Appl Microbiol Biotechnol (2012) 94:527–535
DOI 10.1007/s00253-011-3742-0
ENVIRONMENTAL BIOTECHNOLOGY
Internal loop photobiodegradation reactor (ILPBR)
for accelerated degradation of sulfamethoxazole (SMX)
Ning Yan & Siqing Xia & Linke Xu & Jun Zhu &
Yongming Zhang & Bruce E. Rittmann
Received: 11 February 2011 / Revised: 2 November 2011 / Accepted: 16 November 2011 / Published online: 4 January 2012
# Springer-Verlag 2011
Abstract The internal loop photobiodegradation reactor
(ILPBR) was evaluated for the degradation of the pharma-
ceutical sulfamethoxazole (SMX) using batch experiments
following three protocols: photolysis alone (P), biodegrada-
tion alone (B), and intimately coupled photolysis and bio-
degradation (P&B). SMX was removed more rapidly by
P&B than by either P or B alone, and the corresponding
dissolved organic carbon (DOC) removals by P&B also
were higher. The faster SMX removal probably was due to
a synergy between photolysis and the rapid biodegradation
of SMX by the biofilm. The greater DOC removal was
brought about by the presence of biofilm bacteria able to
biodegrade photolysis products. Ammonium N released
during photolysis of SMX gave more evidence for the
formation of intermediates and was enough in P&B experi-
ments to support bioactivity when no other N was supplied.
Clone libraries performed on the biofilms before and after
the P&B experiments showed profound changes in the
microbial community. Whereas Rhodopirellula baltica and
Methylibium petroleiphilum PM1 dominated the biofilm
after the B experiments, they were replaced by Micrococcus
N. Yan : S. Xia
College of Environmental Science and Engineering,
Tongji University,
Shanghai 200092, People’s Republic of China
N. Yan : L. Xu : J. Zhu : Y. Zhang (*)
Department of Environmental Engineering, College of Life
and Environmental Science, Shanghai Normal University,
Shanghai 200234, People’s Republic of China
e-mail: zhym@shnu.edu.cn
B. E. Rittmann
Swette Center for Environmental Biotechnology,
Biodesign Institute, Arizona State University,
Tempe, AZ 85287-5701, USA
luteus, Delftia acidovorans, and Oligotropha carboxidovor-
ans after the P&B experiments. The changes in microbial
community structure mirrored the change in function in the
P&B experiments: SMX biodegradation (presumably the
roles of R. baltica and M. petroleiphilum) was out-
competed by SMX photolysis, but biodegradation of pho-
tolysis products (most likely by M. luteus and D. acidovor-
ans) became important. The higher removal rates of SMX
and DOC, as well as the changes in microbial community
structure, confirm the value of intimately coupling photolysis
with biodegradation in the ILPBR.
Keywords Biodegradation . Biofilm . Microbial community
structure . Photolysis . Sulfamethoxazole
Introduction
Pharmacologically active compounds are detected in waste-
waters, streams, and groundwater resources across the world
(Heberer 2002; Zühlke et al. 2004; Buser et al. 1998a, b,
1999; Kim et al. 2007). Among them, antibiotics are attract-
ing special attention, as they can lead to persistent microbial
resistance (Overbye and Barrett 2005; Kumarasamy et al.
2010; Sengupta et al. 2011). In some places, drug residues at
the nanograms per liter level were found in drinking water
(Stackelberg et al. 2004; Hirsch et al. 1999).
Among the antibiotics and broad-spectrum drugs, sulfo-
namides are widely used in clinics because they can inhibit
many Gram-positive bacteria and some Gram-negative bac-
teria, such as Nocardia and Chlamydia, and some protozoa
(Zhang et al. 2009; Keizer et al. 2008). Sulfamethoxazole
(SMX), an important broad-spectrum antibacterial with su-
perior stability (Li et al. 2009), is a complex nitrogen-
containing heterocyclic compounds with a molecular
528
formula of C10H11N3O3S. While a benefit in the clinic, the
stability of SMX can be a problem when it is discharged into
the aquatic environment because its residues may induce
bacterial resistance and be an endocrine disruptor
(Thiele-Bruhn and Beck 2005). Even though sulfonamides
normally are detected at very low concentration in nature,
aquatic organisms might experience deleterious effects due
to a concentration build up in areas subject to decades of
unregulated discharge (Nasuhoglu et al. 2011; Akiyama and
Savin 2010; Tao et al. 2010).
Since SMX and other pharmaceuticals normally reach the
aquatic environment in wastewater discharges (Avisar et al.
