Journal of Environmental Biotechnology
Vol. 5, No. 2, 107–109, 2006

Original paper (regular paper)

Improvement of Heavy Oil Degradation by Rhodococcus erythropolis C2
HISAE AOSHIMA1*, TATSUAKI HIRASE1, TAKAKIYO TADA1, NAOYA ICHIMURA1,HIDEUKI YAMAGUCHI2, MITSURU TAGUCHI2 and TAKESHI MYOENZONO2,3
KRI Inc., Kyoto Research Park, 134, Chudoji-minami-machi, Shimogyo-ku, Kyoto, 600–8813, Japan
Kanto Natural Gas Development Co. Ltd., 3–1–20, Nihonbashi Muromachi Chuo-ku, Tokyo, 103–0022, Japan
3Technoearth Co. Ltd., 661, Mobara, Mobara, Chiba, 297–0026, Japan* TEL: +81–3–3288–1131 FAX: +81–3–3288–1132* E-mail: haoshima@kri-inc.jp
12(Received; 3 September, 2005/Accepted; 10 December, 2005)
Rhodococcus erythropolis C2, which is able to degrade several kinds of fossil fuel, was isolated from soil samples. Oilconsumption ratios of light oil, heavy oil (type-A), and heavy oil (type-C) of strain C2 were >80%, 80%, and 60%, respectively. The oil consumption ratio of the type-C oil increased by a maximum of 25% depending on the amount of light oiladded. The oil consumption ratio of the heavy oil (type-C) was improved by decreasing the viscosity of the oil mixture.
This indicated that the viscosity of the oil compound is an important factor to enhance fuel degradation.
Key words: bioremediation, oil degradation, Rhodococcus erythropolis, viscosity

The most widely distributed environmental pollution canbe attributed to hydrocarbon contamination, caused by oiltanker accidents, storage tank ruptures, and transport accidents. The environment pollution by hydrocarbons at oldpetrol stations or factory sites is a serious problem as well,
as not only does the pollution cause damages to the environment, but also the sales value of the land decreases significantly. Physical technologies, such as combustion andsolidification, have been carried out to remove hydrocarbons from contaminated soils. Although physical techniques
may shorten the work period with low costs, plants are notable to grow in these soils. It is well known that microbial
degradation of spilled hydrocarbons is a major technique inthe natural decontamination process8). Therefore, various
bacteria degrading hydrocarbons have been isolated, andbioremediation technologies by those bacteria have been
investigated3,4,6,9,11,12).
Fuels are classified into three classes according to their
physical and chemical properties (Table1). Petroleum, acomplex of individual compounds, and its components are
generally grouped into four classes according to their diﬀerential solubility in organic solvents; (i) the saturates (n- andbranched-chain alkanes and cycloparaﬃns), (ii) the aromatics (mono-, di-, and polynuclear aromatic compounds containing alkyl side chains and/or fused cycloparaﬃn rings), (iii)the resins (aggregates with a multitude of building blocks
such as pyridines, quinolines, carbazoles, thiophens, sulfoxides, and amides), and (iv) the asphartenes (aggregates ofextended polyaromatics, naphthenic acids, sulfides, polyhydric phenol, fatty acids, and metallopophyrins)5). Fuels also
contain hundreds of complicated compounds (Table 1). An

analysis of the hydrocarbon class composition was carriedout by thin layer chromatography with flame ionization
detection (TLC/FID, IATRON MK-6, Iatron, Tokyo,Japan) 13).
In our previous study, oil degradable bacteria (especiallyfor fuels) were isolated from various places in Japan andassessed to their degradation characteristics for some typesof fuel (Aoshima, H. et al., unpublished results). R. erythropolis C2, from Nishinomiya, Hyogo Pref., was found themost eﬀective for degradation of several types of mineral oilof all isolated bacteria (Fig. 1). The oil consumption ratio

Fig. 1. Electron micrograph of R. erythropolis C2 (×5.0 K).

AOSHIMA et al.

108
Table 1.

Chemical properties of Light oils and Heavy oils.
Light oil

TLC/FID

Chemical Properties

ClassHeavy oil

S1

1

23

S3

Flash Point

50<50<50<45<45<

Distilled Attribution (°C)
(90% distilled Temp.)

360>

360>

350>

330>

330>

Pour Point (℃ )+5–2.5–7.5

–20

–30

Setane Index

50<50<50<45<45<Viscosity (mm2/s)

2.7<

2.7<

2.5<

2.0<

1.7<

Sulfur (wt%)

