1. Microbial Kinetics and Substrate utilization in Fermentation

2. Batch culture and Kinetics of Microbial growth in batch culture
After inoculation the growth rate of the cells gradually increases.
The cells grow at a constant, maximum, rate and this period is known as the log or exponential, phase.

3. Growth of a typical microbial culture in batch conditions

4. The rate of growth is directly proportional to cell concentration or biomass-
i.e dx/dt α X
dx/dt = μX
Where,
X is the concentration of microbial biomass,
t is time, in hours
μ is the specific growth rate, in hours -1

5. On integration of equation (1) from t=0 to t=t ,we have:
xt = xo e μt
Where,
Xo is the original biomass concentration,
Xt is the biomass concentration after the time interval, t hours,
e is the base of the natural logarithm.

6. On taking natural logarithms of equation (2) we have :
In Xt = In Xo + μt (3)

7. Therefore, a plot of the natural logarithm of biomass concentration against time should yield a straight line, the slope of which would equal to μ.
During the exponential phase nutrients are in excess and the organism is growing at its maximum specific growth rate, ‘μmax ‘ for the prevailing conditions.

8. Typical values of μmax for a range of microorganisms are given below in the Table.

9. Effect of substrate concentration on microbial growth
Whether the organism is unicellular or mycelia the growth is influenced by consumption of nutrients and the excretion of products. The cessation of growth may be due to the depletion of essential nutrient in the medium (substrate limitatioln), the accumulation of some autotoxic product of the organism in the medium (toxin limitation) or a combination of the substrate limitation and toxin limitation.
The nature of the limitation of growth may be discussed by growing the organism in the presence of a range of substrate concentrations and plotting the biomass concentration at stationary phase against the initial substrate concentration is shown given below in fig 2:
FIG. 2. The effect of initial substrate concentration on the biomass concentration at the onset of stationary phase, in batch culture.

10. From figure 2 it may be seen that over the zone A to B due to an increase in initial substrate concentration gives a proportional increase in the biomass occur at stationary phase. This relation between increase in initial substrate concentration and proportional increase in the biomass may be described by equation:
X = Y(SR - s) (3)
Where,
X -is the concentration of biomass produced,
Y -is the yield factor (g biomass produced g-1 substrate consumed),
SR -is the initial substrate concentration, and
s -is the residual substrate concentration.
Thus, equation (3) may be used to predict the production of biomass from a certain amount of substrate

11. In Fig. 2:-
Over the zone A to B: s = 0; at the point of cessation of growth.
Over the zone C to D an increase in the initial substrate concentration does give a proportional increase in biomass due to the exhaustion of another substrate or the accumulation of toxic products

12. Monod Equation
The decrease in growth rate and the cessation of growth due to the depletion of substrate, may be described by the relationship between μ and the residual growth limiting substrate.
This relationship is represented by a equation given by Monod in1942 is know as Monod equation.
Based upon Michaelish-Menten kinetics.
According to Monad equation-
μ = μmax . S /Ks + S (4)
Where,
S is residual substrate concentration,
Ks is substrate utilization constant, numerically equal
to substrate concentration when μ is half of μmax.
Ks s a measure of the affinity of the organism with substrate
It tell about the relationship between specific growth rate ‘μ’ and growth limiting substrate concentration ‘S’.

13. Fig: 3 The effect of residual limiting substrate concentration on specific growth rate of a hypothetical bacterium.
In the above figure
The zone A to B is equivalent to the exponential phase in batch culture where substrate concentration is in excess and growth is at μmax .
The zone C to A is equivalent to the deceleration phase of batch culture where the growth of the organism is due to the depletion of substrate to a growth-limiting concentration which will not support μmax .

14. Some representative values of Ks for a range of micro-organisms and substrates
Typical values of K, for a range of organisms and substrates are usually very small and therefore the affinity for substrate is high.

15. If the organism has a very high affinity for the limiting substrate (a low Ks value) the growth rate will not be affected until the substrate concentration has declined to a very low level. Thus, the deceleration phase for such a culture would be short.
However, if the organism has a low affinity for the substrate (a high Ks value) the growth rate will be deleteriously affected at a relatively high substrate concentration. Thus, the deceleration phase for such a culture would be relatively long.
The biomass concentration at the end of the exponential phase is at its highest level. Therefore the decline in substrate concentration will be very rapid so that the time period during which the substrate concentration is close to Ks is very short.
The stationary phase in batch culture is that point where the growth rate has declined to zero. This phase is also known as the maximum population phase.