2009; Santos et al. 2010), advanced treatment to remove
them has become an objective (Abellán et al. 2007; Alexy et
al. 2004). Advanced oxidation processes (AOPs), such as
UV/H2O2, UV/O3,UV/TiO2, and UV/Fe2+/H2O2, might be
good methods for SMX degradation (Baran et al. 2006; Hu
et al. 2007). Photolysis based on UV irradiation alone is
even simpler and often more economical. UV irradiation
without or with a catalyst is called photo(cata)lysis.
Perhaps the best use of UV photo(cata)lysis is to partially
transform the compounds so that they are readily biode-
graded (Marsolek et al. 2008; Muñoza et al. 2009; Chong
et al. 2010). This strategy gets around the reality that com-
plete mineralization by an AOP is far too expensive. Photo
(cata)lysis for pre-treatment of recalcitrant compounds most
commonly is carried out sequentially (Reddy et al. 2004;
Suryaman et al. 2006). However, having biodegradation as a
distinct second stage may result in excessive consumption
of UV light and formation of products that are unnecessarily
oxidized in the first stage (Dantas et al. 2008; Kim and
Tanaka 2009; Silva and Faria 2009). A solution to this
problem is intimate coupling, in which photo(cata)lysis
and biodegradation occur together (Marsolek et al. 2008;
Zhang et al. 2010a, b). Then, once a biodegradable product
of photo(cata)lysis is formed, it is immediately biodegraded.
SMX is a photosensitive material that undergoes signif-
icant photolysis when it absorbs light with wavelength less
than 310 nm (Abellán et al. 2007; Beltrán et al. 2008). Thus,
UV photolysis (without TiO2 catalysis) may be a viable
approach for treatment of water containing SMX, particu-
larly since the goal is partial transformation to biodegrad-
able products, not complete mineralization.
Based on the concept of intimate coupling of photolysis
and biodegradation, we developed an internal loop photo-
biodegradation reactor (ILPBR) in which UV irradiation is
coupled with biofilm biodegradation for accelerated treat-
ment of water containing SMX. We used three protocols in
the ILPBR experiments for SMX degradation: photolysis
alone, biodegradation alone, and intimately coupled photol-
ysis and biodegradation. We also used genomics techniques
to examine how intimately coupled photobiodegradation
affected the community structure in the biofilm carriers.
Appl Microbiol Biotechnol (2012) 94:527–535
We tested the hypotheses that intimately coupled photolysis
and biodegradation results in accelerated removal of SMX
and its products (represented by dissolved organic carbon,
DOC) and that the microbial community is altered by
exposure to the conditions of intimate coupling in a
way that emphasizes the biodegradation of SMX-
photolysis products, not biodegradation of SMX.
Materials and methods
Internal loop photobiodegradation reactor
The ILPBR, shown in Fig. 1, was made of quartz glass with
a 100-mL working volume. We installed a baffle in the
center of the ILPBR to create photolysis and biodegradation
zones. The biofilm carriers in the biodegradation zone were
hydroformylated fibers produced by an aldehyde reaction;
they were soft plastic fibers having a rough outer surface
ideal for biofilm attachment. We provided UV light (254 nm
wavelength) to the photolysis zone using a UV light assembly
consisting of three lamps with 8 W per lamp. We used an air-
lift pump (150 mL min−1) to circulate water between the
photolysis zone and the biodegradation zone.
Acclimation and incubation of SMX-degrading bacteria
We obtained activated sludge from the underflow of a sec-
ondary clarifier at the Longhua Municipal Wastewater
Treatment Plant in Shanghai and acclimated it to SMX by
incubating the biomass in an inorganic salt medium to
which we added SMX. The temperature was 20~25°C for
the first 2 weeks, and we replaced the supernatant every day
after allowing the sludge to settle. The inorganic salt medi-
um contained (NH4)2SO4 0.1 g L−1, KH2PO4 0.5 g L−1,
Na2HPO4 0.5 g L−1, MgSO4 7H2O 0.5 g L−1, and yeast
Outlet
Baffle
Biofilm grown on
hydroformylated
UV light
fiber
Inlet
Aeration
Fig. 1 Schematic of the internal loop photobiodegradation reactor
Appl Microbiol Biotechnol (2012) 94:527–535
extract 0.02 g L−1. We gradually increased the SMX con-
centration from 10 to 120 mg L−1 during the 2-week accli-
mation. We judged that the acclimated sludge was suitable
for inoculation in the ILPBR because the SMX removal
