Journal of Environmental Biotechnology
Vol. 8, No. 1, 43–47, 2008

Original paper (regular paper)

Characterization of Burkholderia sp. Y1 Isolated from Oil Polluted Soil
WATARU MURAOKA1 ＊ , TAKUJI NAKASHIMA3, YASUHIRO TABIRA2, HIROSHI EGUCHI2, KAZUYUKI IMAGAWA1,YOSHIMASA MASTUMURA4, SATOSHI TAKESHITA2 and TAKEHIRO TAKEMASA1
1

Graduate School of Science and Technology, Nagasaki University, Bunkyo 1-14, Nagasaki City, Nagasaki 852-8521, Japan
2
Joint Research Center, Nagasaki University, Bunkyo 1-14, Nagasaki City, Nagasaki 852-8521, Japan
3
NITE Biological Resource Centre, National Institute of Technology and Evaluation, Chiba 292-0812, Japan
4
Nagasaki Senkan co., ltd., Nameshi 4-5-35, Nagasaki City, Nagasaki 852-8521, Japan* TEL: +81–(0)95–819–2745 FAX: +81–(0)95–819–2745* E-mail: d705134b@cc.nagasaki-u.ac.jp(Received; 15 September, 2007/Accepted; 12 February, 2008)
Burkholderia sp. Y1 was isolated from oil polluted soil as a lipid-degrading bacterium. Strain Y1 degraded 83.1% of salad

oil when it was cultivated in the basic medium containing 1% (w/v) of salad oil for 2 days at 35°C. When cultivated in medium containing 5 and 10% (w/v) of oil, strain Y1 showed lipid degradation rates of 30.2% and 15.4%, respectively, suggesting that strain Y1 could maximally assimilate 0.15 g/l salad oil. The lipid degradation was dependent on the initial cell density. Thin layer chromatography (TLC) analysis of culture supernatant indicated that strain Y1 first dehydrated triglycerides insalad oil in the degrading process and produced free fatty acids, diglycerides and monoglycerides, then gradually degraded
residues. Also, gas chromatography (GC-FID) analysis indicated that strain Y1 was able to assimilate C16 to C20 of fatty
acids in salad oil equally.
Key words: lipid degradation, Burkholderia sp., waste water treatment

1. Introduction
All facilities discharging waste water containing oils are
required to be equipped with grease traps in Japan. It is estimated that an average of around 1.1 kg of oil is discharged
in a year from one facility which discharges less than 50 m3
of waste water per day4). The waste oil accumulated ingrease traps has to be removed frequently, and it generally
costs ten to thirty thousand yen per month. To reduce thecost of handling grease traps is a significant problem.
Many detailed researches have been reported concerning
lipid-degrading bacteria such as Acinetobacter 17,19) ,Pseudomonas21,5) or Rhodococcus9). Similarly, there havebeen several reports concerning the eﬀectiveness of bacterial
consortium20,1). However, most of them were conducted using media containing only less than 1% (w/v) oil, and it
took a long time for microbes to degrade oil. At an actual
site, a much higher oil density is accumulated and a more
rapid treatment system is required. In this study, we isolated
and characterized a bacterium able to degrade high concentrations of salad oil.
2. Material and methods
2.1. Reagents
Without some exception, all reagents were at ultra puregrade and were purchased from Wako Pure Chemical Industries, Ltd., Tokyo, Japan and salad oil was purchasedfrom a market.
2.2. MediaThe stock solution used for suspending isolated microbes
contained 8.0 g of NaCl, 2.0 g of KCl, 36.3 g of Na2HPO4·
12H2O, 2.4 g of KH2PO4 and 200 ml of glycerol in one literof sterilized water. This solution was adjusted to pH 8.0,
then autoclaved at 121°C for 15 min. The basic medium used
for enrichment culture and estimating lipid degradation
rate contained 1.0 g of NaH2PO4, 2.0 g of K2HPO4, 2.0 g of
(NH 4 ) 2 HPO 4 , 0.1 g of yeast extract and 0.2 g of
MgSO4·7H2O in one liter of sterilized water, (pH 7.0)12).
Standard Method Agar (SMA), Lactobacilli MRS Agar(MRSA) (Becton, Dickinson and Company Sparks, MD,USA) and Yeast Extract Glucose Chrolamphenicol Agar
(YEGCA) (Becton) mixed with victoria blue dye base oil(VBO) were used for isolating lipid-degradation microbes.
VBO was prepared following the procedure of Standard
Method for the Count of Lipolytic Organisms6).
2.3. Isolation of lipid degradation microbes
Suspensions of (1% (v/v)) soil or liquid samples collectedfrom oil polluted sites were inoculated to 10 ml of basic medium containing 10% (w/v) of salad oil and incubated at