16. Growth Curve
Log CFU/ml
Optical Density
Lag

17. Lag phase Three causes for lag: physiological lag low initial numbers
appropriate gene(s) absent
growth approx. = (dX/dt = 0)

18. Exponential phase
Nutrients and conditions are not limiting
growth = 2n or X = 2nX0
Where X0 = initial number of cells
X = final number of cells
n = number of generations 20 21 22 23 24 2n 20 21 22 23 24 2n 20 21 22 23 24 2n 20 21 22 23 24 2n 20 21 22 23 24 2n 20 21 22 23 24 2n

19. This is an increase is 5 orders of magnitude!!
Example: An experiment was performed in a lab flask growing cells on 0.1% salicylate and starting with 2.2 x 104 cells. As the experiment below shows, at the end there were 3.8 x 109 cells.
This is an increase is 5 orders of magnitude!!
How many doublings or generations occurred?
Cells grown on salicylate, 0.1%
X = 2nX0
3.8 x 109 = 2n(2.2 x 104)
1.73 x 105 = 2n
log(1.73 x 105) = nlog2
17.4 = n

20. Calculating growth rate during exponential growth
dX/dt = uX
where u = specific growth rate (h-1)
Calculating growth rate during exponential growth
dX/dt = uX where u = specific growth rate (h-1)
Rearrange: dX/X = udt
Integrate: lnX = ut + C, where C = lnX0
lnX = ut + ln X0 or X = X0eut
y = mx + b (equation for a straight line)
Note that u, the growth rate, is the slope of this straight line

21. Calculating growth rate during exponential growth
dX/dt = uX where u = specific growth rate (h-1)
Rearrange: dX/X = udt
Integrate: lnX = ut + C, where C = lnX0
lnX = ut + ln X0 or X = X0eut
y = mx + b (equation for a straight line)
Note that u, the growth rate, is the slope of this straight line

22. Find the slope of this growth curve
lnX = ut + ln X0 or u = lnX – lnX0
t – t0
u = ln 5.5 x 108 – ln 1.7 x 105
= 2 hr-1

23. What is fastest known doubling time? Slowest?
Now calculate the doubling time
If you know the growth rate, u, you can calculate the doubling time for the culture.
lnX = ut + ln X0
For X to be doubled: X/X0 = 2
or: 2 = eut
From the previous problem, u = 2 hr-1,
2 = e2(t)
t = hr = min
What is fastest known doubling time? Slowest?

24. How can you change the growth rate???
When under ideal, nonlimiting conditions, the growth rate can only be changed by changing the temperature (growth increases with increasing temp.). Otherwise to change the growth rate, you must obtain a different microbe or use a different substrate.
In the environment (non-ideal conditions), the growth rate can be changed by figuring out what the limiting condition in that environment is.
Question: Is exponential growth a frequent occurrence in the environment?

25. Growth Curve
Stationary

26. Stationary phase Death phase
nutrients become limiting and/or toxic waste products accumulate
growth = death (dX/dt = 0)
death > growth (dX/dt = -kdX)
Death phase

27. Monod Equation
The exponential growth equation describes only a part of the growth curve as shown in the graph below.
The Monod equation describes the dependence of the growth rate on the substrate concentration:
u = um S
Ks + S .
u = specific growth rate (h-1)
um = maximal growth rate (h-1)
S = substrate concentration (mg L-1)
Ks = half saturation constant (mg L-1)

28. Combining the Monod equation and the exponential growth equation allows expression of an equation that describes the increase in cell mass through the lag, exponential, and stationary phases of growth:
dX/dt = uX
u = dX/Xdt
u = um S
Ks + S .
Monod equation
Exponential growth equation
dX/dt = um S X
Ks + S .
Does not describe death phase!

29. Ks . There are two special cases for the Monod growth equation
At high substrate concentration when S>>Ks, the Monod equation
simplifies to:
dX/dt = umX
growth will occur at the maximal growth rate. Ks
2. At low substrate concentration
when S<< Ks, the Monod equation
simplifies to:
dX/dt = um S X Ks .
growth will have a first order dependence on substrate concentration (growth rate is very sensitive to S).
Which of the above two cases is the norm for environmental samples?

