Medical Microbiology

Microbial Colonization
When microbes find a new host and start to multiply
– called colonisation
A balance can develop between colonised microbes
and humans – will lead to ‘so called’ normal flora
If microbe causes disease – called an infection
If source of microbe is patient’s own flora –called
an endogenous infection
If source of microbe is flora from outside the
patient’s body – called exogenous infection
 Medical Microbiology Terms

Pathogenicity vs. Virulence Infection vs. Disease

PATHOGENICITY: the quality of producing disease or the INFECTION: the colonization and/or invasion and multiplication of
ability to produce pathologic changes or disease pathogenic microrganisms in the host with or without the manifestation
of disease
VIRULENCE: a measure of pathogenicity in terms of dosage
and LD 50 DISEASE: an abnormal condition of body function(s)
DOSAGE: the number of pathogenic microorganisms entering or structure that is considered to be harmful to the affected individual
the host (host); any deviation from or interruption of the normal structure or
function of any part, organ, or system
of the body
LD 50 = the number of microorganisms required to cause lethality BENIGN: a non-life or non-health threating condition
(death) in 50% of the test host MALIGNANT: a disease tending to become progressively worse
(MORBIDITY = illness) and potentially result in death (MORTALITY
TRUE PATHOGEN: any microorganism capable of causing = death)
disease; an infecting agent
CONTAGIOUS: capable of being transmitted from one host to another;
OPPORTUNISTIC PATHOGEN: a usually
harmless communicable; infectious
microorganism that becomes pathogenic under
conditions causing an opportunistic infection favorable
INFECTIOUS DOSE: number of pathogenic organisms required to cause
disease in a given host
 Kochs Postulates
1. The microorganism must be present in every case of
the disease but absent from healthy individuals:
2. The microorganism must be isolated and grown in pure
culture:
3. The cultured microorganism should cause the same
disease when introduced into a healthy host
4. The same microorganism must be re-isolated from the
newly infected host:
Bacteria Characteristics
1. Complete Set of Life Functions: Bacteria are unicellular
organisms that possess all the essential functions of life,
including metabolism, growth, reproduction, and response
to stimuli.

2. Reproduction by Simple Division: Bacteria reproduce

asexually through a process called binary fission. In
binary fission, a single bacterium divides into two identical
daughter cells, each with the same genetic material as
the parent cell.
 Bacteria Characteristics
3.Formation of Visible Colonies: When bacteria
are cultured on a solid growth medium they often
form visible colonies. These colonies consist of
numerous bacterial cells that have divided and
grown into a visible mass making them useful for
bacterial identification and classification.
4.Genetic Material Transfer: Bacteria can transfer
genetic material both vertically (from parent to
offspring) and horizontally (between different
bacteria).
-Vertical gene transfer when the genetic material
is passed from the parent cell to the daughter
cells.
-Horizontal gene transfer, on the other hand,
Characteristics of Gram Positive and Gram negative Bacteria
 Modes of Disease Transmission
Airborne Some bacteria are carried on air currents in droplet nuclei. Q fever,
tuberculosis, and Legionella travel great distances from their origin.

Droplet When an infection is spread via droplets greater than 5 μm in diameter, this
type of spread is not considered airborne given that the droplet is unlikely to travel
through the air for more than 1 m.

Vectors Typically, the arthropod (mosquito, tick, louse) takes a blood meal from an
infected host (which can be human or animal) and transfers pathogens to an
uninfected individual

Vehicular (including food, water, and fomite transmission) Bacterial infection due to
food and water generally develops when bacteria enter the intestine via the mouth.
Modes of Disease Transmission

