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 Road
12/12/2012 Moradabad (U.P)

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 of
Antibiotic Producing Microorganisms from Soil Samples of
Certain Industrial Areas”.
 The entire study will be divided in appropriate aims and
objectives likely to be initiated, propagate and conclude the entire
work in the due course of time.
12/12/2012

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. 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. Biostimulation is a technique that relies on increasing the
activity of the resident bacteria by adding the factors that are
limiting activity, such as nutrients or air.
Biostimulation technique is first used up by (Oskay M., Tamer
A.U. and Azeri C. (2004) African J Biotechnol, 3 (9), 441- 446).
The medium used to estimate the population density of
phosphate solubilizers show a clear zone around the colonies
indicating phosphate solubilization. According to (Alanis A.J.
(2005) Archives Med Res, 36, 697-705).
12/12/2012

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. These secondary metabolites Penicillin's and
cephalosporin's can affect many metabolic reactions in a
cell in order to render effect.
According to (Newman D.J. and Cragg G.M. (2007) J
Nat Prod, 70, 461-477.) Penicillins and cephalosporins
mode of action is the biosynthesis of the
peptidoglycan present in the bacterial cell wall. They affect
specifically the transpeptidase that forms the peptide cross-
linking.
12/12/2012

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. Over 5,000 antibiotics have been identified from the cultures of
Gram-positive and Gram-negative organisms, and filamentous
fungi, but only about 100 antibiotics have been commercially
used to treat human, animal and plant diseases.
Fungal strains and streptomyces members are extensively used
in industrial antibiotic production. Bacteria are easy to isolate,
culture, maintain and to improve their strains.
Bacillus species being the predominant soil bacteria because of
their resistant endospore formation and production of vital
antibiotics like bacitracin etc, are always found inhibiting the
growth of the other organisms.
12/12/2012

10.  According to (Hube A.E, B.H. Soller and U. Fischer 2009)
Phylogenetic classification of heterotrophic bacteria associated with
filamentous marine cyanobacterial in culture, Systematic and
Applied Microbiology, 32: 256-265. Enhancement in the antibiotic
production 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, and
this work may provide some potential information on the antibiotic
production and the control of microbial strains.
12/12/2012

11. According to Kumar N, Singh RK, Mishra SK, Singh AK.
Isolation and screening of soil Actinomycetes as source of
antibiotics active against bacteria. International Journal of
Microbiology Research 2010; 2: 12-18.
The genus, Streptomycete, is responsible for the formation of
more than 60 % of known antibiotics from the soil. while a
further 15 % are made by a number of related Actinomycetes spp,
Micromonospora, Actinomadura, Streptoverticillium and
Thermoactinomycetes Antibiotics, because of their industrial
importance, are the best known products of actinomycetes.
12/12/2012

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. 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. Another group of gram-positive bacteria present in soil and
responsible for the production of antimicrobial agents For
example, B. subtilis can produce non ribosomal oligopeptides
with antifungal and antimicrobial properties such as surfactins,
inturinics and bacilysin.
(Thajuddin. N & G. Subramanian 2005,) Cyanobacterial
biodiversity & potential applications in biotechnology, Current
Science, 89(1): 47-57. States that Cyanobacteria are known to be
colonized by various heterotrophic bacteria.
12/12/2012

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 of
12/12/2012
antibiotics in soil.

16. In my research study to getting some actinomycetes
strains that produce antibiotics that have not been
discovered yet and active against drug resistant
pathogens.
 So we need to screen more and more actinomycetes
from different habitats for antimicrobial activity.
12/12/2012

17. 6. Components of Media
Macronutrients (C, N, P, K).
Micronutrients (Fe, Mg, Ca, Na).
Vitamins
12/12/2012

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. 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,
Dilution
12/12/2012

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. 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. 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

23. IDENTIFICATION OF
ISOLATED MICROBES
12/12/2012

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 essential
microbiological tool.
12/12/2012

25. Too much or too little… Ideal volume
12/12/2012

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 the
12/12/2012 Laboratory or in Nature

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 the
12/12/2012 Laboratory or in Nature

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 Nature
12/12/2012

29. MONITORING
AND
ENUMERATION
12/12/2012

30. 13. Culture Dependent Measurements of
Microbial Growth
12/12/2012

31. 14. Plate Count
Inoculate Petri
plates from serial
dilutions
12/12/2012

32. 15. Pour Plate
After incubation, count colonies on plates that
have 25-250 colonies (CFUs)
12/12/2012

33. 16. Most Probable Number (MPN) Method
Multiple
tube MPN
test
Count
positive
tubes and
compare to
statistical
MPN table.
12/12/2012

34. 17. Soil Experiment
Sensitivity/Inhibition Testing
Blood Agar Plate Testing
Total Hemolysis
Oxidase Test
(Gram neg. rods): Oxidase positive
Catalase Test
Catalase positive
Triple Sugar Iron Test
12/12/2012

35. 18.Biochemical Analyses
Nitrogen, phosphate, potassium, pH, and humus
levels for the soil were assessed semi-quantitatively
using commercially – obtained test kits (Luster TM
Rapitest TM soil Kit).
19. Biostatistical Analyses
Bivariate correction statistics using the person
coefficient were performed. Comparison statistics of
the distribution of narrow and broad – spectrum
antibiotic producers relative to other microflora, and
coastal versus inland microflora, were made using.
12/12/2012

36. 20. Impact of Proposed Research in
Academic/Industry:
The successful execution of the proposed studies may lead to the
first specific biochemical and biotechnological/Microbiological
hypothesis of “Bioremediation”.
21.Major Inputs (Infrastructure for Research Work):
Necessary infrastructure facilities are available under the umbrella
of School of Biotechnology, IFTM University, Moradabad, U.P.,
and shall be provided to the candidate with adequate facilities for
carrying out the major experimental part of the work embodies in
the proposed synopsis herein. Besides, if required, the candidate
may use Dolphin P.G. Institute of Biomedical and Natural
Sciences, Dehradoon, Uttarakhand which already exists in
collaboration of School of Biotechnology, IFTM University,
Moradabad, U.P. An excellent library and round the clock internet
facilities are available at IFTM University, Moradabad, U.P. that
can be used by the candidate while working on the proposed
12/12/2012
research plan for his doctoral degree.

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 J
Biotechnol, 3 (9), 441- 446
[5] Alanis A.J. (2005) Archives Med Res, 36, 697 705.
12/12/2012

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. [9] Hube A.E, B.H. Soller and U. Fischer 2009 Phylogenetic
classification of heterotrophic bacteria associated with
filamentous marine cyanobacterial in culture, Systematic and
Applied Microbiology, 32: 256-265.
[10] Ramadan M.F, Asker M.M.S, Ibrahim Z.K 2008 Functional
bioactive compounds & biological activities of Spirulina
platensis lipids, Czech. J.
[11] Praveen Kumar R, Vijayan D, Leo Antony M, Muthu
Kumar C and Thajuddin N 2009 Phylogenetic diversity of
cultivable bacteria associated with filamentous non –
heterocystous marine cyanobacteria, J. Algal biomass Utln, 1(1):
86-101.
12/12/2012

40. 12/12/2012