CURRENT TRENDS AND OPPORTUNITIES IN MICROBIOLOGICAL

RESEARCHS

Prof. K. Manjunath

Professor

Department of Microbiology and Biotechnology

Bangalore University

What is Microbiology?

Micro - too small to be seen with the naked eyeBio - lifelogy - study of

Organisms included in the study of Microbiology

1. Bacteria-Bacteriology

2. Protozoans-Protozoology

3. Algae-Phycology

4. Parasites-Parasitology

5. Fungi,Yeasts and Molds -Mycology

6. Viruses-Virology

Golden Age of Microbiology 1857 - 1914

Pasteur•Pasteurization•Fermentation

Joseph Lister•Phenol to treat surgical wounds – 1st attempt to control infections caused by microoganisms

Robert Koch•Koch’s Postulates

Edward Jenner•vaccination

Paul Erlich•1st synthetic drug used to treat infections•Salvarsan - arsenic based chemical to treat Syphilis

“salvation” from Syphilis

Food Microbiology

• Microorganisms in Food• Food Preservation• Food-borne Illness• Fermented Foods

Microorganisms in Food• Factors affecting microbial growth in food

– composition– pH– presence and availability of water– oxidation-reduction potential

• altered by cooking– physical structure– presence of antimicrobial substances– temperature

• lower temperatures retard microbial growth– relative humidity

• higher levels promote microbial growth– atmosphere

• oxygen promotes growth– modified atmosphere packaging (MAP)

• use of shrink wrap and vacuum technologies to package food in controlled atmospheres

Microorganisms in Food

• Antimicrobial substances– coumarins – fruits and vegetables– lysozyme – cow’s milk and eggs– aldehydic and phenolic compounds – herbs and

spices– allicin – garlic– polyphenols – green and black teas

Microorganisms in Food

• Food spoilage– results from growth of microbes in food

• alters food visibly and in other ways, rendering it unsuitable for consumption

– involves predictable succession of microbes– different foods undergo different types of spoilage

processes– toxins are sometimes produced

• algal toxins may contaminate shellfish and finfish

Microorganisms in Food

• Toxins– ergotism

• toxic condition caused by growth of a fungus in grains

– aflatoxins• carcinogens produced in fungus-infected grains and nut

products

– fumonisins• carcinogens produced in fungus-infected corn

Food Preservation

• Removal of Microorganisms– usually achieved by filtration– commonly used for water, beer, wine, juices, soft

drinks, and other liquids

Food Preservation

• Low Temperature– refrigeration at 5°C retards but does not stop

microbial growth– microorganisms can still cause spoilage with

extended spoilage– growth at temperatures below -10°C has been

observed

Food Preservation

• Canning– food heated in special containers (retorts) to 115° C for 25

to 100 minutes– kills spoilage microbes, but not necessarily all microbes in

food

– Spoilage of canned goods• spoilage prior to canning• underprocessing• leakage of contaminated water into cans during cooling

process

Food Preservation

• Pasteurization– kills pathogens and substantially reduces number

of spoilage organisms– different pasteurization procedures heat for

different lengths of time– shorter heating times result in improved flavor

Food Preservation

• Reduced water availability– Drying– Freeze-drying (lyophilization)– Addition of high concnetrations of solutes such as

sugar or salt

Food Preservation

• Chemical-Based Preservation– GRAS

• chemical agents “generally recognized as safe”

– pH of food impacts effectiveness of chemical preservative

Food Preservation

• Radiation– ultraviolet (UV) radiation

• used for surfaces of food-handling equipment• does not penetrate foods

– radappertization• use of ionizing radiation (gamma radiation) to extend shelf life or

sterilize meat, seafoods, fruits, and vegetables• kills microbes in moist foods by producing peroxides from water• peroxides oxidize cellular constituents

Food Preservation

• Microbial Product-Based Inhibition– Bacteriocins: bactericidal proteins active against related

species– some dissipate proton motive force of susceptible bacteria– some form pores in plasma membranes– some inhibit protein or RNA synthesis– e.g., nisin: used in low-acid foods to inactivate Clostridium

botulinum during canning process

Food-borne Illness

• Food-Borne Infection– ingestion of microbes, followed by growth, tissue

invasion, and/or release of toxins

• Food-Borne Intoxications– ingestion of toxins in foods in which microbes

have grown– include staphylococcal food poisoning, botulism,

Clostridium perfringens food poisoning, and Bacillus cereus food poisoning

Food-borne Illness

• Detection of Food-Borne Pathogens– culture techniques– immunological techniques - very sensitive– molecular techniques