Burton’s Microbiology for the Health science

Modes of Disease Transmission -Exapmes

Mode of transmission Disease examples

Streptococcal impetigo (skin-to-
skin), gonorrhea (mucus
membrane-to-mucus
Contact
membrane),
Salmonella (fecal–oral), syphilis
(transfusion)
Airborne Tuberculosis, Q fever, legionella
Pertussis, meningococcus,
Droplet
Haemophilus influenzae
Lyme disease (tick), Shigella
Vectors (fly) epidemic typhus (lice),
bubonic plague (fleas)
Campylobacter (food), trachoma
Vehicular
(fomites)
 Common Bacterial Diseases
S Organism Habitat Disease Prevention
No
1 Acinetobacter baumannii Moist skin, GI tract UTI, sepsis, meningitis, Clean environment,
pneumonia instruments, hands
2 Clostridium difficile GI tract Clostridium difficile Clean environment,,
infection (CDI) hands, use of antibiotics
3 Clostridium tetani Environment Tetanus Sterilisation of
instruments
5 Enterococcus species GI tract, GU tract UTI, sepsis Clean environment, hands,
use of cephalosporins
6 Escherichia coli GI, GU tract UTI, sepsis, neonatal Clean environment, hands,
meningitis use of cephalosporins
7 Helicobacter pylori Gastric mucosa Gastritis Sterile food intake
8 Klebsiella pneumoniae Environment, GI UTI, sepsis, pneumonia Clean hands, use of
tract cephalosporins
9 Mmycobacterium tuberculosis Respiratory tract Tuberculosis Isolation
10 Neisseria meningitidis NP Meningitis Isoaltion, Vaccination
 Virus-Common Characteristics

Living Host Dependency: Viruses are obligate intracellular parasites, meaning they require a living
host cell to replicate and multiply.

Host Range: Viruses can infect a wide range of organisms, including bacteria (bacteriophages or
phages), plants, and animals, including humans.

Genetic Material and Protein Coat: Viruses consist of genetic material, which can be either DNA or
RNA, enclosed within a protective protein coat called a capsid.

Lipid Envelope: While many viruses have a simple protein capsid, some viruses have an additional
lipid envelope surrounding the capsid. This lipid envelope is derived from the host cell's membrane
and contains viral proteins. The presence of an envelope can influence the virus's ability to infect
host cells and evade the host immune system.

Replication: In detail Next slide

 Replication of Virus
. Attachment: The first step in viral infection is attachment. These receptors are typically proteins or
carbohydrates on the cell's surface. The virus attaches to the host cell by binding to these receptors.
. Entry: Once attached, the virus must gain entry into the host cell. The methods of entry vary among different
viruses. Some viruses directly enter the cell by fusing their viral envelope or capsid (outer shell) with the host
cell membrane, allowing the viral genetic material to enter the cell. Others are taken up by the host cell through
endocytosis, where the host cell engulfs the virus in a vesicle.
. Uncoating: After entry, the virus must uncoat its genetic material. This involves the removal or degradation of
the viral protein coat (capsid) or envelope, exposing the viral genome. The exact process of uncoating
depends on the virus type.
. Replication and Transcription: Once inside the host cell, the viral genetic material is replicated and
transcribed. DNA viruses typically use the host cell's machinery to transcribe their genes into RNA and then
replicate their DNA. RNA viruses may directly replicate and transcribe their RNA using the host's cellular
machinery or carry enzymes of their own.
. Translation and Protein Synthesis: The viral RNA or DNA directs the host cell to produce viral proteins,
using the host cell's ribosomes and protein synthesis machinery. These viral proteins are essential for the
assembly of new virus particles.
. Assembly: New virus particles are assembled within the host cell. The viral genetic material is packaged into
capsids, and in some cases, an envelope composed of host cell membrane or viral proteins. The newly formed
viral particles are often referred to as virions.
. Release: Once assembled, new virions need to be released from the host cell to infect other cells and
continue the infection cycle. The release can occur through various mechanisms:
1. Cell lysis: The host cell bursts open, releasing virions into the surrounding tissue.
Common Viral Disease
COVID-19 (Coronavirus Disease 2019): Caused by the novel coronavirus SARS-CoV-2,
COVID-19 rapidly spread globally in 2019 and 2020, leading to a pandemic. It is primarily
transmitted through respiratory droplets.
Influenza (Flu): Seasonal influenza viruses (such as influenza A and influenza B) are highly
contagious and can lead to seasonal outbreaks. They are transmitted through respiratory droplets.

Measles: Measles is one of the most contagious viral diseases known. It can spread through
respiratory droplets and direct contact with an infected person. Vaccination has been highly
effective in reducing measles cases.

Chickenpox (Varicella): Varicella-zoster virus causes chickenpox, a highly contagious disease

primarily affecting children. It is transmitted through respiratory secretions and direct contact.

