, encompassing inoculum development, seed expansion, and production fermentation. The scale down process has specific parameters such as vessel geometries, and operational control strategies must be evaluated for each step.
The terms "similar reactor" or "similar vessel geometries" to describe optimal conditions for a scale-down strategy. Geometric similarity means
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that the overall aspect ratios of each vessel (small vs. large) are close similarity.
Maintenance of equivalent oxygen transfer (k L α) and control of dissolved oxygen is the most important requirement for most fermentation scale-down strategies. More importantly, the impeller (rotor) and sparger designs and placements within the vessel should be identical between scales. These include sample-dilution schemes and measurement times for calculating culture optical densities, wet and dry cell-weights, media metabolite levels, and protein expression.
If the sparger design is different between scales, then agitation, aeration, and oxygen enrichment may need to be adjusted to provide equivalent oxygen transfer in the scaled-down process.
The inoculum development and expansion conserve the vessel geometries, incubation conditions, and working volumes at each step. All process control setpoints and ranges (temperature, shaker speed, stroke
length, pH, and dissolved oxygen) should be the same .
OPERATIONAL SCALE-DOWN process
Sterilize all process vessels and tanks according to the current manufacturing procedure for the process. Sterilization temperatures, procedures for probe and flowmeter calibration, and post-use cleaning protocols should be the same as the large-scale process. Use GMP-
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released raw materials, Lot-to-lot variations between raw materials, including master or working cell-bank vials, as well as all media components, antifoam additions, and acid and base stock solutions, can greatly impact process performance.
All volume-independent operational control-parameter setpoints Such as pH, process temperature, inoculation percentages (v/v) for each step and schedule of feed-media additions should be identical to the operating ranges of the large-scale fermentation process.
The volume-dependent parameter: pre and post-sterilization volumes of growth media parameters should be equal to the initial and final volumes in the manufacturing process.
Feed media delivery rates: Adjust all feed rates based on the scale factor. (Unit weight or unit volume per hour) and the control (flowmeter vs. air pressure). Otherwise to consider other two processes are total air flow and oxygen flow rate at the controlled manner.
Agitation to provide either representative oxygen transfer rate (k L a), tip speed (v T), Reynolds Number (N RE), or power-input per unit volume (P/V), (according to the equations listed in Table) performance parameter
for scale-down qualification.
The final product titer and quality, at a specific rate of production, which is defined as the amount of product expressed per unit biomass weight (wet or dry).
Creation of scale-down models that meet qualification requirements can be challenging, particularly for upstream unit operations. These models can be of great use when performing experimental studies in an efficient and economical fashion. However, there are considerations unique to each unit operation that applies during scale-down.
Productivity, power requirements Basic control theory.
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The Flexibility is need when controlling fermentation. Along with effective coordination, flexibility is crucial when want to minimize waste material and maximize product quality.
Any bioreactor system is it bacterial, fungal, insect or mammalian content. It will be some sort of control System that will monitor the basic parameters inside the vessel: pH, DOT, temperature,Level/foam and mixing speed. All these parameters are controlled by the input of a gas, Liquid or energy. These elements have to be supplied in a controlled manner by pumps, rotameters, and solenoids.
Gas Flow and Delivery Control Devices
The variable area flowmeter assembly is used to measure and adjust gas flow rate to the bioreactor.
The flow rate is obtained by determining the height of the float on a scale. The solenoid valve is controlled by a signal from an external controller example, Dissolved oxygen or pH.
The most accurate, controlling gas flow to a bioreactor is using mass Flow controllers.They allow for the accurate measurement and recording of gas flow into the bioreactor,Temperature Regulation:The fermenter must have an adequate provision for temperature control.
Temperature control may be considered at laboratory scale, and pilot and production scales.
1 .in laboratory scale fermentations, normally little heat is generated. Therefore, heat has to be added to the system; this can be achieved in the
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following ways: (a) the fermenter may be placed in thermostatically controlled bath, (b) internal heating coils may be used, (c) water may be circulated through a heating jacket, or (d) a silicone healing jacket may be
used .
2 .In case of larger fermenters, excess heat is generated, and the fermenter surface becomes inadequate for heat removal. In such cases, internal coils have to be used to circulate cold water through them for removing the excess heat.
Foam Control:Foam is produced during most microbial fermentations. Foaming may occur either due to a medium component, e.g., protein present in the medium,
These proteins may denature at the air-broth interface and form a protein film that does not rupture readily. Foaming can cause removal of cells from the medium. Five different patterns of foaming are recognized. Foaming may lead to several physical and biological problems. Foaming may interfere with the functioning of sensing electrodes resulting in invalid process data, and incorrect monitoring and control of pH, temperature, etc.
(1) A defined medium may be used to avoid foam formation. This may be combined with modifications in physical parameters like pH, temperature, aeration and agitation.
(2) Often the foam may be unavoidable; in such case, antifoam should be used. Antifoams are surface active agents; they reduce surface tension in the foams and destabilize protein film by the following effects: (a) hydrophobic bridges between two surfaces, (b) displacement of the absorbed protein, and (c) rapid spreading of the surface film.
pH Control
Nutrients, anti-foaming agents, temperature, and the microorganisms those are contributing to pH changes. Its control is achieved via acid and base feeds. The pH controlled by peristaltic pumps this will inject a specific amount of acid or base into the vessel .Some fermentation applications would use CO2 instead of acid to control the pH .
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Agitator Control
The purpose of agitation is two-fold. First, it is to maintain a homogenous mixture within the vessel. The second reason being to control the amount of oxygen required by the culture. In cases where paddles are harmful to the culture, agitation is achieved via bubbles this process known as ‘airlift'. it containing a mixture of N2, O2 and CO2and its controlling DO2 and pH.
Feed/Nutrient
During an incubation cycle a nutrient energy source is added and the biomass and end product will increase as this is depleted. .e.g. glucose
Air Flow Control
It's used to control the amount of oxygen required in the fermentation process.
Pressure Control
A high pressure alarm will indicate that the pressure has reached an unsafe level. Ex: bourdan tube pressure gauge, which is used for the direct indication of the pressure condition in the fermenter.
Dissolved Oxygen Control
The amount of dissolved oxygen is a function of the agitator speed, air flow, pressure, pH level, temperature and the fermentation process itself. The most common method of controlling the amount of dissolved oxygen is to used cascade PID loop
UNIT-III: - Industrial Fermentation Products
Bio –Fuels
Ethanol
Ethanol is The Fermentation product of sugar crops after distillation is known as ETHANOL. Ethanol, or ethyl alcohol, has the chemical formula C2H5OH. Ethanol has been used as a fuel for internal combustion engine.
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Ethanol is a flammable & colorless liquid .Boiling point of a ethanol is 78.4 C. Melting point of a ethanol is -114.3 C and Density of a ethanol is .79 gm/cm3 .Ethanol has a high octane number Chemical composition of a ethanol is 52% carbon 13% hydrogen 35% oxygen
1st Generation Biofuels was Bioalcohol that made from Corn and sugarcane while 2nd Generation Biofuels used Cellulosic Biofuels such as Biohydrogen Biomethanol. The 3rd Generation Biofuels are Algae fuel
World ethanol production between 2000 and 2007 from 17 billion to more than 52 billion liters. In 2013 worldwide ethanol fuel production reached 79.7 billion liters. Ethanol made from non-cellulosic material in maize .The United States and Brazil are responsible for 86% of the
world’s ethanol production .
Production of ethanol
Chemistry
Glucose is created in the plant by photosynthesis: 6 CO2 + 6 H2O + light → C6H12O6 + 6 O2 During ethanol fermentation, glucose is decomposed into ethanol and carbon dioxide: C6H12O6 → 2 C2H5OH+ 2 CO2 + heat Combustion, ethanol reacts with oxygen to produce carbon dioxide,
water, and heat: C2H5OH + 3 O2 → 2 CO2 + 3 H2O + heat
Industrial Operations: there are two types of process to produce ethanol from Corn that is a Dry milling process and Wet milling process. Ethanol from cellulose using Gasification that ethanol called as cellulosic ethanol
Corn Ethanol
Ethanol from corn is produced through fermentation, chemical processing and distillation. Corn is the main feedstock; two types of Corn ethanol Production are Dry milling Process and Wet milling Process. Corn Ethanol Corn Kernel: Endosperm: 82% of the dry weight. It’s the source of starch and protein
Dry milling
The corn grain is steeped in a dilute combination of sulfuric acid and water in order to separate the grain. Corn oil is a by-product of this process.
