Friday, 4 May 2012

Computer Applications in Fermentation Technology

Computer Applications in Fermentation Technology

The use of computers for for modelling fermentation processes started in 1960s. Initially the use of computers was restricted because of the cost factor but reductions in the cost and the availability of the cheaper small computers has widened interest in their possible applications. The availability of efficient small computers has led their use for pilot plants and laboratory systems because the financial costs for the online computer systems counts the insignificant part of the whole system. There are three distinct systems areas of computer function postulated by Nyiri in 1972:
a) Logging of Process Data: This is performed by the data acquisition which has both hardware  and software components. there is  an interface between the sensors and the computer. the software should include the computer program for sequential; scanning of the sensor signals and the procedure of data storage.
b) Data Analysis: Data reduction is performed by the data analysis systems which is  acomputer program based on a series of mathematical equations. the analysed information may be put on a print out, fed into data bank or utilized for process control.
c) process control: is also performed by a computer program. signals from the computer are fed tio the pumps, valves or switches via the interface. in addition to this computer program may contain instructions to display devices or teletypes to indicate alrms.
Components of Computer Linked system:
When a computer is linked to a fermenter to operate as a control and recording system, a number of factors must be considered to ensure taht all the components interact  and function satisfactorily for the control a d data logging.An example is DDC (Direct Digital Control) system to explain the computer controlled addition  of a liquid from a resrvoir to a fermenter. 
A simple outline of the main components is as follows:

Sensor S in fermenter produces a signal which may need to be simplified and conditioned in the correct analogue form. at this stage it is necessary to convert the signal to a digital form which can be  subsequently transmitte dto the computer. An interface is placed in the circuit  at this point. The interface serves as  the junction point for the inputs  from the fermenter  sensors to the computers and output signals  from the computer to the fermenter controls such as  a pump T attached to an additive reservoir.
A sensor will generate  a small voltage proportional to the parameter it is going to measure. for example a temperature probe might generate 1V at 10oC and 5V at 50oC. But this signal cannot be understood by the computer  and must be converted  from an analogue to digital converter ( ADC) into digital form.
The accuracy will depend upon the number of bits  it sends to the computer. AN 8-bit converter will work in the range of 0-255 and it is tehrefore  able to divide  a signal voltage into  256 steps. This will give  amaximum accuracy of 100/256, which is pproximately  0.4%. However a 10- bit converter  can give 1024 steps with a n accuracy of 100/1024, which is pproximately 0.1%. therefore when a parameter is to be monitored  very accurately  a converter of the appropraite degree of accuracy will be required. the time taken for an ADC to convert voltage signals to a digital output will vary with accuracy, but improved accuracy will lead to slower conversion  and hence slower control responses. The small computers is often used for one or more fermenters. it is coupled  to a real time clock, which determines how frequently  readings from teh sensor should be taken and possibly recorded. Ancillary equipments  include  a VDU, a data store , a teletype, a graphic display unit, a print out, alarms and barometer.
It is also possible to develop online programs so that online instruments can be checked regularly and recalibrated when necessary.

Next post will be about the details of three distinct areas.
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Thursday, 1 March 2012


Industrial Alcohol
The production of industrial alcohol, ethanol become commercially feasible on a large scale after 1906 when the Industrial Alcohol Act was passed. This act allowed the sale of tax-exempt alcohol, if it has first denatured to prevent its use in various Alcohol beverages. Industrial Alcohol is commonly employed as a solvent and to a lesser extent as a raw material for chemical synthesis. Smaller amounts are also used as a motor fuel like gasoline.
Choice of fermentation microorganism for the alcohol production depends upon the type of carbohydrate employed in the medium. For example if starch and sugar are raw materials in the medium then specially selected strains of Saccharomyces crevisiae are utilized. Production from Lactose of whey is accomplished with Candida pseudotropicalis. If it is sulfur waste liquor fermentation the Candida utilis is the best organism, because of its ability to ferment pentoses. So particular strains of these various organisms actually employed for the fermentation are selected for several properties. They must grow rapidly, have higher tolerance to the high concentartions of sugar but at the same time they must be able to produce much larger amounts of alcohol and be resistant to the produced alcohol.
The media for the commercial production includes:
Blackstrap Molasses / Corn (Blackstrap molasses has greater use)
Sulfite waste liquor
Wood Wastes