Ebola Virus Disease: Ebola is highly infectious and can lead to severe outbreaks in Central and
West Africa. It is transmitted through direct contact with the blood, secretions, organs, or other
bodily fluids of infected people, as well as surfaces and materials contaminated with these fluids.
HIV/AIDS: While not as contagious as some other viruses, HIV is transmitted through contact with
specific body fluids (blood, semen, vaginal fluids, rectal fluids, breast milk) and can lead to a global
pandemic if not controlled.
 Antibiotics

Antibiotics are a class of medications or substances that are

used to treat bacterial infections in humans and animals. They
work by either killing bacteria or inhibiting their growth and
reproduction.
Type of Bacteria Common Antibiotics
Gram-Positive Bacteria

- Staphylococcus aureus Methicillin, Vancomycin, Linezolid

Penicillins (e.g., Penicillin, Amoxicillin), Cephalosporins (e.g.,

- Streptococcus pneumoniae
Ceftriaxone)

Gram-Negative Bacteria

Cephalosporins (e.g., Ceftriaxone), Fluoroquinolones (e.g.,

- Escherichia coli (E. coli)
Ciprofloxacin), Aminoglycosides

- Klebsiella pneumoniae Carbapenems (e.g., Meropenem), Cephalosporins, Aminoglycosides

- Pseudomonas aeruginosa Ciprofloxacin, Meropenem, Ceftazidime

Anaerobic Bacteria

- Clostridium difficile (C. difficile) Metronidazole, Vancomycin

- Bacteroides fragilis Metronidazole, Cephalosporins

Mycobacteria

- Mycobacterium tuberculosis Isoniazid, Rifampin, Ethambutol, Pyrazinamide

Spirochetes

- Treponema pallidum (causes syphilis) Penicillin

- Borrelia burgdorferi (causes Lyme disease) Doxycycline, Amoxicillin

Chlamydia and Rickettsia

- Chlamydia trachomatis Azithromycin, Doxycycline

- Rickettsia rickettsii (causes Rocky Mountain spotted fever) Doxycycline

Mycoplasma and Ureaplasma

- Mycoplasma pneumoniae Macrolides (e.g., Azithromycin), Tetracyclines (e.g., Doxycycline)

- Ureaplasma urealyticum Macrolides, Tetracyclines

Helicobacter pylori Amoxicillin, Clarithromycin, Metronidazole, Proton Pump Inhibitor

Antibiotics Mode of Action
 Antibiotics Mode of Action

1. Inhibition of Cell Wall Synthesis: Many antibiotics, such as penicillins and cephalosporins, work by inhibiting
the synthesis of the bacterial cell wall. Bacterial cell walls provide structural support and protection, and without
them, the bacteria become vulnerable to osmotic pressure and can burst.

2. Disruption of Cell Membrane: Some antibiotics, like polymyxins, target the bacterial cell membrane. They
disrupt the integrity of the cell membrane, leading to leakage of cellular contents and eventually cell death.

3. Interference with Protein Synthesis: Antibiotics like aminoglycosides and tetracyclines interfere with bacterial
protein synthesis. They bind to the bacterial ribosomes, the cellular structures responsible for protein production,
and disrupt the translation of genetic information into proteins. This prevents the bacteria from building essential
proteins, leading to their inhibition and eventual death.

