Microbial Problem Solving with Cost Effective and Biodegradable Biocide in the Oil and Gas Industry

Abstract

Standard fuel contains up to 0.2 ml water per litre, of which is more than enough for microbial activity. The development of microorganisms mainly occurred when aqueous condensate collected in oil tanks. Studies have shown that the troubles of clogged filters, fuel tank corrosion, breakdown of tank coatings and engines are connected to the microorganisms. It is important and necessary to stop the bacterial growth with specific use of biocides. This paper will describe some experiences in overcoming this problem using a cost effective and environmentally friendly biocide as well as preventive measures.

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Copyright 2002, Society of Petroleum Engineers Inc.
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Abstract
Standard fuel contains up to 0.2 ml water per litre, of which is
more than enough for microbial activity. The development of
microorganisms mainly occurred when aqueous condensate
collected in oil tanks. Studies have shown that the troubles of
clogged filters, fuel tank corrosion, breakdown of tank
coatings and engines are connected to the microorganisms. It
is important and necessary to stop the bacterial growth with
specific use of biocides. This paper will describe some
experiences in overcoming this problem using a cost effective
and environmentally friendly biocide as well as preventive
measures.
Introduction
The fact that hydrocarbons from petroleum products can be
used as a source of carbon by various microorganisms is
widely known. H. Kaserer
1
, N.L. Söhngen
2
and K. Störmer
3
reported on the utilization of hydrocarbons by
microorganisms. However, these reports had not indicated the
relationship between occurrence or damage in practice and
microorganisms’ activities.
The discoveries of microbiological phenomenon on direct
or indirect effects in practice gradually increased. C.E.
Zobell
4
described the practical problems occurring as a
consequence of microbiological attack on lubricating oils,
diesel oil, natural gas, cooling oils, asphalt and rubber.
According to A.R. Lansdown
5
, airplane accidents were
attributable to the blockage of filters due to the growth of
bacteria and fungi in propellants. D.G. Parbery
6
reported
similar observation. E.C. Hill
7
and other authors found that
anhydrous oils contain some organisms and active growth took
place as soon as water was present.
Although it has been known in principle for more than
twenty years that microorganisms are disruption factors, often
there is no information on this whatsoever in the service
laboratories of the oil and gas industry.
A typical scenario was the frequent breakdown of the fire
engines at an airport. Analyses often show filter blockage in
the fuel system. Based on this result, firstly the fire-engine
tanks, followed by the storage tanks, were cleaned. The
material causing the filter block was also analyzed further, and
it was shown that the blocking was caused by fibers of organic
material. Therefore, the use of paper towels was prohibited,
since it was suspected that the fibers of these paper towels
somehow got into the fuel system and thereby blocked the
filters. However, when the problems did not cease, a
microbiological control was carried out, which revealed the
actual problem - massive microbiological contamination.
The above example showed that the knowledge of
microbiological attack has not been widespread. It is
suspected that knowledge concerning microbial problems is
intentionally not passed on, so as to avoid claims for
compensation. Nobody wants to accept the responsibility for
ensuring microbiologically perfect quality products.
At present, microbiological purity is not a criterion of
quality in mineral oil standards. Having said that,
microbiological tests should become a standard in the event of
blocked filters and unexplained sludge deposits in land vehicle
engines, ships’ engines or airplane engines. The same applies
to mineral oil storage, irrespective of where these come in the
sales route, whether refinery or end-user filling station.
How is this possible?
Microorganisms are ubiquitous. They colonize the earth; exist
in human, animals and plants. They present in a variety of
forms: fungi, mould, viruses, bacteria and yeast (Figures 1,2).
They are useful in the food and medicine industry; they work
specifically in sewage treatment plants; and also pose an
ecological significance. Their present, however, with their
uncontrolled multiplication often create problems.
SPE 74087
Microbial Problem Solving with Cost Effective and Biodegradable Biocide in the Oil and
Gas Industry
W. Siegert, Schülke & Mayr GmbH and P.H. Lee, Schülke & Mayr Asia Sdn. Bhd.

2 W. SIEGERT, P.H. LEE SPE 74087
Fig. 1Bacteria under the microscope.
