Applied Surface Science 443 (2018) 255–265

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Applied Surface Science

journal homepage: www.elsevier.com/locate/apsusc

Full Length Article

Carbon fibers with a nano-hydroxyapatite coating as an excellent biofilm

support for bioreactors
Qijie Liu a, Chao Zhang a, Yanling Bao b, Guangze Dai a,⇑
a
School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, PR China
b
Aerospace Composites Research Institute, Xi’an 710000, PR China

a r t i c l e i n f o a b s t r a c t

Article history: A biofilm support with high biocompatibility is needed for bioreactors. A nano-hydroxyapatite (HA) coat-
Received 26 October 2017 ing on carbon fibers (CFs) was prepared by electrochemical deposition (ECD). The sludge immobilization
Revised 27 January 2018 assays, bacterial cells adhesion assays and Derjaguin–Landau–Verwey–Overbeek (DLVO) theory were
Accepted 11 February 2018
used to evaluate the capacity of CF supports to immobilize activated sludge and bacterial cells. The sludge
Available online 12 February 2018
immobilization and bacterial cells adhesion assays illustrated that HA coating could enhance the capacity
of CFs to immobilize microorganisms. SEM images showed that HA and bacterial cells formed a dense
Keywords:
film on CFs surface. In addition, HA, acting as a glue, could combine CFs with bacterial cells or between
Hydroxyapatite
Carbon fibers
cells, which helped CFs capture more bacterial cells. DLVO theory illustrated that CFs with HA coating had
Biofilm supports a lower total interaction energy than CFs without handling, explaining the higher capacity of CFs with HA
DLVO theory coating to immobilize bacterial cells. This result was owning to the less negative zeta potential and higher
Bacteria adhesion hydrophilicity of CFs with HA coating, and the hydrophilicity made a greater contribution to the lower
Biocompatibility total interaction energy. Experiments and theory reveal that HA coating could enhance the biocompati-
bility of CFs, and CFs with HA coating could be used as an excellent biofilm support for bioreactors.
Ó 2018 Elsevier B.V. All rights reserved.

1. Introduction
Biofilm supports for microorganisms are widely used in
bioreactors for wastewater treatment [1,2], beer production [3],
methanogenesis [4], etc. Biofilm supports could prevent the out-
flow of microorganisms and decrease the damage to microorgan-
isms caused by changes in environment [5]. The support material
with excellent biocompatibility could improve efficiency of biore-
actors. Accordingly, it is important to optimize the design of sup-
ports for the immobilization and growth of microorganisms.
Activated carbon and synthetic resins were used as support mate-
rial for methanogenic phenol-degrading consortia and possessed
the highest adsorption capacity for Pseudomonas aeruginosa cells
[6]. Tsekova and Llieva [7] used polyurethane foam as support
material for immobilizing Aspergillus niger B-77, and the efficiency
of copper removal reached more than 99%. Zeolite [8] has been
widely used as support material for the removal of ammonium in
anaerobic digestion because of its favorable characteristics for
microorganism adhesion.
Carbon fiber is widely used as biofilm supports owing to its high
chemical durability and good capacity to immobilize microorgan-
⇑ Corresponding author.
E-mail address: daiguangze@swjtu.edu.cn (G. Dai).
isms. Matsumoto and Ohtaki [9] proved that CF is an excellent bio-
film support for wastewater treatment by experiments and theory.
However, CFs without handling has smooth surface, great inertia,
low surface energy and low reactivity, which could not cater for
the growing needs of bioreactor. Consequently, it is significant
for CFs to improve biocompatibility for immobilizing microorgan-
isms. Some researchers were focused on improving the biocompat-
ibility of CF. Bao and Dai [10] used nitric acid oxidized CF as a
biofilm support material, and CF after nitric acid oxidation has a
good biocompatibility. Wang and Liu [11] revealed that nitric
acid-treated CF has enhanced hydrophilicity and could immobilize
more Candida tropicalis cells in xylitol fermentation. In our previ-
ous work [12], carbon nanotubes/carbon fiber hybrid material
was evaluated as a biofilm support material for wastewater treat-
ment. Carbon nanotubes deposited on CF surface could enhance
the biocompatibility of CF to immobilize E. coli and S. aureus cells.
Hydroxyapatite (HA, Ca10(PO4)6(OH)2) exists widely in nature as
an inorganic component of bones and teeth [13]. HA has excellent
biocompatibility, and it has a potential to be a outstanding support
material for microorganisms [14]. Kamitakahara and Takahashi [5]
found that HA has a good capacity for the adhesion of E. coli
because of the high hydrophilicity. Fibers with HA coating have
been applied for tissue engineering application [15,16]. However,