• probes used to detect specific DNA or RNA• sensitive and specific

Food-borne Illness

• Detection of Food-Borne Pathogens– PulseNet

• established by Centers for Disease Control• uses pulsed-field gel electrophoresis under carefully controlled

and duplicated conditions to determine distinctive DNA pattern of each bacterial pathogen

• enables public health officials to link pathogens associated with disease outbreaks in different parts of the world to a specific food source

– FoodNet• active surveillance network used to follow nine major food-

borne diseases• enables public health officials to rapidly trace the course and

cause of infection in days rather than weeks

http://www.cdc.gov/foodnet/http://www.cdc.gov/pulsenet/

Fermented Foods

• Alcoholic Beverages– Alcohol is produced from fermentation by the

yeast Saccharomyces cerevisiae

• Bread• Dairy Products• Other Fermented Foods

Fermented Foods• Beer

– Produced by the fermentation of malted grain• Malted grain: Grain that has been allowed to

germinate, then dried in a kiln & perhaps roasted• Germinating the grain causes the production of a

number of enzymes, most notably α- and β-amylase• Malted grains that may be used are barley, rye, or

wheat• Unmalted grains, such as rice or corn, may also be

used

Fermented Foods• Wine

– Produced from the fermentation of fruit juice, usually from grapes

– The grapes are crushed to form a “must”• For white wines, white grapes are usually used, and the skins

are removed from the must (“pressing”) before fermentation• For red wines, red or black grapes are used, and the skin is

allowed to remain during fermentation• For rosé wines, red grapes are used and the juice is allowed to

remain in contact with the skins just long enough for a rose or pink color to develop

Fermented Foods• Wine

– Secondary fermentation and aging• Takes 3 – 6 months• Done in either stainless steel vessels or in oaken

barrels• The vessel is kept airtight to prevent oxidation• Proteins are broken down, & particles settle

– Blending and bottling http://en.wikipedia.org/wiki/Winemaking

Fermented Foods• Distilled spirits

– Produced by the fermentation of grain mash (similar to beer), followed by distillation to increase the alcohol content

– Different types of grain are used to produce different types of whisky

http://en.wikipedia.org/wiki/Whiskey

http://www.thewhiskyguide.com/

Fermented Foods• Bread

– involves growth of Saccharomyces cerevisiae (baker’s yeast) under aerobic conditions

– maximizes CO2 production, which leavens bread

– other microbes used to make special breads (e.g., sourdough bread)

– can be spoiled by Bacillus species that produce ropiness

Fermented Foods• Yogurt

– Milk is feremented by a mixture of Streptococcus salivarius ssp thermophilus and Lactobacillus bulgaricus (official name Lactobacillus delbrueckii ssp. bulgaricus). Often these two are co-cultured with other lactic acid bacteria for taste or health effects (probiotics). These include L. acidophilus, L. casei and Bifidobacterium species.

– Acid produced from the fermentation causes the protein in the milk (casein) to coagulate into a semisolid curd

– If you want strawberries or peaches, you must add them after the yogurt is made

http://en.wikipedia.org/wiki/Yogurt

Fermented Foods• Cheese

– Milk is treated with lactic acid bacteria and an enzyme called rennin that partially hydrolyses the protein and causes it to coagulate into “curds.” The liquid portion of the milk at this time is called “whey.”

– The whey is separated from the curds, and the curds are aged (“ripened”)

– Different microbes in the early and late stages of processing give rise to cheeses with different characteristics

http://www.realcaliforniacheese.com/

Fermented Foods• Other fermented foods

– sausages– hams– bologna– salami– izushi – fish, rice, and vegetables– katsuobushi – tuna– sauerkraut

Microbial Diversity

AlgaeRed - Rhodophyta

Brown - PhaeophytaGreen - ChlorophytaBlue-green algae are

BACTERIACyanobacteria

AlgaeRed - Rhodophyta

Brown - PhaeophytaGreen - ChlorophytaBlue-green algae are

BACTERIACyanobacteria

ProtozoaMotile / unicellular

PseudopodiaPhagocytosis

ProtozoaMotile / unicellular

PseudopodiaPhagocytosis

FungiMolds

Spores / mycelia / hyphaeYeasts / budding

FungiMolds

Spores / mycelia / hyphaeYeasts / budding

EUKARYOTES

PROKARYOTES

BACTERIA - Eubacteria ARCHAEA - Archaebacteria

How diverse are they?