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The grain is screened to remove debris and ground. During the cooking, the starch in the flour is physically and chemically prepared for fermentation .
Hot Slurry, Primary Liquefaction, and Secondary Liquefaction .
The mixed grain is mixed with water, where the pH is set at 5.8. Amylase enzyme is introduced to hydrolyze the starch. The mixture is then heated to 190 F. This temperature is maintained for 30 to 45 minutes. The slurry is pumped to a jet cooker at 221 F and held for 5 minutes. Mixture is then cooled. The mixture can either be cooled through atmospheric or vacuum flash condenser.
After cooling, the mixture is held for 1 to 2 hours at 180-190 F. This is the time when the amylase enzyme breaks down the starch into more simple dextrins. The Alpha-amylase randomly hydrolyzes the alpha-1,4-linkage. Dextrin is low molecular weight carbohydrates produced by hydrolysis.
A secondary enzyme called glucoamylase is added before the mixture is pumped into the fermentation tank. Glucoamylase is An enzyme that breaks the bonds near the ends of large carbohydrates releasing maltose and glucose.
The enzymes break the dextrins into glucose. Yeast is added to convert the sugar to ethanol from Carbon dioxide. Then allowed to ferment for 50 to 60 hours that will contain 15% ethanol.
Common Yeast: Saccharomyces cerevisiae, S. uvarum, Schizosaccharomyces pombe .
Reaction: C6H12O6 → 2 C2H5OH+ 2 CO2 – Under anaerobic conditions, yeast metabolize glucose to ethanol by the Embden –Meyerhof pathway. The yield obtained does not normally exceed 90%.
Fermentation Conditions are pH 5-7; temperature 30-37 C. if temperature attain Above 50 0C yeast can`t survive, and at low temperatures of 0 to 10 C there is no activity.
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Mash is pumped into a distillation system. The columns utilize the differences in the boiling points of ethanol and water to separate the ethanol.
Types of Distillation Methods
Pressure swing distillation: At high pressure the boiling point will increase, and the azetropic point will shift to lower b.p. with pressure. It must be distill in two columns at different pressures. The azetropic point will be crossed .
Azeotropic Distillation: Introduce an entrainer to form a ternary azetrope.
Wet Milling
Grain is separated into the germ, fiber, and starch in a steeping process – Uses dilute sulfurous acid. Many Byproducts produced in addition to ethanol. Such as Poultry feed, corn oil and syrup, and corn starch.
Steps for Wet Milling
1 .Steeping: soak corn for 24- 48 hours. 2. Coarse milling: separate the germ from the kernel. 3. Fine milling: separate fiber from endosperm 4. Separation: separate starch from gluten. 5. Hydrolysis 6. Fermentation
Biofuels: Hydrogen Hydrogen is the lightest and most abundant element, but it does not normally occur in nature in large quantities. Hydrogen is an alternative of petroleum that offers reduction of CO2 emissions and it can be seen as a good ‘storage’ complement for renewable energy systems.
Hydrogen gas is a one of the hopeful and alternate source for reduction of greenhouse effect Approximately 95% of hydrogen produced is consumed at the site of production, with 1.5 million tons being sold for industrial and chemical uses. The hydrogen can be produced from the various methods for example electrolysis of water and fossil fuels through steam reforming of natural gas or methane (CH4). Carbohydrate rich, solid wastes such as cellulose and starch containing agricultural and food industry wastes and some food industry wastewaters can be used for hydrogen production by using suitable bio-process technologies. The majority of this hydrogen is used to produce ammonia fertilizer, as feedstock for chemical and
petroleum refining areas, plastics, solvents and other commodities .
Enterobacter aerogenes has been using the production of Biofuels hydrogen or Biohydrogen. The hydrogenase enzyme present in anaerobic organisms. This enzyme oxidizes during fermentation and reduced ferrodoxin to produce molecular hydrogen,
Biohydrogen is basically a type of hydrogen that is being produced by the biological processes. The organisms involved for this production may be algae and bacteria. Biohydrogen is a type of the biofuel that is produced by the action of these microorganisms. The multidisciplinary fermentation processes of H2 using numerous and a variety of substrates. Diversity of Microorganisms as H2 Producing Biocatalysts
The Fermentation of hydrogen uses variety of organisms including the archaea, anaerobic and facultative aerobic bacteria, cyanobacteria, and lower eukaryotes produce H2 . The major H2 producing biocatalysts are typical heterotrophs in the fermentation process.
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The natural biological processes are categorized into four primary groups: (1) water-splitting photosynthesis (2) photofermentation; (3) dark fermentation; and (4) microbial electrolysis processing (5) Microbial Electrolysis Cells.
The oxygenic photosynthetic microorganisms such as green microalgae e.g. Chlamydomonas reinhardtii and Chlorella etc. the cyanobacteria such as Anabaena variabilis and Nostoc punctiforme to use this process that requires only water and sunlight.
The biophotolysis is further divided into direct and indirect processes. In direct biophotolysis: the electrons derived from the light energy-mediated water splitting are transferred through photosystem II (PS II) and photosystem I (PS I) to ferredoxin (Fd) as an electron carrier, and subsequently, the reduced Fd reduces a hydrogenase enzyme that is
responsible for H2 production .
2H+ + 2Fd (re) ↔ H2 + 2Fd (ox).
In the case of indirect biophotolysis, photosynthesis converts light energy to chemical energy in the form of a carbohydrate, which is reused to produce H2. at present, indirect processes using green algae and heterocystous cyanobacteria., resulting in the production of H2:
Photofermentation involves the conversion of light energy to biomass with the production of H2 and carbon dioxide (CO2);
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For the process of photofermentation, purple nonsulfur (PNS) photosynthetic bacteria, including Rhodobacter species, are used to convert organic acids such as acetate, lactate, and butyrate to H2 and CO2 in anaerobic and anoxic conditions. Moreover, these bacteria capture solar energy to transform organic acids into H2 using nitrogenases in the absence of ammonium (NH4) ions.
2.4 . Dark Fermentation
Dark fermentation is a method of producing hydrogen by anaerobic bacteria grown on carbohydrate-rich substrates as biomass.
The products of dark fermentation are not pure hydrogen but a multi-component gas where H2 and CO2 are the main constituents, other gases such as CH4 or H2S.
Hydrogen yields can be improved by increasing H2 production through acetate end-product reaction, and decreasing butyrate, ethanol and propionate product reaction. This is through fermentation process with thermophiles or extreme thermophiles, operating at temperatures higher than 333K.
Microbial electrolysis cells (MECs), a new technique to produce H2 from a wide variety of substrates, These are fundamentally adapted microbial fuel cells (MFCs) The MECs are capable of more than 90% efficiency in the production of H2 .
The MEC technology is also called electrofermentation or biocatalyzed electrolysis cells.
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Biofuels: methane
Methane consists of a carbon atom to which is bound four hydrogen atoms known as hydrocarbons. Methane is colorless gas When liquefied, is less dense than water Soluble in water, but very soluble in organic liquids such as gasoline, ether, and alcohol
Methane is an end product of the anaerobic decay of plants, that is, of the breakdown of certain very complicated molecules. As such, it is the major constituent up to97 of natural gas. The leaf tissues of living plants emit methane; plants are absorbing methane from the soil and then emitting it through their leaf tissues. Methane is released from the animal mainly by belching (eructation). The average cow emits around 250 liters of methane per day. Methane is one of the important greenhouse gases, therefore, the methane produced by methanogenesis in livestock is a considerable contributor to global .However, when methane gas is burned, it converts less harmful substances such as CO2 and water. It has many benefits in comparison to fossil fuel sources of electricity generation. Methane fermentation achieved by biochemical breakdown of polymers to methane and carbon dioxide in presence of methanogens. Methane fermentation offers an effective means of pollution reduction, superior to
that achieved via conventional aerobic processes. BIOGAS and biomethane
Biogas is a digester gas arising from the activity of methanogenic bacteria. The biogas to be upgraded to the quality of natural gas – so-called “biomethane” or “bio-natural gas” – .Biomethane can play a central role in the development of Biofuels .The production of first generation Biofuels such as biodiesel, bioethanol and vegetable oil is solely based upon the oil, sugar or starch content of plants. The biogas may be used in various fields of technological processes e.g. in the production of methanol.
