Fermentation
Technologies
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over view
Fermentation technologies are the most prestigious
bioanalytical technique in the study of biotechnology. It is very important for all the biotechnologist to understand the concept of fermentation.It paves way for quality research in industry. This subject is further linked to some of the other key streams of science like bioengineering, microbiology, biophysics, biochemistry, biotechnology, mathematics etc.
The students will learn the following salient aspects of the subject
Unit I
Introduction to Fermentation Technolgy
In this unit the student will learn the historical perspectives of fermentation. This unit can be linked to the basic media preparation experiments in micrbiology.Preparation of media for fermentation will be taught.Various techniques related to strain improvement and assay procedures will be discussed.All the necessary pre requisites for fermentation will be detailed.
Unit II
Structure and Working of a Fermentor
In this unit the student will learn all about fermentor design, about bioreactors and its instrumentation. Principles of sterilization, aeration, agitation, mass and heat transfers will also be discussed. Techniques related to down stream process-ing and product recovery will be explained.
Unit III
Kinetics of Fermentation
This unit is linked to biochemical engineering branch of science The kinetics of various fermentation techniques will be discussed.
Unit IV
Products of Fermentation
The entire unit deals with the protocols needed to produce various commercial products like Ethanol, antibiotics, enzyme, beverages and various fermented food products
Unit V
Biosafety & Future of Fermentation Technology
The knowledge of biohazards in fermentation is a pre requisite before handling fermentor based experiments. Students need to update themselves on the current trends and future pros-pects in fermentation Therefore the main objective of this unit is to increase the awareness of students in current areas of research in fermentation.
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MSC-BIOTECHNOLOGY
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SYLLABUS
Unit I
Introduction to Fermentation Technolgy:
Brief history of fermentation process, Fermentation Media, Screening, Scale up and scale down, Inoculum preparation, Assay techniques, Strain improvement techniques.
Unit II
Structure and Working of a Fermentor
Design of a fermentor, Design of a bioreactor, Sterilization, Aeration and agitation, Mass & Heat transfer, Instrumentation and control, Product recovery and downstream processing
Unit III
Kinetics of Fermentation:
Growth Kinetics in fermentation, Kinetics of fed batch fermentation, Kinetics of continuous Fermentation.
UNIT IV
Products of Fermentation
Microbial biomass production, enzyme, Antibiotic and steroid fermentations, Food & Beverage fermentation, Ethanol production from conventional and non-conventional sub-strates, Industrial wastewater treatment, Bioenergy production.
UNIT V
Biosafety & Future of Fermentation Technology
Biohazards in fermentation, Containment in fermentation and downstream processing, Patent and secret processes, Fermenta-tion economics, Future of fermentaFermenta-tion technology.
Students must ensure to achieve the following learning outcomes during the semester:
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To review the history of fermentation process and to understand all the necessary infrastructure for fermentation techniques•
Study fermentor design and downstream processing•
Interpreting the kinetics of fermentation•
Understand the protocols needed to produce various fermented products•
Understand the biohazards in fermentation. Updating with current and future trends in fermentation technologyOutcomes and Assessment
Criteria
Outcomes Assessment criteria
To achieve each outcome a student must demonstrate the ability to :
1 Review the historical developments in fermentation
technologies - Understand the history of fermentation - Enumerate the steps involved in the
preparation of media for fermentation - Discuss different types of strain
improvement techniques and assay techniques
2 Understand the intricate details of fermentor design - describe the parts of a fermentor - Explain the principles of sterilization,
Aeration, agitation, and mass- heat transfer - Discuss the crucial steps involved in
recovery of desired products and downstream processing - To understand the effective use of
computers in fermentation technologies
3 Undertake the study of fermentation kinetics - Discuss Growth kinetics
- Interpret the kinetics of fed batch and continuous fermentation
4 To understand all the steps involved in the production of various useful fermented products by fermentation technologies
- Describe the steps involved in the production of Microbial biomass ,enzyme, Antibiotic and steroid
-Understand Food & Beverage fermentation - Assess ethanol production from
conventional and non conventional substrates
- Analyze industrial waste water treatment - Discuss bioenergy production.
5 Review various biohazards involved in fermentaion.Study the future prospects in fermentation technologies
- Discuss the biohazards and safety measures observed in fermentation industry - Study the recent and future prospects in
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Unit No. Lesson No. Topic Page No.
Lesson Plan v
Course Requirement ix
Unit 1 Introduction to Biosensors
Lesson 1 Introduction to fermentation process 1
Lesson 2 Fermentation Media 6
Lesson 3 Screening 13
Lesson 4 Scale up and scale down 23
Lesson 5 Inoculum preparation 27
Lesson 6 Assay technique 31
Lesson 7 Strain Improvement 40
Unit 2 Structure and Working of a Fermentor
Lesson 8 Design of a fermentor 49
Lesson 9 Design of a bioreactor 56
Lesson 10 Sterilization 63
Lesson 11 Aeration and agitation 71
Lesson 12 Mass and heat transfer 75
Lesson 13 Instrumentation & Control I 86
Lesson 14 Instrumentation & Control II 91
Lesson 15 Product recovery & downstream processing 95
Lesson 16 Use of computers in fermentation 102
Unit 3 Kinetics of Fermentation
Lesson 17 Growth kinetics in fermentation 108
Lesson 18 Fed batch fermentation 112
Lesson 19 Continuos fermentation 119
Unit 4 Products of Fermentation
Lesson 20 Microbial biomass production 122
Lesson 21 Microbial enzyme production 127
Lesson 22 Microbial enzyme production 136
Lesson 23 Beer and wine fermentataion 146
Lesson 24 Ethanol fermentataion 154
CONTENT
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Lesson 25 Microbial production of health care products I 159 Lesson 26 Microbial production of health care products II 164
Lesson 27 Industrial wastewater treatment 168
Lesson 28 Bioenergy 175
Unit 5 Biosafety and Future of Fermentation Technology
Lesson 29 Biohazards in fermentation 180
Lesson 30 Containment in fermentation 184
Lesson 31 Containment in downstream processing 188
Lesson 32 Patent and secrete processes 194
Lesson 33 Fermentation economics 200
Lesson 34 Future of Industrial fermentations 206
Glossary 214
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Class Participation
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The student should come forward to interact with his classmates in order to share his thoughts.•
To understand the subject better it is very important that there is a healthy classroom discussions.•
Class room participation will help the students to improve their communication skills•
This is a great opportunity for introvert students to get rid of their fear to face the audience.Expectations from Students
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Regularity is the key to make continuous evaluation a success•
Students are expected to read the lesson plan regularly sothat they can participate well in classroom discussions.
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Students should come prepared for the class discussion•
Extra reference and awareness of latest trends is required•
Students are expected to discuss any relevant doubts openlyduring the interaction session.
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The current areas of research related to the topic chosen for discussion should be given more emphasisNeed of their preparation
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For healthy and interactive discussion•
Exchange of thoughts and ideas and to analyze the topic from various perspectives•
To enrich their knowledge•
To make them realize, how the preparation helps them to participate in group discussionMethod of evaluation
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Attendance: 75% attendance is compulsory•
Expression of ideas without any fear or inhibition•
Participation in group discussions, presentations andprojects. Presentation and quality of assignments.