• Diverse range of species• Earliest life on the planet• Anaerobic then aerobic• Three Kingdoms (1977)• 16S rRNA Analysis• Eukaryote Plants &

Animals• Bacteria• Archaea• Extreme living microorganisms

• Diverse range of species• Earliest life on the planet• Anaerobic then aerobic• Three Kingdoms (1977)• 16S rRNA Analysis• Eukaryote Plants &

Animals• Bacteria• Archaea• Extreme living microorganisms

3 – 3.5 billion yearsREDUCED ATMOSPHERE

Eubacteria

Plants & Animals Archaea

OXIDISED ATMOSPHERE

Microbial Diversity. Three Kingdoms

Microbial Diversity. The Third Kingdom - Archeae – Summary of

Differences

Eubacteria Archaebacteria

Peptidoglycan wall Cell wall variantsRibosomal RNA Very differentRNA polymerase Several enzymesMembrane lipids Ether-linked/branchedProtein synthesis Very differentNo methanogenesis Some are methanogensAntibiotic sensitivity Insensitive to many

Eubacteria Archaebacteria

Peptidoglycan wall Cell wall variantsRibosomal RNA Very differentRNA polymerase Several enzymesMembrane lipids Ether-linked/branchedProtein synthesis Very differentNo methanogenesis Some are methanogensAntibiotic sensitivity Insensitive to many

Range of cellular morphologies in eubacteria

Bioinformatics

Bioinformatics:

A subject that teaches application of computational

tools and approaches for expanding the use of

biological, medical, behavioral, health and other

data, including those to acquire, store, organize,

archive, analyze and visualize such data.

Future?• The Indian Bioinformatics market, which is only

2.5% of the global market, has the potential to capture 5% of the global pie, provided the government ushers in necessary changes. According to a report ‘Building Blocks of Bioinformatics: Human Resource Requirements In India’, prepared by CII and DIT, the future seems very bright for the industry since majority of the Indian Bioinformatics companies are planning to increase their scale of operations.

Computer to Digital life to lifeCan human-made systems be made to possess properties

of life?• Digital Systems are used, to perform experiments aimed at revealing the

principles of living systems

• This effort is truly interdisciplinary and runs the gamut from biology, chemistry and physics to computer science and engineering

• Computational effort concerns the search for principles of living systems • Computational experiments consider

life "as it could be" • Many problems in life science have algorithmic aspects. • Among those, the {protein folding problem} is one• Proteins are polymer chains consisting of monomers of twenty different kinds,

which tend to {fold}, to form a very specific and stable geometric pattern, known as the protein's {native state}

• Human diseases are linked to specific genes • Majority of traits and diseases appear to be {polygenic}, in that they involve the

complex interactions, as in a many-input Boolean circuit, of many genes.

Life Science Vs Computer Science

• Life Science is frustratingly holistic?• It emphasizes the importance of the whole and the interdependence of its

parts like in CS• Computer science has provided highly useful tools for collecting,

exchanging and analyzing data• Modeling and simulation of Data• Finding the right data structure or algorithm can give answers to life

science problems • Computer science algorithms made it possible to put together a vast

amount of data from sequencing machines when the human genome was sequenced.

• Computer science’s computational paradigm has shaped new modes of inquiry in life sciences  

 

•

DNA?

Genes?

Protein Sequence?

Structure?

Expression?

Path Way?

EMBL-Bank DNA Sequences

UniProt Protein Sequences

EMSD Macromolecular Structure Data

Array-Express Microarray Expression Data

EnsEMBL Human Genome

Gene Annotation

Molecular medicine

• The human genome has profound effect on the fields of biomedical research and clinical medicine. Every disease has a genetic component.

• This may be inherited (as is the case with an estimated 3000-4000 hereditary disease including Cystic Fibrosis and Huntingtons disease) or a result of the body's response to an environmental stress which causes alterations in the genome (eg. cancers, heart disease, diabetes.).

