Bioresource Technology 131 (2013) 390–396

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Immobilization of Rhodococcus rhodochrous BX2 (an acetonitrile-

degrading bacterium) with biofilm-forming bacteria for wastewater
treatment
Chunyan Li a, Yue Li a, Xiaosong Cheng b, Liping Feng a, Chuanwu Xi c, Ying Zhang a,⇑
a
College of Resource and Environment, Northeast Agricultural University, Harbin 150030, Heilongjiang, PR China
b
College of First Clinical Medicine, Harbin Medical University, Harbin 150001, Heilongjiang, PR China
c
Department of Environmental Health Sciences, University of Michigan School of Public Health, Ann Arbor, MI 48109, USA

h i g h l i g h t s

" A multiple-species biofilm was established.

" The multiple-species biofilm showed strong resistance to acetonitrile loading shock.
" The multiple-species biofilm displayed a typical spatial heterogeneity structure.
" The acetonitrile-degrading bacterium was immobilized in the multiple-species biofilm.
" The multiple-species biofilm improved acetonitrile-containing wastewater treatment.

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

Article history: In this study, a unique biofilm consisting of three bacterial strains with high biofilm-forming capability
Received 17 July 2012 (Bacillus subtilis E2, E3, and N4) and an acetonitrile-degrading bacterium (Rhodococcus rhodochrous BX2)
Received in revised form 20 December 2012 was established for acetonitrile-containing wastewater treatment. The results indicated that this biofilm
Accepted 24 December 2012
exhibited strong resistance to acetonitrile loading shock and displayed a typical spatial and structural
Available online 31 December 2012
heterogeneity and completely depleted the initial concentration of acetonitrile (800 mg L1) within
24 h in a moving-bed-biofilm reactor (MBBR) after operation for 30 days. The immobilization of BX2 cells
Keywords:
in the biofilm was confirmed by PCR–DGGE. It has been demonstrated that biofilm-forming bacteria can
Biofilm
Immobilization
promote the immobilization of contaminant-degrading bacteria in the biofilms and can subsequently
Rhodococcus rhodochrous BX2 improve the degradation of contaminants in wastewater. This approach offers a novel strategy for
Acetonitrile enhancing biological oxidation of toxic pollutants in wastewater.
Wastewater treatment Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction
In recent years, the use of specific contaminant-degrading
microorganisms in the wastewater treatment system has provided
an effective way to enhance the degradation of toxic organic pollu-
tants (Zhang et al., 2012; Schauer-Gimenez et al., 2010; Duque
et al., 2011). For example, some acetonitrile-degrading microor-
ganisms, such as Pseudomonas spp., Nocardia spp., and Rhodococcus
spp., have been successfully used to treat cyanide-containing
wastewater (Feng and Lee, 2009; Sorokin et al., 2007; Okamoto
and Eltis, 2007).
However, due to the loss of degrading microorganisms, washed
out from the system or grazed by protozoa, high degradation rate
⇑ Corresponding author. Tel.: +86 0451 55190993; fax: +86 0451 55191170.
E-mail address: zhangyinghr@hotmail.com (Y. Zhang).
of pollutants did not persist for a long time in wastewater treatment
systems; while the extended persistence of the degrading bacteria in
the system is a prerequisite for maintaining its degrading capability.
The immobilization of microorganisms has been suggested as a
strategy for maintaining efficient degradation in wastewater treat-
ment systems. Different ways of immobilizing degrading bacteria
have been developed which lead to improved effectiveness in the
treatment of wastewater. Guiot et al. (2000) found that the increase
of degradation was attributable to the immobilization of degrading
bacteria within alginate beads when they investigated the bioaug-
mentation of phenol, ortho-cresol, and para-cresol in upflow anaer-
obic sludge bed (UASB) reactors. El-Naas et al. (2009) found that
immobilization of Pseudomonas putida in polyvinyl alcohol (PVA)
gel pellets in a bubble column bioreactor (BC) can enhance the bio-
degradation of phenol. However, these methods may be cost prohib-
itive and/or complex to execute.

