The characterization of oil-degrading microorganismsfrom lubricating oil contaminated (scale) soil

K. JirasripongpunDepartment of Biotechnology, Faculty of Engineering and Industrial Technology, Silpakorn University,

Nakhornpathom, Thailand

65 ⁄ 02: received 25 February 2002, revised 13 June 2002 and accepted 18 June 2002

K . J IRASRIPONGPUN. 2002.

Aims: To isolate and characterize oil-degrading microorganisms from contaminated (scale) soil.

Methods and Results: Oil-degrading microorganisms were isolated from enrichment cultures

of scale soil. Each isolate was identified using 16S rDNA gene and oil degradability was

determined on both unused and used lubricating oil. The weight of the extracted remaining oil

revealed that most isolates degraded unused lubricating oil more than used lubricating oil.

Chemical composition of oil analysed by TLC-FID and GC-MS demonstrated that Nocardia

simplex W9 degraded used oil the best, and resulted in a decrease in saturates, aromatics and

resins to 52Æ46, 38Æ13 and 18Æ81%, respectively.

Conclusions: Nocardia simplex W9 is the best degrader, among all the isolates, on both used

and unused lubricating oil.

Significance and Impact of the Study: The presence of Nocardia simplex W9 in scale soil

enables iron to be recycled by biodegradation.

INTRODUCTION

Lubricating oils are manufactured in various formulations

for different applications. Most formulas generally consist of

two fractions: chemical additives and base fluid. The

chemical additives, about 5–20% (w ⁄ v), are selected com-

pounds added for specific functions (Vazouez-Duhalt 1989).

Base fluid, the main fraction in lubricating oil, is a complex

mixture of hydrocarbons: linear and branched paraffins,

cyclic alkanes and aromatic hydrocarbons (Westlake et al.1974; Goto et al. 1994).

In the iron rolling process at a steel rolling mill,

lubricating oil is normally used to coat an iron sheet as oil

film while subjecting it to a high squeezing pressure and a

high temperature. The iron dust, resembling fish scales, is a

waste product of the process and these oil-contaminated

scales are mixed with soil to produce so called �scale soil�.This scale soil is released, resulting in a great loss of iron

each year. For example the Nippon Steel Company of Japan

at Hokkaido has reported to collect approximately 30 000

tons yearly. Removing oil from scale soil for iron recycling

cannot be simply done by smelting and refining, as the oil

contaminant can create environmental pollution when burnt

(Vazouez-Duhalt 1989). Extracting oil residue to a safe level,

prior to heat treatment, using chemical treatment seems also

to be very difficult due to the high expense and more

additional problems arising from the chemical wastes

(Benton 1997). The cheap, effective and safe method for

iron recycling could possibly be done through microbial

degradation (Venkateswaran and Harayama 1995). There-

fore, the aims of the present study were (i) to isolate oil-

degrading microorganisms from scale soil, (ii) to identify

the isolates using 16S rRNA gene sequence, and (iii) to

study their oil biodegradability compared with the known

degrader, Rhodococcus sp.strain I72 (Shirai et al. 1995).

MATERIALS AND METHODS

Oil and medium

Both unused and used lubricating oils (from a steel company

at Hokkaido in Japan) were studied. Unused lubricating oil

was heated at 100 �C overnight to remove the volatile

hydrocarbons. The used lubricating oil was extracted from

scale soil using dichloromethane, filtered, and subjected to

rota-evaporator (RE121 Rotavapor, Buchi, Switzerland) at

40 �C, 80 rev min)1 for 30 min. The concentrated oil was

finally filtered through a 0Æ2 lm PTFE filter and the

dichloromethane was evaporated in an oven at 40 �C, and

Correspondence to: Dr Kalyanee Jirasripongpun, Department of Biotechnology,

Faculty of Engineering and Industrial Technology, Silpakorn University,

Nakhornpathom 73000, Thailand (e-mail: kalyanee@su.ac.th).

ª 2002 The Society for Applied Microbiology

Letters in Applied Microbiology 2002, 35, 296–300

2000 p.p.m. oil was added to a defined mineral salts

medium, M9, as the sole carbon and energy source when

used. The composition of M9 was 6Æ0 g Na2HPO4, 3Æ0 g

KH2PO4, 0Æ5 g NaCl, 1Æ0 g HN4Cl, 1Æ0 ml of 24Æ6% (w ⁄ v)

MgSO4Æ7H2O and 1Æ0 ml of 1Æ47% (w ⁄ v) CaCl2Æ2H2O in

1000 ml of distilled water.

