Bioresource Technology 293 (2019) 122061

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

Simultaneous sulfamethoxazole biodegradation and nitrogen conversion by T

Achromobacter sp. JL9 using with different carbon and nitrogen sources
⁎
Dong hui Lianga,b, Yongyou Hua,b,
a
School of Environment and Energy, South China University of Technology, Guangzhou Higher Education Mega Centre, Guangzhou 510006, PR China
b
The Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters, Ministry of Education, South China University of Technology, Guangzhou Higher
Education Mega Centre, Guangzhou 510006, PR China

G R A P H I C A L A B S T R A C T

Proposed reaction processes of SMX biodegradation and nitrogen conversion with different carbon and nitrogen resources. (Stape 1: Baterial growth; Stape 2:
Nitrogen conversion; Stape 3: SMX biodegradation).

A R T I C LE I N FO A B S T R A C T

Keywords: This study investigated sulfamethoxazole (SMX) biodegradation and nitrogen conversion by Achromobacter sp.
Carbon source JL9 using different carbon and nitrogen sources. Results showed that SMX and sodium acetate could be co-
Nitrogen source metabolized as carbon sources for bacterial growth and nitrogen conversion with highest removal efficiencies of
Sulfamethoxazole 82.44%, 80.2%, and 79.45% for NH4+-N, NO3−-N, and SMX, respectively. Strain JL9 was able to utilize SMX as
Biodegradation
its sole nitrogen source for growth, with an SMX biodegradation efficiency of 63.10%. In addition, carbon and
Nitrogen conversion
nitrogen balance analyses showed that approximately 35.31% and 63.22% of carbon and nitrogen, respectively,
Biotoxicity
were lost as gaseous products. Finally, medium toxicity gradually decreased during the carbon and nitrogen
dependence experiments. This study, thus, suggests that carbon and nitrogen play vital roles in SMX biode-
gradation and biotoxicity reduction.

1. Introduction However, Considering their potential risks for wildlife and human
(increased microbial resistance), large-scale consumption, wide occur-
Sulfamethoxazole (SMX) were the first antibiotics to be used sys- rence and toxicities on non-target organisms, SMX become one kind of
temically for human and animal medicines both for their antimicrobial emerging pollutants (Martinez, 2009; Yu et al., 2005; Chen et al.,
properties and as growth promoters (Xu et al., 2011; Xiong et al., 2019). 2017), and have attracted worldwide attention (Ahmed et al., 2015).

⁎
Corresponding author at: School of Environment and Energy, South China University of Technology, Guangzhou Higher Education Mega Centre, Guangzhou
510006, PR China.
E-mail address: [email protected] (Y. Hu).

https://doi.org/10.1016/j.biortech.2019.122061
Received 31 July 2019; Received in revised form 22 August 2019; Accepted 23 August 2019
Available online 25 August 2019
0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
D.h. Liang and Y. Hu Bioresource Technology 293 (2019) 122061

