2. Historical development of antibiotics
•PaulVuilemin(1889)wasthefirstandforemostscientistwhovehementlypromulgatedtheveryconceptof‘antibiotic’activitytointroducetheterminology‘influencesantibiotiques’ (orantibioticinfluences)inordertodescribetheprevailingnegativeinteractionsamongsttheanimalsandplants.
•Lateron,Walksman(1940s)eventuallycoinedtheterm‘antibiotic’andalsointroducedaplausibledefinitionas—‘achemicalsubstancederivedfrommicroorganismswhichhasthecapacityofinhibitinggrowth,andevendestroying,othermicroorganismsindilutesolutions’.
4. Terminologies
•Secondary metabolitesareorganic compoundsthat are not directly involved in the normalgrowth,development, orreproductionof an organism.Unlikeprimary metabolites, absence of secondary metabolities does not result in immediate death, but rather in long-term impairment of the organism'ssurvivability,fecundity, or aesthetics, or perhaps in no significant change at all. Secondary metabolites are often restricted to a narrow set of species within aphylogeneticgroup.Secondary metabolites often play an important role inplant defense against herbivoryand other interspecies defenses. Humans use secondary metabolites as medicines, flavorings, and recreational drugs. For example : Small "small molecules"
•Alkaloids(usually a small, heavily derivatized amino acid):
–Hyoscyamine, present inDatura stramonium
–Atropine, present inAtropa belladonna,Deadly nightshade
–Cocaine, present inErythroxylon cocatheCocaplant.
•Terpenoids(come fromsemiterpene oligomerization):Azadirachtin (Neemtree)
Artemisinin, present inArtemisia annuaChinese wormwood tetrahydrocannabinol, present inCannabis
12. Classification of Antibiotics
Classification of Antibiotics is based on different criteria:
a) Based on their spectrum of activity:-
Group-1:Active against Gram-positive bacteria and gram-negative cocci
Ex: Pencillins, Erythromycin
Group-2:Mainly active against Gram-negative bacilli.
a) For systemic infection: -Aminoglycosides, Polymyxins.
b) For urinary tract infections:-Nitrofurantoin, Nalidixic acid
Group-3:Broad spectrum antibiotics
Ex: Sulphanamides, Tetracycline
Group-4: Specific antibacterials
a) Active against anaerobic organisms.
Ex: metronidazole, lincomycin
b) For tuberculosis
Ex: Streptomycin, Isoniazid
c) for Chlamydia, Rickettsia, Mycoplasma infections
Ex: Erythromycin, Tetracyline
13. Group-5:Antifungal, antiviral agents
a) Antifungal agents: Polyenes, Nystatin
b) Antiviral: Idoxuridine, Amantadine
c) Antimalignancy antibiotics: Actinomycin, Mitomycin
b) Based on the structure: Aminoglycosides, Macrolides, Tetracyclines, Polyenes, Nitrofurans.
c) Based on their source:
Natural, Synthetic and Semi-synthetic
Based on whether they are cidal or static:
Ex: Bacteriocidal: Penicillin, Aminoglycosides
Bacteriostatic: Sulphanamides, Tetracyclines.
d) Based on their mechanism of actions:
1) Inhibitors of cell wall synthesis.
2) Inhibitors of cell membrane.
3) Inhibitors of protein synthesis.
4) Inhibitors of nucleic acid synthesis.
14. ANTIMICROBIAL SPECTRUM AND METHODS USED FOR THEIRSTANDARDIZATION
•Microbiology, in particular clinical medical microbiology, is a scientific discipline chiefly concerned with the isolation and subsequent identification of causative disease-producing microorganisms (or pathogens) : bacteria, fungi (including yeast), viruses, rickettsia, and parasites.
•In general, there are well-defined specific as well as non-specific techniques available with regard to the isolation and identification of the ‘suspect organisms’ as stated under :
• Propagation on an appropriate primary culture media,
• Selective isolation on special (specific) culture media,
• Application of appropriate living host material e.g., mouse, embryonated egg, tissue culture and the like,
• Determination of morphological features of the organism,
• Determination of staining characteristics of the organism,
• Confirmation by biochemical analysis, and
• Confirmation by immunochemical analysis.