Fig. 2Moulds and fungi.
Microbial activity and growth are only possible in the
presence of free water. Without water, microbes become
dormant or die. Therefore, in order for microorganisms to
grow and develop in or on a material, the material must be
infected and contain adequate quantity of water.
In general, as little as 100 ppm of water is sufficient for
microorganism’s multiplication. Standard fuel contains up to
0.2 ml water per liter. A third of this is dissolved and the rest
settles at the bottom of the tank. Thus, microorganisms can
live and multiply in diesel fuel easily. This is particularly true
in the condensation water settles at the bottom of the storage
tank or which is finely distributed in the fuels.
In ventilated oil tanks that are exposed to temperature
fluctuations, free water is formed as a result of condensation.
Water vapors are breathed in with air, condense on tank walls
and land at the bottom at some points. Leaking fill caps are
another prime source. Holes in underground tank walls allow
ground water to enter.
Water can also come from other sources. Fresh or
seawater may be put in oil-storage tanks intentionally,
especially during washing of difficult accessible parts of the
systems, or as ballast in tankers. Such additions of water also
bring in inorganic salts, end up as nutrient salts, with other
biological impurities.
Foreign impurities such as soil, sawdust, leaves and many
others, in the storage tanks may also constitute a dangerous
source of infection and good culture media for
microorganisms.
Water may dissolve some water-soluble components out of
the oil phase. Both phases usually contain air, which is of
importance for the development of aerobic microorganisms.
Naturally, spores and bacteria always contaminate the fuel oil
itself because they present everywhere. They are too small to
be removed or filtered practically.
The growth usually takes place in the zone between water
and oil. The phase boundary is the ideal nutrient medium for
organisms. Depending on types of microorganisms and the
micro ecological conditions, the growth can expand in the oil
or the aqueous phase. The contamination of the diesel fuel can
be passed on from the refinery to the storage tanks via the
intermediate tanks, to the customer’s tanks or the filling
station, and from there into the vehicle tanks. The transmission
of bacteria/fungi into the supply chain is easily done.
Under optimum conditions after 20 minutes, as a result of
division, one microorganism becomes two, after a further 20
minutes four, then 8, 16, 32, 64 etc. (Figure 3). After 10
hours, that would already be 1,073,741,824 microorganisms.
1
100
10000
1000000
100000000
10000000000
12345678910
Time (hour)
Number of microorganisms
Fig. 3Rapid multiplication of microorganisms.
The consequences of microbial growth are the formation of
sludge in contaminated tanks. On the tank walls, slimy,
slippery, dark colored coatings of microorganisms may
develop. This leads to diesel fuel that was originally light and
clear becoming dark and cloudy. As a result of the biomass,
filter blockages occur, which in some cases make daily filter
changes in the vehicle necessary. In addition, aluminum and
sheet-steel tanks will also damage by pitting corrosion due to
the acidic metabolic products of microbial growth
(biocorrosion).
Both problems, i.e. filter blockage and corrosion are no
doubt due to microorganisms. This connection is well
understood among experts, and has been discussed for many
years. In order to solve this problem, the use of biocide has
been proven effective in many cases in practice.
How Does One Recognize Microorganism?
Of course, not every blocked fuel filter is attributable to
microbial impurities. It is impossible to see with naked eyes
whether sludge deposits in diesel tanks or filters are
microorganisms, or simply just precipitations or ageing
products from the diesel.
Moulds

SPE 74087 MICROBIAL PROBLEM SOLVING WITH COST EFFECTIVE AND BIODEGRADABLE BIOCIDE IN THE OIL & GAS INDUSTRY 3
Only microbial examination with determination of
bacterial count provides a reliable answer. Schülke & Mayr
has over hundred years of history in preservatives and
industrial hygiene. We have adopted and implemented in-
house experimental and laboratory analysis methods to
examine problematic samples of fuel, water or sludge.