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Processes of methane fermentation
Hydrolysis: the polymerized, insoluble organic compounds, such as carbohydrates, proteins and fats are decomposed to soluble monomers and dimers. This stage of the methane digestion process occurred by extracellular enzymes amylases, proteases and lipases produced by hydrolyzing bacteria.Acidogenesis (acidification phase): During this stage, the acidifying bacteria convert water-soluble chemical substances, carbon dioxide and hydrogen is the source of energy for anaerobic microorganisms. This process may be divided into two types: Hydrogenation and dehydrogenation.
Acetogenesis: In this process, the acetate bacteria convert the acid phase products into acetates and hydrogen which may be used by methanogenic bacteria Methanobacterium suboxydans. The Methanobacterium propionicum has been decomposing propionic acid to acetic acid, As a result of Acetogenesis. Acetogenesis is a phase of biogas production, because approximately 70% of methane arises in the process of acetates reduction.
Methanogenesis: This phase consists in the production of methane by methanogenic bacteria .the only few bacteria are capable to produce methane from acetic acid.
3 ,Developments in bioreactor technology
Methane fermentation has been used since 1900 for treating excess sludge discharged from sewage-treatment plants. This technology has since been developed to treat waste waters, such as those derived from alcohol distillation, antibiotic production, and baker's yeast manufacture.
3.1 Upflow anaerobic sludge blanket (UASB)
The UASB process that is capable of self-granulation (flocculation) of anaerobic microbes. Waste water entering from the bottom of the reactor passes through a sludge bed and sludge blanket where organic materials are anaerobically decomposed. Gas produced is then separated by a gas-solid separator, while the granular sludge naturally settles to the bottom.
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3.2 Upflow anaerobic filter process (UAFP)
The UAFP systems were applied to the treatment of domestic sewage and industrial waste waters containing relatively low levels of organic materials. The reactor contains a "medium", i.e. a microbial support. Hence, a high-density microbial population is retained within the reactor. The treated waste water and the biogas separated at the top of the bioreactor .Pumice was used as a microbial supporter for methanogenesis from methanol-rich waste water of the evaporate condensate from a pulp mill.
An antibiotic is a chemical substance, produced by micro-organisms, which has the capacity to inhibit the growth of and even to destroy bacteria and other micro-organisms.” many antibiotics are used commercially, or are potentially useful, in medicine for activities other than their antimicrobial action. They are used as antitumor agents, enzyme inhibitors including powerful hypocholesterolemic agents, immunosuppressive agents, and anti-migraine agents, etc. The main producers of the microbial metabolites, the actinobacteria, fungi and other filamentous bacteria,
Penicillin was the first naturally occurring antibiotic discovered. It is obtained in a number of forms from Penicillum 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. All penicillins are derived from 6-Aminopenicillanic acid
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
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purposefully modified chemically by removing the acyl group to leave 6-aminopenicillanic acid and then adding acyl groups that produce new properties.
Β-lactam antibiotics have been in clinical use for more than 60 years and are currently the most widely used group of antibiotics utilized to treat bacterial infections. The common moiety of all β-lactam antibiotics is a 2-azetidinone ring, more commonly referred to as the β-lactam ring, which is responsible for their bactericidal capabilities. the beta-lactamases that are synthesized by many penicillin-resistant bacteria.)
They include the
penicillins such as
1, penicillin G produced by the fungus Penicillium chrysogenum .It is relatively unstable in acid, thus the bioavailability is low And is often necessary for the treatment of diphtheria and tetanus.
2,ampicillin (a semi-synthetic) it's similar to penicillin G in the activity against gram-positive organisms
3,amoxicillin (semi-synthetic) EFFECTIVE AGAINST Gram positive and Gram negative bacteria TREATMENT FOR Skin infection ,Sinusitis Urinary tract infections.
There are four sub-families of the β-lactam antibiotic family that are currently used in clinical practice: the penicillins, cephalosporins, carbapenems, and monobactams, which vary in their ring structure around the β-lactam ring to form unique β-lactam nuclei
Beta lactem antibiotic inhibit the final step of bacterial cell wall synthesis and its can work on both gram-positive and gram-negative bacteria.
The first synthetic penicillins-namely, BRL 1241 (" celbenin "). The chemical formula of this compound is
Two β-lactam antibiotics penicillin G and penicillin V, are products of a fermentation process. The majority of β-lactam antibiotics are classified as semi-synthetic because the β-lactam moiety is obtained from the enzymatic hydrolysis of a natural fermentation product; penicillin G, penicillin V, or cephalosporin C, while the acyl side chain is obtained by a chemical or chemoenzymatic synthesis.
Chemical Synthesis
The majority of semi-synthetic β-lactam antibiotics are still synthesized by way of the Dane anhydride process, which can achieve yields as high as 90%
This process is carried out at temperatures as low as -30o C, uses highly reactive pivaloyl choride and silylating protection groups, and requires large volumes of dichloromethane, triethylamine and acetone for solvents.
Biocatalytic Synthesis
The coupling of the β-lactam nuclei with the acyl side chain can be accomplished enzymatically utilizing penicillin G acylase (PGA). Penicillin g is a penicillin derivative commonly used in the form of its sodium or potassium salts in the treatment of a variety of infections. It is effective against most gram-positive bacteria and against gram-negative cocci.
Biocatalysts Used to synthesize β-Lactam Antibiotics
PGA (EC 3.5.1.11) is a very important enzyme in the industrial production of semi-synthetic β-lactam antibiotics; bacteria, actinomycetes, fungi and yeast produce PGA
Streptomycin
Streptomycin is a water-soluble aminoglycoside derived from Streptomyces griseus. It is marketed as the sulfate salt of Streptomycin. The chemical name of Streptomycin sulfate is D-Streptamine. Streptomycin sulfate is a bactericidal antibiotic. It acts by interfering with normal protein synthesis.
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Streptomycin is usually administered for the treatment of drug susceptible tuberculosis. Concomitant uses with other agents are bacterial endocarditis such as Streptococcal endocarditis and Enterococcal endocarditis. Streptomycin is considered a secondline agent for the treatment of gram-negative bacillary bactermia, meningitis, and pneumonia; brucellosis; granuloma inguinale;
chancroid, and urinary tract infection .Streptomycin is the Secondary metabolite produced by Streptomyces griseus. Change in environment condition and substrate availability influence final product.In fermentation a soyabean based medium is used with glucose as carbon sourceNitrogen source is combined in soyabean meal, limits growth. After growth the antibiotic levels in the culture begin to increase.PHASE 1 : Rapid growth producing mycelial biomasss.Little production of Streptomycin is obtained. The medium change to be alkaline when mycelia releasing ammonia during the phase 1 the pH
increased .PHASE 2: Additional production of mycelium. Streptomycin
accumulates in the medium. Incubation periods of this phase are to 24 hrs to 6 to 7 days.PHASE 3 : Process has completed. Finally the mycelium is separated by filtration and antibiotic recovered.
Proteolytic activity of the microbe releases NH3 to the medium from the soybean meal, causing a rise in pH
The glucose and NH3 released are consumed during this phase. The pH remains fairly constant-between 7.6 and 9.0.The extraction and quantification of the intracellular pools of NTPs and of ppGpp were performed by an HPLC method mycelium from 100-200 ml cultures was harvested by filtration on a nitrocellulose filter (pore size 0.2 pm; .streptomycinis diluted with dilute acid. The
diluted streptomycin is then pipetted solvent filtered and dried .
Genetics of streptomycin production in Streptomyces griseus
The nucleotide sequence of a 5.1 kb fragment from the streptomycin biosynthetic gene cluster from Streptomyces griseus have five open reading frames which form part of two convergently oriented
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transcription units such as strDEL and strNB2M. the gene products like StrD , StrE, StrM, and StrL are involved in biosynthesis of the streptose moiety of streptomycin. StrE and StrL are significantly similar in primary structure to each other and to other oxidoreductases (epimerases) involved in hexose metabolism.
CephalosporinsCephalosporins are β-lactam antibiotics containing a dihydrothiazine ring with D-α-aminoadipic acid. Cephalosporins are less toxic and broad-spectrum antibiotics comparable in action to ampicillin. They are effective against Gram-positive and Gram-negative bacteria. Which was first isolated from sea water near a sewage outlet at Cagliari, Sardinia, Italy in1948 1964 the first semi synthetic cephalosporin i.e. cefalothin was launched in the Market by Eli Lilly
and company .