15.
16.
17.
18. Specific Tests to identify pathogens
•There are three specific test that may be used to identify the pathogens, namely :
(a)Enzymatic and Immunological Tests : The introduction of rapid manual enzymatic and immunological test kits have enormously enabled to identify the presence of ‘pathogens’ in the cerebrospinal fluid (CSF) analysis.
(b) CoagglutinationTests : In this specific tests, the particular antibody is bound to protein A on the surface of a staphylococcal cell, and the very presence of antigen causes agglutination, and
(c) Latex-Agglutination Tests : In this particular tests a specific antibody gets coated onto the latex particles and when an antigen is present, the latex particles are visible distinctly.
19. Types of antibiotic spectrum
•An antibiotic's spectrum can be broad or narrow.
•Narrow spectrum antibioticsact against a limited group of bacteria, either gram positive or gram negative, for example sodium fusidate only acts against staphylococcal bacteria.
•Broadspectrum—antibioticsactagainstgrampositiveandgramnegativebacteria,forexampleamoxicillin,ampicillin,tetracycline, chloramphenicol,etc.Broadspectrumantibioticsareproperlyusedinthefollowingmedicalsituations:empirically(i.e.,basedontheexperienceofthepractitioner),priortotheformalidentificationofthecausativebacteriaandwhenthereisawiderangeofpossibleillnessesandapotentiallyseriousillnesswouldresultiftreatmentisdelayed.
•Broadspectrumantibioticsarealsousedfordrugresistantbacteriathatdonotrespondtoother,morenarrowspectrumantibioticsandinthecaseofsuperinfections,wheretherearemultipletypesofbacteriacausingillness,thuswarrantingeitherabroad-spectrumantibioticorcombinationantibiotictherapy.
28. Serological Tests :
•Itisacommonpracticetouse‘blood’forcarryingouttheserologicaltests, butquiterarelyforvirusisolation.
•However,itisabsolutelyimportantandvitalthatbothacuteandconvalescent-phasebloodspecimensshouldbeexaminedthoroughlyinparalleltoestimatepreciselywhether‘antibodies’haveappeared, loweredorenhancedinthe‘titervalue’inthespanofthedisease.
•Examples : A few typical examples of ‘human viral infections’ are as enumerated under :
• Respiratory infections (e.g. Adenovirus group)
• Diseases of the nervous system (e.g., Polio and Coxsackie viruses of the picornavirus group)
• Small pox (Poxvirus group)
• Measles (Paramyxovirus group)
• Chicken pox (Herpesvirus group)
• Influenza (Myxovirus group)
31. Methods Used for Standardization of Antibiotics
•Officialcompendiainvariablymakeuseoftheterminology‘antibiotic’thatessentiallydesignatesa‘medicinalpreparation’,containinganappreciablequantumofachemicalentitywhichiscausedtoproducenaturallybyamicroorganismorbyasemi-syntheticrouteartificially,andthatpossessestheinherentabilitytoeitherdestroy(bactericidaleffect)orinhibit(bacteriostaticeffect) microorganismsinrelativelydilutesolution.
43. Drawback of this technique
(a)Itfailstogiveadefiniteindicationaboutwhichamineororganicacidhasbeenproducedactually.Hence,itshouldbeimmediatelyfollowedbyfurthertestingwiththehelpofcertainwell-knownanalyticalprocedurese.g.,paperchromatography,electrophoresissoastodetermineandestablishwhethertheacidicorbasicproductreallyisoneofinterest.
(b)Importantly,insuchaneventwherecoloniesofmicroorganismsbyvirtueofthisinitialscreeningprocedure, seemtopossess‘appreciablefermentativepotential’mustimmediatelybesubjectedtopurification;and,therefore, subculturedsubsequentlyontoslantsofanappropriateagarmediumtobemaintainedadequatelyas‘stockcultures’duringfurthertestingdevices.
50. Steps for streak plate method
1.Sterilize the wire loop in a flame.