30. Growth in terms of substrate loss
In this case the growth equation must be expressed in terms of substrate concentration. The equations for cell increase and substrate loss can be related by the cell yield:
dS/dt = -1/Y (dX/dt) where Y = cell yield
Y = g cell mass produced
g substrate consumed
Glucose (C6H12O6) Pentachlorophenol (C6Cl5OH) Octadecane (C18H38)
0.4
0.05
1.49

31. Growth in terms of substrate loss
dS/dt = -1/Y (dX/dt)
dS/dt = -1/Y (dX/dt)
Combine with: dX/dt = um S X
Ks + S .
Combine with: dX/dt = um S X
Ks + S . .
dS/dt = - um (S X)
Y (Ks + S)
Which parts of this curve does the equation describe?

32. Fermentation technology for production of various industrial compunds(vitamins,antibiotics,organic acid, etc)

33. Microbial products by fermentation Technology
Primary metabolites
Small molecules of living cells Intermediates or end products of the pathway.
Related to synthesis of microbial cells in the growth phase.Include alcohols, amino acids, nucleotides, organic acids, polyols, vitamins, and enzymes
e.g. Lactic acid,citric acid
Secondory metabolites-
Accumulate following active growth.
Have no direct relationship to synthesis of cell material and natural growth
Include antibiotics and toxins

34. Primary metabolites-
Secondory metabolites-

35. Primary metabolites
They are compounds made during the ordinary metabolism of the organism during the growth phase. A common example is ethanol or lactic acid, produced during glycolysis. Citric acid is produced by some strains of Aspergillus niger as part of the citric acid cycle to acidify their environment and prevent competitors from taking over. Glutamate is produced by some Micrococcus species,and some Corynebacterium species produce lysine, threonine, tryptophan and other amino acids. All of these compounds are produced during the normal "business" of the cell and released into the environment. There is therefore no need to rupture the cells for product recovery.

36. Secondory metabolites-
They are compounds made in the stationary phase; penicillin, for instance, prevents the growth of bacteria which could compete with Penicillium molds for resources. Some bacteria, such as Lactobacillus species, are able to produce bacteriocins which prevent the growth of bacterial competitors as well. These compounds are of obvious value to humans wishing to prevent the growth of bacteria, either as antibiotics or as antiseptics  (such as gramicidin S . Fungicides, such as griseofulvin are also produced as secondary metabolites.[Typically secondary metabolites are not produced in the presence of glucose or other carbon sources which would encourage growth,[8]and like primary metabolites are released into the surrounding medium without rupture of the cell membrane.

37. Vitamin production by Fermentation Technology
Vitamins are defined as essential micronutrients that are required in trace quantity and are very important compounds in diet
Synthesized by prototrophic microorganisms
Microbes excrete vitamins in excess of their metabolic needs under highly specific and artificial condition

38. Vitamin B12
Vitamin B2
Vitamin C
proVitamin A

39. Micro –organisms in industrial production of vit. B12
Streptomyces griseus , S. olivaceus , Bacillus megaterium , B. coagulans , Pseudomonas denitrificans , Propionibacterium freudenreichii , P. shermanii and a mixed fermentation of a Proteus spp and a Pseudomonas sp

40. Vit.B12 production using Streptomyces olivaceus NRRL B-1125
Manufactured by submerged fermentation
Aeration and agitation of medium essential
Fermentation process completed in 3 to 5 days

41. Inoculum prepation
Pure slant culture of Streptomyces olivaceus NRRL B-1125 is inoculated and grown in 100 to 250 ml of inoculum medium.
Seeded flask are kept on shaker for incubation .
Flask cultures are used to inoculate large amount of inoculum media arranged in series of tank .
2 or 3 successive transfers are made to obtain required amount of inoculum cultures.
Inoculum of production tank must be 5% of the volume of production medium

42. Industrial production of vitamins

43. Production medium
Consist of carbohydrate ,proteinaceous material , and source of cobalt and other salts .
Sterilization of medium batchwise or continuously .
Batch – medium heated at 250°F for 1 hr
Continuous – 330°F for 13 min by mixing with live steam.
Components
Amount ( %)
Distillers solubles
4.0
Dextrose
0.5 to 1
CaCO3
0.5
COCl2.6H2O
1.5 to 10 p.p.m.

44. Temperature , pH , aeration and agitation
Temperature : 80°F
pH : At starting of process pH falls due to rapid consumption of sugar, then rises after 2 to 4 due to lysis of mycelium
pH 5 is maintained with H2SO4 and reducing agent Na2SO4 .
Aeration and agitation : Optimum rate of aeration is
0.5 vol air/vol medium/min. Excess aeration cause foaming.