Biosynthesis
Cephalospoium acremonium is the most important source for the production of cephalosporins. Presently major source for the production of cephalosporins include..Cephalosporin C
Most cephalosporins are produced semi synthetically by the chemical attachment of side chains to 7-aminocephalosporanic acid. Cephalosporin C fermentations are a primary source of cephalosporin nucleus that is used in quantity for the preparation of a large number of semi-synthetic cephalosporin antibiotics. Such fermentations generally take place for a period of several days and it has been observed that because cephalosporin C itself in aqueous solution is subject to non-enzymic decomposition by β-lactam hydrolysis, a typical industrial fermentation results in a loss of about 25% of the observed cephalosporin C titre due to non-enzymic decomposition.
It has also been generally observed that about 15% of the cephalosporin nucleus produced during fermentation is present as diacetyl cephalosporin C. the accumulation of cephalosporin C begins to fall off after about six days fermentation, which is the standard period of normal cephalosporin C fermentation.
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Fermentation
The organism Acremonium chrysogenum used by fermentation. Spores suspension of fungal strain was prepared by washing 5 days old culture slants with sterilized saline solution (0.9% NaCl) and shaking vigorously for 1 min. Spores concentration was adjusted to approximately 108 spore's mL-1. A medium of the composition consist of corn steep liquor, soya oil and beet molasses is use as the basal medium.
Batch Fermentation
Inoculums level of 5% is transfer to fermenters. The incubation temperature, initial pH and aeration were Carrie out at 25°C, 6.0 and 6500 L min-1, respectively. The anti forming agent dosed to avoid foaming during the fermentation and supply constant sterile air flow which maintains a constant level of dissolved oxygen in the fermenter. Sterile air was introduced into the fermenters through small spargers to ensure adequate aeration of the fermentation medium. When the elapsed time (180 h) for a fermentation run ended, the fermenter was discharged, thereafter, biomass is isolate by centrifugation at 5000 rpm for 10 min and the supernatants were separated to carry out the necessary analyses.
RECOVERY AND PURIFIC TION OF CEPHALOSPORIN
Crude cephalosporin fermentation broth filtered to remove the mycelia and other insolubles and commingled Mix with 2.33 volumes of acetone. The resulting mixture was allowed to settle for 30 minutes and refilltered. The filtrate pass over a resinous bed, the adsorbate was eluted extracted with a pH 3.5 sodium formate buffer solution. The eluate is concentrated to thick syrup, seeded with a crystal of cephalosporin, and allowed to crystallize. The diluted crystal line slurry was filtered and the crystals of cephalosporin were air dried.
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XanthanXanthan gum is a polysaccharide produced by the fermentation of the bacterium Xanthomonas campestris. This organism is found in nature on the leaf surfaces of green vegetables, particularly the brassica family. Xanthan is biopolymers and more than 20,000 tones of Xanthan have been producing world-wide per annum. About 60 percent of the Xanthan produced is used in foods that used to improve texture of bread and thickening in juice, drinks, chocolates, and pickles. The remaining 40 percent used in industrial applications such as Petroleum Flocculent and lubricant, Adhesives, toothpastes and cosmetics.
Xanthan is made up of beta-D-glucose units which are linked in a way identical to the linkages between the glucose units of cellulose. Xanthan gum is polyelectrolyte with a β-(1- 4)-D-glucopyranose glucan backbone with side chains of –(3-1)-α- linked D-mannopyranose
Xanthan gum is produced by aerobic submerged fermentation using the bacterium Xanthomonas campestris, a micro-organism which is found naturally on cabbages.
There is a multi-stage inoculums build up from agar plate to shake flasks to small seed fermentation vessels to large final fermentation vessel. At all stages of production, fermentation equipment is thoroughly cleaned and sterilized before use and strict aseptic techniques are followed to ensure the culture is pure and without contamination.
The fermentation medium is comprised of glucose syrup derived from maize or wheat, inorganic nitrogen (ammonium or nitrate salts), an organic nitrogen source (protein), and trace elements.
Once the final fermentation is complete, the contents of the vessel are pasteurized to kill all the bacterial cells used in the initial culture and optimize the conformation of the polymers.
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The gums are recovered from the fermentation broth, by addition of alcohol (usually isopropyl alcohol) causing precipitation of Xanthan and gellan fibres.
The resulting fibres are then treated to remove the excess alcohol and dried under careful conditions. The resulting “cake” is ground milled into a powder and packaged in a controlled environment.
Polyhydroxyalkanoates
Polyhydroxyalkanoates (PHAs) are thermoplastic polyesters synthesized by Ralstonia eutropha and other bacteria as a form of intracellular carbon and energy storage and are accumulated as lipid inclusions in the cytoplasm of these bacteria.
Polyhydroxyalkanoates plastics are biodegradeable and are used in the production of bioplastics. They can be either thermoplastic or elastomeric materials, with melting points ranging from 40 to 180 °C. These are gaining more and more importance world over. Different sources (natural isolates, recombinant bacteria, plants) and other methods are being investigated to exert more control over the quality, quantity and economics of poly(3-hydroxybutyrate) (PHB) production. Their biodegradability makes them extremely desirable substitutes for synthetic plastics. The PHB biosynthetic genes phbA, phbB and phbC are clustered and organized in one phbCAB operon
Poly (3-hydroxybutyrate) [P(3HB)] is the most common PHA and was first described by Lemoigne, a French scientist in year 1925 . Since then, various bacterial strains have been identified to accumulate P(3HB) both aerobically and anaerobically.. Among the more than 250 different natural PHA-producers. which are capable of utilizing various carbon sources including plant oils or wastes to produce PHA. C. necator has been the most extensively and commonly used bacterium for PHA production.
PHB is synthesized from acetyl coenzyme A (acetyl-CoA) by a sequence of three reactions catalyzed by 3-ketothiolase (acetyl-CoA acetyltransferase; EC 2.3.1.9), acetoacetylCoA reductase
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(hydroxybutyryl-CoA dehydrogenase; EC 1.1.1.36) and poly (3-hydroxybutyrate) synthase.
Different bacteria consistently produce polymers of different molecular weights (7). Azotobacters, for example, accumulate PHB in the range 8 x 105 to 2 x 106, and A. eutrophus accumulates PHB of 6 x 105. Bacteria that are used for the production of PHA can be classified into two groups. The first group of bacteria requires limitation of essential nutrients such as nitrogen, oxygen and presence of excess carbon source for the efficient synthesis of PHA. The second group of bacteria does not require nutrient limitation for PHA synthesis and can accumulate PHA during exponential growth phase.
Batch and fed-batch fermentations are widely used in the industrial fermentation processes. Fed-batch cultivation is more efficient than batch cultivation in terms of achieving high product and cell concentration because the medium composition can be controlled by substrate inhibition. Fed-batch culture is suitable for bacteria belonging to the first group. A two-stage cultivation method is most often employed, which was initially adopted by ICI for the industrial production of P(3HB-co-3HV)
In the first stage, the bacterial cells are grown until a pre-determined cell mass concentration is reached without nutrient limitation. The cells are then transferred to the second stage medium with limiting nutrients and the carbon substrates fed, are utilized by the cells to make PHA. During this nutrient limitation stage, the cells are unable to multiply and remain almost constant. However, the cells begin to increase in size and weight due to the intracellular accumulation of PHA as a storage product
Recovery and purification
The most common method for the extraction of PHA from biomass is solvent extraction by using chloroform. By using this method, highly purified PHA can be obtained without the degradation of PHA molecules.
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Other halogenated hydrocarbon solvents such as dichloromethane, dichlorethane and chloropropane can be also used to extract and purify PHA from the cell biomass. However, these methods are not suitable for the mass production of PHA as the solvent is potentially hazardous to health and environment Enzymatic digestion technique used to Extraction of PHA to result in high recovery and purification of PHA with no polymer degradation. Enzymes such as protease (trypsin, chymotrypsin, rennin, papain and bromelain),
application
PHA has a wide range of potential applications because of its desired features such as biocompatibility, biodegradability and negligible cytotoxicity to the cells. PHA has been manufactured for non-woven materials, polymer films, , and pharmaceutical products used in surgery, transplantology, tissue engineering, and pharmacology.
