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A.p singh


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A.p singh

  1. 1. Detailed Literature Review On “Comprehensive Studies on Isolation and Biochemical & Microbial Characterization of Antibiotic Producing Microorganisms from Soil Samples of Certain Industrial Areas”. Submitted To IFTM University Moradabad, Lodhipur – Rajput, Delhi Road Moradabad (U.P) For Registration of the Degree of Doctor of Philosophy in Microbial Biotechnology By Mr. Atul Pratap Singh (M.Sc, M.Phil) Department of Microbiology Dolphin (P.G) Institute of Biomedical and natural sciences, Dheradhun (H.N.B Garhwhal University, Shrinagar – Uttarakhand) Under Guidance of Dr. Sanjay Mishra Professor, School of Biotechnology, IFTM University, Lodhipur – Rajput, Delhi Road12/12/2012 Moradabad (U.P)
  2. 2. 1. Proposal of Research Work: Area/Specialization of the Research Work: Microbial Biotechnology. A study on “Comprehensive Studies on Isolation,Biochemical Characterization and Microbial Characterization ofAntibiotic Producing Microorganisms from Soil Samples ofCertain Industrial Areas”. The entire study will be divided in appropriate aims andobjectives likely to be initiated, propagate and conclude the entirework in the due course of time.12/12/2012
  3. 3. 2. Literature Review Of Research Work  In the present Research Study, a soil bacterium with the antibiotic activity is screened studied for morphological as well as Biochemical characteristics which can provide valuable information about the Antibiotic producers microbial strain.  As industrialization expands, petroleum hydrocarbons become a greater potential source of contaminants in the soil and water environments, According to (Duraipandiyan V., Sasi A.H., IslamV.I.H., Valanarasu M. and Ignacimuthu S. (2010) J Mycologie Médicale, 20 (1), 15-20).  The density of living organisms in soil is very high i.e. as much as billions / gm of soil, usually density of organisms is less in cultivated soil than uncultivated / virgin land and population decreases with soil acidity.12/12/2012
  4. 4. Top soil, the surface layer contains greater number of microorganisms because it is well supplied with Oxygen and nutrients. Lower layer is depleted with Oxygen and nutrients hence it contains fewer organisms. According to Ramadan M.F, Asker M.M.S, Ibrahim Z.K (2008). Functional bioactive compounds & biological activities of Spirulina platensis lipids, Czech. J. Food. Sci, 26: 211-222. To remediate oil terminals contaminants in these environments, biostimulation and bioaugmentation are generally considered as techniques by, (Chang and Lin 2006).12/12/2012
  5. 5. Biostimulation is a technique that relies on increasing theactivity of the resident bacteria by adding the factors that arelimiting activity, such as nutrients or air.Biostimulation technique is first used up by (Oskay M., TamerA.U. and Azeri C. (2004) African J Biotechnol, 3 (9), 441- 446).The medium used to estimate the population density ofphosphate solubilizers show a clear zone around the coloniesindicating phosphate solubilization. According to (Alanis A.J.(2005) Archives Med Res, 36, 697-705).12/12/2012
  6. 6. 3. Antimicrobial Agents Antimicrobial agents are natural or synthetic chemical substances which have the capacity of inhibiting or terminating total metabolic cell activity. The major class of antibacterial agents are the class I. Beta- lactams (including penicillins, cephalosporins, monobactams, carbapenems), aminoglycosides, tetracyclines, sulfonamides, macrolides (such as erythromycin), quinones and glycopeptides (vancomycin). According to Butler M.S. and Buss A.D. (2006) Biochem Pharmacol, 71, 919-929.12/12/2012
  7. 7. These secondary metabolites Penicillins andcephalosporins can affect many metabolic reactions in acell in order to render effect.According to (Newman D.J. and Cragg G.M. (2007) JNat Prod, 70, 461-477.) Penicillins and cephalosporinsmode of action is the biosynthesis of thepeptidoglycan present in the bacterial cell wall. They affectspecifically the transpeptidase that forms the peptide cross-linking.12/12/2012
  8. 8. class II. Antimicrobials, known as 6-Aniniluracil, inhibits DNA polymerase.  class III. of antimicrobials like tetracyclines, chloramphenicol and macrolides inhibit protein synthesis.  According to ( Saadoun I. and Gharaibeh R. (2003) J Arid Environ, 53, 365-371). (Baltz R.H. (2007) Microbe, 2,125-131). Both, gram-positive and gram-negative microorganisms, posses these proteins and other antimicrobials, such as the quinolones, noviobioci, inhibit DNA replication.12/12/2012
  9. 9. Over 5,000 antibiotics have been identified from the cultures ofGram-positive and Gram-negative organisms, and filamentousfungi, but only about 100 antibiotics have been commerciallyused to treat human, animal and plant diseases.Fungal strains and streptomyces members are extensively usedin industrial antibiotic production. Bacteria are easy to isolate,culture, maintain and to improve their strains.Bacillus species being the predominant soil bacteria because oftheir resistant endospore formation and production of vitalantibiotics like bacitracin etc, are always found inhibiting thegrowth of the other organisms.12/12/2012