https://doi.org/10.1016/j.apsusc.2018.02.120
0169-4332/Ó 2018 Elsevier B.V. All rights reserved.
256 Q. Liu et al. / Applied Surface Science 443 (2018) 255–265
no studies so far have investigated CFs with HA coating as biofilm
support material for bioreactor.
In this study, a nano-hydroxyapatite coating on carbon fibers
was prepared by electrochemical deposition (ECD). HA-coated
CFs with different deposition times (60 min, 120 min and 180
min) compared with CFs without handling to evaluate them as
support materials for microorganisms. The sludge immobilization
and bacterial cells adhesion tests were used to investigate the
properties of CFs as support material for microorganisms in prac-
tice. We adopted Escherichia coli (E. coli) and Staphylococcus aureus
(S. aureus) here, due to their common representative strains of
Gram-negative bacteria and Gram-positive bacteria, respectively.
Derjaguin–Landau–Verwey–Overbeek (DLVO) theory was used to
calculate interaction energy profiles between support materials
and bacteria cells. The interaction energy profiles would be the
important factor to determinate the properties of CFs as supports
for microorganisms in theory. The results implied that HA coating
on CFs could significantly improve the capacity of CFs to immobi-
lize microorganisms.
2. Materials and methods
2.1. CF supports
PAN-CFs (Commercial T300, diameter:
7 lm, purity: >92%)
was used as CF supports in this study. To remove a hydrophilic siz-
ing agent (mainly hydrophilic surfactant and polyvinyl alcohol), CF
bundles (0.2 g) were immersed into acetone (500 mL), and the
solution was heated to reflux at 60 °C (thermostatic water bath)
for 72 h. Then CF bundles were washed with distilled water and
dried at 120 °C (electric vacuum drying oven) for 4 h (denoted as
CF-0).
For depositing HA coating, CFs (0.05 g) and graphite electrodes
acted as cathode and the parallel anode in ECD, respectively. The
electrolyte solution consisted of 3.80  104 mol/L NH4H2PO4,
6.35  104 mol/L Ca(NO3)2 and 0.1 mol/L NaNO3, with Ca/P ratio
being 1.67 [17]. 0.1 mol/NaNO3 was added to improve the conduc-
tivity of the electrolytes. 3.80  104 mol/L NH4H2PO4 and 6.35 
104 mol/L Ca(NO)3 were used corresponding to the pH value of 6
[18]. DC Power (M8853, Maynuo electronics) was applied to supply
a constant current. The temperature of electrolyte solution was
refined to 98.4 °C in a water bath. To find the optimal constant cur-
rent, CFs were deposited for 60 min at different depositing currents
(1.0 mA, 3.0 mA, 5.0 mA, 7.0 mA and 9.0 mA). After deposition, CFs
were rinsed with distilled water and dried at room temperature.
CFs were deposited for 60 min, 120 min or 180 min (denoted as
CF-HA60, CF-HA120 and CF-HA180, respectively) at 5.0 mA deposit-
ing current. The electrolyte solution was renewed every 60 min.
And HA-coated CFs were used as cathode under the same condi-
tions. After deposition, HA-coated CFs were rinsed with distilled
water and dried at room temperature. CF supports were weighted
before and after deposition to calculate the mass percentage of HA
coating.
A field-emission scanning electron microscopy (FE-SEM, Quanta
FEG 250, FEI) was used to observe the surface morphology of CF
supports. The chemical properties of CFs were presented by fourier
transform infrared (FTIR) spectrometry. A fully computerized Nico-
let 5700 spectrometer was adopted to record FTIR spectra (KBr dis-
persed pellets) in the range of 400–4000 cm1 at a resolution of 4
cm1. Energy-dispersive X-ray analysis (EDS, X-Max 50, OXFORD)
was applied to analyze the elemental content of CFs surface. The
crystal structure of the HA coating was carried out by a X-ray