4. Inhibition of Nucleic Acid Synthesis: Antibiotics such as quinolones and rifampin inhibit the synthesis of
nucleic acids (DNA and RNA) in bacteria. By interfering with the replication and transcription processes, these
antibiotics disrupt the bacteria's ability to reproduce and carry out essential genetic functions.
5. Blockage of Folic Acid Metabolism: Sulfonamides and trimethoprim are antibiotics that interfere with the
bacterial synthesis of folic acid, which is essential for DNA and RNA synthesis. By blocking this metabolic
pathway, these antibiotics starve the bacteria of critical building blocks needed for growth and replication.
1. Interference with DNA Gyrase: Quinolone antibiotics target DNA gyrase, an enzyme that plays a role in DNA
supercoiling and maintenance. By inhibiting DNA gyrase, quinolones prevent proper DNA coiling and repair,
leading to bacterial cell death.
2. Inhibition of RNA Polymerase: Rifampin, a key antibiotic used in tuberculosis treatment, works by inhibiting
RNA polymerase, an enzyme essential for bacterial RNA synthesis. This disruption affects protein synthesis and
overall bacterial viability.
3. Disruption of Metabolic Pathways: Some antibiotics, like metronidazole, target specific metabolic pathways
within bacteria. Metronidazole, for example, is particularly effective against anaerobic bacteria as it interferes with
their energy metabolism.
4. Formation of Reactive Oxygen Species: Certain antibiotics, like nitrofurans, generate toxic reactive oxygen
species (ROS) within bacterial cells. ROS damage cellular components, including proteins and DNA, leading to
bacterial death.
It's important to note that different antibiotics have varying specificities and mechanis
 Antibiotic resistance Mechanism
1. Enzymatic Inactivation: Some bacteria produce enzymes that can chemically modify or degrade
antibiotics
2. Efflux Pumps: Bacteria may have efflux pumps that actively pump antibiotics out of their cells
3. Alteration of Target Sites: Bacteria can alter the target sites that antibiotics bind to
4. Reduced Permeability: Bacteria can change their cell membrane or wall structure to reduce the
penetration of antibiotics.
5. Bypass Pathways: Bacteria may develop alternative metabolic pathways that do not rely on the
target that antibiotics inhibit.
6. Biofilm Formation: Bacteria within biofilms, which are protective layers of cells, can be less
susceptible to antibiotics
7. Horizontal Gene Transfer: Bacteria can acquire resistance genes from other bacteria through
mechanisms like conjugation, transformation, and transduction. This allows them to quickly gain
resistance to multiple antibiotics.
8. Mutational Resistance: Bacteria can acquire resistance through spontaneous mutations in their
DNA. If a mutation provides a survival advantage in the presence of an antibiotic, it can be passed
on to subsequent generations.
9. Cooperative Resistance: Bacteria within a population can work together to share resistance genes
and mechanisms, increasing their overall resistance.
Live-attenuated vaccines contain live
pathogens from either a bacteria or a
virus that have been "attenuated," or
weakened. live-attenuated vaccines are
produced by selecting strains of a
bacteria or virus that still produce a
robust enough immune response but
that does not cause disease

Examples: Measles, mumps, and

rubella (MMR) vaccine, varicella
(chickenpox) vaccine
 Inactivated vaccines take a live
pathogen and inactivate or kill it.
When the vaccine is then
introduced to a human through a
shot, for example, the inactivated
pathogen is strong enough to
create an immune response,
however, is incapable of causing
disease. Multiple doses are often
needed in order to build up
immunity and offer full protection.
Benefits: Inactivated vaccines
can be mass-produced and are
relatively inexpensive to make.
Examples: Polio vaccine,
influenza vaccine

Pfizer Inc
 Subunit vaccines only contain pieces
of a pathogen, not the whole
organism, so they cannot make you
sick or cause infection. This makes
them suitable for people who should
not receive “live” vaccines, such as
young children, older people, and
immunocompromised people.
Examples: Haemophilus influenzae
type B (Hib) vaccine (conjugate

Pfizer Inc.
 Toxoid vaccines use inactivated
toxins to target the toxic activity
created by the bacteria, rather than
targeting the bacteria itself. “The
goal of toxoid vaccines is to give
people a way to neutralize those
toxins with antibodies through
vaccination,” says Dr. Scully.
Benefits: Toxoid vaccines are
especially good at preventing
certain toxin-mediated diseases
such as tetanus, diphtheria, and
pertussis. Booster shots are
typically recommended every 10
years or so.
Examples: Tetanus vaccine,
diphtheria vaccine