The removal of SMX from wastewater is a challenging issue, since The nitrogen-free medium (NFM) consist of a mixed solution of :
conventional processes were not designed for that purpose. While there CH3COONa 1.5 g/L, KH2PO4 0.2 g/L, MgSO4·7H2O 0.1 g/L, CaCl2 0.1 g/
are studies has been indicated that the biodegradation of SMX by iso- L, NaCl 0.5 g/L, SMZ 50 mg/L and 2 mL trace element solution, pH 7.0.
lated and enriched bacteria in lab-scale reactors (Wang et al., 2019), The components of trace elements in the solution were following Su
Mulla et al. (2018) indicated that pure bacteria (Ochrobactrum SA1, et al. (2018): 0.5 g·L−1 MgSO4·7H2O, 1.0 g·L−1 EDTA, 0.2 g·L−1 ZnSO4,
Labrys SC11 and Gordonia SCD14) also completely degraded 3-amino-5- 0.1 g·L−1 MnCl2·4H2O, 0.5 g·L−1 FeSO4·7H2O, 0.5 g·L−1 CuSO4·5H2O
methylisoxazole (SMX) with initial concentration of 4 mg/L. Reis et al. and 0.2 g·L−1 CoCl2·6H2O. All 500 mL erlenmeyer flasks contained
(2018) has reported that Proteobacteria mandelii strains showed the high 300 mL medium, autoclaved for 30 min at 121 °C and immediately
biodegradation efficiency of SMX, and reaching maximal rates (ap- cooling to room temperature at clean bench. Whole experiments were
proximately 5 μM SMX/h) at the declining phase. Xue et al. (2019) conducted at least in triplicate.
states that the sustainment of electrical stimulation contributed to the
rapid removal of SMX in microbial fuel cells (MFCs), and more than 2.2. Physiological and biochemical characteristics of strain JL9 in response
85.1% of sulfamethoxazole (30 mg/L) could be degraded within 60 h. to different carbon sources
Kassotaki et al. (2016) demonstrated the long term experiments (up to
10 weeks) were conducted with 100 mg/L of SMX in the nitrification To investigate the physiological and biochemical characteristics of
sequencing batch reactor, resulting in up to 98% removal. Pure cultures strain JL9 when SMX as the sole carbon source, strain JL9 was in-
of Pseudomonas, Brevundimonas, and Variovorax showed potential for cubated in EM medium with a constant shaking speed of 120 rpm.
SMX usage as the sole carbon and nitrogen source as well as co-sub- When bacterial growth reached the logarithmic phase, bacteria and
strate for cellular growth (Kang et al., 2018). supernatant were separated by centrifugation at 3 °C and 8000 rpm for
The main components of organisms are carbon and nitrogen, and 10 min then bacteria were washed three times with phosphate-buffered
forms the basic molecular structures of the organic acids, amino acids saline (PBS). Bacteria were then added to the same volume of PBS as the
and DNA. Although various organic compounds have already been original BM culture, 15 mL of bacterial suspension (OD600≈1.0) was
studied for SMX biodegradation, an ideal carbon source should be re- added to 500 mL Erlenmeyer flasks containing 300 mL sterile CFM
newable and cheaply available, such as those derived from waste re- medium containing 50 mg/L SMX as the carbon source. Bacteria were
sources (Reyes-Alvarado et al., 2017; Sinharoy and Pakshirajan, 2019). then incubated at 30 °C with shaking at 120 rpm. Samples were with-
Some researcher reports that bacterium (including Microbacterium, drawn periodically to estimate levels of biomass, NH4+-N, NO3−-N,
Pseudomonas, Brevundimonas, and Variovorax) appeared the capable of NO2−-N, and SMX.
utilizing SMX as a sole carbon and nitrogen source (Kim et al., 2019; The physiological and biochemical characteristics of strain JL9 in
Kang et al., 2018). However, little article reported simultaneous SMX the presence of different carbon sources was also investigated. Bacteria
biodegradation and nitrogen conversion with different carbon and ni- were prepared as described, then after resuspension in PBS, 15 mL of
trogen source sources. The mechanism of SMX biodegradation and the bacterial resuspension was added to a 500 mL Erlenmeyer flask con-
toxicity of the by-products when bacterium using with different carbon taining 300 mL sterile CFM medium containing 50 mg/L SMX as the
and nitrogen sources is still unclear. Hence, it is essential to isolate and carbon source. After 48 h, 1.5 g/L CH3COONa was added to culture and
identified of pure bacterium have the ability of SMX biodegradation samples withdrawn periodically to estimate levels of biomass, NH4+-N,
using with different carbon and nitrogen source sources. NO3−-N, NO2−-N, SMX, and TOC.
The main objective of this study was to investigate the dependence
of sulfamethoxazole (SMX) biodegradation on carbon and nitrogen 2.3. Physiological and biochemical characteristics of strain JL9 in response
sources using Achromobacter sp. JL9 under aerobic conditions. to different nitrogen sources
Experiments examining the effects of additional carbon sources on SMX
biodegradation and nitrogen conversion were also conducted. The same protocol for bacterial suspension preparation as in Section
Additionally, the consumption pathways used for carbon and nitrogen 2.1 was used for detecting physiological and biochemical character-
sources were identified by nitrogen and carbon balance experiments. istics of strain JL9 in response to having SMX as the sole nitrogen
Biotoxicity of products generated during carbon and nitrogen source source. 15 mL of the bacterial suspension was added to 500 mL Erlen-
experiments was also determined by calculating the inhibition per- meyer flasks containing 300 mL sterile NFM medium containing 50 mg/
centage (PI) toward E. coli and agar well diffusion method. This study L SMX as the sole nitrogen source. Samples were taken periodically to
overall may provide important information regarding SMX biode- measure levels of biomass and SMX.
gradation and biotoxicity reduction in pharmaceutical wastewater that
lacks carbon and nitrogen sources. 2.4. Carbon balance experiments