Isolation and identification

Scale soils were seeded in 5 ml of M9 containing

2000 p.p.m. of unused oil in stainless steel capped test

tubes under shaking at 100 rev min)1, 20 �C for 7 d.

Cultures were enriched two times and isolates were purified

according to Huy et al. (1999) on M9 agar containing

1000 p.p.m. lubricating oil. The isolates were Gram stained,

mobility tested using 0Æ2% semisolid agar plates containing

10 p.p.m. lubricating oil, and species were identified based

on 16S rRNA gene sequences. For identification, genomic

DNA from pure cultures was extracted and purified using a

DNA isolation kit (Puregene, Gentra systems, Danvers, MN,

USA). The DNA (30–500 ng) was subjected to PCR in a

DNA thermal cycler (Takara PCR thermal cycler MP, Takara

Biochemicals, Shiga, Japan) using prOR forward primer

(5¢-AGA GTT TGA TCC TGG CTC AG-3¢) and 9-rev

reverse primers (5¢-AAG GAG GTG ATC CAG CC-3¢).The amplified DNA fragments were purified using Sea

Plaque GTG agarose (FMC Byproducts, Rockland, ME,

USA) and a Qia quick spin column kit (Qiagen, Stanford,

CA, USA). The purified DNA was concentrated using an

evaporator rotary dryer (TOMY centrifugal concentration

CC101) and 30 ng DNA were submitted to DNA sequen-

cing using FS Prism Ready Reaction Dye dideoxyTM

Terminator cycle sequencing kit (Applied Biosystems,

Foster City, CA, USA). The forward sequencing primers

spanned positions 8–27, 335–355, 786–804 and 1223–1241.

The reverse primers spanned positions 357–377, 1100–1115

and 1522–1541. The nucleotide sequences determined by an

automated fluorescent dye terminator sequencer 377S

(Applied Biosystems, Foster City, CA, USA) were then

used to identify isolates using the GenBank Blast search

(http://www.ncbi.nlm.nih.gov/BALST/).

Degradation of lubricating oil and oil analysis

The oil degradability of each isolate was performed similarly

to Goto et al. (1994). The culture tubes of each strain

including positive control (Rhodococcus sp.strain I72) and

negative control (without inoculum), in triplicate of either

used or unused lubricating oil, were incubated for 30 d on a

100 rev min)1 shaker at 20 �C. The remaining oils from each

isolate were extracted using dichloromethane, then they were

dry weight determined and the oil degradability was

calculated based on weight loss (Shirai et al. 1995) as follows.

ð%Þ oil degradation

¼ oil weight in negative control� oil weight in sample

oil weight in negative control

� �

� 100

The efficient isolates were further examined on their oil

degradability in 10 ml M9 containing 2000 p.p.m. used oil.

The tubes with 6% (v ⁄ v) of a 7-day-old culture of each of

the selected strains, including negative controls and positive

controls were tested similarly. Compositions of the extracted

remaining oil were analysed into component groups of

saturates, aromatics, resins and asphaltenes using the TLC-

FID method (Goto et al. 1994), and into selected com-

pounds of n-alkanes (C14–C34), napthalene derivatives

(C2–C4), phenanthrene derivatives (C1–C7), dibenzothi-

ophene derivatives (C1–C4) and hopane using the GC-MS

method (Ishihara et al. 1996).

The chromatograms of each component were automatic-

ally recorded and computed for concentration. The oil

degradation of each component was calculated based on

hopane as follows:

ð%Þ oil degradation

¼

component in negative control based on hopane

�component in sample based on hopane

component in negative control based on hopane

0BB@

1CCA�100

RESULTS

Table 1 shows 26 isolates selected on their growth ability on

unused lubricating oil, weathered oil and aromatic oil of

culture broth derived after enrichment. The isolates named

after source of growth and order of enrichments are

characterized on their Gram stains, motility abilities, species

and oil-degrading abilities as shown in Table 1. Most

isolates were identified based on 16S rRNA gene sequences

with lengths from 1400 bp to 1500 bp to be bacterial species

with 95–100% homology. Only the sequence of isolate A3,

with 1457 bp using 18S rDNA primer, was identified as

yeast. All isolates showed oil degradability on unused

lubricating oil to a higher extent than the used oil after

30 d incubation; the isolates with efficient degradability

were S1, S6, W9, A1, A3 and A4.