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Contribution to the syndicate to prove team spirit•
Contribution of syndicates will be assessed individually and group wise•
Performance in end semester examination•
Performance of syndicate members will be judged 3-4 times during the semester.•
The weightage of evaluation methods are 1. Class room assessment – 30 %2. Assignment and projects - 20 % 3. End sem exam -50 %
Syndicate
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Syndicate is a group of students who will take up the given task and work as a team.•
The whole class will be divided into syndicates for evaluation.•
Syndicate member will submit the assignment individuallyAssignments
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After completion of each learning outcome (or maximum two), different assignments will be given for each syndicateSyndicate solution
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The students of a particular syndicate should discuss the assignment as a group and arrive at a common solutionIndividual Submission
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However each student should submit assignments individually.•
In the assignment they can put their views separately in addition to the combined solution of the team.•
The student will have an added advantage if he has provided the latest information from books, journals and web site.FERMENT
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•
The student is given a chance to assess himself (self assessment) and also will be evaluated by the peers.Group Presentation
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Syndicates will have to present their work before the class.•
The instructor as well as the peers will evaluate presentation.•
The final assessment will be done considering both theevaluations.
Group Project
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Each syndicate will be given a particular project.•
Different projects will be assigned to different syndicates.•
After completion of the project, the syndicates are expectedto give a presentation on that project.
End Semester Examination
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All the students have to appear in the end semester examination.•
At the end of the semester, the cumulative percentage will be calculated from the continuous evaluation performance, assignments, projects and end semester examination performance.Useful References
1. Bailey and Ollis : Biochemical Engineering Fundamentals 2. Casida L.E.: Industrial Microbiology
3. Hambelton,P., Melling J.&Salusbury T.T.: Biosefty in Industrial Biotechnology
4. Jogdand S.N. : Enviromental Biotechnology
5. Michael J. Waites, Neil L. Morgan, John S. Rockey & Gary Higton(2001): Industrial Microbiology an Intoduction. 6. Prescott & Dun’s : Industrial Microbiology
7. Stanbury P.F. , Whitaker A.,& Hall S.J.: Principal of fermentation technology (Second Edition)
Useful Links for Study
seafood.ucdavis.edu/iufost/lee.htm seafood.ucdavis.edu/iufost/creative.htm www.metkinen.fi/ www.cplpress.com/contents/C1315.htm www.fz-juelich.de/ibt/ferm/ferm.html Fermentation Kinetics www.np.edu.sg/~dept-bio/biochemical_ engineering/lectures bioferm1_main.htm www.np.edu.sg/~dept-bio/questions/ fermentation_1 ferm1_mcq.html www.pubmedcentral.nih.gov/ articlerender.fcgi?artid=106562 www.pubmedcentral.nih.gov/ articlerender.fcgi?artid=380741 www.asbcnet.org/Journal/ abstracts/search/1995 bc1995a15.htm Fermentor www.electrolab.biz www.labkorea.com/products/fermenter/fermenter.html www.bakker.org/cfm/webdoc12.htm www.thefreedictionary.com/Fermenter www.mrc-dunn.cam.ac.uk/facilities/fermenter.html Bioreactors www.labx.com www.electrolab.biz www.frtr.gov/matrix2/section4/4-42.html www.bellcoglass.com/us/bioreactors.shtml www.nbsc.com/ferm_eq/ferm.htm
Fermented Food Products
www.aomori-tech.go.jp/hiro/en/k_hakko.html www.healingcrow.com/ferfun/ferfun.html www.cplpress.com/contents/C1315.htm
store.blackwell-professional.com/0813800188.html www.fbfc.com/scoop/feb01/lacto.html
Beer and wine fermentation
www.homebrewit.com/aisle/1190 www.sp.uconn.edu/~ns166vc/Notes/Beer.html www.beveragebusiness.com/art98/bryson0203.html www.leeners.com/ferment.html www.homecraft.on.ca/instructions.htm Industrial microbiology www.microbes.info/resources/Industrial_Microbiology/ www.cas.muohio.edu/~stevenjr/ mbi630
FERMENT A TION TECHNOLOGIES industrialmicro630.html www.slic2.wsu.edu:82/hurlbert/ micro101/pages Chap19.html www.brockportmicrobiology.com/ www.sc.mahidol.ac.th/scbt/ academics/research_areas/IM.htm bioresearch.ac.uk/browse/mesh/C0021262L0021262.html www.transgalactic.com/publications/ p_industrial_microbiology.htm methanogens.pdx.edu/boone/ courses/BI480/Lectures BI480Lec15.html Enzyme Technology www.lsbu.ac.uk/biology/enztech/ www.irl.cri.nz/get/biocat/ www.aetltd.com/ www.biores-irl.ie/biozone/enzymes.html enzymes.novo.dk/enzymes/enzyme-technology.html
Cell growth in Fermentation
www.spectroscopymag.com/spectroscopy/ article articleDetail.jsp?id=86260 www.np.edu.sg/~dept-bio/questions/ fermentation_1 ferm1_mcq.html www.blackwell-synergy.com/links/ doi/10.1111/j.1365 2672.2004.02331.x/abs/ Bioenergy Production oasys2.confex.com/acs/228nm/techprogram/S12991.HTM www.ieabioenergy.com/events/Brazil2002/bg.php www.ieabioenergy.com/events/Brazil2002/ www.envirofacs.org/bioenergy.pdf www.fsa.usda.gov/pas/publications/ facts Bioenergy03QA.pdf Biohazards in Fermentation www.ipu.ac.in/BTBA115.htm www.cerl-fsi.com/biohaz.htm www.ehs.ucsf.edu/Manuals/BSM/BSMEntireDoc.htm
Future of Industrial Microbiology
www.life.umd.edu/classroom/ bsci424/BSCI223WebSiteFiles Chapter28.htm dwb.unl.edu/Teacher/NSF/C11/C11Links/ ww.gch.ulaval.ca 7Eagarnier/hur_c20.htm highered.mcgraw-hill.com/sites/0072320419/ student_view0 chapter1/study_outline.html www.biocareers.org.uk/IT3cl.htm Antibiotic Fermentation www.fda.gov/ohrms/dockets/ac/ 02/briefing 3841B1_05_PFIZER/sld033.htm www.fda.gov/ohrms/dockets/ac/ 02/briefing 3841B1_05_PFIZER/tsld033.htm books.cambridge.org/0521304903.htm
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Learning Objectives
In this lecture, you will learn•
What is fermentation? What are its types?•
Products of fermentations•
Use of recombinant technology in fermentationsWhat is Fermentation?
