BENUE STATE UNIVERSITY MAKURDI

MICROBIOLOGY UNIT
DEPARTMENT OF BIOLOGICAL SCIENCES

PETROLEUM MICROBIOLOG
LECTURE NOTES
MCB 401 | 2018

COURSE LECTURER: DR. E.M. Mbaawuaga

Petroleum microbiology is a branch of microbiology that deals with the study
of microorganisms that can metabolize or alter crude or refined petroleum products.
These microorganisms, also called hydrocarbonoclastic microorganisms, can degrade
hydrocarbons and, include a wide distribution of bacteria, methanogenic archaea, and
some fungi.
Petroleum is a general term for a mixture of hydrocarbons that exist naturally on Earth in
gaseous (natural gas), liquid (crude oil) and solid (asphalt) states and are characterized by
density lower than that of water. Typical compounds of petroleum contain carbon and
hydrogen (CnHm) with small amounts of oxygen, nitrogen and sulphur. Petroleum occurs
generally in sedimentary rocks formed under marine or terrestrial environments and
buried in the external envelop of the lithosphere. Under natural conditions, petroleum gas
is composed by saturated hydrocarbons (alkanes) with one to three carbon atoms.
Petroleum oil is a complex mixture of alkanes with more than four carbon atoms, such as
paraffin, cycloalkanes and aromatics. Sulfur, nitrogen and oxygen can be present in oil
within complex organic molecules such as resins and asphalts.
Composition of Petroleum

Petroleum, or crude oil as it is now usually referred to when raw, contains several
chemical compounds, the most prolific being the hydrocarbons themselves which give
the petroleum composition its combustible nature.

Although the composition of petroleum will contain many trace elements the key
compounds are carbon (93% – 97%), hydrogen (10% - 14%), nitrogen (0.1% - 2%),
oxygen (01.% - 1.5%) and sulphur (0.5% - 6%) with a few trace metals making up a very
small percentage of the petroleum composition.

The actual overall properties of each different petroleum source are defined by the
percentage of the four main hydrocarbons found within petroleum as part of the
petroleum composition.

The percentages for these hydrocarbons can vary greatly, giving the crude oil a quite
distinct compound personality depending upon geographic region. These hydrocarbons
are typically present in petroleum at the following percentages: paraffins (15% - 60%),
napthenes (30% - 60%), aromatics (3% to 30%), with asphaltics making up the
remainder.

Raw petroleum is usually dark brown or almost black although some fields deliver a
greenish or sometimes yellow petroleum. Depending upon the field and the way the
petroleum composition was formed the crude oil will also differ in viscosity.

At the extreme ranges petroleum can be almost solid, and required a significant
investment of resources to refine into a useable state as anything other than bitumen. At

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the other end of the scale the petroleum composition can be a clear fluid resembling
kerosene or gasoline, needing very little refining to be useable as a fuel.

When discussing the composition of petroleum it is important to note that the compound
of the raw petroleum tends to dictate the usage of the refined product. Petroleum is
generally measured in volume, and for some composition of petroleum it is not cost
effective to refine these into fuel.

A lighter, less dense raw petroleum composition with a compound that contains higher
percentages of hydrocarbons is much more profitable as a fuel source. Whereas other,
denser petroleum composition with a less flammable level of hydrocarbons and sulphur
are expensive to refine into a fuel and are therefore more suitable for plastics
manufacturing and other uses.

Unfortunately the worlds reserves of light petroleum (light crude oil) are severely
depleted and refineries are forced to refine and process more and more heavy crude oil
and bitumen.

In some cases the refining process will need to remove carbon and add hydrogen, adding
an extra, costly step to the refining process. This change in compound of the world's
energy producing petroleum and the associated rise in refining costs has directly affected
the price of gasoline across the world.

Petroleum formation occurs by various hydrocarbons combining with certain minerals

such as sulphur under extreme pressure. Modern day scientists have proven that most if
not all petroleum fields were created by the remains of small animal and plant life being
compressed on the sea bed by billions of tons of silt and sand several million years ago.

When small sea plants and animals die they will sink, they will then lie on the sea bed
where they will decompose and mix with sand and silt. During the decomposition process
tiny bacteria will clean the remains of certain chemicals such as phosphorus, nitrogen and
oxygen.

This leaves the remains consisting of mainly carbon and hydrogen. At the bottom of the
ocean there is insufficient oxygen for the corpse to decompose entirely. What we are left
with is the raw materials for the formation of petroleum.