For Molasses fermentation , molasses must be diluted with water to a sugar conc. between 10 -18%. Concentrations greater than 20% are not employed as they could be detrimental to yeast. The pH of the medium is set between 4 -5 by adding sulfuric acids or lactic acids, or by employing Lactic acid bacteria to bring initial lactic acid fermentation. Microbial contaminants are usually inhibited by the low pH, high sugar conc. and anaerobic conditions of the fermentation and by the high alcohol production by the yeast.
Starchy media such as corn, rye and barley must undergo initial starch hydrolysis. This can be accomplished by mashing with barley malt, by addition of dilute acids or by utilizing fungal amylolytic enzymes (Aspergillus and Rhizopus).In most of the cases, malt is used to accomplish hydrolysis of starch By mixing 30% barley and 70% corn with water and carry on the mashing procedures similar to the wine or nbeer making procedures.
Fermentation is carried out in large reactors at a temperature between 21 - 27oC, but heat evolution might raise the temperature to 30oC, so cooling coils are used to bring the temperature down. Fermentation lasts for about 2-3 days, but actual time period depends upon the substrate utilized and temperature. The fermentation broth at completion of fermentation ranges from 6 -9 percent alcohol by volume and this alcohol reflects the yield 0f 90 -98% theoretical conversion of substrate sugar to alcohol.So yileds should not be confused with "proof" as proofing means alcohol concentartion designation and it will be twice the percentage in vol of ethanol as dissolved in water e.g. 70% ethanol is 140 proof.


Fermentation Economics:
The objective of any successful fermenttaion process is the ability to produce  a fermentation product. Thus the product must be sold to recover all the costs along with desired profit. But manufacturing should be done in accordance with the market demand. So there could be 2 possibilities:
First possibilty is : That the market for so called product already exist because the same or similar product has previously been sold by others.
Second possibilty is : a newly manufactured or discovered product e.g. a new antibiotic  will require a market to be established.
This might include the approval by FDA ( food and drug adminstration)
There are certain obstacles regarding the marketing of a certain product like the semand of the product is low or it has relatively very few its quite obvious that for a product lke this it could be challenging to get patent coverage because of lack of utility.
for the products which are already in the market , there could be a fierce competition. so to succed in the competition the product must be cheap enough that it can be sold at or slightly less than the already existing selling price. So, in the nutshell, the whole fermentation process and its product  must be able to compete on an economically sound basis with the similar products in the market.
The economic position of  a fermentation product is closely tied to the costs associated with its production and distribution. These costs can be categorised into several classes as follows:
Media components:
The competitive postion and expected profits from a fermentation product are closely tied to the costs of the various components of the production medium. usually inoculum medium is less expensive because it is required to provide rapid cell growth only and not for converting large amount of carbon substarte into a fermentation product. However any medium component may be subject to fluctuations in context to availabilty and costs. so it is always advantageous to have a alternative medium for use if any unusual situation happens.
Labor Costs:
labor costs involve technical ad non technical trained personnel at all levels of competence. This includes handling of cultures, inoculum, production, product recovery and purification, packaging, cleaning and adminstartion and so forth. Labor costs vary from fermentation to fermentation.
Contamination and sterlization: 
Contamination always add  costs to any fermentation process. most fermentations cannot survive serious conmtaminations so the medium must be discardedmodertae fermentations does not require the medium to be discarded but it might affect the yields. Certain fermentations are more prone to contamination than others. This involves cases in which foaming is a problem. some are more sensitive to phage infections like bacterial fermentations. So there has to be an alternate method for the contaminant growth. Such methods include low pH of the medium, partial heat treatment of the medium and inclusion of certain chemicals so a sto retad contaminant growth.
Yield and Product Recovery:
Product Yield and Recovery are the prime considertaions of fermentation economics and in that context yield and recovery must be considered together, since high yield id of little value if the product cannot be recovered  properly for sale. 

