Volume 5 Preprint 14
An insight into the mechanism of sludge formation in naphtha storage tank
A. Rajasekar, S. Maruthamuthu, N. Muthukumar, S. Mohanan, P. Subramanian and N. Palaniswamy
Keywords: sludge, water contamination, degradation, bacteria, corrosion, FTIR, NMR, XRD
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Volume 5 Preprint 14
An insight into the mechanism of sludge formation in
naphtha storage tank
A. Rajasekar, S. Maruthamuthu1 N. Muthukumar, S. Mohanan,
P. Subramanian and N. Palaniswamy
Corrosion Science and Engineering Division,
Central Electrochemical Research Institute, Karaikudi -630 006, Tamilnadu, India.
Abstract: Degradation of petroleum product problem arises since hydrocarbon is an
excellent food source for a wide variety of microorganisms. Microbial activity leads to
unacceptable level of turbidity, corrosion of pipeline and souring of stored petroleum
product. In the present study, biodegradation of naphtha in a storage tank has been
investigated. Huge quantity of sludge was noticed in a naphtha storage tank in South
West India. To investigate the reason for the formation of sludge, iron bacteria,
manganese oxidizing bacteria, acid producers, and heterotrophic bacteria were
enumerated and identified in the sludge and in water. Sulphate reducing bacteria (SRB)
could not be noticed either in the sludge or in water. IR spectroscopy study showed the
formation of primary alcohol during degradation process. NMR showed the presence of
heteroatom in the sludge. A mechanism for the formation of sludge has been proposed.
Key wards: sludge. water contamination, degradation, bacteria, corrosion, FTIR, NMR,
Corresponding author: E-mail: firstname.lastname@example.org. Tel.: 04565 – 227550 Fax:914565-227779
This is a preprint of a paper that has been submitted for publication in the Journal of Corrosion Science and
Engineering. It will be reviewed and, subject to the reviewers’ comments, be published online at
http://www.umist.ac.uk/corrosion/jcse in due course. Until such time as it has been fully published it should
not normally be referenced in published work. © UMIST 2004.
Microbial contamination of fuels has been the cause of intermittent operational
problems throughout the world for many years. Even less than 0.1% of water
contamination is enough for microbial activity leading to biodegradation of
hydrocarbons. In order to prevent the effects of microbial growth, several lines of
approaches such as good house keeping practices, treatment with biocides to limit the
growth and use of special tank linings, etc are used. The types and ability of
microorganisms to degrade petroleum hydrocarbons have been widely documented (1-5).
Internal corrosion as a cause for leakage of steel tanks were documented in US, France,
Sweden and Switzerland by various sources (6-9). Oil and Natural Gas Corporation Ltd
(ONGC), India noticed the failure of pipeline by heterotrophic bacteria and SRB in
Indian offshore pipelines, but degradation of product was not noticed (10). Jana et al.
(11) reported the combined effect of CO2, SRB, and chloride in the low velocity area
causing severe corrosion and failure of pipeline in Mumbai offshore.
In the present case naphtha is stored in a tank. The major constituents of naphtha
is presented in Table A1. This stored naphtha is transported through a 22 inch diameter
underground pipeline (5.5 Km) to a nearly thermal power station. Corrosion products of
about 10 Kg is collected once in 2 month at the filters in the naphtha receiving end at the
thermal power station. Large quantity of sludge was noticed in the naphtha storage tank
where the disposal of sludge has to be cleared by the pollution control board. Water
contamination of a minimum of 4 to 5 inches height is found to exist in the naphtha
storage tank at the bottom of the tank. The microbial growth in the sludge often made
severe turbidity and cloudiness of naphtha. Moreover, sludge often changed the actual
chemical properties of naphtha in the storage tank and transporting pipelines. Since no
information is available on the mechanism of naphtha degradation in naphtha storage
tank, the present study has been designed to investigate the reasons for the sludge
formation in the naphtha storage tank in tropical Indian condition.
Materials and methods
Enumeration and identification of microbes
Sludge from the naphtha storage tank and water samples were collected using
sterilized conical flasks. The conical flasks containing the samples were kept in an icebox
and transported from the site to CECRI- Microbiology laboratory. The collected naphtha
sludge and water samples were serially diluted (10 fold) using 9ml of sterile distilled
water blanks and the samples were plated by pour plate technique. The nutrient agar
medium, iron medium, API Broth and Mn-medium (Hi-media, Mumbai) were used to
enumerate heterotrophic bacteria (HB), iron bacteria (IOB), acid producers (AP), sulphate
reducing bacteria (SRB) and manganese oxidizing bacteria (MOB) respectively. Plates in
triplicate were prepared for each dilution. The plates were inverted and incubated at 33°C
for 24 hours.