The partially decomposed remains will form a large, gelatinous mass, which will then
slowly become covered by multiple layers of sand, silt and mud. This burying process
takes millions of years, with layers piling up one atop another.

As the depth of the sediment build up increases the weight of the sand and silt pressing
down on the mass will compress it into a layer which is much thinner than the original.

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Finally, when the depth of the buried decomposing layer reaches somewhere around
10,000 feet the natural heat of the earth and the intense pressure will combine to act upon
the mass. The end result, over time, is the formation of petroleum.

With petroleum formation the actual temperature applied to the original organic mass is
critical in determining the overall properties of the resulting petroleum. Typically lower
temperatures during petroleum formation will result in thicker, darker raw petroleum
deposits, the most solid of which being a bitumen substance.

If the heat applied during the formation of petroleum process fluctuates too much then
gas will be produced, often separating from the petroleum, sometimes remaining mixed
with the raw oil. If temperatures are too high, in the somewhere over 450 degrees
Fahrenheit then the original biomass will be destroyed and no gas or petroleum is formed.

As the mud and silt above the deposit become heavier and the forces placed upon the silt
and mud begin to change the bottom layers of the compressing layer above the petroleum
then it will turn into shale.

As the shale forms the oil will be forced out of its original area of formation. The raw
petroleum then moves to a new rock formation, usually termed a reservoir rock, and lays
trapped until it is accessed in some way.

As we can see, the formation of naturally occurring raw petroleum takes millions of
years, certainly far longer than can be deemed renewable, yet mankind has managed to
almost complete deplete the world supply in little more than a century.

The hydrocarbons in crude oil can generally be divided into four categories:

 Paraffins: These makes up 15 to 60% of crude and have a carbon to hydrogen

ratio of 1:2. These are generally straight or branched chains, but never cyclic
(circular) compounds. Paraffins are the desired content in crude and are used to
make fuels. The shorter the paraffins are, the lighter the crude is.
 Napthenes: These makes up 30 to 60% of crude and have a carbon to hydrogen
ratio of 1:2. These are cyclic compounds referred to as cycloparaffins. They are
higher in density than equivalent paraffins and are more viscous.
 Aromatics: These can constitute 3 to 30% of crude. They are undesirable because
their combustion results in soot. They have a much less hydrogen in comparison
to carbon than is found in paraffins. They are also more viscous. They are often
solid or semi-solid when an equivalent paraffin would be a viscous liquid under
the same conditions.
 Asphaltics: These average about 6% in most crude. They have a carbon to
hydrogen ratio of approximately 1:1, making them very dense. They are generally
undesirable in crude, but their 'stickiness' makes them excellent for use in road
construction.

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Surfactants are compounds that lower the surface tension (or interfacial tension)
between two liquids, between a gas and a liquid, or between a liquid and a solid.
Surfactants may act as detergents, wetting agents, emulsifiers, foaming agents,
and dispersants.

Biosurfactants These are microbial-synthesized surface-active substances that allow for

more efficient microbial biodegradation of hydrocarbons in bioremediation processes.
There are two ways by which biosurfactants are involved in bioremediation. (1) Increase
the surface area of hydrophobic water-insoluble substrates. Growth of microbes on
hydrocarbons can be limited by available surface area of the water-oil
interface. Emulsifiers produced by microbes can break up oil into smaller droplets,
effectively increasing the available surface area. (2) Increase the bioavailability of
hydrophobic water-insoluble substrates. Biosurfactants can enhance the availability of
bound substrates by desorbing them from surfaces (e.g. soil) or by increasing their
apparent solubility. Some biosurfactants have low critical micelle
concentrations (CMCs), a property which increases the apparent solubility of
hydrocarbons by sequestering hydrophobic molecules into the centres of micelles.