Since oils are not soluble in water or culture media, the
bacterial counts, or the colony-forming units (cfu), cannot be
determined by the usual dilution procedure using culture
plates. For this reason, membrane filtration was applied, as
described by M.A. Rogers and A.M. Kaplan
8
. This required
100 ml of fuel oil to be poured into a sterile diaphragm with
0.45 ± 0.02 µm pore width and filtered under vacuum. The
cfu contained in the oil, both of bacteria and fungal spores are
retained on the filter due to their larger diameters. The residue
from the filtration was rinsed with 100 ml of sterilized 0.1%
aqueous solution of a wetting agent (alkyl-aryl polyether
alcohol) that is harmless to microorganisms, and then
rewashed with 30 ml of sodium chloride solution. Each filter
with germs was then laid on a nutrient agar plate that
contained 0.1% meat extract, 0.2% yeast extract, 0.5%
peptone and 0.5% cooking salt, and then incubated for 5 days
at 25
o
C. The number of growing colonies gives the cfu per
100 ml of oil.
What Must Be Done?
M.A. Rogers and A.M. Kaplan
9
have suggested various
biocides to prevent the growth of microorganisms in
petroleum products.
As early as 1971, the Naval Research Laboratory in
Washington investigated the effectiveness of various biocides
for the treatment of navy distillate. With the IP Code of
Practice for examination of light distillated fuel for variable
microorganisms (IP 386/88), the testing of diesel fuel samples
was standardized.
In addition, a standard for sampling was also drawn up by
a working group in the Institute of Petroleum. A guideline for
the investigation of microbial content in fuel boiling below
390
o
C and associated water was outlined in November 1996.
This describes the sampling process in detail and includes a
revised version for microbiological testing.
The SGS (Societe Generale de Surveillance) limit values
are used for microbial load in trade agreements. The total
viable organisms are limited to <3 x 10
3
/l, that is, less than the
limit for drinking water, which in Germany, for example, is
<100 /ml = < 10
5
/l. However, it is important to note that even
at this low level, it is possible to lead to severe problems in
practice. Hence suitable biocide and preventive measures are
necessary.
Good housekeeping and/or continuous use of a chemical
preservative (biocide) can solve the microbial problems
occurring in clean systems.
If an infection is already well established, the microbes
must be killed and removed as soon as possible. Physical or
chemical decontamination or both should be employed. The
chemical biocide selected must be rapidly effective against
large numbers of microbes in the presence of substantial
organic fouling.
Although the details of antimicrobial strategies differ from
system to system, one of the key decisions is the choice of
biocide. Biocide selected has to be able to kill microbes in oil
and water phase, safe to use, and with acceptable
environmental impact.
Schülke & Mayr, together with the affected operators of oil
depot, refinery, tanker lorry, fleet, and etc. have developed
biocide that fulfils the basic criteria; and formed appropriate
strategies in order to kill microorganisms, avoid microbial
growth and prevent recolonisation by means of preventive
measures.
To date, we have developed an effective biocide, which is
based on the chemistry of methylene-bis-oxazolidine,
especially for the application in diesel fuel. This compound
contains no heavy metals or halogens and combusts
completely without ash. It can be added directly to the diesel
fuel and has been tested and approved by leading engine and
vehicle manufacturers
10,11,12
, the oil industry
13,14
and the
NATO
15
. It is reported that the substance has no negative
effects on injectors or combustion processes in engines.
Methylene-bis-oxazolidine is characterized by its broad
spectrum of effect against both aerobic organisms (bacteria,
yeasts, mould fungi) and anaerobic organisms. In particular,
the growth of sulphate-reducing bacteria that lead to severe
corrosion damage is reliably inhibited. On account of its
excellent anti-corrosion properties, it also prevents corrosion
caused by microbial breakdown products and neutralizes any
acids formed. Furthermore, it displays a good immediate
effect.
Methylene-bis-oxazolidine is readily soluble in water and
organic solvents. Only low concentrations are soluble in
nonpolar solvents. Its water/diesel oil distribution coefficient
is determined as 28; hence it is present in greater amounts in
the particularly susceptible water phase.
The substance forms no corrosive combustion products
(approved by the Bundes Immissionsschutzgesetz) and shows
excellent stability in diesel oil and other fuel. This permits
economical long-term protection. It is also been classified into
Group D under OCNS
16
. OCNS is the UK Offshore Chemical
Notification Scheme, which is administered by the Department
of Trade and Industry using scientific and environmental
advice from the Center for Environment, Fisheries &
Aquaculture Science (CEFAS) and the Fisheries Research
Services (FRS) marine Laboratory in Aberdeen. The OCNS
applies to all chemicals, which are used in the actual
exploration, exploitation and associated offshore processing of
petroleum in the UK Continental Shelf.