After 24 hours, the colonies were counted and isolated. The plates
containing bacterial colonies with 30-300 numbers were selected for calculation. The
bacterial colonies were expressed as colony forming units ( CFU) per gm /ml of naphtha
sludge and water samples respectively. SRB was enumerated by MPN technique.
Morphologically dissimilar colonies were selected at random from all plates and
isolated colonies were purified using appropriate medium by streaking methods. The pure
cultures were maintained in specific slants for further biochemical analysis. The isolated
bacterial cultures were identified up to genus level by their morphological and
biochemical characterization viz gram staining, motility, indole, methyl red, Vogurs
pruscur, citrate test, H2S test, carbohydrate fermentation test, catalase test, oxidase test,
starch , gelatin, lipid hydrolysis etc (12).
Chemical properties of sludge and water
5 gms of sample of sludge was mixed with 100 ml of triple distilled water and
agitated for 2 hrs using shaker. After shaking, the samples were filtered and the filtrates
were used for chloride and sulphate analysis. Chloride was estimated by Mohr’s method
and sulphate was estimated by the gravimetric method. Chloride and sulphate were also
estimated in contaminated water collected in the storage tank.
FTIR was used for the analysis of biochemical characteristics of the samples of
sludge and naphtha. The spectrum was taken in the mid IR region of 400– 4000 cm-1 and
recorded using ATR (Attenuated Total Reflectance) technique. The sample was directly
placed in the zinc selenide crystal and the spectrum was recorded in the transmittance
H NMR Spectroscopy (Bruker,300m Hz)) was also used for the analysis of the
sludge samples. The sample of sludge was dissolved in deutrated chloroform solvent and
Tetra Methyl Silane (TMS) was used as a reference standard.
The sample of sludge collected from the storage tank was dried and crushed to a
fine powder and used for XRD analysis to determine the nature of the complex formed in
the sludge. A computer controlled XRD system, JEOL Model JDX – 8030 was used to
scan between 100 and 850 – 2 θ with copper K α radiation (Ni filter) at a rating of 40
KV, 20 mA.
Enumeration and identification of microbes in the sludge and in water
Table.A2 presents the data on the enumeration of bacterial population in naphtha
sludge and water. The heterotrophic bacteria count was in the range of 106 CFU in one
gram of sludge and one ml of water. Iron bacteria, manganese oxidizing bacteria and
acid producers were in the range between 103 and 105, whereas SRB was too low to count
both in the sludge and in water. In all the types of microbes, gram-negative bacteria
Among the heterotrophic bacteria isolated, gram negative bacteria were more
dominant than gram positive bacteria by 80%. Generic distribution was found to be
Pseudomonas sp.(20%), Bacillus sp.(10%), Gallionella sp.(10 %) and Vibrio sp. (10%).
All the manganese-depositing bacteria and iron oxidizing bacteria isolated from the
sludge were completely dominated by gram negative bacteria. Generic
composition was dominated by Gallionella sp. (25%) and followed by Legionella sp.
(12.5%) and Siderocapsa sp. (12.5%). Among iron bacteria, Gallionella sp. (22.22%) and
Thiobacillus sp. were equally shared and followed by Bacillus sp. (11.11%). Acid
producing bacterial isolates were found to be gram negative. Among them Thiobacillus
sp. 28.6% was followed by Thiospira 14.25% and Sulfolobus sp. 14.25%.
Chemical properties of sludge and water
Table A3 presents the data on the sulphate and chloride content present in the
water and sludge. The chloride content was 7 ppm whereas the sulphate content was 155
ppm in water. The chloride and sulphate content in the sludge was about 12ppm and
60 ppm respectively.
The FTIR spectrum (Figure.A1a) of the naphtha shows peaks at 2955 cm-1, 2923
cm-1, 2855 cm-1 indicating the presence of CH-aliphatic stretch. The peaks at 1457 cm-1
and 1378 cm-1, indicate the CH def for methyl group. The peaks in the range between
693 cm-1 and 727cm-1 indicate the presence of substituted benzene. The FTIR spectrum
(Figure.A1b) of sludge shows a broad peak between 3000 and 3500 cm-1 indicating the
presence of OH-band. Another peak at 1033 cm-1 indicates the CO stretching for primary
alcohol group. The peak at 1635 cm-1 indicates the presence of SH stretch.