Used lubricating oil was the target oil for biodegradation.

Degradability on used lubricating oil in the isolates strains

S1, S6, W9, A1 and A3 were repeated using results from

TLC-FID and GC-MS. However, result from GC-MS

could not be determined. Therefore, TLC-FID was used to

determine the biodegradation of oil, using hopane as the

internal standard. Table 2 shows that W9 resulted in the

highest oil weight lost, and it degraded components in used

OIL-DEGRADERS FROM SCALE SOIL 297

ª 2002 The Society for Applied Microbiology, Letters in Applied Microbiology, 35, 296–300

lubricating oil higher than the strain I72; W9 degraded

saturate, aromatic and resin fractions to 52Æ46, 38Æ13 and

18Æ81%, respectively. While strain I72 degraded each

fraction to 42Æ20, 34Æ01 and 0%, respectively.

DISCUSSION

Microbes are the main degraders of petroleum hydrocar-

bons in contaminated ecosystems (Leahy and Cowell

1990). Serial enrichments to isolate oil-degraders using

lubricating oil as sole carbon and energy source provided

various microorganisms from scale soil. However, fewer

species were isolated as the number of enrichments

increased. The isolates were bacteria and yeast belong-

ing to the genera of Nocardia, Acinetobacter, Pseudo-monas, Ralstonia, Gordono, Rhodococcus, Agrobacterium and

Debaryomyces. These genera are classified as the most

common hydrocarbonoclastic microorganisms listed by

Bossert and Bartha (1984). Among them, Nocardia,

Acinetobacter, Pseudomonas and Ralstonia were frequently

isolated, as was found by Austin and coworkers (Austin

et al. 1977). However, these isolates showed no relevance

of their capacity on oil degrading to the ordinal number of

enrichments. Probably the substrate for sequential enrich-

ment was unchanged, unlike the Venkateswaran’s tech-

nique (Venkateswaran and Harayama 1995) where oil

degradability of the isolates was enhanced using residual

hydrocarbon left after the last enrichment as substrate for

the next enrichment. Varying substrates to weathered oil

lead to no new isolate; the oil components in lubricating

oil and weathered oil were similar (Goto et al. 1994).

However, using aromatic oil containing aromatic fraction

Isolates Identified by NCBI as Characteristics Motility

Oil degradation (%)

Used oil Unused oil

S1 Pseudomonas mandelii G –, c, rod + 12Æ70 27Æ60

S2 Ralstonia eutropha G –, b, rod + 7Æ36 11Æ50

S3 Pseudomonas stutzeri G –, c, rod + 5Æ50 10Æ40

S4 Pseudomonas fragi G –, c, rod + 2Æ95 8Æ79

S5 Acinetobacter junii G –, c, rod + 9Æ20 9Æ95

S6 Rhodococcus sp. G +, rod – 9Æ20 25Æ10

S7 Nocadia asteroides G +, rod, hyphae – 5Æ10 10Æ23

S8 Agrobacterium

tumefaciens

G –, a, rod – 2Æ40 7Æ85

S9 Nocardia simplex G +, rod – 6Æ70 10Æ45

2S1 Comamonas testosteroni G –, b, rod – 2Æ10 7Æ80

2S2 Ralstonia sp. G –, b, rod ND 1Æ80 6Æ10

2S3 Pseudomonas mandelii G –, c, rod + 8Æ14 13Æ10

2S4 Acinetobacter junii G –, c, rod + 7Æ80 10Æ95

3S2 Nocardia simplex G +, rod – 7Æ15 12Æ10

3S3 Nocardia simplex G +, rod – 6Æ50 10Æ58

W1 Acinetobacter junii G –, c, rod + 6Æ43 10Æ67

W2 Pseudomonas monteilii G –, c, rod + 5Æ50 10Æ50

W3 Rhodococcus roseus G +, rod ND ND ND

W4 Pseudomonas monteilii G –, c, rod + ND ND

W6 Nocardia asteroides G +, rod, hyphae – 7Æ82 12Æ00

W7 Ralstonia sp. G –, b, rod ND ND ND

W9 Nocardio simplex G +, rod – 24Æ53 50Æ00

A1 Gordona terrae G +, rod – 14Æ15 30Æ00

A2 Nocardia transvalensis G +, rod, hyphae – 6Æ99 10Æ68

A3 Debaryomyces hansenii Yeast – 8Æ49 19Æ30

A4 Gornado terrae G +, rod – 15Æ00 27Æ10

ND: not determined.

a, b, c: type of proteobacteria.