Pfizer Inc
 vector vaccines use a harmless
virus to deliver to the hosts
cells the genetic code of the
antigen you want the immune
system to fight. “They are
basically a gene delivery
system,” says Dr. Scully. In
doing so, information about the
antigen is delivered, which
triggers the body’s immune
response.3
Benefits: Viral vector vaccines
usually trigger a strong immune
response. Typically, only one
dose of the shot is needed to
develop immunity. Boosters
may be needed to maintain
immunity.
Examples: Ebola vaccine,
COVID-19 vaccine
Pfizer Inc (AstraZeneca and Johnson &
Johnson)
 INDUSTRIAL MICROBIOLOGY
The word fermentation
comes from the Latin, which
means to boil.
It originated from the
releasing of gas bubbles during
wine fermentation.
Fermentation in Industrial
microbiology has three
di erent meanings
1.The type of metabolism of a carbon source
in which energy is generated by substrate and
in which
organic function as the final
molecules electron during the
acceptor
carbon-containing break-down of
compounds.
generated
2. Any process in which micro-organisms are
grown on a large scale, even if the final
electron acceptor is not an organic
compound
3. The processing of fermented food which
ORGANIZATIONAL SET-UP IN AN INDUSTRIAL
MICROBIOLOGY ESTABLISHMENT
 Important Characteristics of Industrial Microbes
The organism must be able to grow in a simple medium and should preferably
not require growth factors (i.e. pre-formed vitamins, nucleotides, and acids
The organism should be able to grow vigorously and rapidly in the medium in
use.
Not only should the organism grow rapidly, but it should also produce the
desired materials
Its end products should not include toxic
The organism should have a reasonable genetic, and hence physiological
stability.
The organism should lend itself to a suitable method of product harvest at
the end of the fermentation.
Wherever possible, organisms which have physiological requirements which
protect them against competition from contaminants should be used.
The organism should be reasonably resistant to predators such as
bacteriophages.
organism should not be too highly demanding of oxygen as aeration
 Product Microorganism Major Producer(s)
Penicillin Penicillium fungi Pfizer, Inc.
Streptomycin Streptomyces griseus Merck & Co., Inc.

Insulin (Recombinant DNA) E. coli or Saccharomyces cerevisiae Novo Nordisk, Eli Lilly and Company, Sanofi
Various pharmaceutical companies, including
Various microorganisms or virus GlaxoSmithKline (GSK), Merck & Co., Inc., and
Vaccines strains Sanofi Pasteur
Archer Daniels Midland Company (ADM), Cargill,
Citric Acid Aspergillus niger Inc.
Archer Daniels Midland Company (ADM), Cargill,
Ethanol Saccharomyces cerevisiae Inc., Poet LLC
Xanthan Gum Xanthomonas campestris CP Kelco, ADM, Jungbunzlauer
Corbion, Galactic, Henan Jindan Lactic Acid
Lactic Acid Various Lactic Acid Bacteria Technology Co., Ltd.

Enzymes Various microorganisms Novozymes, DuPont (now part of IFF), DSM

Monosodium Glutamate Ajinomoto Co., Inc., Fufeng Group, Kyowa Hakko
(MSG) Corynebacterium glutamicum Bio Co., Ltd.
 Bioremediation-From the Lab to the Field
What is Bioremediation?
• Bioremediation is a branch of biotechnology that employs the
use of living organisms, like microbes and bacteria to
decontaminate affected areas.
• It is used in the removal of contaminants, pollutants, and toxins
from soil, water, and other environments.
• Bioremediation is used to clean up oil spills or contaminated
groundwater.
• Bioremediation may be done "in situ"–at the site of the
contamination–or "ex situ"–away from the site.
3
 Mechanism of Bioremediation
• Conversion of contaminants to mineralized (e.g. CO2, H2O, and salts) end-
products via biological mechanisms
• Biotransformation refers to a biological process where the end-products are
not minerals.
• Biodegradation involves the process of extracting energy from organic
chemicals via oxidation of the organic chemicals
 What Are the Types of Bioremediation
:

• Biostimulation - Microbes are stimulated to begin the

remediation process via chemicals or nutrients that activate
them.
• Bioaugmentation - Used mainly in cleaning up soil
contamination, this process adds bacteria to the surface of
the affected area, where they are then allowed to grow.
Aerobic vs Anaerobic biodegradation

Aerobic biodegradation
• If oxygen is the terminal electron acceptor, the process is called
aerobic biodegradation

Anaerobic biodegradation
• All other biological degradation processes are classified as anaerobic
biodegradation
 Microorganisms
• Aerobic bacteria:
• Examples include: Pseudomonas, Alcaligenes, Sphingomonas,
Rhodococcus, and Mycobacterium
• Shown to degrade pesticides and hydrocarbons; alkanes and polyaromatics
• May be able to use the contaminant as sole source of carbon and energy.
• Methanotrophs:
• Aerobic bacteria that utilize methane for carbon and energy
• Methane monooxygenase has a broad substrate range
• active against a wide range of compounds (e.g. chlorinated aliphatics
such as trichloroethylene and 1,2-dichloroethane)
• Anaerobic bacteria:
• Not used as frequently as aerobic bacteria
• Can often be applied to bioremediation of polychlorinated biphenyls
(PCBs) in river sediments, trichloroethylene (TCE), and chloroform
• Fungi:
• Able to degrade a diverse range of persistent or toxic environmental
pollutants
How Microbes Use the Contaminant
• Contaminants may serve as:
• Primary substrate
• enough available to be the sole energy source

• Secondary substrate
• provides energy, not available in high enough concentration

• Cometabolic substrate
• fortuitous transformation of a compound by a microbe relying on some
other primary substrate
Requirements for Microbial Growth
Elect ron Accept or
( O2 , NO3 –, SO4 2 - , et c.)