Biopreservative: Lactobacillus sakei
Biopreservative are various types of products derived from lactic acid bacteria and other suitable microorganisms, namely bacteriocins and other antimicrobials, fermentates, bioprotective cultures, and bacteriophages
Everyone wants preservative-free food, but without preservative food that is not preserved. There is thus a strong market need for natural food protection solutions that can ensure both food safety and food shelf-life. One of the few possible solutions is biopreservation based on the concept of using food-grade microorganisms as so-called cell factories.
Food-grade microorganisms can form a multitude of different substances that are inhibitory to other microorganisms. These mechanisms are part of the natural balance in complex microbial ecosystems. The biopreservation principles from food-grade microorganisms can be categorized according to the antimicrobial compound (e.g. bacteriocin, other metabolites, bacteriophages, enzymes)
Lactobacillus sakei is an important food-associated lactic acid bacterium a producer of the bacteriocin sakacin K. Although initially characterized
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from rice wine and isolated from plant fermentations and fermented fish .its main habitat is meat It is widely used as starter culture in the production of fermented meat products and is regarded as a potential meat and fish Biopreservative.
L. sakei resists harsh conditions which often prevail during preservation, such as high salt concentration, low water activity, low temperature and pH . An important property of the bacterium is the production of lactic acid that acidifies the product and both inhibits growth of spoilage bacteria and food pathogens, and confers taste and texture to the fermented products .
The species has also been observed as a transient short time inhabitant of the human gastrointestinal tract.
Sequence analysis of the L. sakei 23K genome has provided valuable information, showing a specialized metabolic repertoire that reflects adaptation to meat products.
Among the few sugars available in meat and fish, L. sakei utilizes glucose and ribose for growth. The two sugars are fermented through different metabolic pathways: sugar hexose fermentation is homolactic and proceeds via the glycolytic pathway leading to lactate, whereas pentoses are fermented through the heterolactic phosphoketolase pathway ending with lactate and other end products such as acetate.
The species can be divided into two subspecies described as L. sakei L carnosus
Bacteriocin biosynthesis
Characteristically, class IIa bacteriocins, are first formed as ribosomally synthesized precursors or pre-peptides, which appear not to be biologically active and contain an N-terminal extension or leader sequence. Subsequent cleavage of the prepeptide at a specific processing site removes the leader sequence from the antimicrobial molecule concomitantly with its export to the outside of the cell. The amino acid sequence of a number of class-IIa-bacteriocin leader peptides, which vary in length from 18 up to 27 residues, One important feature of the majority of these leaders is the presence of two glycine residues in the C-terminus, at positions −2 and −1 relative to the processing site. These leaders are
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believed to serve as signal peptides for the processing and the secretion of class IIa bacteriocins.
Thermostable enzymes:-Proteases
Proteases (EC 3.4) are enzymes that hydrolyze proteins to short peptides or free amino acids and catalyze peptide synthesis in organic solvents or in solvents with low water content. Proteases have a large variety of applications, mainly in the detergent and food industries. These include beer chill proofing, meat tenderization, cheese manufacture, flavor development in fermentation, baking, and manufacture of soy, dietetic, and health products. They are also used extensively in the leather and pharmaceutical industries. Thermostable proteases are advantageous in some applications because higher processing temperatures can be employed, with the consequences of faster reaction rates, increase in the solubility of nongaseous reactants and products, and reduced incidence of microbial contamination from mesophilic organisms. Proteases secreted from thermophilic bacteria this are useful in a range of commercial applications.
The good sources of thermostable proteases, such as thermolysin from Bacillus thermoproteolyticus, aqualysin I from Thermus aquaticus YT-1, and caldolysin from Thermus aquaticus T-351.
Bacterial T Protease
The protease contributes 60% of industrial enzyme contribution of bacterial protease. The all the genus of bacteria Bacillus itself contributes major content of protease for the industrial application.
Fungal Thermostable Protease
Many of researchers have isolated and checked activity of various proteases form fungal sources such as cultures of, A. fumigatus, Aspergillus tamarii
Purified stable alkaline serine-protease from Aspergillus clavatus ES1 was optimally active at 50°C ..
Recombinant Thermostable Protease
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Often the habitats of thermostable microbial strains become problem for the researcher to isolate and purify the enzyme form their native wild source as maintaining those conditions in the laborites. The better option to overcome forms this problems to clone gene and express in the suitable host system which will be convenient to grow. The best host system used for last two decades E. coli which perfect for the expression for the recombinant gene.
Fermentation and purification
B. stearothermophilus strain was grown in medium containing 0.1% w/v yeast extract and 0.25% w/v dried skim milk. The culture was incubated and stirred at 200 rpm in aerated vessels at 65°C for 48 h and then centrifuged at 10,000g for 15 min. The supernatant was assayed for Proteolytic activity.
Purification of Thermostable Proteases
The dried culture supernatant was dissolved to mM Tris– HCl buffer, pH 8.5, and dialyzed overnight against the same buffer. It was reconcentrated by lyophilization and dissolved in 5 ml of the same buffer.
Step 1: Lysine affinity chromatography column. The concentrated protein sample was loaded onto a lysine affinity chromatography column .Protease S was eluted with a solution of lysine and NaCl
Step 2: Strong anion exchange chromatography. Supernatant loaded onto a strong anion exchange column Protease N was eluted in the first fraction. Protease B was eluted. These fractions were pooled and dialyzed overnight against 20 mM Tris–HCl buffer, pH 7.0.
Biosurfectants: a comparative account.
Biosurfactants are biological surface-active agents capable of reducing interfacial tension between liquids, solids and gases, thereby allowing them to mix and disperse readily in water or other liquids. (Bio)surfactants are amphiphilic molecules consisting of a hydrophilic and a hydrophobic moiety that interacts with the phase boundary in
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heterogeneous systems. The non-polar “tail” is typically a hydrocarbon chain whereas the polar “head” appears in many different varieties such
as carbohydrates, amino acids or phosphates.
The large majority of the currently used surfactants are petroleum-based and are produced by chemical means. These compounds are often toxic to the environment and their use may lead to significant environmental problems,
Biosurfactants have several advantages over synthetic surfactants such as higher biodegradability ,lower toxicity ,good bio compatibility with eukaryotic organism, effectiveness at wide range of temperature ,pH value ,and salinities. Moreover, biosurfactants can be produced by fermentation from renewable resources, typically from sugars and vegetable oils.
Factor affecting biosurfactants production is lack of availability of economic and versatile products. Currently there is only a very limited offer of commercially available biosurfactants, e.g., surfactin, sophorolipids and rhamnolipids. Its potential utilization in food-processing, pharmacology, and oil industry .According to recent data, global biosurfactants market was worth USD 1,7 bi in 2011 and is expected to reach USD 2,2 bi in 2018.
Microorganisms produce a variety of surface-active agents (or surfactants). These can be divided into low-molecular-weight molecules that lower surface and interfacial tensions efficiently and high-molecular-weight polymers that bind tightly to surfaces. Microorganisms utilize a variety of organic compounds as the source of carbon and energy for their growth
Biosurfactant production
Biosurfactants are typically produced by microorganisms growing in hydrocarbons as a carbon source,
Cultivation for biosurfactants includes shake flask, batch, fed-batch, continuous, and integrated microbial/enzymatic processes. Bioreactor is often applied in continuous or fed-batch fermentation in biosurfactant production.
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Biosurfactants can be classified in three groups based on their chemical composition: glycolipids (1), oligopeptides and lipopeptides (2), phospholipids, fatty acids and neutral lipids (3) and polymeric biosurfactants
The most promising group of biosurfactants is the glycolipids
2 . Glycolipids
Glycolipids exist of one or more carbohydrates in combination with one or more fatty acids, hydroxy fatty acids or fatty alcohols. The type of glycolipids is rhamnolipids synthesized by Pseudomonas sp. Mannosylerythriol lipids synthesized by Pseudozyma antarctica trehalose lipids synthesized by Rhodococcus sp., Nocardia sp. and Mycobacterium sp. And sophorolipids synthesized by Candida sp.
2.1 Sophorolipids
Sophorolipids are one of the most promising glycolipids commercial developments as they can be produced at yields of over 400 g/L from glucose and vegetable oil with the non-pathogenic yeast Candida bombicola.