2.Cool the hot loop by touching the loop wire into the sterilized agar.
3.Dip the loop into the sample (inoculum) and spread it froth and back across the agar surface.
4.Before continuing to streak the plate, the remaining mo’s on the loop are killed by sterilizing (introducing) the loop in the flame.
5.After cooling the sterilized loop, the loop is dragged through the previous path, picking up a small number of mo’s and spreading them into the new area of the plate.
6.After sterilizing and cooling the loop, the process is repeated once again. With each new path, the loop picks up a smaller number of bacteria, and therefore can spread them farther and farther apart.
7.During incubation the bacteria (mo’s ) produce colonies. The isolated colonies found in the last streak represent the isolated strains.
57. Fermentors (Bioreactors)
•Themostarticulate,manipulativeandprogressiveindustrial(commercial) usageofmicroorganismsinvariablyneedsthattheybeallowedtogrowinlargevesselsessentiallyloadedwithconsiderablequantumofhighlynutritiveculturemedia.Thesespeciallydesignedvesselsareuniversallyandcommonlytermedasfermentorsorbioreactors.
•In the recent past, biotechnological processes (bioprocess technology) is found to use both aggressively and progressively specific cells derived exclusively from higher plants and animals to give rise to several useful and vital products.
•Examples :
(a) Plant Cell Culture : It is largely aimed at the adequate formation of secondary products solely, for instance : drugs (antibiotics), flavours, and perfumes.
(b) Animal Cell Culture (Mammalian Cell Culture) : It is mainly concerned with the production of extremely potent and life-saving products, such as : (i) vaccines ; (ii) antibody formation ; and (iii) protein molecules e.g., interferon, interleukins etc.
58. Fermentor vs bioreactor
•TheprocessoffermentationisknowntomankindforthousandsofyearsbutitsscientificstudieswerefirstconductedbyFrenchscientistLouisPasteurin1850’swhenhestudiedformationoflacticacid.Hefoundthatsouringofmilkwasaresultofactivityoflivingorganisms,andnotachemicalchangeaswasthoughtearlier.Recentadvancesintechniquesinthefieldoffermentationhaveledtothedevelopmentoffermentorsandbioreactorsthatarebothbasedupontheactivitiesoflivingorganisms.Despitesimilaritiesinworkingprinciple,therearedifferencesinafermentorandabioreactorthatwillbediscussedinthisarticle.
•Whileinearliertimes,theprocessoffermentationwasmainlyusedforproductionofbrewingalcoholicbeveragesonly,withtheincreaseinourknowledgeaboutbacteriaandfungi,fermentorshavebeendevelopedthatarebeingputtomoreproductiveuses.Bioreactorsareastepaheadindesignandconstruction.Whilefermentorsaresystemsusedforgrowthandmaintenanceinacontrolledmanner,ofapopulationofbacterialorfungalcells,bioreactorisasystemusedforgrowthandmaintenanceofmammalianandinsectcells.Thusitisclearthattherearenaturaldifferencesbetweenthetwo,andthesedifferencesareenlistedbelow.
60. Fermentationisthetermusedbymicrobiologiststodescribeanyprocessfortheproductionofaproductbymeansofthemasscultureofamicroorganism.
The product can either be:
1. The cell itself: referred to as biomass production.
2. A microorganisms own metabolite: referred to as a product from a natural or genetically improved strain.
3. A microorganisms foreign product: referred to as a product from recombinant DNA technology or genetically engineered strain, i.e. recombinant strain.
Fermentationiscommonlydefinedastheprocessiswhichenergyisformedbytheprocessofoxidationoforganiccompoundslikecarbohydratesandsugars.Thisleadstoconversionoftheseorganiccompoundsintoanacidoranalcoholwhichprovidesenergy.Itcanbecarriedoutbymicroorganismswiththehelpofoxygenaswellaswithoutit.Whenfermentationiscarriedoutinthepresenceofoxygen,itiscalledaerobicfermentationandwhencarriedoutwithoutit,itiscommonlyknownasanaerobicfermentation.