• From Human Genome Project Data Base we can search for the genes directly associated with different diseases and understand the molecular basis of these diseases more clearly.

• This new knowledge of the molecular mechanisms of disease will enable better treatments, cures and even preventative tests to be developed.

Personalized medicine• Clinical medicine will become more personalized with the

development of the field of pharma-co-genomics. • This is the study of how an individual's genetic inheritance affects the

body's response to drugs. • At presentAt present, some drugs fail to make it to the market because a small

percentage of the clinical patient population show adverse affects to a drug due to sequence variants in their DNA.

• As a result, potentially life saving drugs never makes it to the marketplace.

• TodayToday, doctors have to use trial and error to find the best drug to treat a particular patient as those with the same clinical symptoms can show a wide range of responses to the same treatment.

• In futureIn future, doctors will be able to analyse a patient's genetic profile and prescribe the best available drug therapy and dosage from the beginning.

Preventative medicine

• With the specific details of the genetic mechanisms of diseases being unraveled, the development of diagnostic tests to measure a persons susceptibility to different diseases may become a distinct reality.

• Preventative actions such as change of lifestyle or having treatment at the earliest possible stages when they are more likely to be successful, could result in huge advances in our struggle to conquer disease.

Gene therapy

• In the not too distant future, the potential for using genes themselves to treat disease may become a reality.

• Gene therapy is the approach used to treat, cure or even prevent disease by changing the expression of a persons genes.

• Currently, this field is in its infantile stage with clinical trials for many different types of cancer and other diseases ongoing.

Drug development

• At present all drugs on the market target only about 500 proteins.

• With an improved understanding of disease mechanisms and using computational tools to identify and validate new drug targets, more specific medicines that act on the cause, not merely the symptoms, of the disease can be developed.

• These highly specific drugs promise to have fewer side effects than many of today's medicines.

Microbial genome applications

• Microorganisms are ubiquitous, that is they are found everywhere. They have been found surviving and thriving in extremes of heat, cold, radiation, salt, acidity and pressure.

• They are present in the environment, our bodies, the air, food and water. Traditionally, use has been made of a variety of microbial properties in the baking, brewing and food industries.

• The arrival of the complete genome sequences and their potential to provide a greater insight into the microbial world and its capacities could have broad and far reaching implications for environment, health, energy and industrial applications.

• By studying the genetic material of these organisms, scientists can begin to understand these microbes at a very fundamental level and isolate the genes that give them their unique abilities to survive under extreme conditions.

Crop improvement

• Comparative genetics of the plant genomes has shown that the organisation of their genes has remained more conserved over evolutionary time than was previously believed.

• These findings suggest that information obtained from the model crop systems can be used to suggest improvements to other food crops.

• At present the complete genomes of Arabidopsis thaliana (water cress) and Oryza sativa (rice) are available.

Insect resistance• Genes from Bacillus thuringiensis that can control a

number of serious pests have been successfully transferred to cotton, maize and potatoes.

• This new ability of the plants to resist insect attack means that the amount of insecticides being used can be reduced and hence the nutritional quality of the crops is

increased.

Improve nutritional quality • Scientists have recently succeeded in transferring genes

into rice to increase levels of Vitamin A, iron and other micronutrients.

• This work could have a profound impact in reducing occurrences of blindness and anaemia caused by deficiencies in Vitamin A and iron respectively.

• Scientists have inserted a gene from yeast into the tomato, and the result is a plant whose fruit stays longer on the vine and has an extended shelf life.

Development of Drought resistance varieties

• Progress has been made in developing cereal varieties that have a greater tolerance for soil alkalinity, free aluminum and iron toxicities.

• These varieties will allow agriculture to succeed in poorer soil areas, thus adding more land to the global production base.

• Research is also in progress to produce crop varieties capable of tolerating reduced water conditions.

Veterinary Science

• Sequencing projects of many farm animals including cows, pigs and sheep are now well under way in the hope that a better understanding of the biology of these organisms will have huge impacts for improving the production and health of livestock and ultimately have benefits for human nutrition.

Comparative Studies • Analyzing and comparing the genetic material of different species is

an important method for studying the functions of genes, the mechanisms of inherited diseases and species evolution.

• Bioinformatics tools can be used to make comparisons between the numbers, locations and biochemical functions of genes in different organisms.

• Organisms that are suitable for use in experimental research are termed model organisms.