Product Purity:
At one end of the scale some products, like antibiotic preprations must be sterile and free from pyrogens. In contrast other antibiotic preprations  are sold in crude form for mixture with animal feeds. Thus the purity level required for the marketing of  a fermentation product has  a major effect on the costs associated with the product.Specific fermentation products can also be marketed at more than one concentartion as level of purity. For example, lactic acid is sold at strengths ranging from approx 20 - 85% and its purity levels range from crude technical grade to high purity edible  and U.s.P. grades. Each of these grades of lactic acid has a place on the market.
Waste Disposal:
Costs attributed to waste disposal vary from minimal to maximal factor in fermentation process. A critical consideration is the acceptance of waste by Municipal's STW (Sewage Treatment Works); as they might want pre treatment of wastes before the acceptance. In the altter case the fermentation company must have its own waste treatment plant. Disposal of wastes is no longer simple in contrsat to historical disposal in the rivers, streams or other water bodies. Certain fermentations require the waste to be sterlized before disposal.
Research Costs:
Fermentation process must include those expenses incurred in the research that actually discovered the process nad developed it. These costs can be considerable for those fermentations where they provide new products .there are less tangible research costs that must also be considered in the overall cost of fermentation. This type of reserach is pursued in teh hope that teh resulting basic information obtained on the growth and synthetic activities of microbes will be of later value in defining areas of exploration and approaches for discovering new fermentation processed and bettering old processes.
An appraisal of the economic potential is required for the fermentation process which evaluate all the above categories under present and future market protential. but evaluation must be made as early  as possible during process development. Also process must be evaluated at later stages during actual production. It is also important to consider present and future costs and  a selling price for the product that market can bear.. All these points must be evaluated and then decide whether the fermentation product can be sold at an acceptable level of profit or not? If the decisons are negative then the alternatives are to abandon the whole process and carry out further reesarch on the product recovery. After all a great deal of money is at stake in these decisons!!!

Reference: Casida, L.E. Industrial Microbiology

Sunday, 12 February 2012

Media Sterlization

Media Sterlization:

Media can be treated by variety of methods like radiations, filtration, ultrasonic treatment and heat. Practically the universal and most favourable method for sterilization is the steam.

Kinetics of Sterlization:
The destruction of microorganisms by steam or the moist heat can be described by the first order chemical reaction and can be represented by the following equation:
-                                                                                                 dN / dt = kN                               Eq no. 1

N is no. Of viable organisms present
t is the time of sterilization treatment.
k  is the reaction rate constant or the specific death rate

Upon integration of equation no.1 we get:
                                          Nt / N0 = e –kt                           Eq no. 2

N0 is no. of viable organisms present at the start of sterilization
Nis the no. Of viable organisms after treatment period t

Upon taking natural logarithms of the Eq. No. 2 we get:
                                           ln (Nt / N0)  =  -kt                    Eq no.3


 A plot of natural logarithm of  Nt / N0 against time will yield a straight line ; the slope of which is equal to – k (see the fig. ABOVE)
So we can make 2 anomalous predictions from the plot:

1.  An infinite time is required to achieve sterile conditions. ( Nt  = zero)

2.   After a certain period of time there will less than one viable cell present.
But the above plots can be observed only when the sterilization of pure culture is done in one physiological form only and that too under the ideal sterilization conditions. Thus, the value of k is not only species dependent but also on the physiological form of the cell e.g. endospores of genus Bacillus will be more heat resistant than the vegetative cells.
Considering this factor Richards in 1968 has produced a series of graphs (see Graphs below) which illustrate the deviation from the theory which can be expressed in the practice.


      GRAPH 1


Above 3 graphs illustrate the effect of time of heat treatment on the survival of a population of bacterial endospores.
Graph 1 shows the initial population increase resulting from heat activation of spores in early stages of sterilization; followed by the decline in viable spore number. Activation o spores is significantly more than their destruction during early stages of the process and therefore viable number of spores increases before the observation of exponential decline.
Graph2 illustrates the initial stationary period followed by the decline; owing to the fact that the death of the spores is compensated by the heat activation of spores.
Graph 3 shows the population decline at a sub maximum rate due to the death of the spores.


Now we know that with any first order reaction, the reaction rate increases with temperature increase due to increase in the reaction rate constant which is the case of destruction of microorganisms (i.e.specific death rate constant; k).
Thus, the relationship between the temperature and reaction rate constant can be expressed by Arrhenius Equation:

                                                     dlnk/dT  = E/RT2                                                  Eq no 4:

E is the Activation energy
R is gas constant
T is absolute temperature

Upon integration of Eq. no 4 we get:

                                                   k= Ae- E/RT                                                            Eq. no 5:

A is Arrhenius constant
On taking natural Logarithms Eq no. 5 becomes:

                                                     ln k = ln A  - E/Rt                               Eq. No.6:

By combining Eq. no. 3 and 5 following expression can be derived for heat sterilization of a pure culture at a constant temperature:
                                               ln (Nt / N0)  = A.t.e –E/Rt                       Eq. no. 7

Deindoerfer and Humphery used the term  ln (Nt / N0)  as a design criterion for the sterilization which has been called as DEL Factor or NABLA factor.