2.2 Rhamnolipids
Rhamnolipids are produced by Pseudomonas sp. Ps. aeruginosa is the best characterized producer for which yields higher than 100 g/L can be achieved. Rhamnolipids reduce the surface tension of water can be used in tertiary petroleum recovery, decontamination of marine oil pollution, soil remediation and crop protection. They also show antimicrobial activity against Gram-positive and Gram-negative bacteria. Furthermore, rhamnolipids can be applied in the food, cosmetic and pharmaceutical sectors. Pseudomonas aeruginosa may be involved in pathogenesis due to their surface activity cause, to security and regulatory reasons, other, non pathogenic Pseudomonas species are known, but the levels of produced glycolipids are much lower. Examples are Ps. putida, Ps. chlororaphis and Ps. fluorescens
3. Oligopeptides and lipopeptides
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The molecules in this class of biosurfactants in general consist of cyclic peptides linked to a fatty acid. Several bacteria are known to produce those antibiotic-like molecules, among them Bacillus subtilus, which produces surfactin. This is one of the most powerful biosurfactants: it possesses anti-bacterial, antiviral, anti-fungal, antimycoplasma and hemolytic activities.
4. Phospholipids, fatty acids and neutral lipid
Several fungi, yeasts and bacteria, that are able to grow on hydrophobic substrates such as alkanes, secrete large amounts of phospholipids, fatty acids or neutral lipids to facility the uptake of the carbon source. Examples are Acinetobacter sp. and Aspergillus sp.
5. Polymeric biosurfactants
Polymeric biosurfactans are compiled from several components. Emulsan, synthesized by Acinetobacter calcoaceticus. It consists of a heteropolysaccharide backbone to which fatty acids are covalently linked. Another example is liposan, a carbohydrate-protein complex synthesized by the yeast Yarrowia lipolytica.
INDUSTRIAL APPLICATIONS
Industry Application Role of biosurfactants Petroleum Enhanced oil recovery Lowering of interfacial tension, dissolving of oil Environmental Bioremediation Lowering of interfacial tension Food Emulsification and de-emulsification Solubilizer, demulsifier, suspension, wetting, foaming Bioprocessing Downstream processing of Microemulsions, biotransformation, Cosmetic Health and beauty products Foaming agents, solubilizers, wetting agents, cleansers Biological Microbiological Cell–cell competition, plant and animal pathogenesis Pharmaceutical and therapeutics Antibacterial, antifungal Agricultural Biocontrol Parasitism, antibiosis, competition,
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UNIT-IV:-Food and Healthcare products
SCP, various types and processes
The term Single Cell Protein (SCP) refers to the dried microbial cells or total protein extracted from pure microbial cell culture which can be used as food supplement to humans Food Grade or animals Feed grade. The SCP production may to solve the problem of worldwide protein shortage.Various types of SCPSingle Cell Protein - Yeast
Yeast is the source of Single Cell Protein. The pet food industry is a major outlet of SCP yeast .The dog, cat and fish feed is supplemented with yeasts. Torula yeast has been commercially used for this purpose. Example Hickory Smoked Dried Torula Yeast.
Single Cell Protein - Algae
The use of algae as food and feed as part of the diets is East Asian countries as well as the natives in Central Africa. Some of the algae like Chloralla, Soenedesmus, Coelastrum and Spirulina have been found to suitable for mass cultivation and utilization.
Single Cell Protein - Filamentous Fungi
Many filamentous fungi have been reported to produce protein. 3000 fungal isolates "from all over the world" were tested for efficiency of growth, and safety as food. Ex: Mushrooms. Strains of some species of moulds, for example, Aspergillus Niger, A. fumigatus, Fusarium graminearum are very hazardous to human, therefore, use of such fungi should be avoided to use as SCP.
Single Cell Protein - Bacteria
Many species of bacteria have been investigated for use in single cell protein production. Methylophilus Methylotrophus is usually
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and mainly used in animal feed as bacteria. Therefore the large quantities of SCP animal feed can be produced using bacteria.
Processes SCP
Fermentation process: There are many methods available for concentrating the solutions like filtration, precipitation, centrifugation and the use of semi-permeable membranes. The removal of water that is necessary to make for mass storage. Single cell proteins need to be condensed and denatured to prevent spoilage.
The physiological features of microbial organisms recommend the control of the carbon source concentrations, Carbon source used can be n-alkanes, gaseous hydrocarbons, methanol and ethanol, renewable sources like carbon IV oxide molasses. as well as an adequate supply of oxygen for the maintenance of balanced growth under an oxidative metabolic pattern. Production periods as long as six weeks have been implemented in many fungal and yeast.
The biomass from yeast fermentation processes is harvested normally by continuous centrifugation. Filamentous fungi are harvested by filtration. The biomass is then treated for RNA reduction and dried in steam drums of spray driers. The removal or reduction of nucleic acid content of various SCP's is achieved with one of the following treatments:
I, Chemical treatment with NaOH
II, Treatment of cells with 10% NaCl;
III, Thermal shock
During manufacturing, the strain is inoculated into a medium which contains molasses and corn-steep liquor as source of carbon, nitrogen and mineral salts. Temperature 25-26 0C is maintained. It is properly aerated during incubation period. The product is concentrated by centrifugation and finally harvested by a filter press or a rotary vacuum filter
Fermentation
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Cultivation of Single Cell Protein there is two types of fermentation processes which are used for production of single cell protein namely submerged fermentation, Semisolid state fermentation. In the submerged process, the substrate used for fermentation is always in liquid state. The product biomass is continuously harvested from the fermenter by using different techniques then the product is filtered or centrifuged and then dried. In semisolid fermentation, the preparation of the substrate is used in solid state e.g. cassava waste. Carotenoids
Carotenoids belong to the most important components in foods. They are natural colorants, as yellow to red colors, so they have great influence on the acceptability of many foods. Moreover, some carotenoids are precursors of vitamin A; About 600 carotenoids have been isolated from natural sources .They are also added as colorants to many manufactured foods, drinks and animal feeds, either in the form of natural extracts (e.g. annatto) or as pure compounds manufactured by chemical synthesis. Carotenoids are essential to plants for photosynthesis, acting in light-harvesting and, especially, in protection against destructive photooxidation. Without carotenoids photosynthesis in an oxygenic atmosphere would be impossible.
Carotenoids are naturally occurring lipid-soluble pigments, the majority being C40terpenoids, which act as membrane-protective antioxidants scavenging O2 and peroxyl radicals Carotenoids pigments occur universally in photosynthetic systems of higher plants, algae and phototrophic bacteria. The production of carotenoids in seaweed runs to hundreds of million tons per year. Some animals use carotenoids for coloration, especially birds , fish (goldfish and salmon).
Carotenoids are important factors in human health. The essential role of beta-carotene and others as the main dietary source of vitamin A has been known for many years. More recently, protective effects of carotenoids against serious disorders such as cancer, heart disease and degenerative eye disease have been recognized. The carotenoid market is expanding mainly due to increasing demand for food supplements, and food pigments and colors. The rising incidence of life-threatening diseases is also increasing the application of carotenoids in several food supplements.
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The major carotenoid types include beta-carotene, astaxanthin, canthaxanthin, and annatto. Beta-carotene dominated the carotenoid market in 2013, followed by astaxanthin. Beta-carotene is used in various types of food and supplements to prevent or cure certain diseases and disorders such as heart diseases, cataracts, elderly muscular degeneration, Alzheimer’s disease, depression, heartburn, epilepsy, infertility, and high blood pressure.
NOMENCLATURE AND STRUCTURE
In 1831 Wackenroder isolated carotene from carrots. Carotenoids are terpenoid pigments of 40 carbon atoms they are soluble principally in nonpolar solvents. These pigments are grouped in carotenes and xanthophylls. Carotenoids are a class of hydrocarbons (carotenes) and
their oxygenated derivatives (xanthophylls). .
Some of the microbial carotenoids produced by yeasts
β –carotene has been used as food colorant or as a food supplement .It also is added to juices and drinks formulations and others such as butter, margarine and cheese.
•Torulene owns 13 conjugated double bonds, and has an attractive color. Torulene has more antioxidant efficiency than β –carotene.
•Astaxanthin is a red pigment that causes coloration in marine invertebrates, fish and birds .