• They have a number of properties that make them ideal for research purposes including short life spans, rapid reproduction, being easy to handle, inexpensive and they can be manipulated at the genetic level.

• An example ofexample of a human model organism is the mouse.• Mouse and human are very closely related (>98%)(>98%) and for the most

part we see a one to one correspondence between genes in the two species.

ENVIRONMENTAL MICROBIOLOGY

Bioremediation

• A technology that encourages growth and reproduction of indigenous microorganisms (bacteria and fungi) to enhance biodegradation of organic constituents in the saturated zone .

• Can effectively degrade organic constituents dissolved in groundwater and adsorbed onto the aquifer matrix.

• Generally requires a mechanism for stimulating and maintaining the activity of the microorganisms, e.g., addition of an electron acceptor (oxygen, nitrate); nutrients (nitrogen, phosphorus); and an energy source (carbon)

Microbial Metabolism

• Need nitrogen, phosphorus, sulfur, and a variety of trace nutrients other than carbon

• Carbon is often the limiting factor for microbial growth in most natural systems

• Acclimatization period - a period during which no degradation of chemical is evident; also known as adaptation or lag period

• Length of acclimatization period varies from less than 1 h to many months

• Acclimatization of a microbial population to one substrate frequently results in the simultaneous acclimatization to some structurally related molecules

Metabolism Modes

• Aerobic: transformations occur in the presence of molecular oxygen (as electron acceptor), known as aerobic respiration

• Anaerobic: reactions occur only in the absence of molecular oxygen, subdivided into:– Anaerobic respiration– Fermentation– Methane fermentation

Summary of Metabolism Modes

Microbial Reactions and Pathways

• Dechlorination - a chlorine atom is replaced with a hydrogen atom • Hydrolysis - a cleavage of an organic molecule with the addition of water• Cleavage - an organic compound is split or a terminal carbon is cleaved off an organic chain• Oxidation - breakdown of organic compounds using nucleophilic form of oxygen (H2O, OH-, etc); releases electrons• Reduction - breakdown of organic compounds using electrophilic form of hydrogen (H+); takes electrons

Microbial Catalyzed Reactions

• Cultivation dependent - ideal, but has problems!• Cultivation independent:

– Sequence information - eg. 16S rRNA sequences, genome sequences

– rRNA targeted probes, eg. FISH(Fluorescent In Situ Hybridization)Allows a visual inspection of phylogenetic groups of cells in a natural sample

How to study microbial diversity and ecology

Detection of microbial diversity at molecular level

Environmental samples

Enrichment

Specific Medium

Pure Culture

Analysis

Morphology Cell wall Stain Cyst Biochemical

DNA

PCR16S rRNA/ITS/IGS/SSUphylogenetic/functional gene

Cloning

Sequencing

ARDRA/RFLP/DGGE/TRFLP

ISH with probes

AUTORDIOGRAPH

FISH

Tracking and phylogentic analysis

HybridizationWith probe/ genomic dNA

Cultivation dependent

• Pure cultures are the basis of the traditional way of studying bacteria

• Usually only 1% of cells in a natural sample will form colonies on plates

• Different bacteria have different abilities to be cultured; from easy to difficult

• Known examples that cannot be cultured

Bacteria: examples that have not yet been cultured

• Mycobacterium leprae (leprosy)• Treponema pallidum (syphilis)• Epulopiscium fishelsoni• All members of the TM7 phylum

(a major lineage of Bacteria)

Mycobacterium leprae

Treponema pallidum

Epulopiscium

Cultivation independent:

• 16S rRNA sequences, specific genes, mRNAs, whole genome sequences, metagenomes

• Discovered many new groups of Bacteria- but physiologies yet unknown

• Can use sequence information to directly visualise specific bacteria in situ (in their natural state)Fluorescent In Situ Hybridization (FISH) ...

• Permeabilize cells so that the DNA probe can enter Allow it to find its matching sequence on rRNA

- short DNA sequence

- complementary to rRNA

- specific sequence (eg. to genus)

- fluorescent tag attached

rRNA

FISH - Fluorescent In Situ Hybridisation

• Fluorescent DNA probe will bind to rRNA in the cells only if it exactly matches complementary sequence of rRNA target region

• Many different coloured fluors, so can do simultaneous probes for different genera, families....