diffraction (XRD, D8 Advance, BRUKER) instrument. The instru-
ment was operated with a Cu Ka radiation source at 40 kV and
40 mA.
2.2. Sludge immobilization
Activated sludge was obtained from a municipal wastewater
treatment plant. The activated sludge was cultivated in a thermo-
static container with organic synthetic wastewater (peptone, 0.48
gL1; meet extract, 0.32 gL1; urea, 0.08 gL1; NaCl, 0.024 gL1;
NaHPO4, 0.08 gL1; KCl, 0.011 gL1; CaCl2, 0.011 gL1; MgSO4,
0.008 gL1) [9] under aeration at 25 °C for one month.
The mixed liquor suspended solids (MLSS) concentration in the
activated sludge suspension was adjusted to 4000 mg/L. CF sup-
ports were cut into segments of 10 cm in length and grouped into
2 g bundles. Thirty bundles of a support were fixed on one side of a
stick (50 cm long) and immersed into the activated sludge suspen-
sion. At every sampling time point, three bundles of CF supports
were taken out and slightly rinsed with deionized water. Then,
CF supports were dried and weighed. The mean mass of sludge
immobilized on one gram CF supports was calculated.
2.3. Bacterial cells adhesion tests
Escherichia coli and Staphylococcus aureus were purchased from
China General Microbiological Culture Collection Center (CGMCC).
The strains were grown in Beef extract medium (10 g/L peptone, 3
g/L beef extract, 5 g/L NaCl) at 37 °C under shaking at 120 rpm over
night. Bacteria cells in exponential phase were harvested by cen-
trifugation (2500 g, 15 min) and then were suspended in 10-fold
serial dilution phosphate-buffered saline (pH = 7.2) to obtain bac-
teria suspension with an initial optical density (OD) of 0.65 at
600 nm (1.3  109 CFU/mL). 0.02 g of CF supports were immersed
into 10 mL of bacteria suspension to immobilize bacterial cells.
And the suspension was shaken at 200 rpm by a universal shaker.
A visible spectrophotometer was used to measure OD600 value of
the suspension at every time point. For CFU (colony-forming units)
formation assays, the bacteria suspension (1 mL) after 2 h adsorp-
tion was employed. After gradient dilution, the diluted suspensions
(20 lL each in triplicate) were dispersed evenly on nutrient agar
culture medium plates for aerobic incubation at 37 °C for 3 days,
and then the colonies were counted.
For FE-SEM observation of bacteria cells on CFs surface, CF sup-
ports were taken out after 1 h adhesion and soaked in 2.5% glu-
taraldehyde for 12 h to fix bacterial cells. Then CF supports were
dehydrated in graded ethanol (30%, 50%, 70%, 85%, 95%, 99.5%,
20 min for each concentration) and immersed twice into isoamyl
acetate for 20 min. Subsequently, the specimens were treated by
lyophilization and spray-gold.
2.4. Bacterial cells viability tests
The bacterial cells viability tests were conducted to measure the
bacterial viability after adsorption. The bacterial cells were shaking
cultivated over night under 37 °C in beef extract medium. Then the
bacteria cells were harvested by centrifugation and suspended in
phosphate buffered saline with an OD of 0.3 at 600 nm (6  108
CFU/ml). CF supports were soaked into the bacterial suspension.
And the suspension was shaken at 200 rpm for 1 h. The cells were
stained with 5 lM PI and 5 lM SYTO 9 for 1 h in the dark. A laser
scanning confocal microscope (LSCM 510, Carl Zeiss, Germany) was
used to obtain fluorescence images. All samples were tested for
three times, and five images were taken for each test. The Image
J software was used to counted total cells and dead cells. The per-
centage of live bacterial cells was calculated from the quantity
 Q. Liu et al. / Applied Surface Science 443 (2018) 255–265 257