Test conducted shown that methylene-bis-oxazolidine is a
good biocidal compound. Different decontamination concepts
and recommendations are very much depending on the degree
of microbiological contamination. Test results presented a
reduction in the number of microorganisms by > 5 log steps
takes place within a few hours (Figures 4,5,6,7,8,9). Treated
product can usually be sold. The number of residual

4 W. SIEGERT, P.H. LEE SPE 74087
microorganisms is evaluated semi-quantitatively as a function
of time in accordance with the scheme:
0 = no growth (<10
2
germ count/ml)
1 = slight growth (~10
2
-10
3
germ count/ml)
2 = moderate growth (~10
3
-10
5
germ count/ml)
3 = massive growth (>10
6
germ count/ml)
0
1
2
3
G
e
r
m
C
o
u
n
t
S
c
h
e
m
e
0.5 1 2 4 6 24
Cont act t ime ( hour s)
0.05% Grot aMar 71
0.1% Grot aMar 71
0.2% Grot aMar 71
Fig. 4Germ count reduction test: Pseudomonas aeruginosa.
0
1
2
3
G
e
r
m
C
o
u
n
t
S
c
h
e
m
e
0.5 1 2 4 6 24
Contact time (hours)
0.05% GrotaMar 71
0.1% GrotaMar 71
0.2% GrotaMar 71
Fig. 5Germ count reduction test: Pseudomonas putida.
0
1
2
3
G
e
r
m
C
o
u
n
t
S
c
h
e
m
e
0.5 1 2 4 6 24
Cont act t ime ( hour s)
0.05% Grot aMar 71
0.1% Grot aMar 71
0.2% Grot aMar 71
Fig. 6Germ count reduction test: Candida albicans.
0
1
2
3
G
e
r
m
C
o
u
n
t
S
c
h
e
m
e
0.5 1 2 4 6 24
Cont act t ime ( hour s)
0.05% Grot aMar 71
0.1% Grot aMar 71
0.2% Grot aMar 71
Fig. 7Germ count reduction test: Rhodotorula.
0
1
2
3
G
e
r
m
C
o
u
n
t
S
c
h
e
m
e
0.5 1 2 4 6 24
Cont act t ime ( hour s)
0.05% Grot aMar 71
0.1% Grot aMar 71
0.2% Grot aMar 71
Fig. 8Germ count reduction test: Aspergillus niger.
0
1
2
3
G
e
r
m
C
o
u
n
t
S
c
h
e
m
e
0.5 1 2 4 6 24
Cont act t ime ( hour s)
0.05% Grot aMar 71
0.1% Grot aMar 71
0.2% Grot aMar 71
Fig. 9Germ count reduction test: Fusaria.
Environmental Concern
Worldwide, strict demands are made of a modern fuel biocide.
The addition of halogen compounds is generally prohibited, as
in accordance with the Federal Decree on Protection from
Emissions. The efforts to use fuel with the lowest possible
sulphur content have resulted in the demand for biocides that
contain no sulphur. On account of the technical demands, the
biocide must dissolve both in the diesel fuel and in the
condensed water phase. Biodegradability and the absence of
heavy metals are also demanded.

SPE 74087 MICROBIAL PROBLEM SOLVING WITH COST EFFECTIVE AND BIODEGRADABLE BIOCIDE IN THE OIL & GAS INDUSTRY 5
Methylene-bis-oxazolidine based biocide is a fantastic
choice in terms of environmentally acceptability. When it is
used correctly, the substance basically passes via the sales
route to the vehicle fuel tank, together with the fuel. Here it is
converted with the diesel fuel into normal combustion
products. There is no additional environmental contamination
through exhaust emissions. The combustion products are not
hazardous, hence in Germany, it is not restricted by the law to
protect against emissions (decree on chlorine and bromine
compounds as fuel additives) 19
th
. Bim Sch V 1992.