percentage was more than 50% after 2 weeks.
SMX and synthetic wastewater
We purchased SMX from the Shanghai Sinopharm Chemical
Reagent Co., Ltd. and prepared synthetic wastewater by
adding the ingredients of the inorganic salt medium and 10
to 120 mg L−1 of SMX to tap water.
SMX treatment protocols
We used three experimental protocols to evaluate SMX
degradation in batch experiments: photolysis by UV
light alone (P), biodegradation by biofilm alone (B),
and intimately coupled photolysis and biodegradation
(P&B). The temperature of all experiments was 23°C
to 25°C. The UV light intensity was 0.46 mW cm−2
(corresponding to 24 W of UV light on the ILPBR) for the P
and P&B experiments. The SMX concentration was 10, 30,
60, 90, or 120 mg L−1 at the beginning of the batch experi-
ments. To investigate the possibility that N released by SMX
photolysis could be an effective N source, we did not add
ammonium N into the solution for some B and P&B experi-
ments. Liquid samples were taken over time to analyze the
concentration of SMX, total nitrogen, ammonium nitrogen,
and DOC.
Photolysis
The P experiments had constant UV exposure, but no hydro-
formylated fiber and, thus, no biofilm.
Biodegradation
Prior to the B batch tests, we immersed hydroformylated
fibers in the acclimated activated sludge for 24 h to let
microorganisms attach onto the fibers. Then, we transferred
the biofilm-colonized carriers to the biodegradation zone
and began aeration and wastewater circulation between the
photolysis and biodegradation zones. The UV light was off
for the B experiments.
Coupled photolysis and biodegradation
We performed the P&B experiments with the same
biofilm-colonized carriers as in the B experiments. After
the B experiments were completed, we added synthetic
wastewater containing SMX to the ILPBR and turned
on the UV light.
529
Adsorption of SMX
To investigate adsorption of SMX onto biomass, we mixed
200 mL of SMX solution in a 500-mL Erlenmeyer flask
with 20 mL of activated sludge or inactivated sludge at
8 g/L to get 800 mg/L of mixed liquor suspended solids; both
flasks had an initial SMX concentration of about 55 mg/L.
The inactivated sludge was prepared by autoclaving the
activated sludge. The adsorption experiments were carried
out at 25°C, 120 rpm, and for 1 h. We took samples at every
10 min to analyze their SMX concentrations. We also tested
adsorption of SMX onto bare hydroformylated fibers.
Analytical methods
We measured the SMX concentration with a high-performance
liquid chromatograph (model: Agilent 1100, USA) equipped
with a diode array detector at a wavelength of 250 nm and a
ZORBAX SB-C18 column (5 μm, 4.6×250 mm). The mobile
phase was a mixture of acetonitrile water solution (30:70, v/v),
and the flow rate was 1 ml/min. We measured DOC with a
total organic carbon (TOC) automatic detector (model:
Elementar LiquiTOC, Germany), pH with a pH meter
(model: pHs-25c, China), UV light intensity with a light
meter (model: BG-2254, China), ammonium nitrogen with
Nessler reagent spectrophotometry, and total nitrogen (TN)
with alkaline potassium persulfate digestion and ultraviolet
spectrophotometry (Wei 2002). All samples for SMX, TN,
and NH4+-N analyses were filtered through a 0.45-μm
cellulose acetate membrane filter (Jiuding High-Tech filtration,
Beijing).
Community analysis
We performed community analysis on ILPBR biofilm from
the hydroformylated fibers after the B and P&B experiments
were completed. We extracted the DNA from the samples
using the DNAzol reagent and amplified the extracted
DNA through the PCR reaction (Godon et al. 1997).
The reaction volume contained 2× Hotstart-PCR mix 25 μL,
primers (25 pmol μL–1) 0.8 μL×2, template 1 μL, and
ddH2O 22.4 μL. The primers for bacterial 16S rDNA
were: F (5′-AGAGTTTGATCCTGGCTCAG-3′) and R (5′-
CAKAAAGGAGGTGATCC-3′) (Kazuya et al. 1999). Con-
ditions of PCR amplification were an initial denaturation at
94°C for 4 min; 35 cycles of denaturation (30 s at 94°C),
annealing (30 s at 50°C), and extension (1 min at 72°C); and
a final extension at 72°C for 10 min. PCR products were
analyzed on 1% agarose gel by electrophoresis and purified
with a DNA purification kit (Dingguo Co., Ltd., Beijing,
China). Target gene fragments were cloned into pMD-18T-
vector and transferred into Escherichia coli DH5α, and the
positive clones were sequenced (Jierui Co., Ltd., Shanghai,
530 Appl Microbiol Biotechnol (2012) 94:527–535