0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.12.140
 C. Li et al. / Bioresource Technology 131 (2013) 390–396
Biofilm reactors are especially well suited to the treatment of
toxic wastewater containing slowly biodegradable or toxic com-
pounds due to their resistance to toxicity and environmental stres-
ses (Simões et al., 2009; Morton et al., 1998). Compared to
conventional biological treatment systems using activated sludge
(AS) and membrane filtration, the moving-bed-biofilm reactor
(MBBR) is a superior process. The advantages of this process in-
clude: compact units with small footprints, operation at higher
suspended biomass concentrations, low sludge production, less is-
sues with sludge bulking and no fouling issues, a common problem
faced by all membrane systems (Loukidou and Zouboulis, 2001;
Leiknes and Odegaard, 2007).
Coaggregation, recognition, and adherence of genetically dis-
tinct bacteria occur in a variety of ecosystems and are believed
to play a role in the development of multi-species biofilms in many
different environments. Although most degrading bacterial strains
are weak in terms of biofilm-forming capacity on the surfaces of a
carrier, some strains can function as a bridging organism in biofilm
development. It has been reported that some bacteria with strong
biofilm-forming capabilities coaggregate with other microorgan-
isms to develop biofilms and these biofilms are capable of retaining
other microorganisms in biofilms (Rickard et al., 2002, 2004). This
approach provides a simple and inexpensive way for immobilizing
degrading bacteria within wastewater treatment systems.
Acetonitrile is a toxic chemical that can be converted into toxic
hydrogen cyanide and acetaldehyde in living organisms, which
greatly threatens both human and livestock health (Verschueren,
1983). Untreated acetonitrile-containing wastewater discharged
into a natural water area causes the death of aquatic organisms
as well as serious damage to the ecological environment (Zhang
and Jin, 1995).
No studies have been reported regarding the coaggregation of
bacteria with high biofilm-forming capabilities and acetonitrile-
degrading bacteria in wastewater treatment systems. In this study,
strain Rhodococcus rhodochrous BX2 (an acetonitrile-degrading
bacterium isolated from soil) (Li et al., 2012) and three strains with
high biofilm-forming capabilities, Bacillus subtilis E2, E3, and N4,
were investigated in biofilm formation and acetonitrile degrada-
tion. Each biofilm-forming bacterial strain was co-cultured with
BX2, both alone and in combination with others. The resulting bio-
films were compared with respect to their resistance to loading
shock of acetonitrile. The combination of the bacterial strains most
capable of coping with the loading shock was inoculated to a MBBR
and acetonitrile degradation was monitored. Our data demon-
strated that biofilm-forming bacteria can promote the immobiliza-
tion of acetonitrile-degrading bacteria in biofilms and can
subsequently improve the degradation of acetonitrile in
wastewater.
2. Methods
2.1. Bacterial strains and media
Rhodococcus rhodochrous BX2 was isolated from soil and was
found to degrade acetonitrile efficiently. B. subtilis E2, E3, and N4
were isolated from the biofilms in a laboratory-scale acetonitrile
wastewater treatment system and found to have higher biofilm-
forming capacities than other isolates from the same sample
(unpublished data). Mineral medium consisted of KH2PO4
1700 mg L1, Na2HPO4 9800 mg L1, MgSO47H2O 100 mg L1,
CaCO3 2 mg L1, ZnSO47H2O 1.44 mg L1, FeSO47H2O 0.9 mg L1,
CuSO45H2O 0.25 mg L1, and H3BO3 0.06 mg L1was used for ace-
tonitrile utilization assay. Synthetic wastewater consisted of aceto-
nitrile at a concentration of 800 mg L1, glucose 200–300 mg L1,
391
NH4Cl 40.1 mg L1, KH2PO4 9.2 mg L1, CaCl2 10.7 mg L1, MgSO4
8 mg L1, and FeSO4 0.11 mg L1 was used in MBBR reactors.