Oil Recovery
Microbial enhanced oil recovery (MEOR) is a technology in which microbial
environments are manipulated to enhance oil recovery. Nutrients are injected in situ into
porous media and indigenous or added microbes promote growth and/or generate
products that mobilize oil into producing wells. Alternatively, oil-mobilizing products
can be produced by fermentation and injected into the reservoir. Various products and
microorganisms are useful in these applications and each will yield different results. The
two general strategies for enhancing oil recovery are altering the surface properties of the
interface and using bioclogging to change the flow behavior. Biomass, biosurfactants,
biopolymers, solvents, acids, and gases are some of the products that are added to oil
reservoirs to enhance recovery.
Mechanisms of MEOR
The use of microorganisms and their metabolic products to enhance oil production
involves the injection of selected microorganisms into the reservoir and the subsequent
stimulation and transportation of their in-situ growth products in order that their presence
will aid in further reduction of residual oil left in the reservoir after secondary recovery is
exhausted. The MEOR is unlikely to replace conventional EOR methods, because MEOR
itself has certain constraints. This unique process seems superior in many respects,
however, because self-duplicating units, namely the bacteria cells, are injected into the
reservoir and by their in-situ multiplication they magnify their beneficial effects.
Classification of MEOR
Mainly, MEOR is classified as surface MEOR and underground MEOR based on the
place where microorganisms work. For surface MEOR, biosurfactant (Rhamnolipid),
biopolymer (xanthan gum), and enzyme are produced in the surface facilities. These
biological products are injected into the target place in the reservoirs as chemical EOR

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methods. While, for underground MEOR, microorganisms, nutrients and/or other
additives are injected into the reservoir to sustain, grow, metabolize, and ferment
underground.
Depending on the source of microorganisms, underground MEOR are categorized into in-
situ MEOR and indigenous MEOR. While according to procedures of processes,
underground MEOR is sorted as:
 Cyclic Microbial Recovery (Huff and Puff, Single Well Stimulation)
 Wax Removal and Paraffin Inhibition (Wellbore Cleanup)
 Microbial Flooding Recovery
 Selective Plugging Recovery
 Acidizing/Fracturing
a. Cyclic Microbial Recovery
A solution of microorganisms and nutrients is introduced into an oil reservoir
during injection. The injector is then shut in for an incubation period allowing the
microorganisms to produce carbon dioxide gas and surfactants that help to
mobilize the oil. The well is then opened and oil and products resulting from the
treatment are produce
b. Microbial Flooding Recovery
Recovery by this method utilizes the effect of microbial solutions on a reservoir.
The reservoir is usually conditioned by a water preflush, then a solution of
microorganisms and nutrients is injected. As this solution is pushed through the
reservoir by drive water, it forms gases and surfactants that help to mobilize the
oil. The resulting oil and product solution is then pumped out through production
wells
c. Selective Plugging Recovery
Injection of bacterial suspensions followed by nutrients to produce biopolymer and
microbial itself, which may plug the high permeability zone in the reservoir. The
reduction of permeability would change the inject profile and achieve conformance
control.
Microorganisms for MEOR
The microorganisms for MEOR should have the following potential properties:
 Small Size
 Resistant to High Temperatures
 Resistant against High Pressure
 Capability of Withstand Brine and Seawater
 Anaerobic Using of Nutrients
 Unfastidious Nutritional requirements
 Appropriate Biochemical Construction for Production Suitable Amounts of MEOR
Chemicals
 Lack of any Undesirable Characteristics

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 Here, undesirable characteristics mean permeability reduction (damage the formation
where a large amount of remaining exist), corrosion, and souring.

Advantages and Disadvantages of MEOR

a. Advantages of MEOR
 The injected bacteria and nutrient are inexpensive and easy to obtain and handle in the
field
 Economically attractive for marginally producing oil fields; a suitable alternative
before the abandonment of marginal wells
 According to a statistical evaluation (1995 in U.S.), 81% of all MEOR projects
demonstrated a positive incremental increase in oil production and no decrease in oil
production as a result of MEOR processes
 The implementation of the process needs only minor modifications of the existing
field facilities
 The costs of the injected fluids are not dependent on oil prices
 MEOR processes are particularly suited for carbonate oil reservoirs where some EOR
technologies cannot be applied with good efficiency
 The effects of bacterial activity within the reservoir are magnified by their growth
whole, while in EOR technologies the effects of the additives tend to decrease with
time and distance
 MEOR products are all biodegradable and will not be accumulated in the
environment, so environmentally friendly
b. Disadvantages of MEOR
 Safety, Health, and Environment (SHE)
 A better understanding of the mechanisms of MEOR
 The ability of bacteria to plug reservoirs
 Numerical simulations should be developed to guide the application of MEOR in
fields
 Lack of talents

Microorganisms in Petroleum

Despite the drastic physico-chemical conditions existing in oil reservoirs; e.g. high
temperatures up to 190°C, high salinities up to 400 g/l, there is now evidence that
microorganisms inhabit such extreme environments considered as closed systems lacking
oxygen. This is the case of many mesophilic, thermophilic/hyperthermophilic, and
halophilic/hyperhalophilic anaerobic prokaryotes which have been retrieved both by
molecular and cultural approaches at many occasions from such ecosystems

Oilfield microbes have a wide range of metabolisms including fermentative and

respiratory processes.