45. Antifoam agent , prevention of contamination
Antifoam agent : soya bean oil , corn oil,
lard oil and silicones (sterilized before adding) .
Prevention of contamination : essential to maintain sterility ,
contamination results in reduced yields , equipments must be sterile and all transfers are carried out under aseptic conditions .
Industrial production of vitamins

46. Yields
Yield of cobalamin are usually in the range of 1 to 2 mg. per litre in the fermented broth

47. Recovery
Cobalamin associated with mycelium- boiling mixture at pH 5 liberates the cobalamin quantitatively from mycelium.
Broth containing cobalamin is subjected to further work up depending on type of product to be produced

48. Filtration - to remove mycelium.
Recovery contd….
Filtration - to remove mycelium.
Filtered broth treated with cyanide – (cobalamin to cyanocobalamin).
Adsorption chromatography , ion exchange chromatography – adsorbents : activated charcoal , bentonite , fuller’s earth .
Bentonite
Fuller’s earth

49. Elution : water, water-acetone and solution of sodium cyanide or sodium thiocyanate .
further extraction – countercurrent distribution b/w cresol, amyl phenol or benzyl alcohol and water or single extraction into organic solvent (phenol)

50. Industrial production of vitamins
Recovery contd….
To aqueous concentrates , dissolve a Zn salt in a slight acidic solution & then rise the pH to bring about precipitation of ZnOH(impurities are removed) .
Chromatography on alumina & crystallization from methanol-acetone , ethanol-acetone, or acetone-water.
Industrial production of vitamins

51. Recovery contd….
To use as feed supplement , final fermented broth is evaporated to dryness.
Final broth contain 3% solids – in vacuo evaporation (15 to 20 % solid content).
Syrup – drum dried or spray dried.(contain 10 to 30 mg.lb. of cobalamin)

52. Beta- carotene or provitamin A
Provitamin A -----> Vitamin A (intestine)
Fat soluble
Deficiency leads to night blindness
Best source is liver and whole milk also coloured fruits and vegetables
Isoprene derivatives
Tetraterpenoids with eight isoprene residues
400 naturally occurring carotenoids: b-carotene, a-carotene, d-carotene, lycopene, zeaxanthin
Carotenoids Used as food colorants and animal feed supplements for poultry and aquaculture, carotenoids play an increasing role in cosmetic and pharmaceutical applications due to their antioxidant properties.
The pigments are often regarded as the driving force of the nutraceutical boom, since they not only exhibit significant anticarcinogenic activities but also promote ocular health, can improve immune response, and prevent chronic degenerative diseases.

53. Commercial production
Microbial fermentation
Blakeslea trispora (high yeild; 7g/L)
Phycomyces blakesleeanus
Choanephora cucurbitarum
Submerged Fermentation process
Corn starch, soyabean meal, b-ionone, antioxidants
Trisporic acid: act as microbial sex hormone, improves yield
b-Ionone: incr b-carotene syn by incr enzyme activity
Purified deodorized kerosene increases solubility of hydrophobic substrates
stimulators
Recovery: b- carotene rich mycelium used as feed additive
Mycelium is dehydrated by methanol, extracted in methylene chloride and crystallized which is 70-85% pure
DSM Nutritional Products (Switzerland) and BASF (Germany) dominate the market with their chemical synthesis processes, but Chinese competitors are catching up.

54. Halophilic green microalgae Dunaliella salina
Halophilic green microalgae Dunaliella salina. It accumulates the pigments in oil glo- bules in the chloroplast interthylakoid spaces, protecting them against photoinhibition and photodestruction.
Excessive pigment formation in D. salina is achieved by numerous stress factors like high temperature, lack of nitrogen and phosphate but excess of carbon, high light intensity, and high salt concentration, the latter two having the highest impact.
Dried D. salina biomass for sale contains 10–16% carotenoids, mainly b-carotene. In addition crystalline material obtained after extraction with edible oil is also sold.