The 1H NMR spectrum of naphtha shows (Figure -A2) , peak at 0-3 chemical shift
(δ) indicating the presence of aliphatic protons in naphtha compounds. The other peak at
7 chemical shift (δ) indicates the presence of aromatic nuclei in the naphtha compounds.
FigureA3, shows the 1H NMR spectrum of sludge. A peak at 1.5 chemical shift (δ)
indicates the presence of water. Another broad peak between 4 and 6 chemical shifts (δ)
indicates the presence of heteroatom-included proton (Hydrogen). This may be due to
the presence of S-H in the sludge.
Figure A4 presents the details of XRD data corresponding to the phases present in
the sludge sample. α Iron III oxide hydroxide, Iron sulphate and ferric sulphate were
noticed in the sludge.
Microbial activity in oil industries can result in fuel contamination, unacceptable
level of turbidity, filter plugging, corrosion of storage tanks, pipelines and souring of
stored products (13-14). Hence it is quite essential to investigate the nature of degradation
of fuels. The degradation of diesel and crude oil has been studied in oil – spilled soil by
Delille (15). Lloyed Jones et al.(16) isolated alicyclic hydrocarbon utilizing consortia
Rhodococcus sp. Flavobacterium sp. and Pseudomonas sp. isolated from oil refinery soil.
April (17) noticed 64 species of elemental fungi from five flare pits in northern and
western Canada that were tested for their ability to degrade crude oil using gas
chromatography analysis which indicated that the species were capable of degradating
hydrocarbon of the aliphatic fraction of crude oil, nC12 , n-C26. Besides, Roffey (18)
reported on the aerobic and anaerobic degradation in crude oil and in diesel storage
tanks. In the present study, the roles of microbes on degradation of naphtha and the
mechanism for naphtha degradation have been explained .
The enumeration of microbes show the presence of heterotrophic bacteria (HB),
iron oxidizing bacteria (IOB), manganese oxidizing bacteria (MOB) and acid producers
(AP) in the sludge and water. Sulphate reducing bacteria (SRB) could not be noticed in
water and sludge. The sulphate level was about 155 and 60 ppm in water and sludge
respectively. It is surprising that though sulphate is present in water and sludge, sulphate
reducing bacteria could not be noticed. The pH range for the growth of SRB is 6.5 to 8.5,
with optimum being 7.2 to 7.5 (19). The broad ecological classes of sulphur-oxidizing
bacteria can be discerned, among those living at neutral pH and those living at acid pH
(20), whereas many of the forms are living at acid pH. In the present study, the pH of the
sludge was 6.8. At the interface between naphtha and water, the pH was about in the
range between 5.5 and 6.0. The absence of SRB may be due to the domination of acid
producers (AP), iron oxidizing bacteria (IOB) and manganese oxidizing bacteria (MOB).
The acidity creates “syntrophy” for the three communities of microbes namely acid
producers, manganese oxidizers and iron oxidizers which suppress the proliferation of
SRB. Pseudomonas sp., Bacillus sp., Gallionella sp. Thiobacillus sp., Thiospira sp.,
Sulfolobus sp. Legionella sp., and Siderocapsa sp were noticed in the sludge.
Pseudomonas is strictly aerobic, chemoorganotrophic which is able to use other than one
carbon organic compounds as sole carbon and energy sources, catalase positive, usually
oxidase positive. It is aerobic having a strictly respiratory type of metabolism with
oxygen as the terminal electron acceptor; in some cases nitrate can be used as an alternate
electron acceptor, allowing growth to occur anaerobically. Some species are facultative
chemolithotrophs able to use H2 or Carbon as energy sources. Gallionella is a
chemolithotrophic bacteria capable of oxidizing ferrous to ferric ion while assimilating
significant quantitative C14O2 and grow in oligotrophic waters in nature. Iron hydroxide
may make up 90% of dry weight of cell mass. Microaerophilic,optimum pH 6-9 and O2
concentration 1mg/l, found in ferrous ion containing water and in soils. Bacillus has
chemoorganotrophic metabolism with single source of carbon and energy with inorganic
N2. It is strictly respiratory, strictly fermentative or both using various substrates. It is
strict aerobes or facultative anaerobes, gives catalase positive in test. In the absence of
organic compounds it acts as facultative chemolithotrophs using H2 as energy source. It
can tolerate in the temperature range of 25- 750C and pH value of 7.5- 8.0 to 2.