61. •It can be supported with the help of an equation that describes the substrates used in the fermentation process to give the desired products. One of its examples is alcoholic fermentation whose equation is given below. Acetaldehyde + NADH + H+ alcohol Alcohol + NAD+ --------------> dehydrogenase
•Inthisreaction,anaerobicrespirationoccursinthesugarstocausefermentation,withthehelpofthefungusyeast,whichisnotincontactwiththeatmosphereoroxygen.AcetaldehydeandNADHarethesubstratesofthereactionwhicharefermentedalongwithonehydrogeniontoformtheproductwhichisalcohol.ThisreactiontakesplaceinpresenceoftheactiveenzymealcoholdehydrogenasetogivethealcoholandacofactorwhichisoneionofNAD.Thisreactionfollowsthedecarbaxylationofpyruvatewiththehelpoftheenzymepyruvatedecarboxylase,Thiaminepyrophosphate(TPP)andtwoMgions.AfterthisacetyldehydeandCOareformedtogivetheabovementionedreaction.
62. Classification of fermentation process:
(i) Solid-state fermentation,
(ii) Anaerobic fermentation,
(iii) Aerobic fermentation, and
(iv) Immobilized cell bioreactor.
63. (i) Solid-state fermentation,
•Intruesense,suchfermentationproceduresareusuallygovernedbybothmicrobialgrowthandproductformationpredominantlytakingplaceatthesurfaceofthesolidsubstrates,suchas:mold-ripenedcheeses;startercultures; mushroomcultivationsetc.
•Skilfullyextendedfortheproductionofcertainhigh-valueproductsofinterest,namely:extracellularenzymes,valuablechemicalentities,fungaltoxins,andfungalspores(exclusivelyemployedforbiotransformationprocesses).
•The usual traditional substrates essentially comprise of a plethora of ‘agricultural products’ like rice, maize, wheat, soybean etc.
65. Following are some of the special remarks with respect of the solid state fermentation procedures, namely :
(1)they make use of either stationary or rotary trays,
(2) invariably both temperature and humidity controlled air is being circulated through the entire stacked solids,
(3) rotary-drum type fermentorsare used rather less frequently,
(4) they usually offer certain unique advantages besides some vital disadvantages also, and
(5) major commercial application of this phenomenon for the biochemical production is solely confined to Japan.
74. B. Air lift fermentor:
•Theculturesinanair-lifttypefermentorarenotonlysubjectedto‘aeration’butalso‘agitation’bypassingsterilizedcompressedairbubblesintroducedstrategicallyatthebottomofvesselasshowninFig.
Salient Features of Air-lift Type Fermentors: The various vital salient features of an air-lift
type fermentorare as follows :
(1)The fermentorhas an inner draft tube via which the air bubbles as well as the aerated medium rise, because this effectively gives rise to through mixing of the culture and aeration simultaneously.
(2) The air bubbles being lighter lift to the top of the medium and the air subsequently gets
released through on outlet.
(3) In this process, importantly the cells and the medium which eventually lift out of the draft tube usually move downwards outside the tube and are recirculatedduly.
(4) Air-lift type fermentorswith a capacity of 2-90L are invariably available for large-scale
production. However, 2000L fermentorsare being employed specifically for the production
of monoclonal antibodies (MABs).
79. Different types of fermentors (Based on their applications)
Fermentor vessels:
Some have limited applications
Some developed for specific functions
some are of historical developments
1. Packed tower: Historical interest for vinegar preparation
Mainly for effluent treatment
2. The tower fermentor: H/D:-6:1 or 10:1
•For citric acid, brewing industry or beer.
80. 3.The Woldhof-type fermentor:
Yeast production from SWL 7.9 dia & 4.3m ht.
4. Acetators and cavitators:
Vinegar prod. by Acetobacter sp.
5. The cyclone columan:
Mainly for filamentous fungal growth.
Adv: No foam, limited wall growth.
81. 6. Cylindro-conical vessels:
–Brewing of lager and beers
–22-25 m.ht6m. Dia
Adv:
–Reduced process times
–Primary fermentation & maturity in the same vessel
–Ease of removal of sediment
–Maturity time reduction-by washing with CO2
82. 7.Air-lift fermentor:
–For SCP production from MethanolContinuous production.