Fig. 1. SEM images of CFs after 60 min deposition at a constant current of (a) 1.0 mA, (b) 3.0 mA, (c) 5.0 mA, (d) 7.0 mA and (e) 9.0 mA.

ratio of live cells to total cells. The results were averaged out and the surface potential of the CFs, W2 is the surface potential of the
the standard deviations were calculated. bacterial cell, j is the inverse Debye –Hückel length, A is the
hamaker constant and h is the distance of closest approach between
CFs and the bacteria cell. At 20 °C, j could be calculated according
3. Calculation of interaction energy profile
to the relation [20]:
pffiffi
Interaction energy profiles between bacteria cells and CF sup- j1 ðnmÞ ¼ 0:304= I ð2Þ
ports were calculated from the following equation [19].
where I is the ionic strength expressed in molar. The value of I is
Total interaction energy ¼ repulsion energy þ attraction energy 202 mM, when the bacteria cells are suspended in 10-fold serial
GTotal ¼ per e0 a½ðw1 þ w2 Þ2 lnð1 þ ejh Þ þ ðw1  w2 Þ2 lnð1  ejh Þ  Aa=6h dilution phosphate buffered [21].
ð1Þ
According to Young’s equation and Fowkes [22], we obtain
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
where er is the relative permittivity of the suspension medium, e0 is cL ð1 þ cos hÞ ¼ 2 cLW
S cL ; ð3Þ
LW
the vacuum permittivity, a is the radius of the bacteria cell, W1 is
258 Q. Liu et al. / Applied Surface Science 443 (2018) 255–265

where cL is the total surface tension of the liquid, cLW

S is the Lifshitz-
van der Waals surface tension of solid surface, cLW
L is he Lifshitz-van
der Waals surface tension of liquid and h is the contact angle of solid
surface. For water, at 20 °C,cLW
L = 21.8  103 N/m and cL = 72.8 
10 N/m. Therefore, cS could be obtained by measuring the con-
3 LW

tact angle h of CF supports. The hamaker constant (ASLS) for the

interaction between CFs immersed in water is [23]
qffiffiffiffiffiffiffiffi qffiffiffiffiffiffiffiffi
ASLS ¼ 1:44  1018 cLW
S  cLW
L ðJÞ: ð4Þ

The hamaker constant (ASWS) for CFs immersed in water could

be also written as [24]
pffiffiffi pffiffiffiffiffiffiffiffiffiffi2
ASWS ¼ ASS  AWW : ð5Þ

When bacterial cells are interacting with CFs surface in water,

the hamaker constant (ABWS) can be obtained according to [25]
pffiffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffipffiffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffi
ABWS ¼ ABB  AWW ASS  AWW ; ð6Þ