55. Primary Metabolites: Organic Acids
Organic acids are produced by through metabolisms of carbohydrates. They accumulate in the broth of the fermenter from where they are separated and purified.
I. Terminal end products lactic acid
(pyruvate, alcohol) Propionic acid
II. Incomplete oxidation of sugars citric acid
(glucose) Itaconic acid
Gluconic acid
Glycolysis
Krebs cycle
III. Dehydrogenation of alcohol with O2 acetic acid
Manufactured on large scale as pure products or as salts

56. CITRIC ACID: industrial uses
Flavoring agent
In food and beverages
Jams, candies, deserts, frozen fruits, soft drinks, wine
Antioxidants and preservative
Agent for stabilization of
Fats, oil or ascorbic acid
Stabilizer for cheese preparation
Chemical industry
Antifoam
Treatment of textiles
Metal industry, pure metals +citrate (chelating agent)
Pharmaceutical industry
Trisodium citrate (blood preservative)
Preservation of ointments and cosmetics
Source of iron
Acidifyer
Flavoring
Chelating agent
Primary metabolite
Present in all organisms
Detergent cleaning industry
Replace polyphosphates

57. Commercial Production
Aspergillus niger
clavatus
Pencillium luteum
Strains that can tolerate high sugar and low pH with reduced synthesis of undesirable by products (oxalic acid, isocitric acid, gluconic acid)
Glucose
Pyruvate
Acetyl CoA
CO2
OXA
Malate
MITOCHONDRIA
Malate Fumarate Succinyl CoA
citric acid
a-KG
CYTOPLASM
MEDIUM
Pyr carboxylase
Pyr Dehy-
drogenase
Citrate
synthase
100g sucrose --- 112g any citric acid or 123g citric acid-1hydrate

58. Factors for regulation
CARBOHYDRATE SOURCE: sugar should be 12-25%
Molasses (sugar cane or sugar beet)
Starch (potato)
Date syrup
Cotton waste
Banana extract
Sweet potato pulp
Brewery waste
Pineapple waste
High sugar conc incr uptake and production of citric acid
TRACE METALS:
Mn2+, Fe3+, Zn2+ incr yield
Mn2+ incr glycolysis
Fe3+ is a cofator for enzymes like aconitase
pH: incr yield when pH below 2.5, production of oxalic acid and gluconic acid is suppressed and risk of contamination is minimal
DISSOLVED O2: high O2, sparging or incr aeration can affect if interrupted
NITROGEN SOURCE: addition of ammonium stimulates overproduction, molasses is good source of nitrogen

59. Citric acid production
Surface fermentation submerged fermentation
Solid liquid
Stirred Airlift
Bioreactor bioreactor
N alkanes (C9-C23) can also be used to produce citric acid; can result in excess production of isocitric acid

60. ACETIC ACID: industrial uses

61. Acetaldehyde dehydrogenase
ACETIC ACID
Incomplete oxidation of ethanol
Vinegar is prepared from alcoholic liquids since ceturies
NAD+ NADH +H+
NADP+ NADP +H+
CH3 CH2OH---- CH3CHO  CH3CH(OH)  CH3COOH
Ethanol acetaldehyde acetaldehyde hydrate acetic acid
Alcohol
dehydrogenase
Acetaldehyde dehydrogenase
Gluconobacter, Acetobacter with acid tolerant A. aceti
One molecule of ethanol one molecule of acetic acid is produced
12% acetic acid from 12% alcohol
Clostridium thermoaceticum
It is an obligate anaerobe, Gram-positive, spore-forming, rod-shaped, thermophilic organism with an optimum growth temperature of 55–60 o C and optimum pH of 6.6–6.8.

62. VINEGAR: 4% by volume acetic acid with alcohol, salts, sugars and esters
flauoring agent in sauces and ketchups, preservative also
Wine, malt, whey (surface or submerged fermentation process)
Surface: trickling generator; fermentale material sprayed over surface, trickle thro shavings contaning acetic acid producing bacteria; 30oC (upper) and 35oC (lower). Produced in 3 days.
Submerged: stainless steel, aerated using suction pump, production is 10X higher
Clostridium thermoaceticum (from horse manure) is also able to utilize five-carbon sugars:
2C5H10O5 --- 5CH3COOH
A variety of substrates, including fructose, xylose, lactate, formate, and pyruvate, have been used as carbon sources in an effort to lower substrate costs. This factor is also important if cellulosic renewable resources are to be used as raw materials.
Typical acidogenic bacteria are Clostridium aceticum, C. thermoaceticum, Clostridium formicoaceticum, and Acetobacterium woodii. Many can also reduce carbon dioxide and other one-carbon compounds to acetate.