0.20>

Water (vol%)—Ash (wt%)—

Type-A
1

2

Type-C

Type-B

160<2

370<—

5>

10>

—

—

—

50>

250>

400>

400~
1000

3.0>

3.5>

—

—

0.4>

0.5>

0.6>

2.0>

—
20>
0.5>

2.0>
0.3>
0.05>

0.5>

Saturates

—

68.5

—

—

—

62.2

—

—

37.2

—

—

Aromatics

—

27.5

—

—

—

34.9

—

—

55.4

—

—

Resins

—

3.6—

—

—

2.6

—

—

3.7

—

—

Asphartens

—

0.4

—

—

—

0.4

—

—

3.7

—

—of strain C2 was almost 80% for both light and heavy oil(type-A). However, the oil consumption ratio for heavy oil(type-C) of R. erythropolis C2, had a maximum of 60%,
and was lower than that for light oil and type-A oil. Inorder to improve the biodegradability of R. erythropolis C2
for heavy oil (type-C), we examined the eﬀects of detergents
such as food additives and light oil on the biodegradationof heavy oil (type-C).
R. erythropolis C2 was pre-cultured in 0.5% yeast extract
at 35°C for 24 h. The pre-culture solution (0.2 ml) was added
to 20 ml of W medium (2.0 g (NH4)2SO4, 0.2465 g MgSO4,
2.78 g FeSO4, 14.7 g, CaCl2, 0.5 g NaCl, 14.3 g Na2HPO4,5.44 g KH2PO4, 2.01 g ZnSO4, 0.15 g (NH4) 6Mo7O24, 0.2 gCuSO4, 0.4 g CoCl2, 1.49 g MnSO4 per 1 L) containing
20,000 ppm heavy oil (type-C) and 0 to 20,000 ppm of light
oil as sole carbon and energy source or 0.0005% to 0.05%
of detergent, and cultured at 120 rpm, at 35°C for 72 h. Inorder to examine the eﬀectiveness of the detergents, various
types of Triton-X, sodium dodecyl sulphate (SDS), fatty
acid esters, polymer detergents such as casein and carboxymethyl cellulose were used to enhance the degradability ofheavy oil (type-C) by R. erythropolis C2.
The oil consumption ratio by strain C2 was estimated by
the weight measurement method after the chloroformmethanol (=3 : 1 v/v) extraction. Chloroform-methanol (18ml) was added to the culture (20 ml) to extract the remaining oil component, and the mixture of solvent and culture
was stirred at 120 rpm for 15 min at room temperature.
The mixture was transferred to a centrifuge tube and centrifuged at 6,000×g for 10 min at room temperature. The
weight of the extracted oil was measured after the lowerlayer was collected and the solvent had volatilized for
3 days. Oil consumption ratio was calculated from residual

oil components.
Most detergents did not significantly improve oil degradation. The growth of strain C2 was inhibited by the additionof Triton-X series and SDS. While strain C2 was able togrow in the fatty acid ester better than that in other detergents, fatty acid esters consisting of trehalose slightly improved oil degradation. This result suggest that strain C2
might be degrade the fatty acid as a carbon source before
the heavy oil compound. The fatty acid ester is structurally
similar to the biosurfactant such as trehalose 6, 6'-dimycolate
secreted by Rhodococcus sp1). Therefore, the fatty acid ester may slightly contribute in forming stable oil-in-water
emulsions of heavy oil to promote the indigenous biosurfactant of C2. In contrast to detergents, light oils enhanced theheavy oil consumption ratio according to the increased
amount of light oil (Table 2). Most of the contents of light
oil were saturates and aromatics, which are easily degradedby strain C2. Therefore, the amount of bacteria in a liquid
culture containing light oil as a sole carbon source wasmore numerous than that containing heavy oil (type-C). As
a result, light oil increased the production capacity of thebiosurfactant. Simultaneously, the biosurfactant was secreted into the culture solution and was able to stabilize aheavy oil-in-water emulsion. In addition, this phenomenon
can be ascribed to co-oxidation, in which persistent hydrocarbons are oxidized in the presence of hydrocarbons which
can serve as growth substrate such as light oil. Evidence forco-oxidation of recalcitrant substrates was provided by asphaltenes and other aromatic hydrocarbons2,7). In addition,light oil could be helpful to reduce the viscosity of heavy
oil, and increase the aﬃnity of heavy oil for the culture solution. Sugiura et al. have investigated the biodegradationof four diﬀerent crude oil samples, and demonstrated theImprovement of heavy oil degradation
Table 2.

109

Consumption ratios of heavy oil (type-C) under various conditions.

Light oil (ppm)

C-heavy oil (ppm)

Total oil conc. (ppm)

Consumption ratio of total
oil mixture (%)

Consumption ratio of
C-heavy oil (%)

020,000

20,00043.4 (±17.5)

43.4

5,000

20,00025,00062.9 (±4.5)

56.1

10,00020,00030,00080.3 (±11.2)

75.1

15,00020,00035,00085.3 (±2.4)

81.7

20,00020,00040,00087.9 (±3.2)

85.8

20,0000

20,00093.0 (±0.5)—

ACKNOWLEDGEMENTS
We thank Prof. Motoki Kubo for his many useful suggestions.

References

Fig. 2. Relationship between the viscosity proportion of oil compounds and consumption ratio of heavy oil (type-C) consumption by R. erythropolis C2. Viscosity proportion showed valuesof viscosity at 5 rpm per viscosity at 1 rpm.

biodegradability of crude oil was negatively proportional toviscosity10).
The viscosity of each oil sample was determined by a rotary viscometer (type; Programmable DV-III+ Rheometer,Brookfield, USA) at 22°C at a shearing rate of 2/sec2. The
oil consumption ratio of heavy oil was improved by the decrease in the viscosity of the oil mixture (Fig. 2). Oil viscosity and the consumption of heavy oil had a negative proportional relationship. The equation of the calibration curve
was Y=–164.03X+132.85 and the correlation coeﬃcient (r2)
was 0.9833. Hence, a reduction in oil viscosity accelerated
the degradation ability of heavy oil by strain C2. However,in the case of soil contamination, it is diﬃcult for bacteria
to attach to the solidified oil components after weathering.
In order to improve the eﬀectiveness of the contact between
solidified oil and bacteria, dissolution of solidified oil bylight oil will be the eﬀective method to encourage the oil
degradation in soil environment.
In this study, the relationship between fuel degradationand viscosity was demonstrated14). It is highly possible thatthe reduction of viscosity of polluted oils contributes to accelerate degradation of contaminated soil environments. To
further elucidate the advantage of lower oil viscosity forbacteria, we started to analyze the amount of bacteria under various viscosities.

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