2. Materials and methods Carbon balance experiments were carried out in 18 sealable 1 L
glass bottles filled with 400 mL NFM medium. Bottles were fully aerated
2.1. Culture medium with high-purity oxygen for 15 min then sealed with a rubber stopper.
After incubation for 5 days, samples were withdrawn periodically to
For enrichment technique, samples like sediments of pharmaceu- determine the biomass then were filtered through a 0.22 µm filter with
tical was collected from pharmaceutical wastewater area of a disposable syringe to estimate the changes in total organic carbon
Guangdong, China, and the collected sample was used for the isolation (TOC). The gas in the headspace of the bottles was collected daily by
of bacterium (Achromobacter sp. JL9). The components of enrichment inserting a gas-tight syringe daily to measure the concentration of CO2.
medium (EM) were as follows: 10 g·L−1 of peptone, 5 g·L−1 of yeast
extract, 0.5 of NaCl, pH: 7.0. The medium consist of the basal medium 2.5. Biotoxicity
(BM) : CH3COONa 1.5 g/L, NaNO3 0.3 g/L, NH4Cl 0.2 g/L, KH2PO4
0.2 g/L, MgSO4·7H2O 0.1 g/L, CaCl2 0.1 g/L, NaCl 0.5 g/L, SMZ 50 mg/ A commercial toxicity kit was used to assess the biotoxicity changes
L and 2 mL trace element solution, pH 7.0. during SMX biodegradation in the presence of different carbon and
The carbon-free medium (CFM) (pH 7.0) consist of a mixed solution nitrogen sources, The levels were evaluated as the percentage inhibition
of : NaNO3 0.3 g/L, NH4Cl 0.2 g/L, KH2PO4 0.2 g/L, MgSO4·7H2O 0.1 g/ (PI) of Escherichia coli (E. coli) growth. The conventional experimental
L, CaCl2 0.1 g/L, NaCl 0.5 g/L, SMZ 50 mg/L and 2 mL trace element design described in Mirzaei et al. (2018) was followed for this assay.
solution, pH 7.0. Additionally, to further investigate the biotoxicity of samples, agar well

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D.h. Liang and Y. Hu Bioresource Technology 293 (2019) 122061

diffusion assays were conducted. E. coli were heavily plated on the The concentration of NO2−-N also did not change significantly during
entire surface of an agar plate (enrichment medium with 1.0% agar), the experiment. These results indicate that bacterial growth, SMX bio-
three agar wells (5 mm in diameter) were punched into the agar plate, degradation, and nitrogen conversion are inhibited when SMX as the
and samples obtained at different time points were add to the wells. The sole carbon source. Similar to our study, Reis et al. (2014) suggest that
agar plate was incubated for 1 day to allow for the formation of in- SMX is not a readily utilized carbon source for Achromobacter deni-
hibition zones around the wells. Experiments were conducted in tri- trificans PR1.
plicate.
3.2. Effects of additional co-metabolizing carbon sources on SMX
2.6. Analytical methods biodegradation