Fermentation has always been an important part of our lives: foods can be spoiled by microbial fermentations, foods can be made by microbial fermentations, and muscle cells use
fermentation to provide us with quick responses. Fermentation could be called the staff of life because it gives us the basic food, bread. But how fermentation actually works was not understood until the work of Louis Pasteur in the latter part of the nineteenth century and the research which followed. Fermentation is the process that produces alcoholic beverages or acidic dairy products. For a cell, fermentation is a way of getting energy without using oxygen. In general, fermentation involves the breaking down of complex organic substances into simpler ones. The microbial or animal cell obtains energy through glycolysis, splitting a sugar molecule and removing electrons from the molecule. The electrons are then passed to an organic molecule such as pyruvic acid. This results in the formation of a waste product that is excreted from the cell. Waste products formed in this way include ethyl alcohol, butyl alcohol, lactic acid, and acetone-the substances vital to our utilization of fermentation
What is The Role of Fermentation in Industry?
In industry, as well as other areas, the uses of fermentation progressed rapidly after Pasteur’s discoveries. Between 1900 and 1930, ethyl alcohol and butyl alcohol were the most important industrial fermentations in the world. But by the 1960s, chemical synthesis of alcohols and other solvents were less expensive and interest in fermentations diminished. Questions can be raised about chemical synthesis, however. Chemical manufacture of organic molecules such as alcohols and acetone rely on starting materials made from petroleum. Petroleum is a nonrenewable resource; dependence on such resources could be considered short-sighted. Additionally, the use of petroleum has associated environmental and political problems.
The worldwide interest in microbial fermentations is once again growing especially with reference to renewable resources and microbial biocatalysts. Plant starch, cellulose from agricultural waste, and whey from cheese manufacture are abundant and renewable sources of fermentable carbohydrates. Additionally these materials, not utilized, represent solid waste that must be buried in dumps or treated with waste water.
What Other Benefits Microbial Fermentations Offer?
Microbial fermentations have several other benefits. For one, they don’t use toxic reagents or require the addition of
intermediate reagents. Microbiologists are now looking for naturally occurring microbes that produce desired chemicals. In addition, they are now capable of engineering microbes to enhance production of these chemicals. In recent years, microbial fermentations have been revolutionized by the application of genetically-engineered organisms. Many fermentations use bacteria but a growing number involve culturing mammalian cells. Some examples of products currently produced by fermentation are listed in Tables 1 and 2 . Products Produced by Fermentation
Table 1.1 Fermentations by Naturally-Occurring Organisms
PRODUCT APPLICATION ORGANISM
Bacitracin Antiobiotic Bacillus subtilis (bacterium) Chloramphenicol Antiobiotic Streptomyces venezuelae (bacterium) Citric acid Food flavoring Aspergillus niger (fungus) Erythromycin Antibiotic Streptomyces erythaeus (bacterium) Invertase Candy Saccharomyces cerevisiae (fungi) Lactase Digestive aid Escherichia coli (bacterium) Neomycin Antibiotic Streptomyces fradiae (bacterium) Pectinase Fruit juice Aspergillus niger (fungus) Penicillin Antibiotic Penicillium notatum (fungus) Riboflavin Vitamin Ashbya gossypii (fungus) Streptomycin Antibiotic Streptomyces griseus (bacterium) Subtilisins Laundry detergent Bacillus subtilis (bacterium) Tetracycline Antibiotic Streptomyces aureofaciens (bacterium)
Table 1. 2 Fermentations by Genetically Engineered Organisms
PRODUCT APPLICATION ORGANISM
B. growth hormone Milk production(cows) Escherichia coli (E. coli) Cellulase Cellulose E. coli
H. growth hormone Growth deficiencies E.coli Human insulin Diabetics E. coli
Monoclonal antibodies Therapeutics Mammalian cell culture Ice-minus Prevents ice on plants Pseudomonas syringae Sno-max Makes snow Pseudomonas syringae t-PA Blood clots Mammalian cell culture Tumor necrosis factor Dissolves tumor cells E.coli
How Does Fermentation Work in Biotechnology?
In the pharmaceutical and biotechnology industries,
fermentation is any large-scale cultivation of microbes or other single cells, occurring with or without air. In the teaching lab or at the research bench, fermentation is often demonstrated in a test tube, flask, or bottle-in volumes from a few milliliters to two liters. At the production and manufacturing level, large vessels called fermentors or bioreactors are used. A bioreactor may hold several liters to several thousand liters. Bioreactors are
UNIT-1
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LESSON 1:
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equipped with aeration devices as well as nutrients, stirrers, and pH and temperature controls.
At Genentech, Inc., for example, in order to get a product from fermentation, fermentation scientists develop media and test growth conditions. Then, a scale-up must be done to reproduce the process at a large volume. During production, technicians monitor temperature, pH, and growth in the bioreactors to ensure that conditions are optimum for cell growth and product. Bioreactors are used to make products such as insulin and human growth hormone from genetically engineered microorganisms as well as products from naturally-occurring cells, such as the food additive xanthan.
The products being developed by the biotechnology industry have enormous implications for our future health and well-being. All of the exciting discoveries in current biotechnical research and its applications will, of course, have repercussions within human history. Science and politics have always interacted, in both direct and indirect ways.
What are the Various Products Manufactured using Fermentations?
There are five major groups of commercially important fermentations:
(i) Those that produce microbial cells (or bio mass) as the product.
(ii) Those that produce microbial enzymes (iii) Those that produce microbial metabolites. (iv) Those that produce recombinant products.
(v) Those that modify a compound which is added to the fermentation - the biotransformation process.
First we will see the microbial biomass as the fermentation product.
The commercial production of microbial biomass may be divided into two major processes: the production of yeast to be used in the baking industry and the production of microbial cells to be used as human or animal food (single-cell protein). Bakers’ yeast has been produced on a large scale since the early 1900s and yeast was produced as human food in Germany during the First World War. However, it was not until the 1960s that the production of microbial biomass as a source of food protein was explored to any great depth. As a result of this work, a few large-Scale continuous processes for animal feed production were established in the 1970s. These processes were based on hydrocarbon feedstocks which could not compete against other high protein animal feeds, resulting in their closure in the late 1980s (Sharp, 1989). However, the demise of the animal feed biomass fermentations was balanced by ICI pic and Rank Hovis McDougal establishing a process for the production of fungal biomass for human food. This process was based on a more stable economic platform and appears to have a promising future.
This topic has been discussed in more details in one of the subsequent lessons.
Exercis
Study the different types of fermentations where single cell proteins and mycoproteins are produced. Then write briefly what are the limitations of using microorganisms as human food. Use the space provided to express your views.
Exercise
Find out what do you mean by the term ‘probiotics’. Briefly mention what benefits probiotics can offer in human beings, animals and birds. Write your views below.
Let us now see how microbial enzymes can be produced by fermentations.
Microbial enzymes are most widely used in the food and beverage industries and to less extent in clinical and analytical laboratories and as protease detergents in washing powders. The most economical and convenient method of producing these enzymes is by microbial fermentations. Bacillus
stearothermophilus produces amylases as secondary metabolites,
but most other microbes produce enzymes as primary metabolites, during exponential growth.