MURAOKA et al.

44

35°C for 7 days with 100 rpm agitation. The culture showing
the highest lipid degradation rate was chosen for an isolating source. The sample suspension was subjected to enrichment cultivation again under the same conditions for 2 days,
and the culture broth was spread on SMA, MRSA andYEGCA supplemented with VBO for single colony isolation. Each isolated strain was cultured on SMA without
VBO and colonies were suspended in the basic medium
at optical density at 660 nm (OD660) to 1.0 (approximately
109 CFU/ml), then incubated with 1% (w/v) of salad oil at
35°C for 7 days with 100 rpm agitation to estimate the lipid
degradation rate.
2.4. Measurement of lipid degradation
The lipid degradation rate was estimated by the hexane
extraction method5) with a slight modification. Briefly, theculture broth was transferred to a 50 ml centrifuge tube.
The flask was washed three times with 5.0 ml of hexane tocompletely remove salad oil and added to transferred culture
broth. After adding 1.0 g of sodium chloride, suspension
was shaken for 5 min and ultrasonically treated for 10 min
at 50°C, then centrifuged 1,500×g for 10 min. The hexane
layer was transferred to a new tube and the volume wasmade up to 30 ml with hexane. One milliliter of this hexane
solution was used for TLC and GC-FID, and the remaining
solution was transferred to an aluminum cup. After evaporation on a hot plate for 10 min, the hexane extracts were
heated to dry at 80°C for 30 min and cooled down for
30 min in a desiccator. The residues were weighed to estimate
the lipid degradation rate.
2.5. Identification of strain Y1
Strain Y1 was identified by morphological and biochemical
tests16) and 16S rDNA sequence analysis. DNA was extracted from strain Y1 as previously described13) and its 16S
rDNA was amplified by polymerase chain reaction (PCR)
using primers 9F and 1541R (Table 1). The PCR mixture
contained 30 ng of genomic DNA, 3.2 pmol of each primer,
1 fold of buﬀer solution, 25 mmol of dNTPs and 5 unitsof Taq polymerase (TaKaRa Bio Inc, Shiga, Japan). PCR
amplification was carried out at 94°C for 4 min, then 35 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min, andfinally 72°C for 15 min14) and the amplified DNA was purified
by phenol-chroloform method. Sequencing reaction was
carried out with a Big dye terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) according to the manufacturer’s specifications by using sequencing primers 9F,

