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Hydrophobic cell surface and bioflocculation

behavior of Rhodococcus erythropolis

Article in PROCESS BIOCHEMISTRY September 2009

DOI: 10.1016/j.procbio.2009.04.014


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3 authors, including:

Hwai-Shen Liu
National Taiwan University


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Process Biochemistry 44 (2009) 955962

Contents lists available at ScienceDirect

Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Hydrophobic cell surface and bioocculation behavior of Rhodococcus erythropolis

Wei-Nung Chang, Chih-Wen Liu, Hwai-Shen Liu *
Department of Chemical Engineering, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 106, Taiwan, ROC


Article history: An alkane-biodegrading bacterium identied as Rhodococcus erythropolis (NTU-1 strain) was isolated
Received 18 June 2008 from petroleum contaminated soil. The major purpose of the current research was to study the issues
Received in revised form 31 December 2008 regarding biooccules formation and cell surface hydrophobicity of NTU-1. When long-chain alkanes are
Accepted 22 April 2009
supplied as the carbon source, NTU-1 tends to form biooccules and remove signicant amount of
alkanes by biodegradation and physical trapping. Approximately, more than 95% of each alkane could be
Keywords: efciently removed within 4068 h. The bioremediation process was accompanied by formation of
Rhodococcus erythropolis
biooccules with size ranging from 0.1 to 2 cm in diameter. The MATH test and the hydrophobic slide
experiment suggested that NTU-1 might possess a hydrophobic cell surface which is one of the
Hydrophobicity important factors in the formation of biooccules. It provides the interaction of cells with hydrocarbon
Fatty acids droplets effectively and further aggregate into larger clumps. Besides, when grown on n-hexadecane,
Bioocculation experimental results revealed that there were at least 11 different growth-associated fatty acids
produced, with carbon chain length ranging from 12 to 24, and cell surface hydrophobicity was enhanced
via accumulation at the cell surface.
2009 Elsevier Ltd. All rights reserved.

1. Introduction and sometimes benecial to cell growth, such as protection against

protozoan predation, resistance to toxins and better utilization of
Petroleum hydrocarbons pollution in soil, ground, marine carbon sources and energy. Process of bioocculation might be the
and other aqueous environments is commonly encountered and result of following mechanisms: (a) cellcell interactions result in
bioremediation has been recognized as an economical and the cell adhesion to the n-alkanes. (b) Ion adsorption is due to ionic
effective treatment for these contaminations [1,2]. Up to now, it charges from the solution and applied to the cell surface and (c)
was discovered that the mechanisms of hydrocarbon uptake and incorporation of small alkane droplets into cell surface and formed
the microbial catabolic pathways are somehow different a more hydrophobic surrounding at cell wall [911]. Some
according to various microorganisms [35]. Although many investigations observed that an increase in hydrophobicity, lead
reports have shown that the alkane removal is often associated cells to express higher adhesion ability [8,12,13].
with a relatively long bioremediation period (usually 10 days or It was reported that water-insoluble hydrocarbon biodegrada-
longer), the bottleneck and challenge of removing or degrading tion utilizing Rhodococcus is also associated with biosurfactant
hydrocarbons that oat on the water surface are always due to and bioocculant production for efcient occules forming. Most
the low solubility of hydrocarbon in water [6,7]. Rhodococcus of the biosurfactant produced by Rhodococcus contained lipids or
species often play a signicant role in natural degradation of fatty acids [1417]. For example, Rhodococcus rubber produces
hydrocarbons. Therefore, to speed up the biodegradation, three fatty acids as major biosurfactant [18,19]. Fatty acids could
main methods are discussed; namely accelerating dissolution of modify surface tension and increase the contact between cells and
hydrocarbons, the formation of small substrate droplets oil droplets, and these would vary cell surface hydrophobicity
(pseudo-solubilization), and the facilitation of contact between [20]. It is also worth noticing that Rhodococcus cells are
cell and substrate. hydrophobic because of the aliphatic chain of mycolic acid,
The occurrence of biooccules is known to be triggered by glycolipids, fatty acids, and polysaccharides in the cell surface;
environmental factors such as chemical or physical stress, this makes cells contact to oil droplets more easily [21,22]. The
substrate gradient and life stages [8]. Bioocculation is important occulation affects the cell growth and the access of cell to the
substrate during n-hexadecane biodegradation. For example,
bioocculants from Rhodococcus species are useful in bioreme-
* Corresponding author. Tel.: +886 2 33663050; fax: +886 2 23623040. diation processing such as separation of cells in the fermentation
E-mail address: hsliu@ntu.edu.tw (H.-S. Liu). [2325].