Table 1.3 Some commercially valuable microbial secondary compounds
Secondary Metabolite Commercial significance/application
Actinomycin Antitumour Cephalosporin Antibiotic Penicillin Antibiotic Streptomycin Antibiotic
Cyclosporin Immunosuppressant Bestatin Cancer treatment Gibberellin Plant growth regulator
Most of the enzymes in industrial use are extracellular proteins produced by Aspergillus sp. or Bacillus sp. and include alpha-amylase, beta-glucanase, cellulase, dextranase, lactase, lipase,
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pectinase, proteases and others. The extent of purification required for most of these enzymes is minimal, and they can be produced in tons without serious problems.
There are other enzymes required for non-industrial uses. These are intracellular and are produced in much smaller amounts. Some examples are- catalase, asparaginase, cholesterol oxidase, beta-galactosidase, and glucose oxidase and glucose phosphate dehydrogenase. These enzymes have to be greatly purified to homogeneity. They can be produced in kilogram quantities from a few thousands of liters of culture suspension. Also, for isolating these enzymes, cells have to be lysed.
One of the first examples of the industrial use of immobilized enzymes was to produce large volumes of high fructose corn syrup (HFCS; isoglucose). HFCS serves as a good substitute for invert sugar (glucose + fructose) and is prepared from pure glucose syrups with a dextran content of over 95%. Its most important uses are the soft drinks and canning industry where saccharose can be totally replaced. Other important markets are the dairy products, confectionary and baked foods.
Enzyme production is closely controlled in micro-organisms and in order to improve productivity these controls may have to be exploited or modified. Such control systems as induction may be exploited by including inducers in the medium, whereas repression control may be removed by mutation and
recombination techniques. Also, the number of gene copies coding for the enzyme may be increased by recombinant DNA techniques.
Exercise
Find out about the following:
a) Microbial enzymes used for detergent applications
b) Microbial enzymes used for treatment of animal feed (clue: phytase)
c) Significance of invertase in confectionary industry and in sugar industry ( clue: Microbially Induced Sugar Inversion ) Briefly write your answers in the space provided:
All right. Now about the microbial metabolites which are produced by fermentations. First let us see what metabolites are. During exponential growth, microbial cultures produce such essential metabolites as amino acids, nucleotides, proteins, lipids, and carbohydrates. They also produce certain by-products of energy-yielding metabolism such as ethanol, butanol and acetone. Both these’ categories of metabolites are referred to as the primary metabolities. Some examples of commercially important primary metabolites are listed in Table 1.4
Table 1.4 Some commercially important microbial primary metabolites
Primary
Metabolite Producing microbes Commercial application Ethanol Saccharomyces cerevisiae Production of alcoholic beverages Acetone, butanol Clostridium acetobutyricum Solvent Lysine Corynebacteriu
m glutamicum Feed supplement Glutamic acid Corynebacteriu
m glutamicum
Flavour enhancement Polysaccharide Xanthomonas
spp. Food industry, enhance oil recovery
•
Ok. Now tell me about the microbial products where recombinant technology is used.The advent of recombinant DNA technology has extended the range of potential fermentation products. Genes from higher organisms may be introduced into microbial cells such that the recipients are capable of synthesizing ‘foreign’ proteins. These are called heterologous proteins. A wide range of microbial cells have been used as hosts for such systems including E.coli,
Saccharomyces cerevisiae and filamentous fungi. Products
produced by such genetically engineered organisms include interferon, insulin, human serum albumin, factor VIII and somatostatin. Important factors in the design of these
processes include the secretion of the product, minimization of the degradation of the product and control of the onset of synthesis during the fermentation, as well as maximizing the expression of the foreign gene.
Sometimes microbial systems can be effectively used not for the production of any particular product, but for the
transformation of a compound into a structurally similar, higher-value compound. The conversion of ethanol into acetic acid (vinegar) is the oldest such process; more modern processes involve the production of much more highly valuable
substances than vinegar. Various kinds of chemical reactions can be catalyzed to convert a cheaper compound into an expensive product. In many instances the cells or isolated enzymes may be immobilized to improve the efficiency of the reaction.
•
Fermentations are generally carried out in huge fermentors. That means all the fermentations are in liquid phase, isn’t it?Not necessarily. Whilst most industrial processes are indeed carried out in liquid media, there are some, and important ones, which employ a solid medium. These are the solid state fermentations.
The growth and metabolism of microorganisms on moist solid substrates lacking free water is called solid state
fermentation (SSF). They differ in this respect from submerged fermentations where free water is present.
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Historically, SSF processes have been more popular in Oriental, Asian and African countries whereas submerged fermentations were popular in Europe. The presence of some moisture (about 15%) is necessary for SSP to occur but there should be no free water.
Though many microbes can grow on solid substrates, only filamentous fungi can grow to a significant extent in the absence of free water. These fungi can even penetrate within the solid substrate. Bacteria and yeasts grow on solid substrates having moisture levels ranging between about 30-70% (such as compost). Single-celled organisms usually require free water. Thermophilic bacteria grow mainly in the first stage of
composting. Lactic acid bacteria grow in ensiling processes. Yeast grows on solid substrates symbiotically with other microbes in composting, ensiling, and some industrial SSF processes. . SSF has two kinds of applications: (1) Socio-economic and (2) profit-economic. Some examples of the former category include composting of waste and municipal refuse, ensiling of grasses and lignocellulosic materials, and upgrading the quality of food. These processes are fairly simple, not requiring aseptic
techniques, and are mediated by naturally-occurring microbial flora of the substrate.
The examples of profit-economic category are the production of enzymes, organic acids, mould-ripened cheeses, edible mushrooms and fermented foods. All these products generate profits.
•
Can you give some example where solid state fermentations are used?Some notable applications of SSF in industry are stated below: 1. Fermented Foods: Thousands of kinds of fermented
foods are being produced industrially in Japan, Korea, China and other Oriental countries. Miso, shoyu, ontjam, khimchi, beer, tempeh, and fermented fish and meat are good examples, Fermentation often makes the food more nutritious, more digestible, safer, or having better flavour. It also tends to preserve the food, increasing its shelf life and lowering the need for refrigeration. Fermentation can be applied to all kinds of foods. Eight classes of fermented food may be recognized, viz., beverages, cereal products, dairy products, fish products, fruit and vegetable products, legumes, meat products, and starchy products. Of these, dairy products, cereal products, and beverages are the most common. Beer is produced from cereal grains which have been malted, dried and ground into fine powder. The powder is washed in warm water. Fermentation of the washed powder is mediated either by bottom yeast (e.g., Saccharomyces uvarom) or by a top-yeast (S. cerevisiae). The final product (beer) has up to about 8% alcohol. Grapes can be directly fermented by yeasts to wine.
2. Cereals Products: Three popular cereals are wheat, rice and maize. Bread is the commonest type of fermented cereal product. Wheat dough is fermented by S. cerevisiae along with some lactic acid bacteria. Idli, dosa, vada, dhokla and papadam are some common Indian examples of fermented cereal foods. These use mixtures of wheat and legume
flours, which are fermented by Streptococcus, Pediococcus, and Leuconostoc.