8
China). We compared the 16S rDNA sequences with the

TN and NH4 –N (mg/L)

GenBank database based on Basic Local Alignment Search
6
Tool (BLAST) and chose homologs based on an e-value less TN
than 0.001 (Altschul et al. 1990).

+
4 NH4 -N
NH4+-N
+

2
Results
0
0 1 2 3 4 5 6 7 8
SMX biodegradation
T (h)

Figure 2 shows the results for the B and P&B experiments in Fig. 3 TN and NH4+-N changes under UV illumination in the P
the ILPBR. From an initial concentration of 60 mg L−1, experiment
SMX had almost no removal when ammonium N was not
added in the B experiment, but it was removed steadily, achiev- of SMX degradation in the P&B experiments was greater
ing 64% removal over 8 h when 0.53 mgN L−1 was added to a than the sum of the P and B rates, which suggests a synergy
B experiment. In the P&B experiments, the rate of SMX between photolysis and biodegradation.
degradation was accelerated to about twice that of the B
experiment whether or not ammonium N was added. This 1
B
trend suggests that N was released from SMX photolysis in 0.8 P
P&B

C / C0
a high enough concentration to support bacterial growth. 0.6
To quantify N release during P, we illuminated an initial
0.4
SMX concentration of 60 mg/L with UV light and measured
0.2 C0 =10mg/L
the concentration of ammonium N and TN; TN is the sum of a
organic and ammonium N, as no nitrite or nitrate was 0
10
detected. For comparison, we also tested N release during
0.8
biodegradation. Figure 3 shows that about 2.5 mgN/L of
C / C0

ammonium was released over time in the P experiment; no 0.6

C0 =30mg/L
ammonium N was released in the B experiment (not shown). 0.4
The release of 2.5 mgN/L was more than that added 0.2
b
(0.53 mgN L−1) for the B experiments and clearly enough 0
to support biomass growth during the P&B experiments. 10
0.8
C0 =60mg/L
C / C0

Comparison with different protocols for SMX degradation 0.6

0.4
The three protocols were tested for degradation of SMX
0.2
with different initial concentration (10, 30, 60, 90, and c
120 mg L−1), and their removals are shown in Fig. 4. The 0
10
removal rates in the B experiments were the slowest among
0.8
these protocols. The rate was clearly much faster in the P C0 =90mg/L
C / C0

experiments and was accelerated further by P&B. The rate 0.6

0.4
0.2
1 d
B (No N added) 0
0.8 B (N added) 10
P&B (No N added)
P&B (N added) 0.8
0.6
C / C0
C/C0

0.6
C0 =120mg/L
0.4
0.4
–1
0.2 C 0 = 60mg•L
0.2
e
0 0
0 1 2 3 4 5 6 7 8 0 2 4 6 8
T (h)
T (h)
Fig. 2 SMX degradation in B and P&B experiments, including the Fig. 4 Degradation of SMX by photolysis (P), biodegradation (B), and
effect of adding ammonium N photobiodegradation (P&B) in the ILPBR
Appl Microbiol Biotechnol (2012) 94:527–535 531