1359-5113/$ see front matter 2009 Elsevier Ltd. All rights reserved.
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956 W.-N. Chang et al. / Process Biochemistry 44 (2009) 955962

Our previous research [26] concluded that Rhodococcus 2.5. Microbial adhesion to hydrocarbon
erythropolis NTU-1 can biodegrade signicant amount of n- The hydrophobicity of the strain surface was examined by a so-called microbial
hexadecane, pristane, and some fatty acids. Besides, biooccula- adhesion to hydrocarbon (MATH) by using n-hexadecane as solvent. R. erythropolis
tion were also formed while NTU-1 grew on alkanes. These results NTU-1 were suspended in MS medium with pH values at 7 and 4 (adjusted by 1N
provided a new strategy of bioremediation and made an HCl). Two millilitre of bacterial suspension (OD600 = 0.5) was transferred to 4 mL
test cuvette, and followed by the addition of 0.5 mL of n-hexadecane. After
interesting contribution to the biological treatment of hydro- vortexing for 1 min, allowed standing for 10 min to separate the alkane and
carbons. Therefore, in this study, we try to nd out the reasons why aqueous phase at 30 8C, the initial turbidity (OD600) of the aqueous phase was
NTU-1 tends to form biooccules. With the MATH test and the determined every 10 min. The procedures were suggested by [29]. The difference
hydrophobic glass slide experiment, the cell surface hydrophobi- between the optical densities of the aqueous phase before and after mixing with n-
hexadecane was used to quantify hydrophobicity:
city was characterized. Finally, the variations of cell-wall-bound
fatty acids during biodegradation process were analyzed. Hydrophobicity %
OD600 of aqueous phase after mixing with hexadecane
1  100%
OD600 of initial aqueous phase
2. Materials and methods

2.1. Bacterial isolates

R. erythropolis NTU-1 were obtained from petroleum contaminated soil in 2.6. Preparation of hydrophobic slides
Taiwan. NTU-1 was identied based on sequencing of 500 bp-length 16S rDNA, and
Then, glass slides were immersed in 1% octadecyltriethoxysilane (OTE) solution
the partial nucleotide sequence of the alkB has the GenBank accession number
(dehydrated iso-octane as solvent) for 4 h at room temperature. To remove non-
DQ173199 [26]. NTU-1 cells were maintained in the mineral agar supplemented
adsorbed OTE molecules, the glass slides were soaked in CH2Cl2 for 10 s and then in
with pristane as the carbon source in Petri dish at 4 8C and transferred monthly. R.
CHCl3 for 10 s. Finally, the glass slides were dried by nitrogen ux. All the
erythropolis is an aerobic, Gram-positive, non-motile, rod-shape bacterium and
preparations of OTE solution and silanation reactions were preceded in a glovebag
possess mycolic acid in the cell wall.
lled with dry nitrogen to exclude the amount of water traces in the surrounding
2.2. Medium and growth conditions