S ¼ isolates selected from the first enrichment using unused lubricating oil.

2S ¼ isolates selected from the second enrichment using unused lubricating oil.

3S ¼ isolates selected from the third enrichment using unused lubricating oil.

W ¼ isolates selected on weathered oil medium of culture broth from the first enrichment.

A ¼ isolates selected on aromatic oil medium of culture broth from the first enrichment.

Table 1 Characterization of isolates from

scale soil

298 K. J IRASRIPONGPUN

ª 2002 The Society for Applied Microbiology, Letters in Applied Microbiology, 35, 296–300

(Venkateswaran et al. 1995), provided different isolates as

shown in Table 1.

Unused lubricating oil was emulsified under shaking even

in the absence of inoculum, while this was in contrast to the

used lubricating oil. Analysis of the remaining oil showed

more weight loss of unused lubricating oil from each culture

than the used oil. This was probably because the oil

emulsification made available substrate as a microdroplet,

which facilitated hydrocarbons uptake by microorganisms

(Morgan and Watkinson 1994) and resulted in higher

degradation on unused lubricating oil in most isolates.

Chemical composition analysed using TLC-FID of both oils

revealed that unused lubricating oil contained saturates,

aromatics, resins and asphaltenes at 28Æ56, 37Æ02, 21Æ41 and

13Æ02%, while the used oil contained 78Æ42, 12Æ42, 8Æ84 and

0%, respectively (data not shown). The change on oil

components due to high temperature and high pressure in

iron pressing could play a role on the physical property of the

used oil. The analysis of alkanes in both oils using GC-MS

revealed that used lubricating oil contained components of

higher carbon (C > 29) in much higher quantities than

components of the lower carbon (C14–C28) as detected in

unused oil (data not shown). The higher carbon alkane could

result in a low solubility (Eastcott et al. 1988) and is less

degradable than the lower carbon alkane (Westlake et al.1974) as found on used lubricating oil.

However, used lubricating oil was the target for oil

biodegradation in scale soil. Hopane, the most recalcitrant of

oil components present in used lubricating oil, was used as

an internal standard to evaluate oil degradation analysed by

TLC-FID (Prince et al. 1994). The concentration of each

component calculated based on the ratio of peak areas for

each group component and internal standard would give a

more reliable result. Table 2 shows that the isolate strain W9

was the best degrader among the isolates resulting in 34Æ31%

oil weight loss and degraded saturate, aromatic and resin

fractions at 52Æ46, 38Æ13 and 18Æ81%, respectively. The

Rhodococcus sp. strain I72 reported by Shirai et al. (1995) to

be a good degrader on heavy oil with degradability at 46%

less than the isolated strain W9. This was probably due to

the fact that the Rhodococcus sp. strain I72 was a marine oil

degrader, which was grown in suboptimum conditions for

efficient biodegradation to take place. Thus, Nocardiasimplex W9 might be an alternative microorganism used in

biodegradation of oil on scale soil for iron recycling. Other

factors affecting the rate of biodegradation on scale soil, such

as seeding size, available nutrients, the presence of inhibiting

substrates, predation and insufficient movement of seed

organisms within the soil, should be studied in the future to

enhance oil degradation in scale soil.

ACKNOWLEDGEMENTS

This work was financially supported by the JICA (Japan

International Cooperation Agency) on a training course

(course ID: J97-03343). I am indebted to the JICA and the

MBI (Marine Biotechnology Institute, Kamaishi, Japan),

especially Dr Shigeaki Harayama and Dr Hideo Kishira

for their professional advice. Thanks to Dr Nuananong

Jirakarnjanakit and Dr Jureerut Puttanlek for their proof

reading and comments on the manuscript. Thanks also to

Dr Linda Harris and Mr Andrew David Shipley for English

correcting.

REFERENCES

Austin, B., Calomiris, J.J., Walker, J.D. and Cowell, R.R. (1977)

Numerical taxonomy and ecology of petroleum-degrading bacteria.