2.2. Assay for acetonitrile tolerance and utilization
To determine the growth-inhibition effect of acetonitrile on the
biofilm-forming strains, colonies of B. subtilis strain E2, E3, and N4
grown individually on Luria–Bertani (LB) agar plates at 35 °C for
24 h were harvested and then resuspended in the sterile phosphate
buffered saline (PBS) to an optical density of 1.0 at 600 nm
(OD600 nm). Fifty microliter of these suspensions were inoculated
into 5 mL of synthetic wastewater containing 0 or 800 mg L1 ace-
tonitrile to test their tolerance to 800 mg L1 acetonitrile. Mean-
while, suspensions were inoculated into mineral medium
containing 800 mg L1 acetonitrile to determine the acetonitrile-
utilizing capacities of biofilm-forming strains. The cultures were
incubated for 24 h at 35 °C on a rotary shaker at 180 rpm. Bacterial
growth was monitored by reading the absorbance of the culture at
600 nm.
2.3. Growth interaction assay
The growth interactions among four strains (E2, E3, N4, and
BX2) were examined using the method as described by Al-Bakri
et al. (2004) with modification. Briefly, 50 lL of overnight cultures
of each strain was spread onto the LB agar. After drying, three filter
papers soaked with overnight cultures of other three strains were
placed onto the surfaces of the LB agar plates and plates were incu-
bated at 37 °C for 24 h. The growth inhibition among strains was
measured by determining the diameters of inhibition zones around
the filter papers.
2.4. Biofilm formation assay
Biofilm cultures were performed as described previously by
Bechet and Blondeau (2003). The culture system consisted of a
150-mL Erlenmeyer flask (Tian Jin TianBo Glass Instrument Co.
Ltd., Tianjin, China) containing 50 mL of synthetic wastewater,
two pieces of microscope glass slides (JiangSu FeiBo Glass & Plastic
Co. Ltd., Yancheng, China), and 20 pieces of modified polyethylene
carrier (DaLian ShengYuan Water Treatment & Equipment Devel-
oping Co. Ltd., Dalian, China). Erlenmeyer flasks were inoculated
with one of the four strains (BX2, E2, E3, and N4) or a mixture of
fresh overnight cultures (adjusting the 600 nm absorbance to 1.0)
of BX2 and each biofilm-forming strain at ratio of 1:1. The cultures
were then incubated at 35 °C with shaking at 60 rpm.
The biofilm biomass was determined using a method described
by Zhu and Mekalanos (2003) with modifications. Briefly, after
24 h of incubation, the Erlenmeyer flasks were rinsed twice with
distilled water after the synthetic wastewater containing sus-
pended the cells was removed. The remaining attached biomasses
were stained for 30 min with 50 mL 0.1% (w/v) crystal violet in
water. The Erlenmeyer flasks were washed thoroughly with dis-
tilled water and dried at room temperature overnight. The remain-
ing crystal violet was dissolved in 10 mL of ethanol:acetone
(80:20 v/v), and the absorbance was measured at the wavelength
of 570 nm.
2.5. Biofilm shock resistance assay
Biofilms were developed as described in the Section 2.4. After
24 h incubation, biofilms were shocked with acetonitrile by replac-
ing the bulk fluid media with fresh synthetic wastewater medium
supplemented with 800 mg L1 acetonitrile six times at the 8 h
intervals. The viable cell counts in the biofilms attached to the glass
392 C. Li et al. / Bioresource Technology 131 (2013) 390–396
slides before (24 h) and after (72 h) shocking and acetonitrile con-
centration in the replaced synthetic wastewater were determined.
Viable counts of the bacterial cells attached to the glass slides
were determined as following. After removing the glass slides from
the flask and being gently washed three times with sterile PBS, the
glass slides were then placed in a sterile flask containing 10 mL of
sterile PBS. This mixture was then vortexed for 10 min to release
attached cells. A serial dilutions of the suspension was then plated
onto mineral agar (mineral medium with 2% agar) supplemented
with 800 mg L1 acetonitrile as the only carbon and nitrogen
source for the BX2 or onto LB agar for the total bacteria.