All chemicals in this study were of analytical grade (more than 98% purity). The 2.7. Extraction and analysis of cell-wall-bound fatty acids by GC-FID
n-tetradecane (C14), n-hexadecane (C16), n-octadecane (C18), and pristane (C19)
One thousand ppmv of n-hexadecane was supplied as carbon source, and culture
were obtained from SigmaAldrich (U.S.A.); NaCl, K2HPO4, nutrient agar from
conditions were identical with alkane biodegradation test. After 42 and 70 h of
Merck (Germany), and nutrient broth from Difco (France) and all other chemicals
incubation, cells were harvested by centrifugation at 6000 rpm for 15 min and
from Riedel-de Haen (Germany). The bacterial strain was cultivated on mineral
washed three times with 2 mL fresh MS medium. The methods used for extraction
salt (MS) medium consisting of the following ingredients: 1.0 g NaCl, 1.0 g
and preparation of methyl esters of fatty acids were modied from the work of [30].
K2HPO4, 1.0 g (NH4)2SO4, 0.2 g MgSO47H2O, 1 mL trace salt solution in distilled
Around 0.2  0.03 g NTU-1 cells were collected for the fatty acids extraction. After the
water 1 L. The trace salt solution contained 1.0 g FeSO47H2O, 1.0 g MnCl24H2O,
removal of free lipids by extraction with chloroformmethanol (2:1, v/v), cells were
and 1.0 g ZnSO47H2O per liter. The pH before sterilization was adjusted to 7 by
resuspended in 6 mL MS medium. Cell-wall-bound fatty acids were fully hydrolyzed
using 1N HCl. For the preparation of inoculum, NTU-1 was rst picked up from a
for 3 h at 120 8C with equal volume of 25% KOH solution prepared in 50% ethanol. The
stock dish and cultivated in nutrient broth (NB) medium (0.8%, w/v). Cells were
pH was adjusted to 2 by 6N HCl before the aqueous phase was extracted three times
then harvested by centrifugation at 4800 rpm for 15 min after 18 h of cultivation,
with 6 mL of CH2Cl2. After being dried with anhydrous Na2SO4, the dichloromethane
washed twice with MS medium and resuspended in an equal volume of MS
was evaporated by a nitrogen ux. The fatty acids were methylated by adding
medium to 1.2 (OD600).
approximately 5 mL of methanolbenzeneH2SO4 solution (20:10:1, v/v) to 50 mg of
dried dichloromethane extract rst and then by incubating mixture mentioned before
2.3. Alkane biodegradation in sealed tubes for 16 h at 70 8C. The resultant fatty acid methyl esters were extracted
three times with 4 mL n-hexane, washed once with 2 mL of water, and then condensed
To study the effect of biodegradation, 5 mL of inoculum of NTU-1 was added to organic phase with a nitrogen ux. Finally, fatty acids methyl esters were dissolved in
100 mL sterilized MS medium (in 250-mL Erlenmeyer ask) and followed by the small volume of n-hexane.
addition of 10003000 ppmv (v/v) of n-tetradecane, n-hexadecane, n-octadecane, Fatty acids methyl esters (FAME) were analyzed by using GC-FID. GC conditions
and pristane. The asks were then incubated at 30 8C on an orbital shaker at were: the injector and detector temperature were held at 360 and 410 8C,
100 rpm. Samples were taken at 20, 30, 44, 56, 66, and 78 h after inoculation. In respectively. The carrier gas was N2 at the ow rate 5 mL min1. The temperature
early stage of incubation, without bioocculation, residual hydrocarbon which was program was: 180 8C for 3 min, 3 8C min1 to 290 8C, 10 8C min1 to 320 8C. The
not consumed by NTU-1 after incubation in each ask was recovered by extracting fatty acids methyl esters were identied by comparing their retention time with
the culture medium with 30 mL of ethyl acetate directly. To determine the those of standards. The peak height of the carboxylic acids in chromatogram was
efciency of the extractions, n-dodecane (100 mL) was added to each ask as an used to determine each relative amounts [31]. The relative amount of various fatty
internal standard. After biooccules forming, these occules were separated and acids was compared with inoculum.
collected from culture medium through a stainless steel sieve (60 mesh). Both the
culture medium and occules were extracted with ethyl acetate separately. The
ethyl acetate phase was collected after 2 h of extraction. Residual hydrocarbon was 3. Results
determined on a PerkinElmer autosystem gas chromatograph equipped with a
ame ionization detector [27]. A 30 m fused silica capillary column (i.d.: 0.53 mm,
lm thickness: 0.25 mm) with a temperature program of 180240 8C at 30 8C min1 3.1. Bioocculation behavior of R. erythropolis NTU-1
was used. The injection temperature was 250 8C and detector temperature was set
at 300 8C. The amount of each sample injection is 5 mL. Individual hydrocarbon was R. erythropolis NTU-1 was previously found to have obvious
identied through the comparison with standards and quantied with the peak biodegradability of alkanes [26], and therefore was selected for
area against standard curve. The cell growth was directly measured by cell dry
further investigation. Experimental data such as pH value, cell dry
weight. The cells and biooccules were collected through ltration with 0.3 mm
glass-ber lter paper, washed with 30 mL of ethyl acetate, and dried at 110 8C for weight, and alkane biodegradation (n-alkanes and pristane) at
24 h. The method was adopted because the determination of cell growth by different sampling points were collected and presented. Fig. 1A
measuring OD600 was inaccurate as a result of the formation of bioocculates. illustrated the NTU-1 growth with various 1000 ppmv alkanes at
30 8C. It was observed that the cell density of NTU-1 in straight
2.4. Scanning electron microscopy (SEM) alkanes achieved around 0.027 g/100 mL after 40 h of incubation.
R. erythropolis NTU-1 was incubated in MS medium, which supplies with n- In batch cultures, biodegradation was always accompanied with a
hexadecane as carbon source. An appropriate amount of cells was taken after 44 h signicant drop in pH. The declination of pH value, that from 7 to
incubation (after biooccules formed). And biooccules were xed with 2.5% around 45, in the MS medium was quite coincident with cell
glutaraldehyde solution for 1 h and then the samples were dehydrated in series of
growth (Fig. 1B). The growth characteristics were similar to each
20, 40, 60, 80, and 95% ethanol solution for about 30 min, following the SEM sample
preparation procedures by [28]. The major steps included the cleaning, specimen, other when grown on n-alkanes because of there was only two
xation, dehydration and sample coating. Then, sample was scanned using a SEM carbon atoms divergence. Nevertheless, the results of cell growth
machine (JEOL JSM-5310). and pH value revealed an exceptional diversity between straight
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Fig. 1. (A) Growth of R. erythropolis NTU-1 on 1000 ppmv alkanes and (B) pH declination of MS medium. (Initial MS medium acidity: pH 7, 30 8C, 100 rpm).