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 (i) Sulfate-reduction by various prokaryotes
(ii) CO2 reduction by hydrogenotrophic methanoarchaea
(iii) Sulfate-reducers
(iv) Methanoarchaea having the ability to grow chemolithotrophically.

The sulfate-reducing prokaryotes

Many new SRP species responsible for biocorrosion, oil souring, etc have been isolated
from oilfield ecosystems. They include seven mesophilic (optimum range temperature
between 30 and 40°C) Desulfovibrio species within the Deltaproteobacteria and one
mesophilic halophilic Desulfotomaculum species (D. halophilum), recovered from low
temperature oil reservoirs. They share the ability to use hydrogen and to incompletely
oxidize their organic substrates, e.g. lactate, ethanol, etc. to acetate.

This is also the case for thermophilic SRP originating from hot oil reservoirs. They
comprise three Desulfotomaculum spp. (Firmicutes) and two Desulfacinum spp
(Deltaproteobacteria), growing optimally at temperatures around 60°C, together with
two Thermodesulfobacterium spp. (Thermodesulfobacteria) with optimal growth
occurring at 65°C (T. thermophilum) and 70°C (T. commune). Besides all these bacterial
species, only one archaeal thermophilic sulfate-reducing species (optimum temperature
for growth 76°C), Archaeoglobus fulgidus strain 7324, was isolated from a North Sea oil
reservoir.

The methanoarchaea

Hydrogenotrophic and acetoclastic methanoarchaea which are known to compete with

SRP are also of geomicrobiological significance in oil field environments. Mesophilic
hydrogenotrophic MA which are known to participate actively in hydrocarbon oxidation
in oil field waters comprise Methanobacterium bryantii, Methanocalculus halotolerans,
and Methanoplanus petrolearius growing optimally at 37°C whereas Methanobacterium
ivanovii, Methanothermobacter thermoautotrophicus, and Methanoculleus receptaculi
are considered as moderate thermophiles to thermophiles. Methanothermobacter crinale
recently isolated from the Shengli oil field in China showed the highest temperature for
growth (85°C) and is considered as hyperthermophilic archaeon. Only one aceticlastic
non hydrogenotrophic MA species was isolated from oil reservoirs so far,
Methanosarcina mazei, which grows under mesothermic conditions. Acetate can be
directly used by this archaeon, but there is evidence that it can be also oxidized in high
temperature reservoirs by syntrophic bacteria using hydrogenotrophic MA as biological
electron acceptors to produce methane. Besides hydrogenotrophic and acetoclastic MA,
numerous archaeons producing methane only from methylated compounds (e.g.
methylamines, methanol) were isolated from saline to hypersaline oil reservoirs.

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Microorganisms as indicators in prospecting for
hydrocarbon deposits

In view of the connection between bitumen deposits and microorganisms,

microbiological research has been a part of complex geochemical and geological
prospecting methods in oil exploration for many years. The Oil and Gas Institute has
conducted research using the microbial well survey technique and a surface method based
on the isolation of bacteria which use hydrocarbons as their sole carbon source. The first
technique – the microbial well survey technique is based on the isolation of indicator
microbes from oil and gas-bearing zones of cores representing different geological
deposits using specialized microbiological media. The method allows the determination
of the distribution of particular microbial groups and their level of activity in a geological
profile by observing their hydrocarbon-oxidizing activity. Moreover, it allows an
assessment of the potential areas of interest, and the geo-microbial data confirm the
geochemical data on the distribution of an organic substance in materials. The advantage
of this method is its high sensitivity, which allows us what allows to detect trace amounts
of hydrocarbons.