where Aij is the Hamaker constant of between materials i and j (i, j

= B, W, S) and subscripts B, W and S represent bacteria, water and
CFs surface, respectively. For water and E. coli, AWW = 4.0  1020 J
[26] and ASS = 4.173  1020 J [27]. According to Eq. (3)–(6), the
hamaker constant (ABWS) can be obtained by measuring the contact
angle of CF supports.
The contact angle of carbon fibers cannot be directly measured
due to the small diameter. Therefore, a dynamic contact angle ten-
siometer (DCAT 15, Dataphysics, Germany) was adopted to mea-
sure the contact angle. The deionized water was conducted as
test liquid. The reported contact angles were the average of at least
10 repeated measurements. An electrokinetic analyser SurPASS 3
(Anton Paar KG, Graz, Austria) was used to measure the zeta poten-
tials of CF supports. CF supports were cut into pieces (2 mm in
length and 0.5 g in weight) and dispersed in 100 mL deionized
water with ultrasonic technique. The suspension was poured into
the measurement cell. A hydraulic pressure was provided and
the resulting electric potentials were measured by two electrodes.
More details are described elsewhere [9].
4. Results and discussion
4.1. CF supports
To optimize the electrophoresis conditions, HA crystals were
deposited on CFs surface at the different constant currents (1.0
mA, 3.0 mA, 5.0 mA, 7.0 mA and 9.0 mA). Fig. 1 shows SEM images
of CFs with HA coating after 60 min deposition at the different con-
stant currents. Only a few HA crystals appeared on the surface of
CFs at 1.0 mA depositing current (Fig. 1(a)). A large number of
rod-shaped HA crystals were deposited on CFs surface at 3.0 mA
depositing current (Fig. 1(b)). But there were still some areas of
CFs surface which was not covered by HA crystals because of the
low depositing current. CFs were covered with HA crystals evenly
after deposition at 5.0 mA, 7.0 mA and 9.0 mA depositing current
(Fig. 1(c), (d) and (e)). HA crystals at 5.0 mA depositing current
formed a better three dimensional (3D) structure in comparison
with 7.0 mA and 9.0 mA, which could increase the cell-surface con-
tact area. In addition, HA crystals were easy to form clusters under
9.0 mA depositing current (Fig. 1(e)). The results indicated that 5.0
mA current was suitable for HA crystals formation and deposition
on CFs surface.
Fig. 2(a) illustrates the typical deposition voltage-time curves of
CFs after 0–180 min deposition at 5.0 mA. Because the electrolyte
solution was renewed every 60 min, the electrophoresis deposition
repeated every 60 min. The voltage showed an upward trend with
Fig. 2. (a) Deposition voltage–time curves for the ECD of HA coating on CFs and (b)
the mass percentage of HA coating on CFs.
the deposition time and remained relatively stable at 2.37 V for 0–
60 min deposition, 2.45 V for 60–120 min deposition and 2.62 V for
120–180 min deposition. The relatively constant voltage increased
with the repeated times of deposition, because the increase of HA
coating thickness resulted in the intense electric resistance. Fig. 2
(b) shows the mass percentage of HA coating on CFs. With the
increasing deposition time, the mass percentage of HA coating
increased from 9.43% for 60 min to 26.15% for 180 min.
Fig. 3 shows SEM images of CFs without handling and CFs with
HA coating with different deposition time (60 min, 120 min and
180 min). CF-0 had smooth surface and shallow ridges along the
longitudinal direction (Fig. 3(a)). A uniform and dense HA coating
on CFs surface could be obtained by ECD (Fig. 3(b), (c) and (d)).
The HA coating on CFs surface got thicker with the deposition time.
The crystal morphology of HA also transformed with the deposi-
tion time. The crystals was needle-like in the first 60 min deposi-
tion. Then, the crystals transformed into rod after 120 min
deposition. Finally, the crystals changed into hexagonal prisms
after 180 min deposition. During 0–180 min deposition, the aver-
age diameter of HA crystals was about 60 nm, 150 nm and 170
nm for 60 min, 120 min and 180 min, respectively. The increasing
grain size indicated that crystals grew in both the longitudinal
and radial directions. The results were consistent with some other
studies [17,28]. The supersaturation around CFs surface was the
driving force which governed the formation of HA crystals [18].
In the initial stage, HA coating was directly prepared on CFs sur-
face, which resulted in the increasing density of HA crystals. And
 Q. Liu et al. / Applied Surface Science 443 (2018) 255–265
Fig. 3. SEM images of (a) CF-0, (b) CF-HA60, (c) CF-HA120, (d) CF-HA180. The larger images are at the top right corner.
Fig. 4. FITR spectra of CF supports.
needle-like HA crystals took shape under high supersaturation,
according to the classical crystal growth theory [29]. With the
deposition time, HA was deposited on the HA crystals, resulting
in the HA crystals size enlarging in both the radial and longitudinal
directions [30].
The FITR spectra of CF supports is depicted in Fig. 4. Compared
with CF-0, CFs with HA coating had the characteristic peaks
including the stretching vibration of OH and PO3 4 groups. Peaks
appearing at 605 cm1 and 564 cm1 were assigned to P-O bending
259
1
vibrations in PO34 [31]. And the peak at 1033 cm reflected P-O
stretching vibrations in PO3 4 [32]. With the deposition time, the
PO34 adhesion peak became much stronger, which indicates HA
coating of CFs was thicker with the deposition time. In addition,
the peaks at 1384 cm1 and 1637 cm1 represented AOH bending
modes and AC@O stretching vibration, respectively [33]. Com-
pared with CF-0, CFs with HA coating contained more AC@O and
AOH groups, as a small amount of PO3 4 groups in HA were
replaced by CO23 [28,34].
The EDS elemental spectrums of CF supports are shown in Fig. 5.
Compared with CF-0, CFs with HA coating contained P, Ca and Na
elements. So it deduces that HA grew on CFs surface by ECD. The
relative contents of Ca and P increased with the deposition time.
It demonstrates that HA content also increased with the deposition
time. The Ca/P molar ratios of HA were 1. 31, 1.52 and 1.57 for 60
min, 120 min and 180 min, respectively. And the content of Na ele-
ment in CFs with HA coating was quite low (0.3%, 0.84% and 0.80%
for 60 min, 120 min and 180 min in atom percentage, respectively).
This suggested that HA coating was Ca-deficient with a few Na
incorporation, which might enhance the resorbability of HA coat-
ing [35].
The XRD patterns of CF supports are shown in Fig. 6. A board
diffusion diffraction peak at about 25.7° was assigned to (0 0 2)
crystallographic plane of graphite crystallites [36]. Unlike CF-0,
CFs with HA coating had the apparent diffraction peaks at 2h of
32.3°, 33.3°, 34.4°, 40.0°, 47.1°, 48.5° and 49.8°, which represented
HA crystal face with (2 1 1), (3 0 0), (2 0 2), (3 1 0), (2 2 2), (3 1 2)
and (2 1 3), respectively [37]. These apparent diffraction peaks
implied that HA crystals grew successfully on CFs surface by
260 Q. Liu et al. / Applied Surface Science 443 (2018) 255–265