After filtration of mixed liquor through the 0.22 mm cellulose The relationship between the inclusion of additional carbon sources
acetate syringe filters, the concentrations of TN, NH4+-N, NO3−-N and and SMX biodegradation by strain JL9 is shown in Fig. 2. During the
NO2−-N were determined according to standard methods (APHA, first 48 h of biodegradation, SMX was the sole source of carbon and
1998). TOC was taken by the TOC analyzing instrument (Elementa, energy for bacterial growth and nitrogen conversion and the biomass
Germany), the biomass of bacterium was monitored by an UV spec- value of strain JL9 culture increased slightly from 12.88 to 16.09. In
trophotometry (DR 5000, HACH, USA). The concentration of SMX was addition, the average concentrations of SMX, NH4+-N, and NO3−-N
detected by a Waters Acquity UPLC with a UV detector (Singapore). The decreased slightly from 52.38 mg/L to 49.9 mg/L, 50.64 mg/L to
detailed fractionation of dissolved organic carbon (DOC) were mea- 48.12 mg/L, and 50.02 mg/L to 49.38 mg/L, respectively. These results
sured by three-dimensional EEM (Hitachi High Technologies, Japan). confirm that strain JL9 cannot efficiently utilize SMX as the sole source
The TN, NH4+-N, NO3−-N, NO2−-N and SMX biodegradation ratio of carbon and energy for bacterial growth and nitrogen conversion.
(rate) were calculated : Y1=(C0 − Cn)/C0 × 100% (Y2 = (C0 − Cn)/h), From 48 h to 120 h of culture growth, both SMX and sodium acetate
Y1 and Y2 were the degradation ratio (rate) of TN, NH4+-N, NO3−-N, served as carbon and energy sources and the fate of SMX and nitrogen
NO2−-N and SMX. C0 : Initial concentration of pollutants. Cn : the were investigated. There were clear relationships between the presence
concentration of pollutants (n hours). All data were conducted by Mi- of additional carbon sources and SMX biodegradation, bacterial growth,
crosoft Excel (2010) and Origin 2017. Error bars represented the and nitrogen conversion (Fig. 2). When sodium acetate was present, the
standard deviation from triplicate experiments. SMX biodegradation efficiency was 78.46% (0.575 mg·L−1·h−1) and
biomass of strain JL9 increased sharply from 16.1 mg/L to 150.9 mg/L.
Previous study indicated that some bacterium occurred very fast SMX
3. Results and discussion
biodegradation rate of 2.5 mg·L−1·d−1 (Herzog et al., 2013). These re-
sults thus indicate that comparison of previous studies, strain JL9 show
3.1. Effects of carbon source on SMX biodegradation
higher SMX biodegradation capacity. And compared to SMX, sodium
acetate is a valuable carbon source for JL9 growth. In addition, sodium
Experiments were conducted to investigate the dependence of SMX
acetate supplementation, perhaps due to the resulting increase in bio-
biodegradation on available carbon sources. Fig. 1 shows evidence that
mass, led to a higher SMX biodegradation efficiency than when SMX as
the biodegradation of SMX and nitrogen conversion are influenced by
the sole carbon source. In support of our results, a previous study
the presence of an additional carbon source. When SMX was the sole
claimed that the supplementation of additional carbon sources can
carbon source, the biomass of strain JL9 initially increased from 13.88
promote increased biomass and faster SMX depletion than when SMX is
to 19.81 mg/L then decreased. During this part of period, the con-
the sole carbon source (Reis et al., 2014). Additionally, in the presence
centrations of SMX, TN, NH4+-N, and NO3−-N slowly over the course of
of both sodium acetate and SMX, the concentration of NH4+-N and
120 h. The average concentration of SMX decreased slightly from
NO3−-N decreased from 48.12 mg/L to 10.12 mg/L and 49.38 mg/L to
53.38 mg/L to 46.70 mg/L with a biodegradation ratio of 12.5% within
8.78 mg/L with removal efficiencies of 78.97 and 82.22%, respectively.
120 h. Furthermore, the concentration of NH4+-N and NO3−-N de-
Furthermore, only a small amount of NO2−-N was detected during the
creased slightly from 50.64 mg/L and 51.78 mg/L to 41.56 mg/L and
experiment, with 0.79 mg/L NO2−-N at 96 h and no detectable NO2-N
47.72 mg/L with removal efficiencies of 17.9% and 7.8%, respectively.
at 120 h post-inoculation. Fig. 2c showed the characteristics of gas
composition were different between control group (SMX as sole carbon
source) and experimental group (SMX and CH3COONa as co-metabolic
carbon sources). In the initial two day, the N2 concentration maintain at
initial level in the control and experimental group. It was due to the fact
that denitrification efficiency was inhibited when SMX as sole carbon
source both in control and experimental group. The concentration of N2
sharply increased from 5.90 to 32.86 mg/L when additional carbon
sources were added in experimental group, and the N2 concentration in
the control group still maintain at initial level. These data indicated that
denitrification had occurred, and the supplementation of additional
carbon sources can promote denitrification efficiency. These data thus
demonstrate that strain JL9 can conduct heterotrophic nitrification/
aerobic denitrification without NO2−-N accumulation and that the
NH4+-N and NO3−-N removal efficiency increase sharply with the
addition of sodium. Similarly, He et al. (2015) reported that Pseudo-
monas tolaasii Y-11 is able to remove ammonium and nitrate without
nitrite accumulation. Sodium acetate is an effective carbon source for
biological nitrogen conversion, and carbon sources are likely a crucial
complex factor impacting nitrogen conversion systems, as indicated by
previous studies (Wang et al., 2018; Yang et al., 2018). During bacterial
Fig. 1. Baterial growth, nitrogen removal and SMX biodegradation perfor- growth and heterotrophic nitrification/aerobic denitrification, strain
mance with SMX as the sole carbon source. JL9 consumed a large amount of carbon source and the concentration of