341F, and 515F. Sequencing was automatically performedon an ABI Prism 3100-Avant genetic analyzer (Applied
Biosystems). The sequences were compared with the compilation of 16S rDNA genes available in the database (DDBJ,
EMBL and GenBank).
2.6. TLC analysis
The hexane extracts were analyzed by TLC using TLC plastic sheets of silica gel 60 F254 (Merck, Darmstadt, Germany),
with a mobile phase of hexane, diethyl ether and acetic acid
(80 : 30 : 1, v/v/v). Mono-, Di- and Triglyceride Mix (SUPELCO) was spotted for the standard. The spots of lipids andresidual products were detected using sulfuric acid steam2).
2.7. GC-FID analysis
The hexane extracts were esterified by the Boron Trifluoride-Methanol Method7). Esterified substrates were dissolved
in dichloromethane up to 1 ml. n-Dodecane (C12) was addedin each sample as the internal standard. One microliter of thesuspension was analyzed with a gas chromatograph (AutoSystem XL GC, Perkin Elmer Corporation, CT 06859-0010,
USA) equipped with a flame ionization detector (FID) anda capillary column (DB-23, length 30 m, I.D. 0.25 mm, film
thickness 0.1 μm, J&W Scientific, USA). The carrier gas washelium, and injector and detector temperatures were set at
260°C. The column temperature was first held at 60°C for
2 min, then raised to 140°C at the rate of 30°C min–1,
continuously raised to 240°C at the rate of 2°C min–1, andfinally held for 10 min at 240°C. Oil contents were sequenced
by comparing to Supelco 37 Component FAME Mix (FAME37) (SUPELCO, Bellefonte, PA).
3. Result and Discussion
3.1. Isolation and identification of lipid degrading microbes
Oil contaminated soil and water samples were collectedfrom 60 sites. In the first screening, over 10% lipid degradation rate was observed with 10 samples. Eighty strains
were isolated from the sample exhibiting the highest lipid
degradation rate in the first screening. Restriction fragment
length polymorphism of 16S rDNA revealed that isolates
were classified into 4 diﬀerent groups. Among them, strain
Y1 showed the highest lipid degradation rate, approximately
80% after cultivating with the 1% (w/v) of salad oil in thebasic medium at 35°C for 2 days. The morphological andbiochemical tests showed that strain Y1 was a gram nega-

Table 1. PCR and sequencing primers used in this study.
Primer No.

E. coli numbering

Base sequence

Reference

9F
341F
515F
1541R

9–28
341–357
515–534
1521–1541

5’-GAGTTTGATCCTGGCTCAG-3’
5’-CCTACGGGIGGCIGCA-3’
5’-GTGCCAGCMGCCGCGGTAA-3’
5’-AAGGAGGTGATCCANCCRCA-3’

11
8
15
18

Lipid degrading Burkholderia sp. Y1

tive, catalase reaction positive, oxidase reaction positive andsmall rod shape bacteria. Strain Y1 grew and degradedsalad oil well at temperatures from 28°C to 35°C. Optimal
temperature for oil degradation was 30°C. Strain Y1 also
grew on YEGCA, indicating that strain Y1 is tolerant tochloramphenicol (Table 2).
The carbon source assimilating pattern of strain Y1 wasanalyzed by BioLog automated microbial identification/
characterization system (Biolog, Inc., Hayward, CA, USA)for the gram-negative microplates in aerobic conditions10).
Strain Y1 was able to metabolize 62 of 95 substrates in thepanel, indicating that the strain Y1 is most closely related to
Burkholderia pyrrocinia with 60.8% similarity (data notshown). In addition, 16S rDNA analysis was performed inorder to reconfirm the identification of strain Y1 as B.
pyrrocinia. A 1031-bp DNA fragment containing the 16S
rRNA gene was amplified by PCR and sequenced. Similarity
search with the BLAST program found that the 16S rRNA
gene of strain Y1 was 100% identical to that of Burkholderia
cepacia strain ESR63 (EF602558). Although tere is discrepancy between Biolog and 16S rRNA analysis, both analysis
classified strain Y1 to Burkholderia, we identified strain Y1
as Burkholderia sp. strain Y1 (EMBL/GenBank/DDBJ
accession Number AB376079).