Fig. 5. EDS elemental spectrum of (a) CF-0, (b) CF-HA60, (c) CF-HA120 and (d) CF-HA180.

Fig. 7. Time-courses of amount of adhered activated sludge to CF supports. Mean

value and standard deviation out of triplicate independent experiments are shown.

Fig. 6. XRD patterns of CF-0 and CFs with HA coating under different deposition
time. 4.2. Sludge immobilization

ECD. The HA peaks intensity increased gradually with increasing The activated sludge is a biological floc composed of microor-
deposition time, suggesting that the HA coating thickness ganisms, extracellular polymeric substances, organic substances
increased with the deposition time. and inorganic substances. Supports are usually used to adhere to
 Q. Liu et al. / Applied Surface Science 443 (2018) 255–265 261

activated sludge for wastewater treatment in bioreactors. To eval-

uate the capacity of CF supports for immobilizing activated sludge,
CF supports were immersed into activated sludge suspension for
0–36 h. The time courses of amount of adhered activated sludge
to CF supports are shown in Fig. 7. CFs with HA coating had a high
capacity for immobilizing activated sludge in comparison with CF-
0. For CF-0, the amount of immobilized activated sludge reached
the maximum at initial 20 h and remained almost stable below
1.8 g/g (g-sludge/g-CFs). However, the amount of activated sludge
immobilized on CFs with HA coating still had been rising before 28
h and reached nearly 2.6 g/g for CF-HA120, 2.9 g/g for CF-HA120 and
3.8 g/g for CF-HA180, respectively. In addition, the amount of
immobilized activated sludge increased with the deposition time.
In a previous study, CFs after nitric acid oxidation were used to
immobilize activated sludge. And the amount of activated sludge
arrived at 2.27 g/g after 24 h immobilization [10]. Compared with
aromatic polyamide fiber (AP), preoxidized polyacrylonitrile fiber
(PAN) and polyethylene fiber (PE), CF supports have a higher
capacity for immobilizing activated sludge and nitrifying bacterial
sludge [9]. Thus, CFs with HA coating were shown to be an excel-
lent support which can efficiently immobilize activated sludge
for bioreactors.