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D.h. Liang and Y. Hu Bioresource Technology 293 (2019) 122061

Fig. 2. Baterial growth, nitrogen removal and SMX biodegradation performance with different carbon source (a, b), the gas composition produced under different
carbon source (c), CV curve in different reaction stage (d).

TOC decreased sharply from 340.45 mg/L to 66.89 mg/L within 72 h. scanning showed two reduction reaction peaks (Reduction peak A and
Previous studies (Drillia et al., 2005) have shown that while in the B) and two oxidation reaction peaks (Oxidation peak A and B). Gen-
presence of acetate and ammonium nitrogen (alternative carbon and eration of CV curves over time clearly revealed that ORR activities cycle
nitrogen sources, respectively), sulfamethoxazole remained intact. between increasing to a peak value then decreasing, which may be SMX
However, these results suggests that Achromobacter sp JL9 could si- improved with the sodium acetate was added, and then absence of
multaneous sulfamethoxazole biodegradation and heterotrophic ni- carbon sources after the peak of reaction activities. These results in-
trification/aerobic denitrification. The efficiency of biodegradation of dicate that weak ORR activities can occur in liquid medium when SMX
SMX and heterotrophic nitrification/aerobic denitrification is linked to as the sole source of carbon and energy for bacterium growth, and that
the co-metabolism of SMX and sodium acetate as carbon sources for supplementation of additional carbon sources can promote the increase
bacterial growth. Nguyen et al. (2017) also demonstrated that co-me- of ORR activities. Xu et al. (2018) also reported that the addition of
tabolic degradation is important for biodegradation of SMX. sodium acetate as a supplemental carbon source leads to enhanced ni-
The oxygen reduction reaction (ORR) activities of the samples were trogen removal. Vidal et al. (2002) also showed that the available
examined using the cyclic voltammetry (CV) technique (Fig. 2d). CV carbon source is crucial for reaction progress and can promote the

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D.h. Liang and Y. Hu Bioresource Technology 293 (2019) 122061

Fig. 3. EEM spectra for DOM during the experiment of SMX biodegradation with different carbon sources (a: 0 h; b: 48 h (SMX as sole carbon resource); c: 96 h d:
120 h (co-metabolizing)).