Toxicant s Carbon/ Ener gy

Source

Environment al Nut rient s ( N, P)

Condit ions
( Temp, pH, Eh) Trace Element s
Electron Exchange
Forms of Bioremediation
• In situ Bioremediation
• Bioventing
• In situ biodegradation
• Biostimulation
• Biosparging
• Bioaugmentation
• Natural Attenuation

• Ex situ Bioremediation
• Land farming
• Composting
• Biopiles
• Bioreactors
• Phytoremediation
• Phytoextraction or phytoaccumulation
• Phytodegradation or phytotransformation
• Phytostabilization
• Rhizodegradation
• Rhizofiltration
In Situ Bioremediation
• Bioventing
• One of the most common approaches in soil
• Supply air and nutrients via wells
• Takes advantage of indigenous microorganisms
• In situ biodegradation
• Supply air and nutrients by circulating aqueous solutions through
contaminated soils or groundwater
• Biosparging
• Injection of air below the water table increases groundwater
oxygen concentrations and mixing in saturated zone
• Bioaugmentation
• Addition of indigenous or exogenous microorganisms
• Limits to use: competition and necessity
• Biostimulation
• Natural Attenuation or Intrinsic Bioremediation
Bioventing
Biosparging
Five Steps of In Situ Bioremediation
1. Site investigation

2. Treatability studies

3. Recovery of free product and removal of the

contamination source

4. Design and implementation of the in situ

bioremediation system

5. Monitoring and performance evaluation of the in situ

bioremediation system
Ex situ Bioremediation
• Land farming
• Contaminated soil is excavated and spread over land
• Soil is periodically tilled to improve aeration
• Remediation due to indigenous microorganisms, as well as chemical and physical
processes
• Generally limited to the superficial 10–35 cm of soil
• Can reduce monitoring and maintenance costs

• Composting
• Combines contaminated soil with nonhazardous organic amendants (e.g. manure or
agricultural wastes)

• Biopiles
• Combination of landfarming and composting
• Control physical losses of contaminants

• Bioreactors
• Soil and water pumped up from a contaminated plume and processed through an
engineered containment system
• Degradation in a bioreactor is generally greater than in situ because the contained
environment is more controllable and predictable
Phytoremediation
• Phytoextraction or phytoaccumulation
• Plants used to accumulate contaminants in the roots and aboveground
biomass
• Can be a relatively low cost option for a large area
• Results in biomass that must be properly disposed of or reused
• Phytotransformation or phytodegradation
• Uptake of contaminants and transformation to more stable, less toxic,
or less mobile forms
• Eg. metal chromium can be reduced from hexavalent to less mobile (and
non-carcinogenic) trivalent chromium
• Phytostabilization
• Mobility and migration of contaminants are reduced through sorption
onto or into the plant
• Rhizodegradation
• Breakdown of contaminants through activity of the rhizosphere
• Rhizofiltration
• Water remediation technique
• Used to reduce contamination in natural wetlands and estuary areas.
Environmental Factors –
Optimum for biodegradation
 Factors that limit the potential for Bioremediation

Contaminant-related limitations:Synthetic vs. natural

1)
contaminants
-bioremediation potential greater for natural compounds
▪ Physical characteristics

▪ Molecular structure of the contaminant-extent of

chlorination, linear vs. branched structure, saturated vs.
 Factors that limit the potential for Bioremediation
2) Environmental conditions:
▪ Hydrogeology: permeability/hydraulic conductivity, heterogeneity, fracture bed rocks
soil properties, pH
▪ Nutrients: C:N:P-100:10:1
▪ Electron acceptor: oxygen (3 parts of oxygen to converts 1 part of hydrocarbon to CO2
nitrate, sulfate, ferric iron
3) Microorganisms presence:
▪ Assessment of microbial activity, introduced microorganisms. Competence with
indigenous microorganism