Single-cell protein(SCP) typically refers to sources of mixedproteinextracted from pure or mixed cultures ofalgae,yeasts,fungiorbacteria(grown on agricultural wastes) used as a substitute for protein-rich foods, in human and animal feeds.
8. The deep-Jet fermentor:
–For large scale prod. of yeast from whey
(continuous or batch processes)
9. Rotating disc fermentor:
–For effluent treatment and also for citric acid production
83.
84. Sterilization of production media:
1.Usually production media are sterilized before they are inoculated with the desired fermentative pure culture.
2. It depends on the chemical composition of the media.
3. Media containing sugars cannot be sterilized by prolonged heating, due to the caramelizationof sugars.
4.Media containing sugars & phosphates can be sterilized separately, because of the reaction of sugars with phosphates.
5.Sterilization can be done by 3 methods 1. Boiling 2. Passing live steam 3. Autoclaving (Steam under pressure).
85. •The classical technique of sterilization is the use of steam. Two types: 1. Batchwise in the fermentor
•The vessel is equipped with a coil or jacket for heating & cooling.
•Interconnecting pipeline must also be sterilized before making use of it for transfer purposeAdvantages: Saves the production time.
•Disadvantages: 1. Occupies increased plant space 2. Higher cost for additional equipment 3. Involves increased steam usage
86. •Continuous Sterilization: 1. It offers more flexibility in the temperature conditions. 2. It involves passing of production medium through a heat exchanger, a holding coil and a cooler. 3.The medium is finally cooled by counter circulating it against cool input medium and then against cool water. 4. It allows the sterilization at higher temp. without affecting nutritional values. 5. Continuous retention tube sterilizer is widely used in antibiotic field
87. Advantages: 1. Saves production time and plant space 2. Improves quality of medium 3. Economical 4. Allows the use of lower sterilizing temp. Disadvantages:
Media containing heat resistant bacterial spores, vitamins, enzymes etc may not be sterilized.
88. •Sterilization of air: Methods 1. Filtration 2. Heat 3. Electrostatic precipitation4. U.V. light and 5. Chemical agents. Sterilization of air in fermentation industries is widely done by filtration methods.
89. A series of fermentors used for lab scale process development
90. Laboratory process developmentLab scale fermentor Experiments
•Batch process
•Fed-batch process
•Continuous process
•Semi-continuous process
94. Fermentor body construction
Laboratory , Pilot , Industrial
1.Materials of construction:
Glass: For lab scale (smooth, non-toxic,
non-corrosive and transparent)
Mild steel/SS: for pilot & industrial (7mm thick sides; 10mm bottom)
Wood, plastic, concrete: Rarely
95. 2.Temp. Control:Outer jackets/coils
3. Aeration & agitation: for oxygen + mixing
Imp. Components:
A) Impellers (agitators): Types-
disc turbine,
vaned disc,
open turbine,
marine propeller
96. B) Stirrer glands & bearings: Satisfactory seals for asepsis
Types: Packed-gland seal (stuffing box)
Simple brush seal (lab scale)
Mechanical seal (Commonly used)
Magnetic drive
C) Baffles: Prevent vortex & Improves efficacy of mixing
Metal strips 1/10th of D/4 Nos
A gap of 1 to 3.5 cm preferable
.