The supernatants from the replaced liquid media was obtained
by centrifuging for 1 min at 12,000g to remove bacteria cells and
other debris then used for gas chromatography analysis directly
to determine concentrations of remaining acetonitrile. The acetoni-
trile peak area was then measured and analyzed using a SHIMADZU
GC-14C gas chromatograph (GC) equipped with a flame ionization
detector (FID) and a fused silica chromatographic column packed
with 14% OV-1701 (30 m  0.53 mm i.d.). The column temperature
was held at 110 °C, and the injector and detector temperatures
were held at 200 and 200 °C, respectively. The concentrations of
acetonitrile were then calculated based on a standard curve.
2.6. Reactor setup
The schematic of the laboratory-scale MBBR used in this study
is shown in Fig. 1. In order to test whether the co-cultures would
improve acetonitrile-degrading efficiency, three parallel reactors
were set up and inoculated with different inoculums. The activated
sludge used for the inoculation was collected from a municipal
sewage treatment plant in Harbin, China, bacteria BX2, E2, E3,
and N4 were collected from pellets of overnight cultures. The Reac-
tor NO. 1 (R1) was seeded with 3000 mg L1activated sludge; the
Reactor NO. 2 (R2) was seeded with 2400 mg L1 activated sludge
and 600 mg L1 acetonitrile-degrading bacterium BX2; and the
Reactor NO. 3 (R3) was seeded with 1800 mg L1 activated sludge,
600 mg L1 BX2, 600 mg L1 three biofilm-forming bacteria
(E2:E3:N4 = 1:1:1). Three identical reactors, each with 10 L effec-
tive volume, were fed with synthetic wastewater equivalent to
400 mg L1 COD. Each reactor was operated under the same condi-
tions: 25 °C, approximately 30% carrier fill ratio (modified polyeth-
ylene), and the hydraulic retention time (HRT) was maintained for
24 h. The MBBRs were run continuously for approximately 30 days.
The supernatant was obtained by centrifuging the effluent for
1 min at 12,000g and was used to determine acetonitrile concen-
trations, using the same method as described in Section 2.5. Chem-
ical oxygen demand (COD) was analyzed according to Standard
Methods (APHA et al., 2005).
inflow
water pump
Synthetic wastewater
air flow meter
air pump
Fig. 1. Schematic diagram of the MBBR reactor used in this study. Ten liter of effective volume were fed with the synthetic wastewater equivalent to 400 mg L1 COD.
Operational conditions were 25 °C, with approximately 30% carrier fill ratio (modified polyethylene), and the hydraulic retention time (HRT) was maintained at 24 h.
2.7. Microscopic examination of biofilm structures
Biofilm developed on the carriers was examined using the scan-
ning electron microscopy (SEM). Biofilms were placed in fixative
(2.5% glutaraldehyde v/v in PBS) overnight. The samples were
rinsed in PBS (2  10 min) and subsequently dehydrated in a series
of ethanol washes (50% for 10 min, 70% for 10 min, 90% for 10 min,
100% for 20 min), and the samples were then rinsed in additional
washes (ethanol:tertiary butanol, 1:1 for 15 min, tertiary butanol
for 15 min). Finally, samples were freeze-dried at 20 °C for
20 min. The samples were then coated with gold/palladium (40%/
60%). After processing, the samples were observed in an SEM (S-
3400N, Hitachi, Japan) in a high vacuum mode.
2.8. PCR–DGGE analysis
DNA extraction from biofilm samples of BX2 and biofilm-form-
ing bacteria was performed as described by Griffiths et al. (2000),
and samples were further purified using an E.Z.N.A™ Gel Extraction
Kit (OMEGA Bio-tek Inc., Doraville, GA, USA) according to the man-
ufacturer’s instructions. These DNA preparations were used as tem-
plate DNA for PCR–DGGE analysis. The variable V3 region of 16S
rRNA was amplified by PCR with the primer sets: 338f/518r, and
a 40-bp GC-clamp was added to primer 338f as described by Muy-
zer et al. (1993) to increase the separation of the DNA bands during
DGGE analysis. The V3 region of 16S rRNA was amplified using the
following protocol: 5 min at 94 °C; 35 cycles of 1 min at 94 °C;