45

density), respectively (Fig. 1). This indicates that strain Y1
is able to maximally degrade 1.5% (w/v) of oil. Since thesalad oil physically attached to the flask wall during cultivation, the lipid degradation rate on the condition of 1% (w/v)
oil might not reach to 100%. Then, eﬀects of the initial celldensity on the oil degradation were investigated. The initialcell density (OD660) was adjusted to 0.01, 0.1 and 1.0 andthe lipid degradation rate at 1% (w/v) of salad oil was
determined after cultivation for 5 days. The highest degradation rate was observed when the initial OD660 was 1.0 (Fig.
2). In that cultivation, the lipid degradation reached 77.4%
on 1 day after the start of cultivation and gradually increased
to 90% on 2 days. The degradation rates with the initial
OD660 of 0.01 and 0.1 were lower than that with the initial
OD660 of 1.0 and the maximum degradation of 80–87% was
observed on 4 days after the start of cultivation. Thus, thelipid degradation by strain Y1 is dependent on the initialcell density.

3.2. Characterization of lipid degradation ability ofstrain Y1
Further experiments were performed to investigate theability of strain Y1 to degrade salad oil. First, eﬀects of oilconcentrations on the degradation rate of strain Y1 were
investigated. Strain Y1 was cultivated in the basic medium
containing 1%, 5% and 10% (w/v) salad oil at 35°C for
2 days. The initial cell density (OD660) was adjusted to 1.0.
The lipid degradation rate at 1, 5 and 10% (w/v) were
83.1%, 30.2% and 15.4% (0.083, 0.151 and 0.154 g/l oil

Fig. 1. Effects of oil concentrations on lipid degradation by strain
Y1.
Strain Y1 was cultivated in the basic medium containing 1%,
5% and 10% (w/v) salad oil at 35°C for 2 days. The initial celldensity (OD660) was adjusted to 1.0.

Table 2. Characteristics of strain Y1.
Gram stain
Bacterial shape
Colony color (on SMA without VBO)
Catalase reaction
Oxytase reaction
Growth on:
SMA aerobic
anaerobic
MRSA anaerobic
YEGCA aerobic
anaerobic
Growth at:
28°C
30°C
35°C
37°C

–
Rod
Dark cream
+
+
++
+
–
+–
+
+
++
+
+

++, strongly positive; +, positive; +–, weakly positive and –,negative.

Fig. 2. Effects of the initial cell density on lipid degradation bystrain Y1.
The initial cell density (OD660) was adjusted to 0.01 (squares),
0.1 (triangles) and 1.0 (circles) and the lipid degradation rate
at 1% (w/v) of salad oil was determined after cultivation for
5 days.

46

MURAOKA et al.

3.3. Mechanism of lipid degradation by strain Y1
We then characterized the salad oil degradation processof strain Y1. Strain Y1 (the initial OD660=1.0) was cultivatedin the basic medium with 1% (w/v) salad oil at 35°C.
Culture broth was sampled everyday and its supernatants
were subjected to TLC and GC analysis. Strain Y1 was able
to dehydrate triglyceride in salad oil quite well, and produced
free fatty acids, diglycerides and monoglycerides on the firstday (Fig. 3). After dehydration of triglyceride, strain Y1

Fig. 3. TLC analysis of culture supernatants of strain Y1.
Strain Y1 (OD660=1.0) was cultivated in the basic medium
with 1% (w/v) salad oil at 35°C.
Laine M, Fame37; arrow A, triglyceride; arrow B, diglyceride;arrow C, free fatty acid; arrow D, monoglyceride.

Fig. 4. GC-FID analysis of nonculture (A) and culture (B) supernatants of strain Y1.
Strain Y1 (OD660=1.0) was cultivated in the basic medium
with 1% (w/v) salad oil at 35°C. Culture broth was sampled
after 5 days cultivation and its culture supernatants were esterifed for GC analysis equipped with flame ionized detector. nDodecane was used as the internal standard.

assimilated all residual oil components. Culture broth on
the fifth day was analyzed by GC-FID. The salad oil used inthis study contained C12 to C20 fatty acids. The GC-FID
showed that strain Y1 was able to degrade about 80% of allcomponents in salad oil during 5 day cultivation (Fig. 4).
The area calculation found 89.0% total reduction rate, andthis is strongly related to the hexane-extracted lipid degradation rate of 88.3%.

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