alkanes and pristane. The declination of pH when grown in alkane in culture medium. Furthermore, the formation and the
pristane was less marked than grown in straight alkanes. This is appearance of biooccules are similar when culturing on n-
mainly because straight chain alkanes are much easier to be tetradecane, n-hexadecane, n-octadecane, and pristane. The results
degraded, which is a common feature of many alkane-utilizing of scanning electron micrograph (Fig. 3B) provide the evidence that
microorganisms. the formation of occule is due to specic cellcell interaction. This
Using straight alkane as a growth substrate, R. erythropolis NTU-1 phenomenon will be discussed later.
attained stationary phase between 40 and 50 h of cultivation, and
tended to form alkane-cell occules. The phenomenon of NTU-1 3.2. Microbial adhesion to hydrocarbon
bioocculation was further tested with other hydrocarbons. We
found that NTU-1 did not occulate when NB, aromatic compounds, In this part of the experiment, we tried to observe the
and short-chain alkanes were supplied directly as carbon sources biooccules forming and the interaction between cells and alkane.
(data not shown). Biooccules were present when NTU-1 was As mentioned in Fig. 2, NTU-1 was capable of utilizing alkanes and
cultured on 1000 ppmv n-tetradecane, n-hexadecane, n-octadecane, causes declination of pH value of culture medium. The declination
and pristane. Details regarding the amounts of alkane biodegraded of pH value, that from 7 to 45, in the MS medium was quite
and trapped in bacterial occules are summarized in Fig. 2A. coincident with cell growth and alkane biodegradation (Fig. 1B). It
Experimental results showed that biooccules were formed at is considered that pH might inuence the hydrophobicity and
approximately between 40 and 50 h for straight alkanes, and 68 h for bioocculation. The MATH tests presented in Fig. 4 are indexes
pristane. During the incubation, NTU-1 not only degraded but also showing the afnity of cell adhesion to n-hexadecane. Some
trapped both n-alkanes and pristane by forming biooccules. reports indicated that cell surface with absorption percentage
Residual alkane which was not consumed by NTU-1 would be (cellular hydrophobicity) up to 70% might be considered as highly
trapped via bioocculation. It is worth to note that, though acidic pH hydrophobic [32]. The results at two pHs also showed that R.
value might hinder biodegradation and cell growth entered erythropolis NTU-1 cells tended to migrate to the oil/water
stationary phase after alkane-cell occules forming, the results interface. The percentage of absorption increased from 40 to
revealed that cell density would increase slightly at lower pH 68% within 3 h of incubation at the pH 7 condition. Higher
condition and the trapped alkane still could be biodegraded by NTU- absorption percentage in acidic condition (pH 4) was noted, up to
1 slowly. Before alkanes were consumed completely, alkanes could 84% after 36 h of incubation. Although pH inuenced the afnity of
be removed via bioocculation and physical separation. Through the cell hydrophobicity, the absorption percentage in these two pHs
biodegradation and separation of occules from the medium, were high and similar, based on the MATH test.
alkanes in solution can be removed efciently (approximately 95% Fig. 5 displays the phenomena of occulation occurred again
or more). Furthermore, biooccules formation has also provided and become bigger one after the addition of extra alkane. As
high removal efciency when the initial alkane concentration was mentioned in Fig. 2A, NTU-1 would trap almost all of residual
doubled or tripled. Bioocculation occurred at roughly 30 h for 2000 alkane (n-hexadecane in solution 0) after 44 h with 1000 ppmv n-
and 3000 ppmv n-octadecane. Thus, signicant amount of alkane hexadecane as a sole carbon source, and then all of the cell occules
could be trapped within bacterial occules (Fig. 2B). Due to the tended to sink in the bottom of the ask (shown in Fig. 5A-1). All
combination of the biodegradation and the removal of the occules, cell occules were resuspended immediately after adding extra
the remaining alkane in solution was 5% or less within a short period 100 mL n-hexadecane into culture medium. It was attributed to the
of cultivation. adhesion of small cell-to-cell aggregates to n-hexadecane droplets.
Different physical appearances of biooccules formed at different Furthermore, these cell occules started to adhere to each other
n-octadecane concentrations are shown in Fig. 3A. Bioocculation and became to form new occules (re-occulation) during 60 min
formed at low substrate concentrations was rm enough and yellow incubation at 30 8C 100 rpm shaking (Fig. 5A-2). After 90 min of
in color. The size of cell occules was about 0.10.5 cm roughly. incubation, extra 100 mL n-hexadecane was added again. It was
While the concentration of substrates was doubled or tripled, the discovered that most of cell occules aggregated to form a bigger
appearance and size were white in color and rounded in shape, with one after 13.5 h of incubation (Fig. 5A-3).
size ranging from 0.5 to 2 cm in diameter. The morphology in Fig. 3A Although the n-hexadecane was not soluble in aqueous phase
seems to be associated with cell density and amount of residual and oated to the top of MS medium immediately after stopping
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958 W.-N. Chang et al. / Process Biochemistry 44 (2009) 955962