View cells (in situ) under fluorescent microscope, and see what cells fluoresce, showing they have bound the probe

FISH - Fluorescent In Situ Hybridisation

FISH - Fluorescent In Situ Hybridisation

•final note: the vast majority of bacteria are not pathogens. They work for us, in the environment

DNA ISOLATION

ENVIRONMENTAL SAMPLE MICROBIAL ANIMAL TISSUE

PLANT MATERIALSSOILWATER AIR

FUNGUSBACTERIAACTINOMYCETSCYNOBACTERIAALGAE

HAIRBLOODCLINICAL

Plant material Soil Water

ENVIRONMENTAL SAMPLE

Air

• Root, shoot, leaves etc• Grind the material in liquid

Nitrogen with mortar and pestal

• Perform CTAB method for isolating gDNA

Materials:CTAB

(cetyltrimethylammonium bromide) buffer

Microfuge tubesMortar and PestleLiquid NitrogenMicrofugeAbsolute Ethanol (ice cold)70 % Ethanol (ice cold)7.5 M Ammonium Acetate55o C water bathChloroform : Iso Amyl

Alcohol (24:1)Water (sterile)

• Soil or sediment• Bead beating with

Phosphate buffer saline

Materials:Extraction buffer (pH 8.0)* 5% SDS (autoclave to sterilize) Dithiothrietol 1 M†

Phenol (Tris-saturated)Chloroform:isoamyl (24:1) Choroform Sodium acetate 3M Isopropanol Ice-cold 70% EtOH10% PVPP solution

• Centrifugation of water sample .

• Filter water through membrane vaccume filter to filter microbial community

• Take out membrane cut into small pieces to isolate DNA

air is drawn by a suction pump through a narrow inlet tube into a small flask containing the collection medium

MICROBIAL

Bacteria/archea Fungi Actinomycetes Algae

Lyophilize young mycelia

Use CTAB method further

CTAB extraction buffer 0.1M Tris, 1% CTAB0.7M NaCl10mM EDTA1% beta-mercaptanolWater

DNA extraction from algae and seagrass is hampered by the large quantity of polysaccharides and polyphenolics produced within the thalli (leaves) of many species.

CTAB method•Lysozyme /•SDS Lysis•Phenol chloroform extraction•Na salt precipitation•Ethanol concentration

Fellowships for higher studies in India:• DST SC Bose fellowship

• CSIR Jawahar Lal Nehru Post Doctoral Fellowship

• UGC Dr. D.S. Kothari Post Doctoral fellowship

• DST young scientist Award (SERC Division)

• DST Woman Scientist fellowship

• DBT Post Doctoral Fellowship

• CSIR Research Associate scholarship

• K.S. Krishnan Research fellowship BARC

• UGC SAARC Fellowship

• Rajiv Gandhi Science Talent research fellowship

• JRF/SRF awarded by – UGC, CSIR, DBT, ICMR, ICAR

• Junior research scholarship for cancer biology TATA memorial centre and TATA memorial hospital.

• Homi Bhaha centre for science education scholarship

Fellowships for higher studies in Foreign countries:

1. DAAD Fellowship: Indo German Fellowship for Ph.D. and Post Doctorate students

2. JSPS Fellowship: Indo- Japanese fellowship for Post Doctorate students

3. Jawahar Lal Nehru Full bright Fellowship: Indo- US fellowship for PhD and Post Doctorate students.

4. Turkish Biotech/Agriculture research for pursuing Ph.D. in Turkey

5. DBT-TWAS Biotechnology fellowship for Post Doctorate Research Overseas

6. Belgium Govt. Scholarship from External Division ministry of Human Resource and Development

Research GroupDr. Pranjali Vishwakarma Soil microbial diversity analysis using(D.S. Kothari Post Doc fellow) metagenomic tools

Dr. Arun Jyoti Mathews Bioaerosols and occupational health hazard(Associate Prof.)

Ms. Rachna Garg Isolation of bioactive compounds from(CSIR-SRF) medicinal plant

Alaknanda Sarkar Isolation of bioactive compunds from(CSIR-SRF) marine microorganism

Pawan R. Assesment of airquality(Research scholar)

Ms. Ponama Plant Tissue culter and secondary (Teacher fellow) metabolite

Ms. Vijaya Rahmnolipids from microbes(Teacher fellow)