1 min at 62 °C and 1 min at 72 °C; 10 min at 72 °C. DGGE of the
amplified products was performed on the Dcode Universal Muta-
tion Detection System (Bio-Rad Laboratories, CA, USA). Fifteen
microliter of PCR products were electrophoresed on 8% (w/v) poly-
acrylamide gels (40% acrylamide/bisacrylamide solution, 37.5:1) in
1 TAE buffer (40 mM Tris, 20 mM acetate, 1.0 mM Na2-EDTA).
Polyacrylamide gels with a denaturation gradient ranging from
30% to 60% were run at 60 V and 60 °C for 12 h. After electrophore-
sis, the gels were stained with AgNO3, rinsed briefly with ddH2O
and images were taken by a digital imaging system (Alpha Innotech
Corporation, San Leandro, CA). The images were analyzed using the
software Quantity One 4.0 (Bio-Rad Laboratories, CA, USA).
3. Results and discussion
3.1. Three biofilm-forming strains can tolerate but can not utilize
acetonitrile
The three biofilm-forming strains, E2, E3, and N4, were cultured
in the synthetic wastewater containing 0 and 800 mg L1 of aceto-
nitrile at 35 °C for 24 h. The absorbance (OD600 nm) of all cultures
outflow
carrrier
 C. Li et al. / Bioresource Technology 131 (2013) 390–396
were in the range of 0.79–0.93. The biomasses of each strain in
synthetic wastewater containing 800 mg L1 of acetonitrile were
close to those measured in 0 mg L1 of acetonitrile (see Fig. S1 in
the supplementary document), indicating that 800 mg L1 of ace-
tonitrile had no obvious inhibitory effects on the growth of these
biofilm-forming strains. However, the three biofilm-forming
strains cultured in the mineral medium containing 800 mg L1 of
acetonitrile showed no growth (see Fig. S1 in the supplementary
document), indicating that the three biofilm-forming strains can-
not utilize acetonitrile. The results indicated that three biofilm-
forming strains have ability to protect cells from toxic effects of
acetonitrile and can be used in the acetonitrile wastewater treat-
ment system and engineering.
3.2. No growth inhibition among biofilm-forming strains and
acetonitrile-degrading strain
The acetonitrile-degrading strain, BX2 and biofilm-forming
strains, E2, E3, and N4 will be applied together to wastewater
treatment system; thus, functions of each strain could be disturbed
if growth inhibition exists among them. Therefore, the antagonism
of these strains was tested before inoculating them into the
reactors.
No inhibition zones were observed of all four tested strains
from the growth inhibition assay (see Fig. S2 in the supplementary
document), suggesting no growth-inhibitory effects of four strains
against each other. Hence, these strains with acetonitrile-degrad-
ing or high biofilm-forming capabilities were used in the reactors.
3.3. Immobilization of acetonitrile-degrading strain BX2 in biofilms
and resistances of the biofilms to loading shock of acetonitrile
The biofilm biomasses of BX2 and the three biofilm-forming
bacteria, along with the co-cultures of BX2 and each of the bio-
film-forming strains, were measured after culturing in Erlenmeyer
flasks for 24 h at a shaking speed of 60 rpm (see Fig. S3 in the sup-
plementary document). The results indicated that the biofilm bio-
masses formed by all of the mixed cultures were higher than those
of each single culture. The mixed culture of BX2 with E3 formed a
visible biofilm at the air–liquid interface of the flask wall, and their
biofilm biomass was the highest among all combinations (7.4
times that formed by the single BX2 culture).
The resistance of established biofilms to acetonitrile and degra-
dation of acetonitrile were tested (Fig. 2). After biofilms were
developed in the flasks for 24 h, the supernatants were replaced
with the synthetic water containing 800 mg L1 acetonitrile at a
predetermined time intervals (8 h). The acetonitrile degradation
percentages of the mixed cultures (ranging from 47% to 72% at
72 h) were generally higher than that of BX2 alone (27% at 72 h).
The multiple-species biofilm, BX2 with E2, E3, and N4, had the
strongest resistance to acetonitrile loading shock during successive
replacements of synthetic wastewater, and the degradation rate
ranged from 64% to 72% within 8 h of each replacement. In con-
trast, the level of resistance against acetonitrile loading shock of
the BX2 single-species biofilm was relatively low when subjected
to loading shock by successively replacing the synthetic wastewa-
ter, with acetonitrile degradation percentages ranging from 9% to
27%.