If the substance passes into the environment via a
biological clarifying plant, it can be broken down biologically.
If higher concentrations are passed directly into a biological
clarifying plant, they can be inactivated by sodium bisulphate.
Methylene-bis-oxazolidine based biocide is rapidly and
completely biodegradable. Safe disposal of waste biocides or
biocide spillages can be achieved by substantial dilution with
water.
Preventive Measures
A fuel oil could be kept free of microorganisms only by
completely aseptic handling. This is usually not possible on
practice. Hence, even anhydrous fuel oil always contains a
limited number of microorganisms as a consequence of
infection from air, container walls, or by impurities.
It is understood that active propagation of the organisms
only occurs in the presence of water. In addition, the presence
of a free-water phase under the fuel also causes rust to form in
the iron storage tanks. If the storage has no free water, neither
problem will exist.
There is only one sure way to keep a fuel system dry:
“Check for the presence of water on a regular basis, and
remove it when found”. The water checking should be a direct
visual observation of a sample from the lowest point in the
tank. Detergents in the fuel can deactivate the thieving
compounds used as water indicator. Therefore, it is not a
reliable compound for water contents determination. Water
checking and removal from underground storage can be
accomplished by using a hand pump and a long suction pipe.
When buying fuel on the spot market, care must be taken
to ensure that one’s own storage systems are not contaminated
with specially adapted organisms. Sampling systems must
ensure that the microbiological findings are already available
when the fuel is received. Contaminated material must never
be introduced into the tanks without treatment. When taking
fuel from a tanker into the tanks, homogeneous distribution of
the biocide can be ensured. Dosing can take place either via
an injector or via dosing pumps. Of course, care must also be
taken to ensure careful draining.
It should be mentioned here that modern filter/water
separating systems could easily achieve the necessary draining
to < 60-ppm water in order to go below the growth limit for
microorganisms. Experience has shown that with < 60 ppm
water in diesel fuel, further multiplication of organisms can be
prevented. This is because water is mostly present in
dissolved form, and the a
w
(active water) value necessary for
microorganism growth is not reached.
However, these filters are not meant to replace the use of
biocides since microorganisms can easily inactivate the filters
by means of rapid growing and blocking.
Further to avoid recontamination, planning principles for
the new design of tanks are useful and necessary. The
possibility of convenient, economical and regular drainage is
one of the key factors for consideration.
The tank design must permit complete drainage. Drainage
pipes must really be fitted at the lowest point in the system.
Water pockets in the piping system and in slanting filters must
be avoided. The additional incorporation of filter water
separating systems has proven useful, for example in the
German navy.
Unnecessary contamination, such as occurs as a result of
ballast water during transport by river shipping, must be
avoided. In transport via pipelines, care must also be taken to
ensure that there are drainage facilities at the lowest points.
When designing tanks and transport systems, maintenance
of the microbiological quality of the fuel must be included as a
planning principle.
In practice, the following decision tree has proved useful
when determining the measures to be taken:
A. Base sample from the water phase is sludge and
heavily contaminated.
Empty the storage tank and clean with a
suitable disinfectant system cleaner.
Biocide should be added when the tank is
refilled. Shock treatment with biocides is
often necessary. Filtering to remove
particles of dirt should be an automatic part
of the process. The contaminated fuel is
sometimes returned to the refinery for
redistillation.
B. Base samples from the water phase are heavily
contaminated, but no striking sludge formation.
Clean the tank system by means of careful
drainage and subsequent dosing of biocide.
If necessary, adequate mixing can achieve
by pumping the fuel round. In the first week
after adding the biocide, the filters must be
carefully checked because increased sludge
formation can occur as a result of the
microorganisms being killed.
With regard to the economic factors, the
costs of biocide dosing, compared with the
cleaning costs, are of lesser significance, so
that in borderline cases a “killing dose” of
biocide can often be used, even though there
is the risk that cleaning must nevertheless be
carried out later on account of the dirt load
being too great.
C. Fuel and water phases are only moderately
contaminated.
Careful draining of the fuel systems and
subsequent dosing of biocide.