Del factor is a measure of fractional reduction of viable organism count produced by a certain heat and time regime. Therefore:

                                                    =    ln (Nt / N0)
           But;         ln (Nt / N0) = kt
                  &                kt = A.t. e –E/Rt           
               Thus,           = A.t. e –E/Rt                                    Eq. no. 8

On rearranging equation no. 8 becomes :

                        ln t = E/RT +  ln  (  /A)                 eq. no 9

Thus, a plot of the natural logarithm of the time required to achieve a certain value against the reciprocal of the absolute temperature will yield a straight line, the slope of which is dependent on the activation energy.( see fig below)


So a regime of time and temperature can be determined if we want to achieve the desired Del factor. However , as we know fermentation medium is not an inert mixture of components so it is very likely that deleterious reactions occur , resulting in loss of nutrient quality. Two types of reactions occur during sterilization as follows:
(i) Interactions between nutrient components of the medium. A common occurrence during sterilization is the Maillard-type browning reaction which results in discoloration of the medium as well as loss of nutrient quality. These reactions are normally caused by the reaction of carbonyl groups, usually from reducing sugars, with the amino groups of amino acids and proteins. Problems of this type are normally resolved by sterilizing the sugar separately from the rest of the medium and recombining the two after cooling.
(ii) Degradation of heat labile components. Certain vitamins, amino acids and proteins may be degraded during a steam sterilization regime. In extreme cases, such as the preparation of media for animal-cell culture, filtration may be used.

P.S I do not owe any of the above text.

 Shuler, M.L. and Kargi, F. (2002) Bioprocess Engineering: Basic Concepts. 2 nd Edition. Prentice Hall, 
Stanbury, P. , Whittaker, A. (2004) Principles of Fermentation Technology