2
Absorption appeared to dominate during the first 10 min a YP&B = 0.46x - 0.32 YB = 0.41x - 0.37
2 2
of each B experiment because the SMX concentration de- R = 0.92 R = 0.91
1.5
clined sharply at first but then leveled off before gradually

V0(mg/L•min)
YP = 0.03x + 0.15
P 2
declining after 1 h due to biodegradation. None of these R = 0.73
1 B
phenomena appeared during the P experiments, which had P&B
no biofilm or carrier.
0.5
The adsorption experiments, reported in Fig. 5, prove that
SMX was rapidly and significantly adsorbed by activated
0
sludge and inactivated sludge in the first 10 min, and its 10 30 60 90 120
concentration was stable for the remainder of the 60-min Initial SMX concentration (mg/L)
experiment. The fact that the SMX concentration was stable 0.5
over time and almost the same for experiments with activat- b

VRR(mg/L•min)
ed and inactivated sludge shows that biodegradation was 0.4

negligible compared to adsorption over 60 min. Also, the P

adsorption to the bare hydroformylated fibers was minor
compared adsorption to biomass.
Figure 6a shows the relationship between the initial SMX
concentration and its initial removal rate (V0); the initial rate
is for the first 10 min. V0 for the P&B and B experiments
increased with initial SMX concentration, but the P rate was
relatively stable. This supports that initial adsorption of
SMX to the biomass was kinetically controlled by the
SMX concentration. Figure 6b presents the volumetric re-
moval rates (VRR) for the three protocols over their entire
480 min. The VRR by P&B was the highest among the three
protocols for all initial SMX concentrations and greater than
the sum of the individual P and B rates, again illustrating the
synergy of intimate coupling. B always had the lowest VRR,
which demonstrates that biodegradation of SMX was much
slower than photolysis. Also, the B rate did not change
much with initial concentration, although the VRR for the
highest initial concentration was low, which may indicate a
degree of inhibition by SMX at its highest concentration.
For all protocols, the average reaction rate constants (k) for
different initial concentrations, based on zero-order kinetic
model, were 0.12 mg L−1 min−1 for P, 0.06 mg L−1 min−1 for
B, and 0.19 mg L−1 min−1 for P&B, and the respective mean
squared deviations were calculated based on the formula:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ffi
Pn
2
ðxi xÞ
i¼1
n1 to get 0.06 for P, 0.04 for B, and 0.04 for P&B.
60
55
Hydroformylated fibers
SMX (mg/L)
0.3 B
P&B
0.2
0.1
0
10 30 60 90 120
Initial SMX concentration (mg/L)
Fig. 6 Relationships between initial concentration and SMX removal
rates. a V0 is for the first 10 min, and the lines are linear regression of
V0 versus initial SMX concentration. b VRR is for the entire 480-min
experiment
Here x is zero kinetic constant corresponding to different
initial SMX concentration, x is average value, and n is the
sample number. The SMX removal rate clearly was higher
by P&B than that by P or B.
Mineralization of SMX
Full mineralization of SMX is a goal for the treatment of
wastewater containing biologically recalcitrant and inhibi-
tory organics, since the effluent quality normally needs to
have a low content of DOC or BOD. DOC removal was the
index we used to measure the degree of SMX mineralization
in triplicate P, B, and P&B experiments with 60 mg/L SMX.
The average DOC removal percentages after 8 h are shown
in Fig. 7. The average DOC removal percentage by P&B
was by far the highest (57%), compared to 26% for B and
7% for P. The levels of DOC removal were less than the
levels of SMX removal, e.g., 100% SMX removal and 57%

Activated sludge
DOC removal of P&B, and 80% SMX removal and 7%
50 Inactivated sludge DOC removal of P. Figure 7 also shows that average DOC
removal percentage of SMX by B was higher than that by P,
45
although SMX removal rate was slower by B than that by P.
Thus, the main process occurring in the P experiment was
40
0 15 30 45 60 SMX transformation into its intermediates, but the P&B
T (min)
experiments allowed greater SMX transformation (due to
Fig. 5 SMX adsorption with activated, inactivated sludge, and hydro- the synergy) and mineralization of the majority of the
formylated fibers intermediates.
532 Appl Microbiol Biotechnol (2012) 94:527–535