  10. 10.  According to (Hube A.E, B.H. Soller and U. Fischer 2009)Phylogenetic classification of heterotrophic bacteria associated withfilamentous marine cyanobacterial in culture, Systematic andApplied Microbiology, 32: 256-265. Enhancement in the antibioticproduction is studied under various parameters like temperature, pH,carbon source concentration, and Sodium nitrate concentration,which may help in the industrial production.Though a large list of antibiotics are commercially available, andthis work may provide some potential information on the antibioticproduction and the control of microbial strains. 12/12/2012
  11. 11. According to Kumar N, Singh RK, Mishra SK, Singh AK.Isolation and screening of soil Actinomycetes as source ofantibiotics active against bacteria. International Journal ofMicrobiology Research 2010; 2: 12-18. The genus, Streptomycete, is responsible for the formation ofmore than 60 % of known antibiotics from the soil. while afurther 15 % are made by a number of related Actinomycetes spp,Micromonospora, Actinomadura, Streptoverticillium andThermoactinomycetes Antibiotics, because of their industrialimportance, are the best known products of actinomycetes.12/12/2012
  12. 12. 4. Soil Microbial Interactions According to (Brun YV, Skimkets LJ. Prokaryotic development.ASM Press, 2000; 11-31). Competition is an interaction encountered in all habitats since the organism’s present need to do so in order to survive”. According to (Pandey B., Ghimire P. and Agrawal V.P. (2004) International Conference on the Great Himalayas): Climate, Health, Ecology, Management and Conservation. Besides providing these nutrients, plant secondary metabolites that are generally toxic to microorganisms will need to be degraded or detoxified by certain microbes.12/12/2012
  13. 13. 5. Soil Antimicrobial Agent Producing Microbes: Pandey et al.(2009), states that the top cultivable antimicrobial agent producers present in soils are the Actinomycetes.  About 10% - 33% of the total bacterial community present in soil is comprised by these bacteria, being the genera Streptomyces and Nocardia the most abundant Actinomycetes found in soil. clinical importance like, for example, amphotericin, erythromycin, streptomycin, tetracycline, and rifamycin etc.12/12/2012
  14. 14. Another group of gram-positive bacteria present in soil andresponsible for the production of antimicrobial agents Forexample, B. subtilis can produce non ribosomal oligopeptideswith antifungal and antimicrobial properties such as surfactins,inturinics and bacilysin.(Thajuddin. N & G. Subramanian 2005,) Cyanobacterialbiodiversity & potential applications in biotechnology, CurrentScience, 89(1): 47-57. States that Cyanobacteria are known to becolonized by various heterotrophic bacteria.12/12/2012
  15. 15. Nowadays, the drug resistant strains of pathogen emerge more quickly than the rate of discovery of new drugs and antibiotics. Because of this, many pharmaceutical industry have actively involved in isolation and screening of actinomycetes from different untouched habitats, for their production of antibiotics. States by (Duraipandiyan V., Sasi A.H., IslamV.I.H., Valanarasu M. and Ignacimuthu S. (2010) J) Mycologie Médicale, 20 (1), 15-20.  According to him Staphylococcus aureus, for instance, a virulent pathogen that is responsible for a wide range of infections, has developed resistance to most classes of12/12/2012 antibiotics in soil.
  16. 16. In my research study to getting some actinomycetesstrains that produce antibiotics that have not beendiscovered yet and active against drug resistantpathogens. So we need to screen more and more actinomycetesfrom different habitats for antimicrobial activity.12/12/2012
  17. 17. 6. Components of MediaMacronutrients (C, N, P, K).Micronutrients (Fe, Mg, Ca, Na).Vitamins12/12/2012
  18. 18. 7. Types of Microbiological Media Chemically defined media • The exact chemical composition is known Complex (undefined) media • The exact chemical composition is not known • Often consist of plant or animal extracts, such as soybean meal, milk protein, etc.12/12/2012
  19. 19. 8. Methods for Determining Microbial Biomass and Activity (1) Field Samples Profile or Depth Basis Plant Association Composite samples Replicate samples Transportation, Mixing, Grinding, Subsampling, Dilution12/12/2012
  20. 20. 9. Soil Sampling Developing a Sampling PlanWhat sampling strategy (ex: random vs zone vs grid) should I use?What depths should I sample?How many samples should I collect?12/12/2012