Applied and Enviromental Microbiology 34, 60–68.

Benton, C.I. (1997) Lubricants. In Environmental Technology in the Oil

Industry ed. Orszulik, S.T. pp. 367–380. Glasgow: Blackie.

Bossert, I. and Bartha, R. (1984) The fate of petroleum in soil

ecosystem. In Petroleum Microbiology ed. Atlas, R.M. pp. 434–476.

NewYork: Macmilliam.

Eastcott, L., Shiu, W.Y. and Mackary, D. (1988) Environmentally

relevant physical-chemical properties of hydrocarbons: a review of

data and development of simple correlations. Oil Chemical Pollution

4, 191–219.

Goto, M., Kato, M., Asaumi, M. and Venkateswaran, K. (1994)

TLC ⁄FID method for evaluation of the crude oil degrading capacity of

marine microorganisms. Journal of Marine Biotechnology 2, 45–50.

Huy, N.Q., Jin, S., Amada, K., Haruki, M., Huu, N.B., Hang, D.T., Cam

Ha, D.T., Imanaka, T., Morikawa, M. and Kanaya, S. (1999) Charac-

terization of petroleum-degrading bacteria from oil-contaminated sites

in Vietnam. Journal of Bioscience and Bioengineering 88, 100–102.

Ishihara, M., Sugiura, K., Asaumi, M., Goto, M., Sasaki, E. and

Harayama, S. (1996) Oil degradation in microcosms and mesocosms.

Microbial Processes for Bioremediation 15, 101–116.

Table 2 Used lubricating oil degradability of the selected degraders

Isolates

Degradation (%) of used lubricating oil

Weight loss

(%)* Saturates� Aromatics� Resins�

S1 3Æ01 9Æ00 22Æ45 2Æ11

S6 9Æ90 25Æ98 29Æ81 0

A1 7Æ86 13Æ88 4Æ46 0

A3 3Æ92 3Æ64 6Æ09 4Æ75

W6 1Æ00 5Æ22 6Æ49 0

W9 34Æ31 52Æ46 38Æ13 18Æ81

I72 20Æ59 42Æ20 34Æ01 0

*Weight of oil (negative control) minus weight of oil (degraded)

divided by weight of oil (negative control) multiplied by 100.

�(%) oil degradation ¼ (component in negative control based

on hopane ) component in sample based on hopane) ⁄ component

in negative control based on hopane · 100.

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ª 2002 The Society for Applied Microbiology, Letters in Applied Microbiology, 35, 296–300

Leahy, J. and Cowell, R.R. (1990) Microbial degradation of

hydrocarbons in the environment. Microbiological Reviews 54, 305–

315.

Morgan, P. and Watkinson, R.J. (1994) Biodegradation of components

of petroleum. In Biochemistry of Microbial Degradation ed. Ratledge,

C. pp. 1–31. The Netherlands: Kluwer Acadaemic.

Prince, R.C., Elmendorf, D.L., Lute, J.R., Hsu, C.S., Halth, C.E.,

Senlus, J.D., Dechert, G.J., Douglas, G.S. and Butler, E.L. (1994)

17a (H), 21b (H) -Hopane as a conserved internal marker for

estimating the biodegradation of crude oil. Environmental Science and

Technology 28, 142–145.

Shirai, K., Hanzawa, N. and Katusta, M. (1995) Heavy oil degrading

bacteria isolated by long term enrichment in alumina columns

containing heavy oil C. Bioscience of Biotechnology and Biochemistry

59, 2159–2161.

Vazouez-Duhalt, R. (1989) Environmental impact of used motor oil.

Science of the Total Environment 79, 1–23.

Venkateswaran, K. and Harayama, S. (1995) Sequential enrichment of

microbial populations exhibiting enhanced biodegradation of crude

oil. Canadian Journal of Microbiology 41, 767–775.

Venkateswaran, K., Hoaki, T., Kata, M. and Maruyama, T. (1995)

Microbial degradation of resins factioned from Arabian light crude

oil. Canadian Journal of Microbiology 41, 418–424.

Westlake, D.W.S., Jobson, A., Phillipe, R. and Cook, F.D. (1974)

Biodegradability and crude oil composition. Canadian Journal of

Microbiology 20, 915–928.

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ª 2002 The Society for Applied Microbiology, Letters in Applied Microbiology, 35, 296–300