Removal percentage (%)

100 DOC
SMX
amplified and cloned into pMD18-T to build clone
80
libraries. The biofilm after the B experiments was the
60
same as the biofilm at the start of the P&B experiments.
40
20
Thus, changes to the community composition represent
0
the effect of intimately coupled P&B. To characterize
P B P&B
Protocol
the flora composition, we sequenced 86 and 94 clones
from B and P&B samples, respectively, and analyzed
Fig. 7 Average DOC and SMX removal percentages with UV photo- them by a BLAST search according to the GenBank
degradation (P), biodegradation (B), and coupled UV photobiodegra- database. Tables 1 and 2 present the strain distributions of
dation (P&B). The averages are for triplicates of each experiment. The
error bars indicate the range of values, in which the standard devia- the two samples. The 16s rDNA sequences of the strains
tions of DOC removals were, respectively, 0.35 for P, 1.6 for B, and 1.0 were submitted to GenBank, and the accession numbers
for P&B, while the deviations of SMX removal were, respectively, 1.2 were shown in Tables 1 and 2. The biofilm after the B
for P, 1.1 for B, and 0 for P&B experiments contained 29 unique strains, while the biofilm
after P&B had 22 unique strains.
In the biofilm after the B experiments (Table 1), Rhodopir-
Community analysis ellula baltica was the greatest portion (26%), and Methyl-
ibium petroleiphilum PM1 was 14%. In the biofilm after the
Genomic DNA extracted from biofilm on the hydroformy- P&B experiments (Table 2), R. baltica was reduced to 7%,
lated fiber after the B and P&B experiments was PCR and M. petroleiphilum was eliminated. The dominant strains

Table 1 Sequencing statistics of

samples B based on 16S rDNA Unique name Closely related strain Similarity (%) Accession number

OTU-1(1%)a Acidiphilium cryptum 90 JF312870

OTU-2 (7%) Acidovorax ebreus 98 JF312871
OTU-3 (4%) Aromatoleum aromaticum 91 JF312872
OTU-4 (1%) Beijerinckia indica 91 JF312873
OTU-5 (1%) Burkholderia 99 JN392927
OTU-6 (2%) Candidatus Accumulibacter phosphatis 94 JF312874
OTU-7 (2%) Candidatus Nitrospira defluvii 92 JF312875
OTU-8 (1%) Caulobacter segnis 96 JF312876
OTU-9 (4%) Cytophaga hutchinsonii 85 JF312877
OTU-10 (1%) Dehalococcoides 81 JF312878
OTU-11 (4%) Flavobacterium johnsoniae 86 JF312879
OTU-12 (2%) Gemmatimonas aurantiaca 85 JF312880
OTU-13 (1%) Geobacter 82 JF312881
OTU-14 (1%) Hyphomicrobium denitrificans 96 JF312882
OTU-15 (6%) Mesorhizobium 92 JF312883
OTU-16 (14%) Methylibium petroleiphilum PM1 97 JF312884
OTU-17 (1%) Nitrosospira multiformis 90 JF312885
OTU-18 (1%) Oligotropha carboxidovorans 98 JF312886
OTU-19 (1%) Paracoccus denitrificans 95 JF312887
OTU-20 (1%) Pedobacter heparinus 83 JF312888
OTU-21 (1%) Pelobacter carbinolicus 80 JF312889
OTU-22 (1%) Pirellula staleyi 82 JF312890
OTU-23 (1%) Pseudomonas fluorescens 98 JF312891
OTU-24 (26%) Rhodopirellula baltica 84 JF312892
OTU-25 (1%) Rhodopseudomonas palustris 90 JN392955
OTU-26 (1%) Sphingobium japonicum 94 JF312893
OTU-27 (10%) Stenotrophomonas maltophilia 97 JN392957
OTU-28 (1%) Thauera sp. MZ1T 97 JF312895
a
Values in brackets represent a OTU-29 (2%) Xanthobacter autotrophicus 93 JF312896
percentage
Appl Microbiol Biotechnol (2012) 94:527–535 533