Examples in practice have shown that biocide can be
added at the beginning of the sales route, e.g. in the refinery,

6 W. SIEGERT, P.H. LEE SPE 74087
so that it goes through all the trade stages until the end user’s
tank, e.g. the vehicle tank. A loss of biocide via the sales
route happened due to the consumption for killing
microorganisms and of migration into the water phase of
inadequately drained storage tanks. Hence, correct additional
use-concentrations of biocide have to be determined.
Conclusion
A few examples are employed to illustrate how micro-
organisms in fuel oil grow in the presence of water (e.g.
condensed water in tanks) and the degradation of
hydrocarbons by microorganisms can cause failures in the
form of corrosion clogging of oil burner pipes, filters, engines,
fuel distribution systems, etc.
Diesel fuel produced needs to be kept free of
microorganisms by carefully kept free of water during
transportation and storage. When necessary, contaminated
diesel fuel must be treated with biocide.
Experience has showed that methylene-bis-oxazolidine
based compound is effective in protecting the petroleum
products, especially diesel fuel. Some preventive measures
have also been discussed.
References
1. Kaserer, H.: Cbl. Bakteriol. Parasitenk. Infekt. Abt. II, 15 (1906)
573-576.
2. Söhngen, N.L.: “Benzin, Petroleum, Parafinöl und Paraffin als
Kohlenstoff-und Energiequelle für Mikroben. (Benzene,
petroleum, paraffin oil and paraffin as a source of carbon and
energy for microbes.)” Cbl. Bakteriol. Parasitenk. Infekt. Abt II,
37 (1913) 595-609.
3. Stömer, K.: Über die Wirkung von Schwefelkohlenstoff und
ähnlicher Stoffe auf den Boden. (On the action of carbon
disulphide and similar substances on the soil.) Cbl. Bakteriol.
arasitenk. Infekt. Abt. II, 20 (1908) 282-286.
4. Zobell, C.E.: “Action of microorganisms on hydrocarbon.”
Bacteriol. Rev. 10 (1946) 1-49.
5. Lansdown, A.R.: “Microbiological attack in aircraft fuel
system.” J. Roy Aeronautical Soc. 69 (1965) 763-767.
6. Parbery, D.G.: “Biological problems in jet aviation fuel and the
biology of Amorphotheca resinae.” Material u.Organismen 6
(1971) 161-298.
7. Hill, E.C.: “Microbial degradation of lubricant oils and
emulsions and its engineering significance.” In: Walters, A.H.
and Elphick, J.J. (Editors): Biodeterioration of materials.
Microbiological and allied aspects. Amsterdam, London and
New York (Elsevier Publ. Co. Ltd.) 1968, 381-385.
8. Rogers, M.R. and Kaplan, A.M.: “A survey on the
microbiological contamination in a military fuel distribution
system.” In: Society for Industrial Microbiology: Developments
in Industrial Microbiology, vol. 6, Baltimore, Maryland
(Garamond Pridemark Press) 1965, 80-94.
9. Rogers, M.R. and Kaplan, A.M.: “Screening of prospective
biocides for hydrocarbon fuels.” In: In: Society for Industrial
Microbiology: Developments in Industrial Microbiology, vol. 9,
Baltimore, Maryland (Garamond Pridemark Press) 1968, 448-
476.
10. Mercedes Benz AG: “GrotaMar 71 tested and approved for
diesel fuels.” Service Information, 17 March 1989.
11. Daimler Chrysler: “Freigabe für lhr Microbiocide zum
Dieselkraftstoff – GrotaMar 71.” EP/MPO sch, 30 May 2000.
12. MAN Nutzfahrzeuge AG: “GrotaMar 71 approved for microbial
control.” Service Information, 9 July 1998.
13. Shell Netherlands uses GrotaMar 71 for the preservation of
diesel fuel.
14. Duda, A., Skret, I., Hill, E.C.: “Industrial experience in control
and elimination of microbial contamination of petroleum fuels”
2
nd
International Colloqulum (20-21 Jan 1999) FUELS 1999,
Technische Akademie Esslingen, page 105-109.
15. NATO stock no. 685017-9179543.
16. DTI, UK: Letter of Notification – GrotaMar 71 under Group D
of the Revised Offshore Chemical Notification Scheme. 10 June
1997.