63. 1mol
2moles
1mol
1mol
CODH
These enzymes are metalloproteins; for example, CODH contains nickel, iron, and sulfur; FDH contains iron, selenium, tungsten, and a small
quantity of molybdenum; and the corrinoid enzyme (vitamin B12 compound) contains cobalt. C. thermoaceticum does not have any specific amino acid requirement; nicotinic acid is the sole essential vitamin

64. LACTIC ACID: industrial uses
Technical grade
20-50%
Ester manufacture
Textile industry
Food grade
>80%
Food additive (sour flour and dough)
Pharmaceutical grade
>90%
Intestinal treatment
(metal ion lactates)
Glucose
G3P NAD+
NADH +H+
1,3-biphosphoglycerate
G3P dehy-
drogenase
Pyruvate
Lactic acid
LDH
(Lactate dehydrogenase)

65. LACTIC ACID
2 isomeric forms L(+) and D(-) and as racemic mixture DL-lactic acid
First isolated from milk
Toady produced microbial
Heterofermentation Homofermentation
Other than lactate products only lactate as product
Lactobacillus
L. delbrueckii Glucose
L. leichmanni
L. bulgaricus
L.helvetii Whey (lactose)
L.lactis Maltose
L.amylophilus Starch
L.pentosus Sulfite waste liquor
Mostly one isomer is produced

66. LACTIC ACID: production process
1mol of glucose gives 2 moles of lactic acid; L lactic acid is predominantly produced
Fermentation broth (12-15% glucose, N2, PO4, salts micronutrients)
pH /temp 45-50oC/75h
Heat to dissolve Ca lactate
Addition of H2SO4
(removal of Ca SO4)
Filter and concentrate
Addtion of Hexacyanoferrant
(removes heavy metal)
Purification (Ion exchange)
Concentration
Lactic acid

67. Antibiotics by Fermentation Technology
Antibiotics are produced industrially by a process of fermentation, where the source microorganism is grown in large containers (100,000 – 150,000 liters or more) containing a liquid growth medium.

68. Streptomycin 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 source.
Nitrogen source is combined in soyabean meal, limits growth.
After growth the antibiotic levels in the culture begin to increase.
Source:

69. Phases during fermentation of streptomycin
PHASE 1: Rapid growth producing mycelial biomasss.Little production of Streptomycin is obtained.
PHASE 2: Additional production of mycelium.Streptomycin accumulates in the medium.
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.

71. PRODUCTION OF PENICILLIN
Penicillin was the first important commercial product produced by an aerobic, submerged fermentation
First antibiotic to have been manufacture in bulk.
Used as input material for some semi synthetic antibiotics.
It is fermented in a batch culture

72. When penicillin was first made at the end of the second world war using the fungus Penicillium notatum, the process made 1 mg dm-3.
Today, using a different species (P. chrysogenum) and a better extraction procedures the yield is 50 g dm-3.
There is a constant search to improve the yield.

73. The yield of penicillin can be increased by:
Improvement in composition of the medium
Isolation of better penicillin producing mold sp. Penicillium chrysogenum which grow better in huge deep fermentation tank
Development of submerged culture technique for cultivation of mold in large volume of liquid medium through which sterile air is forced.

74. Primary and Secondary Metabolites
Primary metabolites are produced during active cell growth, and secondary metabolites are produced near the onset of stationary phase.

75. Commercial Production Of Penicillin
Like all antibiotics, penicillin is a secondary metabolite, so is only produced in the stationary phase.

76. INDUSTRIAL PRODUCTION OF ANTIBIOTIC- PENICILLIN
The industrial production of penicillin was broadly classified in to two processes namely,
Upstream processing
Downstream processing

77. UPSTREAM PROCESSING
Upstream processing encompasses any technology that leads to the synthesis of a product. Upstream includes the exploration, development and production.

78. DOWNSTREAM PROCESSING
The extraction and purification of a biotechnological product from fermentation is referred to as downstream processing.

79. UPSTREAM PROCESSING INOCULUM PREPARATION
The medium is designed to provide the organism with all the nutrients that it requires.
Inoculation method- submerged technique
Spores -major source of inoculum

80. RAW MATERIALS
CARBON SOURCES: Lactose acts as a very satisfactory carbon compound, provided that is used in a concentration of 6%. Others such as glucose & sucrose may be used. NITROGEN SOURCES:
Corn steep liquor (CSL)
Ammonium sulphate and ammonium acetate can be used as nitrogenous sources. MINERAL SOURCES: Elements namely potassium, phosphorus, magnesium, sulphur, zinc and copper are essential for penicillin production. Some of these are applied by corn steep liquor.
Calcium can be added in the form of chalk to counter the natural acidity of CSL
PAA- precursor

81. FERMENTATION PROCESS
The medium is inoculated with a suspension of conidia of Penicillium chrysogenum.
The medium is constantly aerated and agitated, and the mould grows throughout as pellets.
After about seven days, growth is complete, the pH rises to 8.0 or above, and penicillin production ceases

82. STAGES IN DOWNSTREAM PROCESSING
Removal of cells
The first step in product recovery is the separation of whole cells and other insoluble ingredients from the culture broth by technique such as filtration and centrifugation.