Monday, 6 February 2012

Facts About DNA

Facts About DNA
  1. DNA stands for deoxyribonucleic acid.
  2. DNA is part of our definition of a living organism.
  3. DNA is found in all living things.
  4. DNA was first isolated in 1869 by Friedrich Miescher.
  5. James Watson and Francis Crick figured out the structure of DNA.
  6. DNA is a double helix.
  7. The structure of DNA can be likened to a twisted ladder.
  8. The rungs of the ladder are made up of “bases”
  9. Adenine (A) is a base.
  10. Thymine (T) is a base.
  11. Cytosine (C) is a base
  12. Guanine (G) is a base.
  13. A always pairs with T in DNA.
  14. C also pairs with G in DNA.
  15. The amount of A is equal to the amoun tof T, same for C and G.
  16. A+C = T+G
  17. Hydrogen bonds hold the bases together.
  18. The sides of the DNA ladder is made of sugars and phosphate atoms.
  19. Bases attached to a sugar; this complex is called a nucleoside.
  20. Sugar + phosphate + base = nucleotide.
  21. The DNA ladder usually twists to the right.
  22. There are many conformations of DNA: A-DNA, B-DNA, and Z-DNA are the only ones found in nature.
  23. Almost all the cells in our body have DNA with the exception of red blood cells.
  24. DNA is the “blueprint” of life.
  25. Chromosomal or nuclear DNA is DNA found in the nucleus of cells.
  26. Humans have 46 chromosomes.
  27. Autosomal DNA is part of chromosomal DNA but does not include the two sex chromsomes – X and Y.
  28. One chromosome can have as little as 50 million base pairs or as much as 250 million base pairs.
  29. Mitochondrial DNA (mtDNA) is found in the mitochondria.
  30. mtDNA is only passed from the mother to the child because only eggs have mitochondria, not sperm.
  31. There’s a copy of our entire DNA sequence in every cell of our body with one exception.
  32. Our entire DNA sequence is called a genome.
  33. There’s an estimated 3 billion DNA bases in our genome.
  34. One million bases (called a megabase and abbreviated Mb) of DNA sequence data is roughly equivalent to 1 megabyte of computer data storage space.
  35. Our entire DNA sequence would fill 200 1,000-page New York City telephone directories.
  36. A complete 3 billion base genome would take 3 gigabytes of storage space.
  37. If unwound and tied together, the strands of DNA in one cell would stretch almost six feet but would be only 50 trillionths of an inch wide.
  38. In humans, the DNA molecule in a non-sex cell would have a total length of 1.7 metres.
  39. If you unwrap all the DNA you have in all your cells, you could reach the moon 6000 times!
  40. Our sex cells–eggs and sperm–have only half of our total DNA.
  41. Over 99% of our DNA sequence is the same as other humans’.
  42. DNA can self-replicate using cellular machinery made of proteins.
  43. Genes are made of DNA.
  44. Genes are pieces of DNA passed from parent to offspring that contain hereditary information.
  45. The average gene is 10,000 to 15,000 bases long.
  46. The segment of DNA designated a gene is made up of exons and introns.
  47. Exons have the code for making proteins.
  48. Introns are intervening sequences sometimes called “junk DNA.”
  49. Junk DNA’s function or lack thereof is a source of debate.
  50. Part of “junk DNA” help to regulate the genomic activity.
  51. There are an estimated 20,000 to 25,000 genes in our genome.
  52. In 2000, a rough draft of the human genome (complete DNA sequence) was completed.
  53. In 2003, the final draft of the human genome was completed.
  54. The human genome sequence generated by the private genomics company Celera was based on DNA samples collected from five donors who identified themselves only by race and sex.
  55. If all the DNA in your body was put end to end, it would reach to the sun and back over 600 times (100 trillion times six feet divided by 92 million miles).
  56. It would take a person typing 60 words per minute, eight hours a day, around 50 years to type the human genome.
  57. If all three billion letters in the human genome were stacked one millimeter apart, they would reach a height 7,000 times the height of the Empire State Building.
  58. DNA is translated via cellular mechanisms into proteins.
  59. DNA in sets of 3 bases, called a codon, code for amino acids, the building blocks of protein.
  60. Changes in the DNA sequence are called mutations.
  61. Many thing can cause mutations, including UV irradiation from the sun, chemicals like drugs, etc.
  62. Mutations can be changes in just one DNA base.
  63. Mutations can involve more than one DNA base.
  64. Mutations can involve entire segments of chromosomes.
  65. Single nucleotide polymorpshisms (SNPs) are single base changes in DNA.
  66. Short tandem repeats (STRs) are short sequences of DNA repeated consecutively.
  67. Some parts of the DNA sequence do not make proteins.
  68. Genes make up only about 2-3% of our genome.
  69. DNA is affected by the environment; environmental factors can turn genes on and off.
  70. There are many ways you can analyze your DNA using commercially available tests.
  71. Paternity tests compare segments of DNA between the potential father and child.
  72. There are other types of relationship testing that compares DNA between siblings, grandparents and grandchild, etc.
  73. DNA tests can help you understand your risk of disease.
  74. A DNA mutation or variation may be associated with a higher risk of a number of diseases, including breast cancer.
  75. DNA tests can help you understand your family history aka genetic genealogy.
  76. DNA tests can help you understand your ethnic make-up.
  77. DNA can be extracted from many different types of samples: blood, cheek cells, urine.
  78. DNA can be stored either as cells on a cotton swab, buccal brush, or frozen blood or in extracted form.
  79. In forensics, DNA analysis usually looks at 13 specific DNA markers (segments of DNA).
  80. The odds that two individuals will have the same 13-loci DNA profile is about one in one billion.
  81. A DNA fingerprint is a set of DNA markers that is unique for each individual except identical twins.
  82. Identical twins share 100% of their genes.
  83. Siblings share 50% of their genes.
  84. A parent and child share 50% of their genes.
  85. You can extract DNA at home from fruit and even your own cheek cells.
  86. DNA is used to determine the pedigree for livestock or pets.
  87. DNA is used in wildlife forensics to identify endangered species and people who hunt them (poachers).
  88. DNA is used in identify victims of accidents or crime.
  89. DNA is used to exonerate innocent people who’ve been wrongly convicted.
  90. Many countries, including the US and UK, maintain a DNA database of convicted criminals.
  91. The CODIS databank (COmbined DNA Index System) is maintained by the BI and has DNA profiles of convicted criminals.
  92. Polymerase chain reaction (PCR) is used to amplify a sample of DNA so that there are more copies to analyze.
  93. We eat DNA every day.
  94. DNA testing is used to authenticate food like caviar and fine wine.
  95. DNA is used to determine the purity of crops.
  96. Genetically modified crops have DNA from another organism inserted to give the crops properties like pest resistance.
  97. Dolly the cloned sheep had the same nuclear DNA as its donor mom but its mitochondrial DNA came from from the egg mom. (Does that make any sense?)
  98. People like to talk about DNA even if it bears no relation to science or reality.
  99. A group of bloggers who write regularly about DNA and genetics have banded to gether to form The DNA Network.
  100. Lists about DNA can get a little boring.