3. Dairy Products: Cheeses constitute one of the largest groups of fermented dairy products, besides yogurt. Cheese is formed when the casein in milk is coagulated as the pH drops to 4.5. This happens when acid is produced by the lactic acid bacteria which convert the milk lactose to lactic acid. Streptococcus lactis is the principal microorganism involved. Yogurt is made from milk by lactic acid bacteria, a mixture of Lactobacillus bulgaricus and Streptococcus thermophilus. 4. Fruits and vegetables Products: Fermentation of fruits
and pickles is usually carried out alongwith the addition of salt and acid for their preservation. In these products, salt-resistant lactic acid bacteria initially of Leuconostoc species and Lactobacillus brevis, soon to be replaced by Lactobacillus plantarum and Pediococcus spp. Some coliform bacteria (Escherichia coli), Enterobacter spp. and Klebsiella spp are also involved. With the release of acids and drop in pH, yeastsbecome prominent.
5. Enzymes: Large quantities of the enzyme Koji are produced in Asian countries. Koji consists of moulded solid substrate for Use in food fermentations. Koji contains mixtures of different enzymes such as alpha-amylases, proteases, maltase, sucrase, lipase, phosphatase deaminase, and cellulase. Different proportions of the various enzymes yield specific types of Koji for specific foods.
6. Organic Acids: Citric acid is being industrially produced by SSF. Itaconic acid and gallic acid can also be so produced. For citric acid, Aspergillus niger is grown on moistened wheat or rice bran at pH 4.0-5.0 at 28C for about a week. Citric acid from the fermented solids is then leached using hot water and the extract so obtained is subjected to further downstream processing.
7. Mushrooms: The quality and flavour of mushrooms produced by SSF are better than those by submerged fermentations. Composting of the substrates, spawn preparation, and mushroom cultivation all involve SSF. Two mushrooms being widely produced by SSF are: the Button mushroom- Agaricus bisporus, and Shiitake Lentinus edodes. In addition, some other genera of edible fungi cultivated in various parts of the world include Volvariella, Pleurotus and Tremella.
The compost for mushroom cultivation is traditionally prepared from mixtures of wheat straw and horse manure. Phase I of composting involves the wetting and thorough mixing of the materials in long stacks (about 2 meters in cross section) on a concrete yard. The stacks are shaken and re-stacked at 2-3 day intervals over about 2 weeks. The temperature in the stacks can rise to 80°C by microbial oxidation of organic material. Microbial activity enriches the nitrogen content of the substrate, and a complex called nitrogen-rich lignin-humus complex which is rich in nutrients, is formed.
At the start of the composting phase, such fungi as Absidia cylindrospora, Mucor hiemalis, M. mucedo, Thamnidium elegans and Zygorhynchus moelleri are common and active.
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They are soon outcompeted by Aspergillus nidulans and some yeasts. Later, thermophilic species of Rhizomucor. Hunicola and Chaetomium become dominant. During early phase, several mesophilic bacteria, e.g., Flavobacterium, Pseudomonas and Serratia are also present. With the rise in compost temperature, these tend to be replaced by
thermophilic, spore-forming bacteria.
After 1-2 weeks, Phase-II (pasteurization) commences. During this, the pests and diseases of mushrooms are eliminated /minimized by placing the Phase-I compost in trays or shelves in bulk pasteurization tunnels and exposed to heat aerobically. During this second phase, thermophilic fungi lower the content of ammonia and improve the quality of the compost. Species of Aspergillus, Chaetomium, Thermophilus and other fungi play important role in Phase-II. Some actinomycetes are also important in this context. The compost is then inoculated with mushroom spawn
which is a culture of mushroom mycelium on moist, autoclaved cereal grains. Rye and millets are the grains of choice for growing the mycelium.
The colonized compost is then “capped” or “cased” to a depth of 3-5 cm with a layer of peat neutralized with limestone. Mushroom mycelium readily colonizes this casing layer. The casing layer induces fruit formation in large numbers. The fruits (sporophores) are finally harvested. 8. Cheeses: Throughout the world, cheeses are ripened by
fungi through SSF to impart distinct flavours. Soft cheese (Camembert) is formed by growing Penicillium camembertii on the surface of curd cake. When P. roquefortii grows through the body of raw, processed curd, marbled cheeses such as green and blue-veined varieties are formed.
9. Fodder Preservation: Ensiling of straw and fodder plants involves the growth of naturally occurring lactic acid producing bacteria on grasses or straw. Lingnocellulosic materials are upgraded and preserved for the utilization at times when fresh fodder is scarce.
10.Insecticides: Diverse microbial insecticides and pesticides are produced commercially by SSF. Two good examples are Biotrol XK and Metauino.
11. Biodegradation and pollution control: Undesirable, pollution materials can be destroyed by SSF. Sludge farming involves soil solid substrate on which organic
biotransformations are affected either by promoting indigenous microbes or by supplying specific culture.
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Learning Objectives
In this lecture, you will learn•
Nutritional requirements•
Media requirements – major and minor•
Media compositionHaving learned about what fermentations are, let us now move towards the fermentation media. What, in the first place, are the fermentation media?
Fermentation media is a blend of natural and synthetic substrate specifically designed to promote the growth and production of the fermentation product. Thus, a fermentation medium must support the growth of the desired organism AS WELL AS the production of the fermentation product. This is especially important because many times the production of the desired metabolite is not directly linked with the growth of the organism.
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Ok, so first the growth of the organisms. What are the basic requirements for the growth of any living organism?We all need a carefully balanced diet for normal growth and metabolism. Microbes and animal cells are no exceptions. For all heterotrophic organisms, the general nutritional requirements could be remembered using a simple formula:
C. HOPKINS, Canteen Manager
Where,
C stands for Carbon, H for Hydrogen, O for Oxygen, P for Phosphorous, K for Potassium, I for iron, N for Nitrogen, S for Sulfur and, Canteen Manager stands for Calcium and Magnesium! Thus these are the ten elements required for growth by any heterotrophic organisms. In addition to this, many fastidious organisms require specific growth factors like vitamins for their growth and production of the desired metabolite.
The fermentation medium therefore must contain ALL these ingredients IN THE RIGHT PROPORTION!
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So the fermentation medium must contain all above constituents. Will the composition of all fermentation media be the same then?No. The particular composition of a fermentation medium can be simple to complex depending on the particular
microorganism and its fermentation. Autotrophic
microorganisms require only the simplest of inorganic media. They require a few inorganic salts, water, and a nitrogen source; their carbon requirement is fulfilled by the carbon dioxide of the air or by carbonates. Thus, from simple inorganic nutrients, autotrophs are able to synthesize all of the complex organic compounds required to sustain life and to allow growth and multiplication of their cells, and they meet their total energy requirements by oxidation of some particular inorganic
component of their medium. At the opposite end of the scale are the highly fastidious microorganisms, such as some of the lactic acid bacteria, which lack the ability to synthesize many of their sustenance and growth requirements. These organisms require the presence of many types of simple to complex preformed nutrients in the medium, and they must have an organic carbon supply to provide for synthesis of cell substance and release of metabolic energy. These are the two extremes and, obviously, microorganisms exist with requirements intermediate between these extremes. However, in addition to these considerations, fermentation growth conditions impose a metabolic stress on microorganisms, as for instance, the high aeration rates and high substrate levels commonly employed, so that additional nutrients and growth factors may be required as compared to the usual laboratory culture of the organisms. Thus, enzyme systems that normally are not limiting factors in metabolic sequences, because of a lack of sufficient levels of coenzymes or for other reasons, may become limiting under the stresses of fermentation growth: under these conditions more complex media are required than would normally be employed.