4.3. Bacterial cells adhesion

The bacterial cells adhesion assays were used to evaluate the

capacity of CFs to immobilize bacterial cells. The OD600 curves with
time are shown in Fig. 8(a) and (b). The OD600 value of bacterial
suspension decreased with time, which indicated that CF supports
had the ability to immobilize both E. coli and S. aureus. However,
after the adhesion of CFs with HA coating, the lower OD value of
bacterial suspension implied that HA coating could enhance the
ability of CFs to immobilize bacterial cells. In addition, CFs with
HA coating could immobilize more bacterial cells with the deposi-
tion time. It meant that the greater amount of HA coating on CFs
surface, the more bacterial cells CFs immobilized. In the initial
two hours, the concentration of bacterial cells decreased rapidly,
which illustrated that a great quantity of cells were immobilized
on CFs surface. Then, OD600 value declined gradually between 2
and 6 h, suggesting CFs immobilized a small amount of cells during
this period. After 6 h, CFs almost no longer immobilized cells based
on the constant OD600 value. It proved that the adsorption reached
equilibrium at 6 h.
To remove the influence of dead bacterial cells and their
released organisms, CFU formation assay was used to determine
the residual amount of bacterial cells after 2 h adhesion. Fig. 8(c)
shows the proportion of CFUs referenced to the control (PBS). After
2 h adhesion, the number of suspended cells declined for CF sup-
ports. CFs with HA coating brought about more declines than CF-
0. Besides, with the deposition time, CFs with HA coating immobi-
lized more bacterial cells. The results were in agreement with the
previous experiment. The residual amount of E. coli was more than
that of S. aureus, which suggested that S. aureus was immobilized Fig. 8. Time courses of OD600 of bacterial suspension after CFs adhering to (a) E. coli
and (b) S. aureus. And (c) residual amount of E. coli and S. aureus after 2 h adsorption
more easily than E. coli. It might be due to the lower negative sur-
of CFs. Mean value and standard deviation out of triplicate independent experi-
face potential of E. coli [9,38]. ments are shown.
SEM was used to observe bacterial cells on CF supports after 1 h
adhesion. Fig. 9 shows SEM images of E. coli and S. aureus immobi- The adhesion and growth of bacterial cells could be influenced
lized on CFs surface. CF-HA180 immobilized a large number of E. coli by surface topography of supports [39]. The bacterial cells pre-
and S. aureus cells, while CF-0 only immobilized a small amount of ferred to adhere to the three-dimensional (3D) surface such as
bacterial cells. The result indicated that HA coating could enhance scratches [40]. The attachment of bacterial cells on surface should
the ability of CFs to immobilize bacterial cells. The structure of HA be attributed to the increase of cell-surface contact area [41].
crystals was destroyed by bacterial cells (Fig. 9(c) and (e)). How- Accordingly, the 3D HA crystals might have stronger adhesion
ever, HA and bacterial cells formed a dense film on CFs surface. property than the plane HA. In addition, the 3D HA crystals could
HA coating, acting as a glue, could combine CFs with bacterial cells be destroyed easily. The broken HA fragments facilitated the com-
or between cells, which helped CFs capture more bacterial cells bination of bacterial cells, resulting in a cells-HA film on CFs
(Fig. 9(d) and (f)). surface.
262 Q. Liu et al. / Applied Surface Science 443 (2018) 255–265

Fig. 9. SEM images of E. coli and S. aureus on CF supports: E. coli immobilized by CF-0 (a); S. aureus immobilized by CF-0 (b); E. coli immobilized by CF-HA180 (c) and (d); S.
aureus immobilized by CF-HA180 (e) and (f).

4.4. Bacterial cells viability tests
Nontoxicity is essential for biofilm supports. To test the toxicity
of CF-HA180 for bacterial cells, the viabilities of immobilized E. coli
and S. aureus on CF-HA180 were evaluated by fluorescence-based
assays. Both live and dead bacterial cells were strained by SYTO
9 dye (green dye), while only dead bacterial cells were strained
by PI dye (red dye). The representative LSCM images of E. coli
and S. aureus are shown in Fig. 10. Bacterial cells were immobilized
on CF-HA180 surface and had relatively high activity (Fig. 10(d) and
(e)). And few bacterial cells were inactivated by CF-HA180 (Fig. 10
(c) and (f)). The percentage of live cells to the total is shown in
Fig. 10(g). The percentage of live E. coli and S. aureus after adhesion
was
94.2% and
95.8%, respectively. It revealed that CFs with HA
coating did not show toxicity for both E. coli and S. aureus. Kurtjak
and Vukomanovic [42] proved that pure HA was non-toxic for
E. coli, S. epidermidis and P. aeruginosa. Other studies [43,44] also
implied that pure HA possessed non-toxicity for bacteria.
4.5. Calculation of interaction energy profile
To understand the bacterial adhesion to CF supports in theory,
the total interaction energy between CF supports and E. coli was
calculated by DLVO theory (Formula (1)). The contact angles, zeta
potentials and hamaker constants of CF supports are shown in
Table 1. CFs with HA coating had lower water contact angles than
CF-0 because of the high hydrophilicity of the HA surface [5]. The
hamaker constants were obtained by the contact angles (Formula
 Q. Liu et al. / Applied Surface Science 443 (2018) 255–265 263

Fig. 10. Cells viability assay for E. coli and S. aureus immobilized on CF-HA180. LSCM images of (a) total E. coli in control, (b) total E. coli with CF-HA180, (c) dead E. coli with CF-
HA180, (d) total S. aureus in control, (e) total S. aureus with CF-HA180 and (f) dead S. aureus with CF-HA180. (g) Correlation of the % of live E. coli and S. aureus after being
immobilized by CF-HA180. Bacterial cells were strained with PI and SYTO 9.