Fig. 2. Biodegradation and trap of alkanes in cultures of R. Strain NTU-1. (A) Initial concentration was 1000 ppmv. (B) 2000 and 3000 ppmv n-octadecane. MS medium acidity
was pH 7 (30 8C, 100 rpm).

shaking, the dispersed alkane droplets might contact the cells more 3.3. NTU-1 biooccules attach to hydrophobic slides
frequently and easily during the biodegradation process. In
general, the size of small alkane droplets, observation by a light Another experimental design to investigate NTU-1 forming
microscope, might be divided into two groups in MS medium: (1) cell occules was also performed. Octadecyltriethoxysilane was
0.51.2 mm and (2) 210 mm in diameter without cells. The size of employed to half of glass slide. As shown in Fig. 6A, the slide was
R. erythropolis NTU-1 cell ranges from 1.5 to 3 mm in diameter, the divided into two regions with different hydrophobicties. In
cell might coat with the smaller oil droplets. Then the oil droplet addition, the distribution of n-hexadecane in MS medium
served as bridges between the cells [10]. This phenomenon shown without cells was observed rst. Fig. 6B shows clearly that
in Fig. 5A inferred that the biooccules forming was related to two alkane (2000 ppmv n-hexadecane) dyed with oil-soluble Sudan I
steps: (1) cells attached and consumed hexadecane rst, resulting adhered to hydrophobic region in static condition. Fig. 6C
in hydrophobic cell surfaces. (2) Hexadecane regarded as a linker indicated that large insoluble n-hexadecane droplets, due to its
induced bioocculation via cell-droplet coagulations stacking up low density (0.76), always adhered to the upper part of the
during biodegradation (Fig. 5B). hydrophobic slide when shaken in orbital shaker at 30 8C and
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Fig. 3. (A) Appearance of biooccules formed at 1000, 2000 and 3000 ppmv n-octadecane (incubated at 30 8C; 44 h). (B) SEM micrographs of R. erythropolis NTU-1 after
occulation (44 h; 1000 ppmv n-hexadecane). Bars: 30 mm (left), 8 mm (middle), 6 mm (right).