Thiobacillus sp. is strictly autotrophic. Energy derived by the oxidation of thiosulphate to
sulphate, sulphur granules and polythionates may accumulate depending on culture
conditions, Elemental sulphur is slowly oxidized. also oxidizes, other partially reduced
sulphur compounds including H2S, tetrathionate and in some strains thiocyanate. It is
strictly aerobic- Growth occurs between pH 4.5- 7.8. Sulfolobus is gram negative and
aerobic which uses elemental sulphur as energy source. It is facultative autotrophs and
prefers pH optimum being 2-3; maximum 5.8; minimum 0.9. Thiospira are usually with
pointed ends, with sulphur inclusions Siderocapsa are spherical to ovoid cells embedded
in a common capsule, partially encrusted with iron and/ or manganese compounds which
is aerobic but can grow under reduced oxygen tension. Legionella are nutritionally
fastidious. They require iron and L-Cysteine- HCl and iron salts are required for growth.
It is chemoorganotrophic, using aminoacids as carbon and energy sources. Vibrio is
chemoorganotrophs, having both respiratory (O2) and fermentative. Metabolism of CHO
is fermentative with mixed products, but no CO2 or H2. On the basis of heterotrophic
bacteria and autotrophic bacterial physiology, it can be assumed that heterotrophic
bacteria utilizes energy from naphtha and the conversion of elemental sulphur in naphtha
has been utilized by acid producing bacteria viz Thiobacillus sp and Sulfolobus sp to
sulphuric acid and dissolved as sulphate in water and sludge. Hence it can be concluded
that the presence of sulphate in water is due to the activity of microbes on elemental
sulphur. Since naphtha with water is transported through the pipeline under pressure the
supply of oxygen may be sufficient for the aerobic bacteria. The concentration of chloride
was about 10 ppm in the water and in the sludge samples. This data reveals that chloride
contribution on corrosion may be nil.
Naphtha is not a single compound, it has many organic constituents viz n- Butane,
Isopentane, 2- Methyl Pentane, n- Hexane, Benzene, n-Heptane, Methyl cyclohexane,
Toluene, 3-Methyl Heptane,3,5 Di Methyl cycloheptane and n- Nonane with sulphur.
IR results (Fig.1b) indicate that CH-aliphatic stretch was degraded by microbes .The
absence of peaks in the sample of sludge in the range between 674 and 727 indicates that
benzene ring is consumed by microbes since the major components of n-heptane (-CH2CH2-)n, toluene and benzene in naphtha are degraded as R-CH3 by consumption of
hydrogen. Heterotrophic bacteria break the organic constituents by consumption of
hydrogen and convert into R-CH3 where as autotrophic bacteria increases the addition of
oxygen and convert as R-CH2-OH (primary alcohol). The addition of oxygen or sulphate
(4-5 chemical shifts (δ)) heteroatom can be seen in NMR spectrum and the spectrum
indicates that both aliphatic and aromatic group are degraded by microbes. The addition
of oxygen is due to the very rapid addition of oxygen by Gallionella sp and Legionella sp
which supports with the observation made by Muthukumar et al (21). Moreover
Gallionella .being chemolithotrophic, microaerophilic and acidophilic species, produces
toxic oxygen products (H2O2) as an outcome of its metabolism and it needs the
scavenging mechanism to overcome the toxic product. Here the role played by
manganese oxidizing bacteria and iron bacteria seems to be vital. The manganese and
iron oxidizers were used for scavenging the produced toxic oxygen product. This is
where the syntrophic relationship between Thiobacillus and Gallionella could be
appreciated. Mars et al (22) studied the effect of trichloro ethylene on the competitive
behaviour of toluene- degrading bacteria viz Pseudomonas putida and Burkholderia sp.
Besides Shim (23) also reported the trichloroethylene degradation by toluene-o-xylene
monooxygenase of Pseudomonas sp. Microbial degradation of nitrobenzene was reported
by Sten et al (24) by Comamonas testosterone and Acidovorax delafieldii and noticed the
broad degradation ability towards nitrobenzene. Zhu (25) characterized the microbial
communities like Proteobacteria sp. and Comamonas denitrificans in gas industry
pipelines and mentioned about the importance of microbially influenced corrosion. In
composite crude oil (26) where the aqueous and oil phases co-exist, the potentially
limited bacterial corrosion may be enhanced in a matrix population of crude oil degraders
and non degraders. Even where the corrosion causing bacteria do not utilize hydrocarbon
as energy sources, the intermediate degradation will boost the energy available to the
corrosion causing bacteria to sustain the corrosion reaction. Jobson et al (27) also
reported that intermediate hydrocarbon degradation products make available energy
sources for the physiological activities of the corrosion bacterium Desulfovibrio sp. This
supply of utilizable hydrocarbon degradation products explaines why corrosion was
intensive in the Pembiana oil pipeline. Besides Westlake et al (28) reported corrosion by
ferric reducing bacteria isolated from oil production system at Pembiana oil pipeline.