To estimate the numbers of BX2 cells fixed in the biofilms, the
CFU of the total and BX2 cells in the dual-species/multiple-species
biofilms established on the glass slides for 24 and 72 h were
counted (Fig. 2). Before the successive loading shock, it was found
that the total CFU and BX2 CFU of the biofilms formed by BX2 with
E2, E3, and N4 were significantly higher than those of the other
dual-species/multiple-species biofilms. In these biofilms, after
being shocked six times, the BX2 CFU and the total CFU per slide
393
were 103.96 (±100.03) and 107.80 (±100.04) CFU, respectively. Addi-
tionally, the relatively high abundance of the acetonitrile-degrad-
ing strain BX2 in the biofilm indicated that strain BX2 can be
well maintained in these multiple-species biofilms over time.
3.4. Acetonitrile degradation in the MBBR inoculated with mixed
cultures of acetonitrile-degrading strain BX2 and biofilm-forming
strains
Degradation of acetonitrile by the mixed cultures was tested in
MBBR with different inocula. The performance of the reactors was
monitor for 30 days and data were presented in Fig. 3. In R1, the
acetonitrile concentration in the effluent was high during the first
9 days with an average degradation of 7.10%, suggesting very low
degrading activity at the beginning. Decrease of acetonitrile in
effluent was observed from day 10 and reached 35.5 mg L1 (an
acclimation degradation) on the 30th day, suggesting microbes
from the initial activated sludge capable of degrading acetonitrile
may have emerged Meanwhile, the concentration of COD in the
effluent at day 30 was 85.32 mg L1.
In R2 and R3, acetonitrile concentrations in the effluent were
lower than those in R1 in the first initial period (1–9 days), indicat-
ing higher removal efficiency in these two reactors (45.95% in R2,
64.93% in R3). On the 30th day after running, acetonitrile concen-
trations in the effluent were 7.59 and 0 mg L1, and COD concen-
trations were 31.13 and 19.68 mg L1 in R2 and R3, respectively,
indicating a complete degradation of acetonitrile in R3. These re-
sults suggest that the biofilm-forming strains facilitate the immo-
bilization of acetonitrile-degrading strains in biofilms, therefore
improve the degrading efficiency.
Acetonitrile could be decomposed in the following two ways
(Lou et al., 2001)
NHase amidase
Acetonitrile ƒƒ! acetamide ƒƒƒ! acetic acid þ ammonia
nitrilase
Acetonitrile ƒƒƒ! acetic acid þ ammonia
Feng et al. (2008) evaluated the effects of intermediate product
inhibition in the NHase/amidase enzyme system of an acetonitrile-
degrading strain, Mesorhizobium sp. F28. They demonstrated that
the accumulation of catabolite decreased the activities of the
NHase and amidase, suggesting that the appropriate operation of
a bioreactor which can degrade acetonitrile continuously would
be beneficial for Mesorhizobium sp. F28 to improve the reactor
performance.
In this study, in addition to acetonitrile, acetamide and acetic
acid were detected by gas chromatography in the effluent of R1
after running 30 days (see Fig. S4 in the supplementary document),
demonstrating that the microorganisms are capable of degrading
acetonitrile in activated sludge over a long period of acclimation
but acetonitrile could not be decomposed thoroughly to non toxic
products. The microorganisms in sludge only converted acetoni-
trile into acetamide and acetic acid, whereas were incapable of uti-
lizing these by-products further. However, the intermediate
metabolite, acetamide, is not only carcinogenic to animals but also
influenced the degradation rate of acetonitrile (Feng et al., 2008).
Thus, the performance of reactors with raw activated sludge for
wastewater treatment was far less than that of adding targeted
degradation bacteria.