(3)–(6)). The radius and zeta potential of E. coli was estimated to be
0.66 lm and 3.90 mV [9], respectively. Accordingly, the total
interaction energy curve was plotted as shown in Fig. 11. No
energy barrier appeared when E. coli cells approached CF supports
surface, which meant that E. coli cells could be immobilized on CF
supports surface without experiencing repulsion. CFs with HA
coating had lower total interaction energy than CF-0. And the total
interaction energy became lower with the deposition time. The
results indicated that HA could strengthen the capacity of CFs to
immobilize bacterial cells and the biocompatibility of CF with HA
coating was better with the increase quantity of HA in theory.
The results consisted well with the bacterial adhesion assays.
The adhesion mechanism of microorganisms is important for
optimize the design of support materials. Some researchers proved
that the adhesion could be affected by physicochemical and biolog-
ical factors [38,45]. When bacterial cell is regarded as a non-living
microparticle, the adhesion mechanism could be explained by DLVO
theory. According to DLVO theory and Formula (1), the total interac-
tion energy between supports and bacterial cells in water is deter-
mined by zeta potentials and hamaker constants of supports. And
the hamaker constants made a greater contribution to the total
interaction energy [9]. Furthermore, the hamaker constants could
be obtained by the water contact angles of supports. Accordingly,
the hydrophilicity of supports might be a decisive factor for immo-
bilizing bacterial cells. Some researchers proved the importance of
hydrophilicity for bacteria adhesion. Kamitakahara and Takahashi
[5] showed that the high hydrophilicity of HA surface might be a
suitable factor for immobilizing E. coli. Liu and Zhao [46] investi-
gated E. coli adhesion on the Ni–P–PTFE coatings with various sur-
face energies by the extended DLVO theory, which demonstrated
that the number of cells attached to the surface increased with the
increasing hydrophilicity of supports. In summary, DLVO theory
explained that CF with HA coating had the high capacity to immobi-
lize bacterial cells because of its high hydrophilicity.
5. Conclusions
A dense and uniform HA coating was prepared directly on CFs
surface by ECD. With the deposition time, the thickness of HA
264 Q. Liu et al. / Applied Surface Science 443 (2018) 255–265

Fig. 10 (continued)

Table 1
Contact angles, zeta potentials and Hamaker constants of CF supports.

Material Contact angle (°) Zeta potential (mV) Hamaker constant (J)
CF-0 78.5 12.8 7.602  1022
CF-HA60 54.3 20.2 1.247  1021
CF-HA120 49.8 21.6 1.325  1021
CF-HA180 38.3 22.8 1.501  1021

coating increased and HA crystals grew in both the longitudinal

and radial directions. The sludge immobilization assays proved
that CF with HA coating can be an excellent support for immobiliz-
ing activated sludge in bioreactors. The bacterial cells adhesion
assays illustrated that HA coating can enhance the ability of CFs
to immobilize E. coli and S. aureus cells. In addition, SEM images
showed that HA and bacterial cells formed a dense film on CFs sur-
face. HA, acting as a glue, could combine CFs with bacterial cells or
between cells, which helped CFs capture more bacterial cells. DLVO
Fig. 11. Interaction energy between E. coli and CF supports.
theory revealed that CF with HA coating had greater capacity to
immobilize bacterial cells because of the lower interaction energy
between bacterial cells and CF supports. The lower interaction tribution. In practice and theory, HA coating can enhance the bio-
energy was attributed to the less negative zeta potential and compatibility of CFs and CF with HA coating can be used as an
higher hydrophilicity, and the hydrophilicity made a greater con- excellent support material for biofilm reactor.
 Q. Liu et al. / Applied Surface Science 443 (2018) 255–265
Acknowledgement
This research did not receive any specific grant from funding
agencies in the public, commercial, or not for profit sectors.
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