The second method, the surface-prospecting method is based on the detection of

anomalies in microbial distribution in soil samples. It is a method based on the premise
that hydrocarbons are generated and/or trapped in subsurface oil reservoirs (at depth) and
migrate upward in varying but detectable quantities. The seepage of hydrocarbons is a
longlasting process, but it is recognisable by the presence of analytically detectable
(anomalous) concentrations of light hydrocarbons (C1 -C5) in soils and waters. A higher
concentration of these hydrocarbons is often correlated with an increased concentration
of hydrocarbon-oxidizing microbes, such as methane-oxidizing bacteria and propane- and
butaneoxidizing microbes, in the area above hydrocarbon reservoirs. Hence, the
discovery of a surface geochemical anomaly can establish hydrocarbon accumulation in
the area. Traps and structures along such pathways should be considered as significantly
more promising than those not associated with such anomalies. The results of
microbiological analysis of soil samples have been applied in geological studies which
concern concentrations of methane and the bacteria that oxidize gaseous hydrocarbons.
The advantages of the surface-prospecting method are many and include:

 evidence of the presence of hydrocarbon generation and migration

 the low cost, ease and rapidity of sample collection and analysis,
 high sensitivity, which allows detection of even “discrete” anomaly state on the
assumption that the threshold value is estimated properly
 the detection of hydrocarbons in both soils and on the sea-floor,
 the possibility of prospecting before conducting detailed seismic surveys,
 having little or no negative environmental impact and having the ability to
evaluate areas where seismic surveys are impractical or ineffective due to
geological factors

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  providing methods applicable to both stratigraphic traps and structural traps, with
the ability to locate traps invisible or poorly imaged with seismic data or due to
environmental factors
 reproducibility of results,
 providing methods applicable in many different climate conditions
 establishing a clear distinction between gas reservoirs and oil-bearing structures
with gas caps.

Methanogenesis

Methanogenesis or biomethanation is the formation of methane by microbes known

as methanogens. Organisms capable of producing methane have been identified only
from the domain Archaea.

Methanogenic degradation of crude oil hydrocarbons is an important process in

subsurface petroleum reservoirs and anoxic environments contaminated with petroleum.
There are several possible routes whereby hydrocarbons may be converted to methane: (i)
complete oxidation of alkanes to H2 and CO2, linked to methanogenesis from CO2
reduction; (ii) oxidation of alkanes to acetate and H2, linked to acetoclastic
methanogenesis and CO2 reduction; (iii) oxidation of alkanes to acetate and H2, linked to
syntrophic acetate oxidation and methanogenesis from CO2 reduction; (iv) oxidation of
alkanes to acetate alone, linked to acetoclastic methanogenesis and (v) oxidation of
alkanes to acetate alone, linked to syntrophic acetate oxidation and methanogenesis from
CO2 reduction.

Microbial Corrosion

Microbial corrosion, also called bacterial corrosion, bio-corrosion, microbiologically

influenced corrosion, or microbially induced corrosion (MIC), is corrosion caused or
promoted by microorganisms, usually chemoautotrophs. Microbial corrosion is
recognized as one of the major problem in various fields, The biological growth cause
fouling and corrosion problems. A large variety of bio-organisms can enhance the
corrosion rate of metals through their metabolic processes. When bacteria grow on the
walls of the pipe, they develop into colonies containing many types of bacteria which
help each other in life. Well-known types which cause corrosion include; Acid producing
bacteria (APB), sulphate reducing bacteria (SRB), iron bacteria (IB) and Manganese
oxidizing bacteria (MoB). SRB and acid producing bacteria are the two types of bacteria
most commonly found in oil and gas pipelines. SRB produce hydrogen sulphide while
APB generate acetic acid/sulphuric acid, both of which are highly corrosive to the pipe.
Iron bacteria precipitate the iron present in the solution arising from corrosion and form a
tubercule on the top of the corrosion pit.

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Microorganisms in aqueous environment form biofilm on solid surfaces, Biofilms consist
of populations of microorganisms and their hydrated polymeric secretions. Numerous
types of organisms may exist in any particular biofilm, ranging from strictly aerobic
bacteria at the water interface to anaerobic bacteria such as sulphate reducing bacteria at
the oxygen depleted metal surface. The presence of biofilm can contribute to corrosion in
three ways;

1. Physical deposition
2. Production of corrosive by products
3. Depolarization of the corrosion cell caused by chemical reaction

Any by-products of microbial metabolism including organic acids and hydrogen

sulphide are corrosive. These materials can concentrate in the biofilm causing
accelerated metal attack. Corrosion tends to be self limiting due to the build up of
corrosion reaction products. However, microbes can absorb some of these materials
in their metabolism thereby removing them from the anodic and cathodic sites. The
removal of reaction products termed deplorization stimulates further corrosion.

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