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What other parameters the fermentation mediumshould satisfy?
In addition, the fermentation medium must satisfy several other conditions like
1. Ability to produce the maximum yield of product or biomass per unit substrate used.
2. Ability to produce the maximum concentration of product or biomass.
3. Ability to promote the product formation at the maximum rate.
4. Ability to produce the minimum of undesired products. 5. To cause minimal problems during media preparation and
sterilization
6. Retain consistent quality and readyavailability throughout the year.
7. To cause minimal problems in other aspects of the production process particularly aeration and agitation, extraction, purification and waste treatment.
Plus, the designed fermentation medium must facilitate easy scale up from the laboratory to the pilot scale, and subsequently to the industrial scale. A medium with a high viscosity will also need a higher power input for effective stirring. This, in turn, affects the profitability of the fermentation process. In addition, several other factors like pH variation, foam formation, change in the oxidation-reduction potential and the morphological form of the organism must be taken into account before arriving at the composition of a fermentation medium. The ease with which the end product could be recovered and the
LESSON 2:
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CONSIDERATIONS
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treatment of effluent generated out of this process also needs careful consideration.
Finally, the fermentation medium has to be economically viable. Therefore, most fermentation media employ industrial and/ agricultural by products as their major ingredient. Some typical examples are cane molasses, beet molasses, sulfite waste liquor, cereal grains, corn steep liquor, soya bean meal, slaughter-house waste etc. While the incorporation of these ingredients considerably reduces the cost of production, in many cases this may not be possible because of one or more of the above mentioned reasons. This is especially true in case of
fermentations where recombined organisms are used as they are likely to be nutritionally more demanding than their native counterparts.
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Tell me more about molasses.Beet and cane molasses are by-products of the sugar industry. These molasses are the concentrated syrups or mother liquors recovered at any one of several steps in the sugar-refining process, and different names are applied to the molasses depending on the particular step from which it was recovered. Of these, blackstrap molasses prepared from sugar cane normally is the cheapest and the most used sugar source for industrial fermentations. In the commercial production of sugar, the juice from crushed sugar cane is concentrated to allow crystallization of its sucrose.In addition to sucrose, blackstrap molasses contains small amounts of complex polysaccharides and invert sugars. The presence of the invert sugars is attributed to the action of the “invertase” enzyme present in the original cane juice. Blackstrap molasses also contains various noncarbohydrate materials. Thus, dark colored, nitrogen-containing; polymeric substances result from “browning,” a reaction of the sugars with amino acids because of the heat and alkali used in processing. Inorganic ions are present in high concentrations and include most of the ions of the original cane juice which were concentrated till the mother liquor during sugar crystallization. Calcium also is present, being added during processing. Organic-acid constituents include aconitic, malic, citric, lactic, formic, acetic, and propionic acids. The nitrogen-containing compounds (other than the polymeric forms) are mainly amino acids and, particularly, aspartic and glutamic acids resulting from the deamidation of the asparagine and glutamine of the cane juice. A few heat and alkali stable vitamins are present, such. as myo-inositol. niacin, pantothenic acid, riboflavin, and small amounts of biotin. Also present are organic phosphorus compounds such as inositol hexaphosphate and its calcium-magnesium salt known as “phytin.”
The overall compositions of the various molasses differ according to the specific geographic areas of production. This is particularly true for their contents of certain metal ions and, in fact, for certain fermentations, such as that for citric acid, the molasses is pretreated with cation exchange resins or potassium ferrocyanide before use so as to remove
interfering cations.
The crystallized sugar is then separated from its mother liquor, and the mother liquor is further concentrated to allow recovery of additional crops of crystalline sugar. This procedure is repeated several times until crystallization inhibitors accumulate to such a concentration that further recovery of sucrose is not economical. At this point, the mother liquor still contains approximately 52 percent total sugars calculated as sucrose (30 percent sucrose, and 22 percent invert sugars) and is known as black-strap molasses. When this molasses is used as a fermentation medium component, it is considered to contain 50 percent
fermentable sugars. Refinery blackstrap molasses is a similar product that differs from black-strap molasses only in that it is the residual mother liquor that has accumulated in the recrystallization refining of crude sucrose.
High-test or invert molasses contains approximately 70 to 75 percent sugar, and it is produced in a manner different from that previously described. The whole cane juice is partially inverted to prevent sugar crystallization; that is, the sugar is partially hydrolyzed to monosaccharides with heat and acid then neutralized and concentrated without the removal of any of the sugar. Thus, high-test or invert molasses contains much of the original sugar of the cane juice, although it has been partially hydrolyzed to D-glucose and D-fructose. It is preferred to blackstrap molasses because of the lower shipping charges on a sugar concentration basis and because of its lower levels of nonfermentable solids including salts and unfermentable sugars. In blackstrap molasses, the unfermentable sugars result from the action of heat on the sugars, particularly fructose, during the refining process. High-test molasses is produced only during years of sugar cane overproduction and, hence, its availability at any one time may be somewhat questionable.
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And how are beet molasses different from cane molasses?Beet molasses are produced by procedures resembling those for sugar cane. However, beet molasses may be limiting in biotin for yeast growth so that Ii small amount of cane blackstrap or other source of biotin should be added for growth of these microorganisms. “Hydrol” is a molasses resulting from the manufacture of crystalline dextrose from corn starch. It contains approximately .60 percent sugar, but it also contains a relatively high salt concentration that must be considered if this molasses is to be used as a medium component.
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Any other industrial by product that can be used as a substrate for fermentation?Good that you asked. Corn steep liquor and Sulfite waste liquor are used for many industrial fermentations. Corn steep liquor is the water extract by-product resulting from the steeping of corn during the commercial production of corn starch, gluten, and other corn products. The used or spent steep waters are concentrated to approximately 50 percent solids, and this concentrate, known as corn steep liquor, is used in the commercial manufacture of feedstuffs and as a medium adjunct in the fermentation industry. It was first extensively employed in fermentation media for the
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manufacture of penicillin. Of the 50 percent solids of corn steep liquor, approximately half is lactic acid. The rest includes amino acids, glucose and other reducing sugars, salts, vitamins, and precursors such as those for the penicillin molecule. Although corn steep liquor does contain this high lactic acid content, the acid is not necessarily utilized by microorganisms during growth in industrial fermentation processes. The high lactic acid content of corn steep liquor results from the growth of lactic acid bacteria and of mycoderma, which are film forming, asporogenous, yeast like fungi. Thus, the lactic acid is not a component of corn but results from a natural fermentation of the corn steep liquor. In other words, corn steep liquor in itself actually is a natural fermentation product and, as such, it can vary greatly in composition for lots from a single supplier, or between lots received from various suppliers. This variation in composition, at times, can lead to poor reproducibility of an industrial fermentation. Thus, if the corn steep liquor is supplying certain medium components (such as a particular amino acid, vitamin, or precursor) at low but critical levels, it may be necessary to determine the specific level of this compound as it is present in each lot of corn steep liquor is to be used.