They identified oil degraders viz Bacillus, Aeromonas, Cornebacteria in the oil wells and
suggested that heterotrophic bacteria converted oil into lactic acid which was utilized by
Pseudomonas and Clostidium and encouraged the formation of FeS. In Indian refined
diesel product pipeline, the benzene in the diesel was utilized by heterotrophic bacteria
Brucella sp and converted into aliphatic compound (21) which supported the theory by
Traxler and Flamming (29) whereas Gallionella sp took energy from only aliphatic
compounds in diesel. Toluene and ethyl benzene were used as sources of carbon and
energy by microbes where as the ethyl benzene was degraded by monooxygenase enzyme
(30). Besides Rhodococcus rhodochrons S-2 produces extra cellular polysaccharides that
help to live in aromatic fraction (31). XRD results reveal the presence of ferrous and
ferric sulphate in the sludge which indicate the role of iron/manganese bacteria while acid
producers cause the formation of sulphate in the water and sludge. In the inner side of the
tank severe corrosion was noticed, where there is no paint. The paints peeled off from
the surface at many spots and this severe corrosion may be due to the abrasion caused by
the movement of the floating roof top followed by the exposure of the surface to the
atmosphere. In can be concluded that Fe++ comes from the storage tank/ pipes and
combines with organic degraded products, whereas organic compound is degraded by
heterotrophic bacteria and Fe++ can be converted as Fe+++ by autotrophs viz. Gallionella
sp and Legionella sp, which was noticed in XRD data (21).
(- CH2- CH2-)
( R CH3-) n
R- CH2 OH
Figures A5 and A6 indicate that degradation starts from naphtha –water interface and it
can be understood that the presence of inorganic products (iron oxide and iron sulphate)
from the storage tank combine with naphtha degraded products at the interface and
settles down in the storage tank. The aromatic and aliphatic compounds in naphtha are
degraded by heterotrophic bacterial activity and subsequently autotrophic bacteria
converts ferrous and manganese on the metal into oxides. The degradation may favour
the microbiologically influenced corrosion of pipeline and at the bottom of the storage
tank. It can be concluded that in fuel transporting pipelines/storage tank, water can
stratify at the bottom of the line / storage tank , which encourage the degradation.
Hence, the degradation accelerate the corrosion by the formation of Fe2O3. A simplest
model can be envisaged.
Mechanism for sludge formation in naphtha storage tank
AP, IOB, MOB
Fe++ +2e(Anodic reaction)
Fe+++ ¼ O2 +
(CH2 – CH2 )n
Fe+++ + ½ H2O
S2O32-(Thiosulphate) Addition of Oxygen
(Acid Producers )
(H2SO4 in water)
H+ + e(Cathodic reaction)
R-CH2 – SH
Iron- Organic Complex (Sludge)
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Table A1. Major constituents in Naphtha
Name of the constituents
Percentage by Weight
2- Methyl Pentane
Methyl Cyclo Hexane
3,5 Di Methyl Cyclo Heptane
Table A2. Enumeration of Bacterial Population in Sludge and Water
Total Viable Count (CFU/gm)
7.2 x 106
1.62 x 104
3.8 x 103
1.02 x 104
Total Viable Count (CFU/ml)
4.8 x 107
6.3 x 105
4.2 x 105
a = One tube blackening at 10-2 dilution, HB = Heterotrophic Bacteria, IB = Iron Bacteria
MOB = Manganese Oxidizing Bacteria, AP=Acid producing bacteria, SRB = Sulphate
Table A3. Physiochemical Properties of a Sludge and Water
Settlement of degraded
Naphtha and iron
Figure 1: FTIR spectrum of naphtha degradation.
Figure 2: 1H NMR spectrum of naphtha (control).
Figure 3: 1H NMR spectrum of sludge.
Figure 4: XRD pattern of the sludge sample
1 and 2 - Ferric oxide, 3 - silicon-di-oxide,
4 and 5 - manganese and its oxides
Figure.5: Sludge formation between Water-Naphtha interface
Figure.6: Sludge formation at the bottom of water-naphtha system