100 rpm. This appearance demonstrated that oil droplets always of biooccules on the glass slide is similar to previous experiments
oat around upper part (roughly 3 cm) of the MS medium with in Fig. 3.
Fig. 6D illustrates that glass slide pre-treated with OTE was put 3.4. Cell-wall-bound fatty acids extraction
into the ask in order to investigate the attachment of NTU-1 to the
hydrophobic or hydrophilic region with 2000 ppmv n-hexadecane Results in previous experiments seemed to suggest that the cell
incubation. The variation of cell occulation on the slide was surface hydrophobicity enhancement might be involved with
observed during cultivation. Cells were capable of attaching to hydrocarbons biodegradation. Table 1 shows the fatty acid proles
hydrophobic region after 40 h cultivation. The increase in the of cell-wall-bound extract analyzed by GC. The peak height of the
amount of biooccules attached on the glass slide depended on carboxylic acids in chromatogram of n-hexadecane biodegradation
incubation time. It is worth to note, though previous results was compared with the fatty acids prole of inoculum for the
exhibited that NTU-1 owned hydrophobic surface originally, cells determination of its relative amount. Results indicated that, NTU-1
could not occulate and attach to hydrophobic region on the glass contained about at least 11 kinds of major fatty acids originally and
slide in the lag phase or without supplying any long-chain alkanes the fatty acids prole did not change much during n-hexadecane
(e.g., supplied with NB, phenol, and octane). This suggested that biodegradation process. Most relative amount of each fatty acid in
the cell surface hydrophobicity was enhanced signicantly only by cell surface increased signicantly after 42 h of incubation. The
a period of alkane degradation process. Moreover, the morphology relative amount ranged from 2 to 16 folds for 42 h and 2 to 22 folds
for 70 h. It was also found that the variations of relative amount
had only subtle change for those with carbon chain length more
than 20. The ratio of relative amount of fatty acids increasing at 42
and 70 h were similar, and only different in the proportion of
individual fatty acid.

4. Discussion

Although many hydrocarbon-degrading bacteria have been

reported for decades, there have been few microorganisms which
can perform effective hydrocarbons removal via combination of
biodegradation and physical separation. The major purpose of this
study is to nd out the reason why the biooccules formed and cell
surface hydrophobicity increased during growth on alkanes. Some
researches exhibited the specic cellcell interactions which
involved hydrophobicity also contributed to bioocculation
[3,10,12]. For instance, occules formation of R. erythropolis DCL
was mainly due to hydrophobic cell surface [33].
From the MATH test, results illustrated that NTU-1 itself
Fig. 4. Hydrophobicity test (MATH) of R. erythropolis NTU-1 to n-hexadecane at pH 7 possesses the hydrophobic cell surface. The observation in Fig. 5
and pH 4. provided the evidence that the formation of biooccules was
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Fig. 5. (A) The phenomena of cell re-occulating by adding 100 mL n-hexadecane. (B) Brief sketch of bioocculation. Cells attach to oil droplet rst, and cell-droplet
coagulations stack up during biodegradation process.

Fig. 6. (A) Hydrophobic region on the glass slide. (B) 2000 ppmv n-hexadecane dyed with Sudan I adhered to hydrophobic region of glass slide in static condition. (C)
2000 ppmv n-hexadecane droplets attached to hydrophobic region with the liquid depth of about 3 cm (orbital shaking at 100 rpm). (D) NTU-1 attached to hydrophobic
region of the glass slide during 2000 ppmv n-hexadecane biodegradation. (Orbital shaking at 100 rpm, 30 8C).

likely due to the oil droplets that served as linker between the the alkanes, which were more easily degraded, such as aliphatic
cells occules when treating long-chain alkanes as the carbon hydrocarbons of lower molecular weight [34]. These hydro-
source. Moreover, the results in Fig. 6D also elucidated that phobic components might accumulate while microorganisms
NTU-1 could attach to the hydrophobic region of glass slides were cultured on alkanes [20,22], and the carboxylic group of
with the aid of n-hexadecane droplets (Fig. 6C), roughly about them had afnity to alkanes. For example, R. erythropolis tends
3 cm in depth from the liquid level. These results inferred that to accumulate alkane and fatty acids in intercellular and cell
the formation of biooccules might be associated with the surface in order to increase the transport rate for hydrocarbons
contact between suspended cells and alkane-aqueous interface biodegradation [35]. This is consistent with the ndings in many
or alkanes droplets. reports. (1) The properties of the cell surface are related to the
In fatty acid analysis, the results indicated that accumulation environment in which microorganisms exist and the time of
of fatty acids might enhance cell surface hydrophobicity and incubation [8,25,31]. (2) It is considered that the hydrophobic
made the contact between cells and oil droplets more easily. The cell surface changes in order to make microorganisms utilize
increase of fatty acids probably derived from the oxidation of hydrophobic carbon sources more easily [36,37]. (3) During the
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W.-N. Chang et al. / Process Biochemistry 44 (2009) 955962 961

Table 1 [5] Yamada-Onodera K, Mukumoto H, Katsuyama Y, Tani Y. Degradation of long-

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