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Ok. Any other by product?Then there is sulfite waste liquor. Sulfite waste liquor is the spent sulfite liquor from the paper-pulping industry. It is the fluid remaining after wood for paper manufacture is digested to cellulose pulp with calcium bisulfite under heat and pressure and, as such, it presents a serious disposal problem for the paper-pulp manufacturers. Disposal in streams, as is the usual practice, causes stream pollution, and in several states legislation has been enacted against this method of disposal. Sulfite waste liquor can be employed as a dilute fermentation medium, being used in the production of ethanol by Saccharomyces cerevisiae and in the growth of Torula uti/is cells for feed. The economics of these
fermentations dictate that the fermentation plant is located in close proximity to the pulping operation so that the cost of transporting the waste liquor is not a factor. The waste liquor contains 10 to 12 percent solids of which sugars make up about 20 percent. Thus, sulfite waste liquor is a dilute sugar solution containing approximately 2 percent sugar. These sugars include the hexosc5 glucose, galactose and D-mannose, and the pentoses D-xylose and L-arabinose. However, the relative amounts of these sugars present in sulfite waste liquor depend, to some extent, on the woods being digested, with soft woods being higher in hexoses and hardwoods higher in pentoses. This is important if yeast such as Saccharomyces cerevisiae is to be employed as the fermentation organism, since it uses only hexoses. Torula utilis, however, can ferment both hexoses and pentoses. In any event, regardless of which type of organism is being considered, the sugars of sulfite waste liquor cannot be fermented directly; the free sulfur dioxide or sulfurous acid of the waste liquor must first be removed by steam stripping or precipitation with lime.
Wood-waste residues hydrolyzed by acid provide sugars similar to those of sulfite waste liquor. The hydrolyzed material is partially neutralized and filtered before use in a fermentation medium. Thus, wood wastes are a virtually untapped source for fermentation carbohydrate nutrients.
Formulation of a Fermentation Medium–What is our objective?
Unless the end product of fermentation is the biomass itself, we are not interested in the growth of organisms during fermentation. We would ideally prefer to have the minimum of growth and the maximum of product synthesis. Unfortunately, this is not always possible. In a fermentation process, therefore, attempts are made to keep the cell growth at its bare minimum.
How to formulate a fermentation medium?
Medium formulation is an essential stage in the design of successful laboratory experiments, pilot-scale development and manufacturing processes. The constituents of a medium must satisfy the elemental requirements for cell biomass and metabolite production and there must be an adequate supply of energy for biosynthesis and cell maintenance. The first step to consider is an equation based on the stoichiometry for growth and product formation. Thus for an aerobic fermentation:
Carbon + Oxygen + Nitrogen + Other requirements = biomass + products + CO2 + H2O + heat
We should be able to express this equation in quantitative terms. i.e. it should be possible to calculate the minimal quantities of nutrients which will be needed to produce a specific amount of biomass and it should be possible to calculate substrate concentrations necessary to produce required product yields. But it is not always easy to quantify all the factors very precisely.
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Then how do we know how much of which nutrient is needed?I will tell you that. First, different organisms will have different nutritional requirements. This is quite elementary. Secondly, since we want to keep the biomass to the minimum, we have to know the basic composition of the organisms being used so that the fermentation medium can be designed accordingly. Based on the general composition of commonly used cells in fermentation, we can formulate the fermentation medium.
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Are there any other considerations to be made? Yes. We will see what are the various parameters to be considered with reference to individual constituents of the medium. Let us start from water, which is the commonest of all the ingredients in a fermentation medium.Clean water of consistent composition is therefore required in large quantities from reliable permanent sources. When assessing the suitability of a water supply it is important to consider pH, dissolved salts and effluent contamination. The mineral content of the water is very important in brewing, and most critical in the mashing process, and historically influenced the location of breweries and the types of beer produced. Hard waters containing high CaS04 concentrations are better for the English Burton bitter beers and Pilsen type
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lagers, while waters with high carbonate content are better for the darker beers such as stouts. The reuse of water in media also must be considered. ICI, a giant SCP producer, realized that very high costs would be incurred if fresh purified water was used on a once through basis, since operating at a cell concentration of 30 g biomass (dw) dm3 would require 2700 X lO6 dm3 of water per annum. Laboratory tests to simulate the process showed that the Methylophilus methylotrophus could be grown successfully with 86% continuous recycling of supernatant with additions to make up depleted nutrients. This approach was therefore adopted in the full scale process to reduce capital and operating costs and it was estimated that water used on a once through basis without any recycling would have increased water costs by 50% and effluent treatment costs 10 fold.
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What is the next nutrient that should be considered? After water let’s learn about the Carbon and Energy source. Most industrial micro-organisms are chemo-organotrophs; therefore the commonest source of energy will be the carbon source such as carbohydrates, lipids and proteins. Some micro-organisms can also use hydrocarbons or methanol as carbon and energy sources.The rate at which the carbon source is metabolized often influences the formation of biomass or production of primary or secondary metabolites. Fast growth due to high concentrations of rapidly metabolized sugars is often associated with low productivity of secondary metabolites. At one time the problem was overcome by using the less readily metabolized sugars such as lactose but many processes now use semi-continuous or continuous feed of glucose or sucrose. Alternatively, carbon catabolite regulation might be overcome by genetic modification of the producer organism.
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If this is the range of carbohydrates available, how do we select the right substrate?What we want to produce from fermentation often tells us what we can use to produce that. In other words, the main product of a fermentation process will often determine the choice of carbon source, particularly if the product results from the direct dissimilation of it. In fermentations such as ethanol or single-cell protein production where raw materials are 60 to 77% of the production cost, the selling price of the product will be determined largely by the cost of the carbon source. But most companies involved in the business of fermentation are continuously looking out for alternative substrates which could be used as a carbon sources. This enables a company to use alternative substrates, depending on price and availability in different locations, and remain competitive. Up to ten different carbon sources have been or are being used by Pfizer Ltd for an antibiotic production process depending on the geographical location of the production site and prevailing economics. The purity of the carbon source may also affect the choice of substrate. For example, metallic ions must be removed from carbohydrate sources used in some citric
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Acid ProcessesThe method of media preparation, particularly sterilization, may affect the suitability of carbohydrates for individual fermentation processes. It is often best to sterilize sugars separately because they may react with ammonium ions and amino acids to form black nitrogen containing compounds which will partially inhibit the growth of many micro-organisms.
Starch, when heated in the sterilization process, gelatinizes, giving rise to very viscous liquid so that only concentrations of up to 2% can be used without modification. The local regulations and the prices of raw material also affect the choice of carbon source.