growth and metabolism of heterotrophic bacteria. metabolites (including fulvic acid and humic acid) were released to the
Three-dimensional excitation-emission matrix (EEM) spectra reaction system. He et al. (2017) demonstrated that some metabolites
showed changes in dissolved organic matter (DOM) during SMX bio- are released when bacteria reached the decline phase.
degradation and nitrogen conversion using different carbon sources
(Fig. 3). Scanning emission and excitation spectra were obtained from
200 to 600 nm in 5-nm increments. Regional division of EEM spectra 3.3. Effects of nitrogen sources on SMX biodegradation
was performed according to Chen et al. (2003): Four chemical com-
ponents were identified in the reaction system, Region I-V included Experiments were also designed to investigate the dependence of
tyrosine, tryptophan (Peak A, Region II), fulvic acid (Peak B, Region SMX biodegradation and bacterial growth on nitrogen sources. As
III), soluble metabolites (Peak D, Region IV), and humic acid (Peak C, showed in Fig. 4, SMX biodegradation and bacterial growth were not
Region V), respectively. Tryptophan, fulvic acid, and humic acid ap- dependent upon the present of an additional nitrogen source. Fig. 4
peared in the reaction system before the addition of sodium acetate and shows that the biomass of strain JL9 increased from 14.65 mg/L (0 h) to
levels of the three peaks corresponding to these compounds were no- 80.9 mg/L (88 h) and then remained stable when SMX was the sole
ticeably higher at 48 h than at 0 h post-inoculation. These results thus nitrogen source, indicating that strain JL9 can utilized the SMX as the
showed that the metabolic activity of strain JL9 was inhibited and that sole nitrogen source for growth. Reis et al. (2014) reported that sulfo-
dead bacteria in the reaction system may have been a source of soluble namide can be used as the sole nitrogen source by Achromobacter de-
metabolites and humic acid. Upon sodium acetate addition, peak in- nitrificans PR1. Kang et al. (2017) also showed that some heterotrophic
tensities of tryptophan (Peak A), fulvic acid (Peak B), and humic acid bacteria in activated sludge can use SMX-N as their sole nitrogen
(Peak C) substantially decreased and a new peak (soluble metabolites, source. Nevertheless, biomass yields and growth rates of strain JL9
Peak D) appeared. This suggests that strain JL9 can utilize various were lower when SMX was the sole nitrogen source compared to ad-
metabolites (tryptophan, fulvic acid, and humic acid) to promote SMX ditional nitrogen source group. This may be because SMX is not a
biodegradation and nitrogen conversion during the logarithmic phase readily utilized nitrogen and energy source by strain JL9. Additionally,
(64–96 h), and that soluble metabolite levels dramatically increased the biodegradation of SMX was also inhibited as SMX concentration
during this phase due to stronger bacterial metabolic activity. Su et al. gradually decreased from 53.38 mg/L to 19.70 mg/L with a removal
(2016) reported larger soluble metabolite peaks correspond to more ratio of 63.09%, likely due to lower biomass yields compared to after
vigorous metabolism. He et al. (2017) demonstrated that humic acid- the addition of another nitrogen source.
like substances can promote the degradation abilities of microbes. To further investigate JL9 growth when SMX as the sole nitrogen
Nevertheless, the peak intensities of tryptophan aromatic protein (Peak source, the agar well diffusion method was proposed to investigate the
A), fulvic acid (Peak B), and humic acid (Peak C) reached their max- dependence of bacterium growth on nitrogen sources. The surface of an
imum values at 120 h (lag phase). Bacterial metabolic activity was agar plate was completely covered in strain JL9 was coating on the
significantly reduced during the lag phase and a large number of entirely surface of an agar plate lacking a nitrogen source. Three agar
wells were punched into the agar plate and 20 mg/L, 35 mg/L, and

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growth and nitrogen conversion.

3.4. Nitrogen and carbon balance experiments

To confirm that different paths are used for consuming carbon

sources during nitrogen dependence experiments, carbon balance ana-
lysis was performed (Table 1). The initial concentration of TC was
486.75 ± 2.01 mg/L, including 10.86 ± 0.25 mg/L biomass-C and
475.89 ± 2.76 mg·L−1 organic-C. After biomass and metabolite
synthesis, organic-C and TOC concentrations rapidly decreased to
242.9 ± 1.89 mg/L and 311.66 ± 1.48 mg/L, respectively. Mean-
while, the concentration of biomass-C increased from
10.86 ± 0.25 mg/L to 68.76 ± 0.59 mg/L and approximately 35.31%
of organic carbon was converted to inorganic carbon. These data
showed the biomass gradually increases with decreasing levels of or-
ganic carbon source, suggesting that bacteria can utilize various carbon
sources for growth when SMX is the sole nitrogen source. Nevertheless,
a lack of nitrogen sources results in low carbon source utilization effi-
ciency in a reaction system.
The different paths of nitrogen consumption during carbon depen-
dence experiments by strain JL9 were investigated (Table 2). The initial
concentration of TN was 110.62 mg/L and after reactions utilizing SMX
as the sole carbon and energy source, the concentration of TN was
108.23 mg/L and thus showed almost no change, likely because growth
and nitrogen conversion activities of strain JL9 are inhibited when SMX
as the sole nitrogen source. However, during co-metabolic degradation,
the concentrations of NH4+ and NO3−-N were decreased to 8.78 mg/L
and 10.12 mg/L, respectively. Compared the initial and final nitrogen
concentrations shows that some 18.78 mg/L of nitrogen source was
converted into biomass-N and almost 63.22% of nitrogen was lost by
the end of the experiment. These data indicate that the main mechan-
isms for nitrogen conversion were nitrification and denitrification ra-
ther than assimilation, likely because nitrogen conversion activities of
strain JL9 were inhibited when SMX was the sole carbon source.
Fig. 4. Baterial growth and SMX biodegradation performance (a), CV curve (b)
when SMX as the sole nitrogen source. 3.5. Biotoxicity assessment