97. D) Spargers: To introduce air.
Porous type: sintered glass/metal/ceramics
Orifice type: perforated pipes as rings or crosses
Nozzle type: most modern (for lab or large scale)
Combined type: sparger-agitator (for small scale)
98. Ideal Characteristics of Medium
•Should provide good Carbon, nitrogen and minerals source
•Should maintain proper C/N ratio
•Good growth and high yield
•Buffer capacity
•Allow to maintain proper pH and temperature
•Allow proper aeration and mixing
•Less viscous
•No foam
•Neutralization of growth products
•Genetic stability
•Good aeration and agitation
•Easy product recovery
•Precursor provision
•Allow or prevent sporulation
•Rapidly available components throughout the year
•Economical/cheap
•Consistent quality
99. Design of a fermentation medium
Choice of a good medium for success of fermentation:
Points to be considered while designing fermentation medium
•Desirable properties of a fermentation medium
•Ideal requirements of a good production medium
•Categories of media: synthetic, organic, crude & complex
100. Merits & demerits of synthetic media
•Advantages
–Ease of preparation
–Easy to follow metabolic pathways
–Recovery simple
–No foam formation
–Less impurities or less by-products
–Less pigment formation
–Ease of purification
•Disadvantages
–Highly expensive
–Low yields
102. Process control and monitoring
•Process parameters to be monitored
Sugar consumption
pH
Temperature
Fermentation time (h)
Agitation
Cell Dry Weight
Product
Computer softwares have been developed to monitor and change the process on line
103. Scale Up:
•Scale up: The transfer of a process from small-scale laboratory equipment to large-scale commercial equipment
•Pilot experiment
–To test the feasibility of the lab scale fermentation process in a semi-industrial scale
–Pilot fermentors normally have a size ranging from 100 L to 10,000 L, depending on the products to be mass produced later.
104. Fermentor sizes for various purposes
Fermentor sizes for various industrial processes
105. Problems emerging during the scale up
•As the size of the equipment is increased, the surface area-volume ratio changes
•Large fermentor has much more volume for a given surface area
•It is obviously more difficult to mix the big tank than the small flask
106. Criteria for Scale up
•Similar or same O2supply rate (mass transfer)
•Similar turbulance rate
•Similar physical & biological parameters
•Similar substrate utilization
•Additional conventional criteria
107. Isolation of mutants
•Inmolecularbiologyandgenetics,mutationsarechangesinagenomicsequence:theDNAsequenceofacell'sgenomeortheDNAorRNAsequenceofvirus.Theycanbedefinedassuddenandspontaneouschangesinthecell.Mutationsarecausedbyradiation,viruses,transposonsandmutagenicchemicals,aswellaserrorsthatoccurduringmeiosisorDNAreplication.
•Mutantsmaybedefinedas—‘variationsofgeneticstructuresthateventuallybreedtrue’.OR
•Anorganismthathascharacteristicsresultingfromchromosomalalterationoranorganismthatresultsduetomutation.
130. Design of fermentation process
•Thevery‘design’offermentationprocessesessentiallyrequirethemostpredominantcomponentofthemediai.e., water,whereinthemicroorganismstendtogrow.
•Thereareseveralvitalandcriticalfactorsthatinvariablygovernaswellasplayanimportantroleinthemediadesignofvariousfermentationprocesses,suchas:
(i)Qualityofwater,
(ii)Qualitycontrolofrawmaterials,
(iii)Nutritionalrequirements,
(iv)Sterilizationpractices,and
(v)Mediapreparation.
131. (i) Quality of water
•Theprevalentqualityofwaterisobviouslyofthegreatestimportancebyvirtueofthefactthatitnotonlyaffectspredominantlythemicrobialgrowth,butalsotheessentialproductionofspecificbioproducts.
•Inthepast,itwasactuallyapracticetoerectandestablishthesocalled‘traditionalbrewingcentres’particularlyinsuchlocationsthatmoreorlessprovidednaturalsprings(i.e.,naturalsources)soastoobtainveryhighqualityofsoft,sweetandpotablewaterwithoutthecumbersomeneedtoresorttoextensiveandexpensive‘pretreatment’.
•However,thepresentdaypracticeessentiallyneedstheutilizationofcommercial-scaledemineralizedwaterplants(i.e.,DM-Plants),reverse- osmosisplants(i.e.,RD-Plants)etc.,toobtainpurewaterrequiredforthefermentationprocesses
132. (ii) Quality control of Raw materials
•Besides,watertheotherchemicalconstituentse.g., pasteurizedwort(maltextractsolution),salts,acidsetc.,mustbeofrelativelybettergradeandqualitysoastoobtainflawlessoptimizedfermentationyieldingspecificbioproducts.