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The carbon source then must be easily degradable, sterilizable and cheap, right?Right! And that laves us with one obvious choice – carbohydrates.
It is common practice to use carbohydrates as the carbon source in microbial fermentation processes. The most widely available carbohydrate is starch obtained from maize grain. It is also obtained from other cereals, potatoes and cassava. Maize and other cereals may also be used directly in a partially ground state, e.g. maize chips. Starch may also be readily hydrolyzed by dilute acids and enzymes to give a variety of glucose preparations (solids and syrups). Hydrolyzed cassava starch is used as a major carbon source for glutamic acid production.
Barley grains may be partially germinated and heat treated to give the material known as malt, which contains a variety of sugars besides starch. Malt is the main substrate for brewing beer and lager in many countries. Malt extracts may also be prepared from malted grain.
Sucrose is obtained from sugar cane and sugar beet. It is commonly used in fermentation media in a very impure form as beet or cane molasses which are the residues left after crystallization of sugar solutions in sugar refining. Molasses is used in the production of high-volume /low-value products such as ethanol, SCP, organic and amino acids and some microbial gums. Molasses or sucrose also may be used for production of higher value/low-bulk products such as antibiotics, specialty enzymes, vaccines and fine chemicals The cost of molasses will be very competitive when compared with pure carbohydrates. However, molasses contains many impurities and molasses-based fermentations will often need a more expensive and complicated extraction/purification stage to remove the impurities and effluent treatment will be more expensive because of the unutilized waste materials which are still present. Some new processes may require critical evaluation before the final decision is made to use molasses as the main carbon substrate
Corn steep liquor is a by-product after starch extraction from maize. Although primarily used as a nitrogen source, it does contain lactic acid, small amounts of reducing sugars and complex polysaccharides. Certain other materials of plant origin, usually included as nitrogen sources, such as soybean
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meal and Pharmamedia, contain small but significant amounts of carbohydrates.
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That was about carbohydrates. Any other substrate? Sure. Vegetable oils (olive, maize, cotton seed, linseed, soya bean, etc.) may also be used as carbon substrates, particularly for their content of the fatty acids, oleic, linoleic and linolenic acid, because costs are competitive with those ofcarbohydrates. A typical oil contains approximately 2.4 times -the energy of glucose on a per weight basis. Oils also have a volume advantage as it would take 1.24 dm3 of soya bean oil to add 10 kcal of energy to a fermentor, whereas it would take 5 dm3 of glucose or sucrose assuming that they are being added as 50% w /w solutions. Ideally, in any fermentation process, the maximum working capacity of a vessel should be used. Oil based fed-batch fermentations permit this procedure to operate more successfully than those using carbohydrate feeds where a larger spare capacity must be catered for to allow for responses to a sudden reduction in the residual nutrient level Oils also have antifoam properties which may make downstream
processing simpler, but normally they are not used solely for this purpose.
Pfizer antibiotic process operated with a range of oils and fats on a laboratory scale. In the UK, when both technical and economic factors are considered, soybean oil or rapeseed oil is the preferred substrates. Glycerol trioleate is known to be used in some fermentations where substrate purity is an important consideration. Methyl oleate has been used as the sole carbon substrate in cephalosporin production.
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Carbohydrates, oils. Anything else?There has been considerable interest in hydrocarbons. Development work has been done using n-alkanes for production of organic acids, amino acids, vitamins and co-factors, nucleic acids, antibiotics, enzymes and proteins. Methane, methanol and n-alkanes have all been used as substrates for biomass production.
In processes where the feedstock costs are an appreciable fraction of the total manufacturing cost, cheap carbon sources are important. In the 1960s and early 1970s there was an incentive to consider using oil or natural gas derivatives as carbon substrates as costs were low and sugar prices were high. On a weight basis n-alkanes have approximately twice the carbon and three times the energy content of the same weight of sugar. Although petroleum-type products are initially impure they can be refined to obtain very pure products in bulk quantities which would reduce the amount of effluent treatment and downstream processing. At this time the view was also held that hydrocarbons would not be subject to the same fluctuations in cost as agriculturally derived feedstock because it would be a stable priced commodity and might be used to provide a substrate. The scenario, however, has changed now. Now, not only the agricultural derivatives are cheaper to petroleum products, but their prices are steadier, enabling the fermentation to be economically more predictable.
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Ok and the next nutrient?Nitrogen. Most industrially used micro-organisms can utilize inorganic or organic sources of nitrogen. Inorganic nitrogen may be supplied as ammonia gas or ammonium salts Ammonia has been used for pH control and as the major nitrogen source in a defined medium for the commercial production of human serum albumin by Saccharomyces cerevisiae. Ammonium salts such as ammonium bisulphate will usually produce acid conditions as the ammonium ion is utilized and the free acid will be liberated. On the other hand nitrates will normally show an alkaline drift as they are metabolized. Ammonium nitrate will first cause an acid drift as the ammonium ion is utilized, and nitrate assimilation is repressed. When the ammonium ion has been exhausted, there is an alkaline drift as the nitrate is used as an alternative nitrogen source.
One exception to this pattern is the metabolism of Gibberella fujikuroi. In the presence of nitrate the
assimilation of ammonia is inhibited at pH 2.8-3.0. Nitrate assimilation continues until the pH has increased enough to allow the ammonia assimilation mechanism to
restart.Organic nitrogen may be supplied as amino acid; protein or urea. In many instances growth will be faster with a supply of organic nitrogen, and a few microorganisms have an absolute requirement for amino acids. Amino acids are commonly added as complex organic nitrogen sources which are non-homogeneous, cheaper and readily available. In lysine production, methionine and threonine are obtained from soybean hydrolysate since it is relatively inexpensive. Other proteinaceous nitrogen compounds serving as sources of amino acids include cornsteep liquor, soya meal, peanut meal, cotton-seed meal, Distillers’ solubles, meal and yeast extract. Chemically defined amino acid media devoid of protein are necessary in the production of certain vaccines when they are intended for human use.
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Ammonia, its salts, urea, proteins, amino acids… again, how do we select the right source?Ammonia or ammonium ion is the preferred nitrogen source. Not only because it is inexpensive but also because of the fact that nitrate reductase, an enzyme involved in the conversion of nitrate to ammonium ion, is repressed in the presence of ammonia. In fungi that have been investigated, ammonium ion represses uptake of amino acids by general and specific amino acid permeases. In Aspergillus nidulans, ammonia also regulates the production of alkaline and neutral proteases. Therefore, in mixtures of nitrogen sources, individual nitrogen components may influence metabolic regulation so that there is preferential assimilation of one component until its concentration has diminished. It has been shown that antibiotic production by many micro-organisms is influenced by the type and concentration of the nitrogen source in the culture medium. Antibiotic production may be inhibited by a rapidly utilized nitrogen source and may start only after most of the nitrogen has been consumed.
In shake flask media experiments, salts of weak acids (e.g. ammonium succinate) may be used to serve as a nitrogen source and eradicate the source of a strong acid pH change