  21. 21. 10. Sampling Strategies Random Zone Grid X X X X X X X X X X X X Figure 1. (A) Aerial photograph (w/ random sampling), (B) Management zones, and (C) Field grids . Advantages or disadvantages of each?12/12/2012
  22. 22. 11. How to Collect Soil Sample depth – plowed fields 6 inches to plow depth – no-till or pastures 4 inches deep Composite cores, mix well. Fill a 1-pint soil test bag, clearly labeled with the field identification. 12/12/2012
  24. 24. 12. Identification and Pure Culture of Streptomyces MaterialsIn nature microbial cultures are mixed.Identification relies upon isolating individual colonies.Testing requires pure cultures.As a result isolation technique provides an essentialmicrobiological tool.12/12/2012
  25. 25. Too much or too little… Ideal volume12/12/2012
  26. 26. Cultural Studies in Determining Microbial Biomass and Activity Count and Identify Culturable Organisms, Nutrient Analyses Characteristics or Biomass of Cultural Organisms or ATP, etc. of Soil Samples Organisms Activity and Biomass in the12/12/2012 Laboratory or in Nature
  27. 27. Microscopic Studies in Determining Microbial Biomass and Activity Light and Electron Microscope, Electron Probe, Autoradiography Morphology and Biovolume of Bacteria, Protozoa, Fungi, Algae, etc. Organisms Activity and Biomass in the12/12/2012 Laboratory or in Nature
  28. 28. Process Studies (Chemical Analyses) in Determining Microbial Biomass and Activity Activity Measurements, CO2, NO3-, Nutrient Turnover Rates, Enzyme Studies Metabolic Activity of Sample Organisms Activity and Biomass in the Laboratory or in Nature12/12/2012
  30. 30. 13. Culture Dependent Measurements of Microbial Growth12/12/2012
  31. 31. 14. Plate Count Inoculate Petri plates from serial dilutions12/12/2012
  32. 32. 15. Pour PlateAfter incubation, count colonies on plates that have 25-250 colonies (CFUs)12/12/2012
  33. 33. 16. Most Probable Number (MPN) MethodMultiple tube MPN testCount positive tubes and compare to statistical MPN table. 12/12/2012
  34. 34. 17. Soil ExperimentSensitivity/Inhibition TestingBlood Agar Plate Testing Total HemolysisOxidase Test(Gram neg. rods): Oxidase positiveCatalase TestCatalase positiveTriple Sugar Iron Test12/12/2012
  35. 35. 18.Biochemical AnalysesNitrogen, phosphate, potassium, pH, and humuslevels for the soil were assessed semi-quantitativelyusing commercially – obtained test kits (Luster TMRapitest TM soil Kit). 19. Biostatistical AnalysesBivariate correction statistics using the personcoefficient were performed. Comparison statistics ofthe distribution of narrow and broad – spectrumantibiotic producers relative to other microflora, andcoastal versus inland microflora, were made using. 12/12/2012
  36. 36. 20. Impact of Proposed Research in Academic/Industry:The successful execution of the proposed studies may lead to thefirst specific biochemical and biotechnological/Microbiologicalhypothesis of “Bioremediation”.21.Major Inputs (Infrastructure for Research Work):Necessary infrastructure facilities are available under the umbrellaof School of Biotechnology, IFTM University, Moradabad, U.P.,and shall be provided to the candidate with adequate facilities forcarrying out the major experimental part of the work embodies inthe proposed synopsis herein. Besides, if required, the candidatemay use Dolphin P.G. Institute of Biomedical and NaturalSciences, Dehradoon, Uttarakhand which already exists incollaboration of School of Biotechnology, IFTM University,Moradabad, U.P. An excellent library and round the clock internetfacilities are available at IFTM University, Moradabad, U.P. thatcan be used by the candidate while working on the proposed12/12/2012research plan for his doctoral degree.
  37. 37. 22. References and Sources[1] Margesin and Schinner (2001)[2] Chang and Lin (2006)[3] Rahman et al. (2003).[4] Oskay M., Tamer A.U. and Azeri C. (2004) African JBiotechnol, 3 (9), 441- 446[5] Alanis A.J. (2005) Archives Med Res, 36, 697 705. 12/12/2012
  38. 38. [5] Newman D.J. and Cragg G.M. (2007) J Nat Prod, 70, 461-477. [6] Butler M.S. and Buss A.D. (2006) Biochem Pharmacol, 71, 919-929. [7] Duraipandiyan V., Sasi A.H., IslamV.I.H., Valanarasu M. and Ignacimuthu S. (2010) J Mycologie Médicale, 20 (1), 15-20. [8] Ramadan M.F, Asker M.M.S, Ibrahim Z.K (2008). Functional bioactive compounds & biological activities of Spirulina platensis lipids, Czech. J. Food. Sci, 26: 211-222.12/12/2012
  39. 39. [9] Hube A.E, B.H. Soller and U. Fischer 2009 Phylogeneticclassification of heterotrophic bacteria associated withfilamentous marine cyanobacterial in culture, Systematic andApplied Microbiology, 32: 256-265.[10] Ramadan M.F, Asker M.M.S, Ibrahim Z.K 2008 Functionalbioactive compounds & biological activities of Spirulinaplatensis lipids, Czech. J.[11] Praveen Kumar R, Vijayan D, Leo Antony M, MuthuKumar C and Thajuddin N 2009 Phylogenetic diversity ofcultivable bacteria associated with filamentous non –heterocystous marine cyanobacteria, J. Algal biomass Utln, 1(1):86-101.12/12/2012
  40. 40. 12/12/2012