We investigated the characteristics and pathway of acetonitrile
degradation by R. rhodochrous BX2 (Sun et al., 2012). Results
showed that the degradation of acetonitrile by R. rhodochrous
BX2 possibly included both nitrile hydratase (NHase) and nitrile
hydrolase pathways. In addition, the cells also grew with the pro-
duction of acetic acid. Combined effects of NHase and Nitrilase
394 C. Li et al. / Bioresource Technology 131 (2013) 390–396

Fig. 2. Biofilm biomass and acetonitrile degradation in the batch reactors. The concentration of acetonitrile before and after replacement of synthetic wastewater (every 8 h)
was shown in lines and bacterial counts (Log10CFU/slide) of BX2 and total bacteria before and after the shock (24 and 72 h) were shown in vertical bars. The bars represent SD
of the assay performed in triplicate.
 C. Li et al. / Bioresource Technology 131 (2013) 390–396
Fig. 3. Performance of the MBBR reactors over 30 days of operation. The concentrations of acetonitrile (a) and COD (b) in the MBBR effluent were monitored. R1 was seeded
with the activated sludge only; R2 was seeded with BX2 and the activated sludge; and R3 was seeded with BX2, three biofilm-forming bacteria, and activated sludge.
Fig. 4. The DGGE fingerprints of 16S rRNA genes of biofilm samples from three
reactors 1, 2 and 3 represent the samples from R1, R2 and R3, respectively. E2, E3,
N4, and BX2 represent the V3 region amplified respectively from corresponding
strain.
pathways accelerated the degeneration of acetonitrile and de-
creased the accumulation of intermediate metabolites.
This may explain why BX2 was capable of thoroughly degrading
acetonitrile within 24 h in the MBBR reactor. With BX2 present, no
extra operation is needed in this acetonitrile wastewater treatment
system; this provides the advantages of rapid degradation of aceto-
nitrile and simplification of the equipment.
The biofilm developed on the carrier in R3 at day 30 was a typ-
ical slimy biofilm, about 2–3 mm in thickness, covers all the carrier
surfaces and possesses flocking shapes with larger surface areas.
SEM observations provided useful information that biofilms con-
sisted of dense networks of cells of all morphologies deeply
embedded in a matrix consisting of exopolymeric material and cir-
cinate water-channels could be observed in images (see Fig. S5 in
the supplementary document).
The presence of degrading strain BX2 and biofilm-forming
strains in biofilms on the carriers was determined with the PCR–
DGGE method. The biofilm samples were taken from the carriers
from each of three reactors on the 30th day after running. The
395
PCR–DGGE analysis was done on these samples (Fig. 4). The three
biofilm-forming strains are subspecies of B. subtilis; thus, their
bands are at the same position. The sample from R3 had bands at
the same positions of the biofilm-forming bacteria sample and
BX2, which indicates that both biofilm-forming bacteria and BX2
were immobilized successfully in biofilms in R3. The samples from
R2 and R3 had a band at the same position as that of BX2, and the
relative intensity of this DNA band, reflective of the relative bio-
mass of this bacterium, from the sample from R3 was higher than
that of the sample from R2, suggesting higher abundance of the
BX2 strain in biofilms. This was in agreement with the perfor-
mance data of both reactors, in which a complete degradation of
acetonitrile was observed in R3.
4. Conclusions
In this study, biofilm containing three biofilm-forming bacteria
and one acetonitrile-degrading bacterium was established and
exhibited strong resistance to acetonitrile loading shock. The bio-
film displayed a typical spatial structural heterogeneity and pre-
sented strong effectiveness for treating acetonitrile-containing
wastewater. According to the literatures, this is the first report
where degrading bacteria were immobilized in biofilms to improve
the treatment of acetonitrile-containing wastewater. This method
that immobilizing specific-pollutant degrading strains into bio-
films formed by biofilm-forming bacteria in the wastewater treat-
ment system was not confined to specific pollutant. So this method
has more obvious advantages and broader application prospects
for toxic wastewater treatment.
Acknowledgements
This research was supported by National Natural Science Foun-
dation of China (41271504), Natural Science Foundation of Hei-
longjiang (No. C201021), Specific topics of National Water
Environment Special Item, China (2012ZX07201003-003), Postdoc-
toral Science and Research Foundation of Heilongjiang (No. LBH-
Q09158).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.
2012.12.140.
396 C. Li et al. / Bioresource Technology 131 (2013) 390–396

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