83. ISOLATION OF BENZYL PENICILLIN
The PH is adjusted to with the help of phosphoric or sulphuric acids.
In aqueous solution at low PH values there is a partition coefficient in favor of certain organic solvents such as butyl acetate.
This step has to be carried out quickly for penicillin is very unstable at low PH values.
Antibiotic is then extracted back into an aqueous buffer at a PH of 7.5, the partition coefficient now being strongly in favor of the aqueous phase. The resulting aqueous solution is again acidified & re-extracted with an organic solvent.
These shifts between the water and solvent help in the purification of penicillin.

84. The main stages of Penicillin production are:

87. Fermented Meat
                                   

88. What is fermented sausage?
1.Introduction Meat is the flesh (muscle tissue ) of warm-blooded animals,but fermented specialties from poultry ( sausages as well as cured and smoked fermented poultry) are available.
What is fermented sausage?
A sausage is fermented if
-its pH below 5.6 and D-lactic acid content above 0.2%
-its colour is heat-stable
-its texture is no longer crumble
-its aroma is typical
-lactic acid bacteria predominate
-Enterobacteriaceae counts are low
       

89. a) Nutritional Role of Meat in the Human Diet:
essential component of the human diet to ensure optimal growth and development.
as a concentrated source of a wide range of nutrients.
high digestibility required relatively smaller guts.
meat and meat products has increased with the affluence of the consumer.
fat content of meat as consumed is around 2to5%.
protein of high biological value.
micronutrient such as iron, zinc, vitamin B1, niacin equivalents, and vitamin B12     significantly contribute to the nutritional value of meat.
red meat contains 50-60% of iron in the hame from (from hemoglobin and          myoglobin).

90. Table. 2 Classification of fermented sausages

91. 2. The history and culture related to fermented meat.
Meat is extremely susceptible to microbial spoilage.
meat as a substrate are optimal for the growth of bacteria.
water activity and pH are 0.96 to 0.97 and 5.6 to 5.8, respectively
nutrients and growth factors are abundantly available.
storage and preservation of meat is necessary for the suppression of microbial      growth or the elimination of microorganisms and prevention of recontamination.

92. 2. The history and culture related to fermented meat
The traditional methods which comprise reduction -
1) water activity ( drying, salting) and/ or pH (fermentation, acidification)
  2) smoking, storage at refrigeration or freezing temperatures,
 3) use of curing aids (nitrite and nitrate)
meat may also contain bacterial food pathogens.
meat has to be of high quality with regard to hygiene and microbial counts.

93. 3. The fermentation process
Fermentation process : two types
-foods from a comminuted matrix
-whole meat products.

94. A. Fermentation of a Comminuted meat matrix
a) Variables in sausage production
Variables include:
The particle size of the comminuted meat and fatty tissue (1 and 30 mm)
The selection of additives (curing salt, nitrate, ascorbic acid, sodium glutamate and    glucono-∂-lactone -source glucose.
The temperature /humidity (below 2to 3℃, the temperature is raised usually to ＞20℃ and ＞28℃, but maximum higher temperatures (32 to 38℃).

95. The diameter of the sausages
The nature of the casings smoking
Heating after fermentation
Supporting the development of mold growth on the surface or establishing a         special tight surface film (e. g. coating with a titanium dioxide film)
Dipping in antifungal preparations ( sorbic acid or pimaricin)
pH-4.8 to 5.4

96. Table. 3

97. Species Employed in Meat Starter Cultures
Bacteria: Lactic Acid Bacteria such as Lactobacillus acidophilus, Lb. alimentarius, Lb. curvatus, Lb. plantarum etc, Lactococcus lactis, Pediococcus acidilactici, P. pentosaceus
Actinobacteria : Kocuria varians, Streptomyces griseus, Bifidobacterium spp.
Staphylococci: Staphylococcus xylosus, S. carnosus ssp. 
Halomonadaceae : Halomonas elongata
Fungi: Penicillium nalgiovense, P. chrysogenum, P. camemberti
Yeasts: Debaryomyces hansenii, Candida famata