Canthaxanthin is a keto-carotene that used to food and cosmetic industries, mainly
Biological properties of carotenoids
Commonly, carotenoids are distinguished as a vitamin A precursor; Lack of carotenoids consequences are deficiency on tears production, blindness, principally in children, plus premature death . .
Natural carotenoids have similar molecular structure than synthetics; .Industrial production of natural carotenoids can be carried out through to
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i) Biotechnological processes using filamentous fungi, yeasts, bacteria or microalgae, and
ii) solid-liquid extraction from plants .
Microbial carotenoid production
The commercial carotenoids are obtained by extraction from vegetables and chemical synthesis. The chemical synthesis generates hazardous wastes that can affect the environment. Microbial production has the advantage to use low-cost substrates, resulting in lower costs of production. Carotenoids are widely distributed in microorganisms including bacteria, yeast, fungus and algae. Blakeslea trispora to produce β-carotene. Another mould, Phycomyces blakesleeanus is also to produces β-carotene at industrial scale, In general there is a vast of microorganisms that can biosynthesize carotenoids;
The general pathways for biosynthesis of carotenoid by yeasts involves three general steps:
1 (Synthesis begins with conversion of acetyl CoA to 3-hidroxy-3-methyl glutaryl-CoA (HMG-CoA), catalyzed by HMG-CoA synthase. Then, HMG-CoA is converted in mevalonic acid (MVA. MVA is phosphorylated by MVA kinase and decarboxylation; into isopentenyl pyrophosphate (IPP).
2 (IPP is isomerized to dimethyllayl pyrophosphate (DMAPP) with the addition of three IPP molecules to DMAPP, catalyzed by prenyl transferase into geranyl. geranyl pyrophosphate (GGPP). Condensation of two molecules of GGPP produces the phytoene (the first C40 carotene of the pathway); which is subsequently desaturated to form lycopene.
3 (Many cyclic carotenoids are derived from lycopene, as β -carotene, γ -carotene, Torulene, Torularhodin and Astaxanthin when it undergoes many reactions.
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Amino acids:-Lysine
Lysine, or L-lysine, is an essential amino acid, it is necessary for human health, but the body cannot make it. Lysine gets from food or supplements. Lysine is the building blocks of protein and it plays an essential role in the production of carnitine, a nutrient responsible for converting fatty acids into energy and helping lower cholesterol. Lysine appears to help the body absorb calcium, and it plays an important role in the formation of collagen, a substance important for bones and connective tissues including skin, tendons, and cartilage.
It has been isolated from casein in1889; Lysine was commercially introduced as a feed additive around 1960. L-lysine has been manufactured using bacterial fermentation of carbohydrates. Corynebacterium glutamicum and related strains are useful microorganisms. The now dominant method of industrial production of lysine is based on fermentation of beet and cane sugar or starch sugars. China was the largest consumer for lysine .Animal feed is the largest application segment for lysine and accounted for 92.4% of total market volume in 2013. The global lysine was 1,902.3 kilo tons in 2013 and is expected to reach 2,854.9 kilo tons by 2020.
Natural LYSINE sources
The best food sources are fish, chicken, beef, lamb, wheat germ, milk, cottage cheese. Lysine has been used in alternative medicine as an aid to prevent cold sores around the mouth
CHEMICAL PROPERTIES Lysine exists as stereoisomers and a racemic mixture of L- and D-forms is obtained by a pure chemical synthesis. Because only the naturally occurring L-lysine form is physiologically active, it is necessary to introduce a biological step in the manufacturing process. This part is
carried out by fermentation in bioreactors .
RAW MATERIALS Sugar from beet and cane using to replace the traditional sugar and
molasses as carbohydrate source in the amino acid fermentation .
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PROCESS
First step is liquefaction of the substrate by acid or alpha-amylase. This step will hydrolyze the starch into shorter chains of polysaccharides.
The liquefaction is followed by saccharification to hydrolysate with amyloglucosidase .
Corynebacterium glutamicum bacteria are grown aerobically in a liquid nutrient medium with the starch sugar syrup as the carbohydrate constituent and ammonia as the nitrogen source. The fermentation is carried out as a batch process. Filtered sterile air is supplied throughout the digestion. A temperature of about 30 to 35 C is maintained during the fermentation period. It is preferred to maintain pH in the range of about 6.5 to 7.0 throughout the fermentation cycle typical lasting 3 - 5 days. For adjusting pH, conventional agents, such as inorganic or organic acidic or alkaline substances may be used .
The bacterium excretes the amino acid the lysine accumulates in the fermentation broth as an aqueous solution of lysine. The solution is condensed by evaporation and Spray drying method have been using when a powder is required. Crystallization applied for isolation and purification, but also modern ion exchange resins and membranes are applied in high grade products.
Glutamic Acid
The amino acid business is a multi-billion dollar enterprise. Glutamic acids are used as flavor enhancers. The largest volume is the food flavoring industry. Glutamic acid is a 'non-essential' classified amino acid that is very common in plants and animals. Besides being a building block of protein, glutamic acid is vital in the transmission of nerve impulses, and is even manufactured in the brain.
A typical human contains 4.4 pounds of glutamate or Glutamic acid. It is a main component of proteins and peptides, and present in most tissue. . It's a major component of protein rich food like milk, and cheese, Glutamate or Glutamic acid is also ubiquitous in grain, beans, vegetables, mushrooms, fruits, AND nuts.
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At the present time, glutamic acid is largely produced through microbial fermentation because the chemical method produces a racemic mixture of glutamic acid (D- and L-glutamic acid). glutamic acid excretion by various micro-organisms; however, most of them were not food-grade micro-organisms. Lactic acid bacteria (LAB) are well known to produce a variety of primary metabolites. A type of soil bacteria produced large amounts of glutamic acid. The Micrococcus glutamicus were the most effective producers and its Wild-types cultures produced up to 10 g/l glutamic acid. The mutants Corynebacteriwn glutamicum have been Yielding excess of 100 g per liter. The addition of penicillin to growing cells of Corynebacteriwn glutamicum triggers the excretion of high levels of glutamic acid.
There are two common forms of glutamic acid, L-glutamic acid and D-glutamic acid. L-glutamic acid found in protein is referred to as 'protein bound' glutamic acid. D-glutamic acid 'outside of protein' or 'free glutamic acid' is artificially and chemically produced outside of the body. This is known as monosodium glutamate or MSG.
Processes
In case of the microbial glutamic acid production, a number of different nutrients sources using, carbon sources such as glucose, sucrose, fructose, ribose, etc. a type of protein hydrolysate is used as carbon- and nitrogen source in fermentation that improve the quality of the fermentation broth.
pH plays an important role in glutamic acid production. Concentration of ammonium nitrate and a pH 4.5 improved glutamic acid production.
Two-stage of fermentation .The bacteria produce α-ketoglutaric acid and subsequently converted to glutamic acid by transamination or reductive amination by glutamic acid dehydrogenase.
After fermentation time of between 1 and 4 days the biomass is separated from the fermentation medium, the medium is then passed over an ion-exchange column. When the purified medium take place enzymatically dimidiate so, that obtain glutamic acid. The glutamate can be obtained by direct crystallization or by chromo-separation followed by crystallization.
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Riboflavin vitamin B2
Riboflavin was first isolated by Blyth in 1879 from whey Riboflavin is a water-soluble and yellow, fluorescent material was named lactochrome. According to IUPAC rules, riboflavin is called 7, 8- dimethyl-10- isoalloxazine, also known as vitamin B2 or lactoflavin. The daily human demand for riboflavin is around 1.7 mg, and deficiencies lead to various symptoms such as, e.g., versions of dermatitis. The vitamin cannot be stored in the body and a constant intake is required. Riboflavin is used to prevent riboflavin deficiency and to treat ariboflavinosis.
Food Sources
Most plant and animal tissues contain at least small amounts of riboflavin. Other sources of riboflavin are organ meats. Milk is both a rich source of riboflavin and a commonly consumed food. Riboflavin loss occurs if it is exposed to the light, for example, if milk is stored in clear glass under light
Chemical synthesis was the first production method to be established and is still dominating, but in recent years the production is shifting more and more to fermentation. Green plants, most bacteria, and moulds, however, can produce their own riboflavin. At present, three organisms are used for the industrial production of riboflavin by fermentation: the filamentous fungus Ashbya gossypii , the yeast Candida famata And genetically engineered strains of Bacillus subtilis.