Table 2 Sequencing statistics of

samples P&B based on 16S Unique name Closely related strain Similarity (%) Accession number
rDNA
OUT-30 (2%) Acidovorax ebreus 98 JF312871
OUT-31 (2%) Arthrobacter aurescens 92 JF312897
OUT-32 (4%) Beijerinckia indica 91 JF312873
OUT-33 (1%) Burkholderia 99 JN392927
OUT-34 (1%) Candidatus Accumulibacter phosphatis 94 JF312874
OUT-35 (1%) Caulobacter segnis 96 JF312876
OUT-36 (1%) Comamonas testosteroni 96 JF312898
OUT-37 (19%) Delftia acidovorans 99 JF312899
OUT-38 (1%) Desulfatibacillum alkenivorans 83 JF312899
OUT-39 (1%) Herbaspirillum seropedicae 87 JF312901
OUT-40 (1%) Klebsiella variicola 95 JF312902
OUT-41 (1%) Leptothrix cholodnii 96 JF312903
OUT-42 (2%) Mesorhizobium 92 JF312883
OUT-43 (25%) Micrococcus luteus 92 JF312904
OUT-44 (1%) Nitrobacter hamburgensis 98 JF312905
OUT-45 (2%) Nitrosospira multiformis 90 JF312885
OUT-46 (19%) Oligotropha carboxidovorans 98 JF312886
OUT-47 (2%) Ralstonia eutropha 98 JN392952
OUT-48 (7%) Rhodopirellula baltica 84 JF312892
OUT-49 (2%) Sphingobium japonicum 94 JF312893
OUT-50 (2%) Sphingomonas wittichii 94 JF312907
OUT-51 (1%) Stenotrophomonas maltophilia 97 JN392957