98. B. Fermentation of Whole Meat Products (HAM)
curing by salting (with or without the use of nitrite and/or nitrate)
to achieve a water activity of ∠0.96 (equivalent to 4.5% sodium chloride)
temperatures (50C)―the salt will diffuse to the deepest part of meat
overcoming the food poisoning through Clostridium botulinum contamination.
after equilibrating the salt concentration and flavor development, the temperature     is raised to 15 to 250C to ripen the ham.
optimum flavor has no changed at least 6 to 9 months, maximum 18th month.
at the end of ripening step, the moisture has been reduced by 25% and salt 4.5 to    6%)

99. 4. Composition and changes during fermentation
growth of LAB and concomitant acidification of the product.
reduction of nitrates to nitrites and formation of nitrosomyoglobin
solubilization and gelification of miofibrillar and sarcoplasmic proteins
degradation of proteins and lipids
dehydration

100. a) Fermentation Microflora
sausage minces favor the growth of Micrococcacea and Lactobacilli (5×108 to  109 CFU/g)
Micrococcacea such as Kocuria varians, Staphylococcus carnosus or S. xylosus       grow to cell counts 106 to 107 CFU/g, when nitrate cure is applied.
inhibited the growth of organism
the predominant microorganism is isolated
growth of Staphylococcus occurs
Penicillium constituted 96% of the microflora
the nontoxigenic species Penicillium nalgiovense was most frequently isolated
the halotolerant yeast (Debaryomyces hansenii) is the predominant

101. b) Acidification, Dehydration, and Microbial Antagonism
isoelectric point of meat proteins (pH 5.3 to 5.4)
increase the ionic strength
sodium chloride and lactate in fermented sausages develop taste of the product.
acidification and drying are importance for inhibition of the growth of pathogens.
low pH and water activity exert an inhibitory effect towards pathogens.
lactic and acetic acids are the major fermentation products
the dry matter content 50-75%
the water activity values depend on ripening

102. c) Proteolytic and Lipolytic Degradation during fermentation
Peptides and amino acids accumulate to levels of about 1% dry matter
Peptides and amino acids act as flavor enhancers and synergists.
excess proteolysis may result in bitter and metabolic off-flavor
amino acids and peptides are utilized by microorganisms for the conversion to       flavor volatiles
the bioactive peptides is influenced by lactic fermentation
Kocuria varians is inhibited by environmental conditions
Lb. casei utilizes peptides released from pork muscles
fat content 40-60% of dry matter
long chain fatty acids are released from triglycerides and phospholipids
free fatty acids are found 5% of the total fatty acids.
polyunsaturated fatty acids is higher than saturated fatty acids.  

103. d) Generation of Flavor volatiles
Routes:
by lipolysis and hydrolysis of phospholipids, followed by oxidation of free fatty      acids.
microorganisms produce organic acids: convert amino acids and peptides to         flavor-active alcohols, aldehydes, and acids
modify products of lipid oxidation
aroma is determined by the addition of spices, smoking, or surface-ripening with     yeasts or molds.

104. e) Biogenic amines
histamine, tyramine, phenylethylamine, tryptamine, putrescine and cadaverine not    exceeding 100mg/kg.
are mainly derived from bacterial decarboxylation of amino acids
putrescine and cadaverine are produced by the Gram-negative spoilage flora
starter cultures inhibit rapidly metabolism of Gram negative bacteria
effectively reduce tyramine levels in fermented sausages

105. Product Diversity and Sensory Properties
The main desirable effects of starter micro-organisms on flavor and taste of fermented meats are
formation of lactic acid
transformation of compounds from abiotic breakdown of lipids
degradation of peptides and amino acids formed by meat proteases
Indirect effects are
consumption of oxygen
reduction of nitrate
protein degradation by mould proteases

106. Sucuk
One of the most important and widely consumed traditional Turkish meat product,
Dried, uncooked, cured and fermented sausage,
Produced from beef or buffalo meat Consist of ground meat and sheep tail fat and curing agents (nitrite and nitrate), with various spices including cumin, garlic, salt, and black and red pepper

107. Sucuk processing stages
Stuffing sausage mixture into natural sausage casings
Fermentation at 22-23ºC by either microorganisms naturally present or added starter cultures
Drying for several weeks at ambient temperature and humidity
due to fermentation, the final product has an increased shelf life as a consequence of the inhibition of the pathogenic and spoilage bacteria,