Production Process: A batch process using E. Ashbyii. Upstream processing consists of preparation of medium and associated continuous counter-current sterilization. Feed components are: 70% glucose syrup, yeast and malt extract, sunflower oil, sulfuric acid, and concentrated salt solution at room temperature. Fermentation is operated batch-wise with 10% inoculum ratios. Downstream processing starts with harvesting followed by crystallization, centrifugation (decanter), and final drying (spray dryer). The requested purity of riboflavin is 70%. The residual 30% consists of salts and biomass. The product is obtained as dry powder or as granulate.
Upstream process
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The upstream processes include preparation and sterilization of the medium. The medium’s composition does not allow sterilization of all components mixed together and using classical batch conditions (121◦C, 20 minutes). Therefore, the medium would be divided into several groups: I. glucose and sunflower oil ,
II. Peptone, yeast and malt extracts ,
III. Salts in water IV. methionine. The latter is sterilized by filtration. Sulfuric acid does not require sterilization.
Fermentation
Fermentation In several steps the necessary seed cultures are prepared in different seed fermenters. The last seed culture is the start inoculum for the main fermentation. The duration of a seed-fermentation is around 50 hours, while the main fermentation lasts about 500 hours. During this time the strain produces 27 g/L riboflavin. Fermentation requires aeration accomplished by a gas compressor and a sterile filter .Exhaust gases are filtered by a second filter . A small fraction of the harvested broth is put into another tank and is used as inoculum for the next batch
Downstream
After fermentation the broth is harvested into the harvest tank. Part of the product crystallizes in the fermenter and also in the harvesting tank. Crystallization is completed in the crystallizer by evaporation of some of the water. Afterwards the suspension is stored in tank
To achieve higher purity, a washing step is used with a second separation. The last step is drying, either using a spray dryer to obtain a powdered product or applying a spray granulation to obtain granulate. Granulate can be dosed more precisely
Vit.B12 Cynocobalamine
Vitamin B12, vitamin B12 or vitamin B-12, also called cobalamin, is a water-soluble vitamin with a key role in the normal functioning of the brain and nervous system, and for the formation of blood. It is one of the
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eight B vitamins. Microbial source of Cynocobalamine extracted from Streptomyces griesus was demonstrated in 1948
Vitamin B12 helps in the production of healthy red blood cells that carry oxygen around the body. Lack of vitamin B12 deficiency called as
anemia .
Industrial production of B12 is through fermentation of selected microorganisms. Streptomyces griseus would be use to the commercial source of vitamin B12 for many years. The species Pseudomonas denitrificans and Propionibacterium shermanii are more commonly used today.
Microbial Cynocobalamine production
Media preparation
Carbon source as
1. Corn steep glucose
2. Beet molasses
3. Soyabean meal/Glucose
Nitrogen source-as
1. Ammonium phosphate
2. Ammonium hydroxide
Formulation of medium
Cobalt salt Medium Cobalt salt Sterilization
Starter culture of Propioni bacterium shermanii Inoculate in Anaerobic fermentation for 3 days Aerobic fermentation for 4 days .Centrifugation Harvested Broth Cell Harvest broth treatment with acid and heating that Released Pseudo-vitamin B12 Bakers coenzyme
When the Addition of cyanide solution with pseudo B12 that release Cynocobalamine.
Starter culture
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The following microbes were suitable for Industrial fermentation of Cynocobalamine. 1. Bacillus megaterium
2. Streptomyces olivaceous
3. Propionibacterium shermanii
4. Pseudomonas denitrificans
5. Rhodopseudomonas palustris
Inoculum
Wild strains Mutant strains of Propionibacterium shermanii
Inoculum Improved strain produce that 50,000 times more vitamin B12 than wild strain of Streptomyces olivaceous.
Batch fermentation
Most fermentation is batch processes Nutrients and the inoculum are added to the sterile fermenter .Anti-foaming agent may be added. Once the desired amount of product is present in the fermenter the contents are drained off and the product is extracted.
Anaerobic Fermentation
It is a Batch fermentation Continuous fermentation also found to be effective. Sterilized medium is filled in stirred tank fermenter 1% of inoculum (Starter culture) is added in to the fermenter. Anaerobic condition is maintained to encourage the production of 5, 6-dimethyl benzimidazole cobalamide (DBC) by P.shermanii.
Aerobic Fermentation
Aerobic Fermentation is over sterile air is pumped in to the fermenter. The culture is stirred well for proper aeration. Aerobic fermentation is performed for 4 days. During this process some amount of DBC and pseudo vitaminB12 (adeninyl cobalamine) are produced. DBC and pseudovitaminB12 (adeninyl cobalamine) are immediate precursors of Cynocobalamine.
Recovery of vitamin B12
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Inside the microbial cells the cyanocobalamin exits in the form of natural substances such as DBC and pseudo vitaminB12. The cultured broth contains 10-23mg vitB12 per liter. It is harvested and centrifuged at high speed to get a concentrated mass of cells. The cell mass is treated with a dilute acid and heat stock at 10-30o C. During this treatment precursors of pseudo vitamin B12 are released free.
Then it is treated with Cyanide solution to split the DBC and pseudo vitamin B12. As a result cyanocobalamin (Vit B12) is released free in the solution. Cynocobalamine in the liquid is separated by using an adsorption column chromatography with IRC-50 resin. The adsorbed cyanocobalamin is then eluted out of the column using phenolic solvent. The solvent fraction is evaporated by exposing it to atmospheric air. As result crystals of cyanocobalamin is let in the vessel. It is stored for future use.
USES
•It is a food preservative
• It is a co-factor
•It is a protective medicine
Fatty acids Palmetate, oleate
Fatty acids are almost entirely straight chain aliphatic carboxylic acids. Fatty acids, esterified to glycerol, are the main constituents of oils and fats. The industrial exploitation of oils and fats, both for food and oleo chemical products
Palmitate Palmitate is a synthetic salt of palmitic acid produced by a process of esterification or alcoholoxidation of the acid. A type of lipase derived from Candida sp is suitable for transesterification of fats and oils to produce fatty acid
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Palmitic acid is a saturated fatty acid found in a variety of plant and animal sources. The most prolific of these natural sources are palm oil, Palmitate strong antioxidant and source of vitamin A, A Many low fat foods lose much of their vitamin A component ex: when they undergo milk fat removal of milk. Palmitate is commonly added to these products as a vitamin A fortification agent. Excessive use of palmitate cause health risks including an increase in LDL cholesterol levels and a decrease in HDL cholesterol concentrations with an associated risk of arterial diseases . Vitamin A palmitate,
Vitamin A palmitate, also called retinyl palmitate or retinol palmitate, it is used as a fortifying agent in dairy products, food additive and use as dietary supplement. It accelerates cell renewal and stimulates the fibroblast and collagen in the skin, thereby reducing wrinkles and fine lines. Due to its anti-oxidant properties, it is also a great anti-aging
ingredient, and helps promote a softer smoother skin .
Oleate
Oleate (C18:1) is, besides palmitate (C16:0), the most abundant fatty acid in the human diet and a common dietary fat found in olive oil,
Oleate is the first unsaturated fatty acid that is generated from the saturated fatty acids produced by the fatty acid synthase. It's name is derived from the olive tree, since it makes up 55-80% of olive oil and 15-20% of grape seed oil and sea buckthorn oil. Oleate is rarely found in its free form. It is usually found as either oleoyl-[acp], oleoyl-CoA, or incorporated into a lipid. Retinol Oleate using for the production of vitamin A.
Bacterial biosynthesis of Oleate does not require oxygen and involves elongation of fatty acids. The obligate anaerobic bacterium Clostridium beijerinckii and the saprophytic bacterium Aerococcus viridans produce the unusual monounsaturated fatty acids -hexadec-7-enoate and Oleate .
In the case of bacterial Oleate production, the key step in the pathway is a similar isomerization. The enzyme catalyzing the isomerization reaction in Aerococcus viridans is a unique hydratase/isomerase. The desaturase enzymes that produce Oleate in membrane bound systems of fungi, yeast,
and mammalian liver. The Stearoyl-CoA present in plant and its specific for desaturase enzyme. Oleate is produced via a stearoyl acp in plants the enzyme responsible for the isomerization in Clostridium beijerinckii is still unknown,