in the P&B biofilm were Micrococcus luteus (25%), Delftia
acidovorans (19%), and Oligotropha carboxidovorans
(19%).
Discussion
SMX is a heterocyclic compounds containing nitrogen and
sulfur (C10H11N3O3S); SMX has three nitrogen atoms in its
structure. Trovó et al. (2009) proposed photolysis degrada-
tion pathways of SMX and listed several degradation prod-
ucts, e.g., C6H6NO2S, C6H6NO, C6H6NO, C6H6N, and
C6H6NO2S. Figure 3 shows that nitrogen was released as
ammonium under UV illumination, but the released amount
was less than one third of total nitrogen, which suggests that
SMX has a stable structure. However, the more nitrogen was
released in the P&B experiments, which shows that the N-
containing intermediates were readily biodegraded.
SMX was removed more rapidly by intimate coupling in
P&B than by either P or B alone in ILPBR batch experi-
ments (Figs. 4 and 6b), and the corresponding DOC remov-
als by P&B also were higher (Fig. 7). The accelerated SMX
removal probably was due to a synergy between photolysis
and the rapid biodegradation of SMX by the biofilm. This
situation would occur if the photolysis kinetics occurred
with a reaction order less than first order. The P results in
Fig. 6a clearly shows that the photolysis kinetics did not
increase in a linear manner with the SMX concentration but
had a reaction order much smaller that first order. Thus,
rapid biodegradation of SMX by the biofilm allowed the
photolysis kinetics to be faster when normalized to the total
SMX in the system, leading to a synergy between biodeg-
radation and photolysis.
The SMX concentration in the P&B experiments (Fig. 4)
did not exhibit the adsorption saturation effect of the B
experiment (Fig. 4) or the adsorption experiment (Fig. 5).
It appears that SMX removal in the first 10 min of the P&B
experiments was a combination of adsorption to biomass
and photolysis, with adsorption becoming more important
for the higher SMX initial concentrations, probably because
of the low reaction order for photolysis. After 10 min, the
P&B experiments showed substantially faster removal ki-
netics than did the P or B experiments due to the synergistic
effect of photolysis and biodegradation.
The higher removal of DOC in the P&B experiments was
brought about by the presence of biofilm bacteria able to
biodegrade photolysis products. This is the objective of
intimate coupling, in which photolysis products are biode-
graded immediately as the solution circulated between pho-
tolysis and biodegradation zones. During the P experiment,
photolysis transformed SMX to intermediate products, but
provided little mineralization (Fig. 7). This was supported
534
by the release of about 40% of the organic N as ammonium
N (Fig. 3), which also supported bacterial metabolism and
DOC mineralization in the P&B experiments.
Clone libraries performed on the biofilms before and after
the P&B experiments showed profound changes in the
community that are consistent with intimate coupling of
photolysis and biodegradation. Whereas OTU-24 (related
to R. baltica) and OTU-16 (M. petroleiphilum PM1) domi-
nated the biofilm after the B experiments, they were mostly
replaced by and OTU-43 (M. luteus), OTU-37 (D. acid-
ovorans), and OTU-46 (O. carboxidovorans) after the
P&B experiments.
R. baltica is a marine, aerobic, heterotrophic bacterium
that seems to be involved in the first steps of biodegradation
of complex macromolecules produced by autotrophic organ-
isms like algae and cyanobacteria (Glöckner et al. 2003). M.
petroleiphilum PM1 is utilized in bioremediation of MTBE
and at aromatics-contaminated sites because of its ability to
degrade a wide variety of normally toxic organic com-
pounds (Hanson et al. 1999; Hristova et al. 2003). Based
on their metabolic characteristics and dominance, R. baltica
and M. petroleiphilum PM1 probably played important roles
in biodegrading SMX in the acclimated activated sludge.
The most likely explanation for the large drops in R. baltica
and M. petroleiphilum is that SMX was degraded so rapidly
by photolysis that the SMX degraders lost the competition
for its substrate.
M. luteus is known to biodegrade hydrocarbons and
olefinic compounds, use biphenyl as a carbon source, and
degrade phthalates, and it harbors a plasmid capable of
degrading malathion and chlorpyrifos (Zhuang et al. 2003;
Guha et al. 1997; Eaton 1982). D. acidovorans is part of a
bacterial consortium used to completely biodegrade com-
mercial linear alkylbenzenesulfonate (Schulz et al. 2000). O.
carboxidovorans OM5T (DSM 1227, ATCC 49405) is a
chemolithotrophic bacterium, originally isolated from
wastewater, with the capability to utilize carbon monoxide,
carbon dioxide, and hydrogen (Meyer et al. 1993). The
numerical importance and metabolic characteristics of M.
luteus and D. acidovorans suggest that they were biodegrad-
ing photolysis products. The role of O. carboxidovorans is
less obvious, but it probably also gained advantage from
being part of the biofilm.
Not all strains possess the biodegradation capacity for
SMX or its photolytic intermediates, and it is reasonable for
these strains to co-exist in the same biofilm. The other
bacteria can accumulate through their biodegradation of
soluble microbial products released by the degraders of
SMX and its photolytic products. These strains are impor-
tant to the overall good performance of the process, as they
biodegrade general organic matter, measured by COD,
whose removal was accelerated in P&B process through
action of the microbial community.
Appl Microbiol Biotechnol (2012) 94:527–535
In summary, SMX adsorption occurred in biofilm rather
than the carrier (Fig. 5). SMX photolysis products also could
have been adsorbed by biofilm during P&B process, but the
increased DOC removal in the P&B experiments (Fig. 7)
increased release of N (Fig. 3), and the significant changes
in microbial community structure are strong indicators that
the photolysis products were biodegraded. In particular,
SMX biodegradation (presumably the roles of R. baltica
and M. petroleiphilum) was out-competed by SMX photol-
ysis in P&B experiments, making biodegradation of photol-
ysis products (most likely by M. luteus and D. acidovorans)
important. The experimental results indicated that the higher
removal rates of SMX and DOC, as well as the changes in
community structure, confirm the value of intimate coupling
of photolysis with biodegradation in the ILPBR.
Acknowledgments The authors acknowledge the financial support
by the National Natural Science Foundation of China (50978164), the
Special Foundation of Chinese Colleges and Universities Doctoral
Discipline (20070270003), Innovation Fund for Key Projects of
Shanghai Municipal Education Commission (10ZZ82), the Shanghai
Leading Academic Discipline Project (S30406), and the United States
National Science Foundation (0651794).
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