Many toxic intermediates were likely produced during SMX biode-

50 mg/L of SMX were added to the wells as a nitrogen source. Fig. 4b gradation, therefore the toxicity of intermediate compounds during
shows that the growth zone of strain JL9 appeared around the three different reaction stages of SMX biodegradation was determined.
agar wells increased with increasing SMX concentration. These results Fig. 6(a) shows the percentage inhibition (PI) of samples during the
indicate that a lack of nitrogen sources represses biomass yields of carbon dependence experiments. The initial sample containing 50 mg/L
strain JL9 and that strain JL9 can utilize SMX as the sole nitrogen SMX showed high PI towards E. coli. Due to inhibition of SMX biode-
source for growth. In addition, biomass yields of strain JL9 are not gradation, changes in sample toxicity can be ignored when SMX is the
inhibited at an SMX concentration of 50 mg/L and that higher con- sole carbon source. However, the SMX concentration sharply decreased
centrations of SMX-N increased biomass rates and the growth zone of from 49.98 mg/L to 10.77 mg/L and the concentration of intermediate
strain JL9. compounds gradually increased when a carbon supplement source was
Changes in electrochemical characteristics of samples during the added. Meanwhile, the toxicity of sample also sharply decreased with
nitrogen dependence experiment were monitored by the CV technique. decreasing levels of SMX (PI = 43.89% (48 h), PI = 8.79% (120 h)),
Fig. 4c shows the resulting two reduction reaction peaks (Reduction and the PI fell to non-toxic levels by the end of the experiment. Results
peak A and B) and single oxidation reaction peak (Oxidation peak A) indicated that SMX is highly toxic to E. coli, which matches previous
from the entire nitrogen dependence experiment. Initially, there was reports by Xiong et al. (2019) of high SMX toxicity towards micro-
three small redox peaks appeared when the reactions conducted, and organisms and aquatic organisms. Additionally, the intermediates pro-
then, the redox peaks showed a increases trend. However, the redox duced by biodegradation of SMX appear non-toxic (−10% < PI <
peaks of nitrogen dependence experiment were lower than that of ad- 10%) and SMX biotoxicity decreases with increases in SMX biode-
ditional nitrogen source. These results illustrate that in the absence of gradation time. Treatment times of up to 120 h retained sample bio-
additional nitrogen source, the redox activities of strain JL9 are in- toxicity within the toxic inhibition region boundaries. To further eval-
hibited, although not eliminated. Additionally, redox activities of strain uated toxicity of samples during SMX biodegradation, the agar-well
JL9 increased with increasing reaction time. Müller et al. (2013) diffusion method was conducted. The initial sample (50 mg/L SMX) was
showed that SMX is co-metabolized with acetate and that SMX biode- added to one well, an experimental sample obtained 48 h after treat-
gradation is enhanced when a readily degradable carbon source is ment (during which SMX was the sole carbon source) was added to
provided. The proposed mechanism of simultaneous SMX biodegrada- another well, and sample after 120 h treatment (after the addition of
tion and nitrogen conversion using different carbon and nitrogen additional carbon source) was added to the third well. A clear inhibi-
sources is shown in Fig. 5. Sodium acetate and SMX may be used as co- tion zone appeared around the initial sample and 48 h sample wells
metabolic carbon and nitrogen sources for cell growth. However, SMX while E. coli colonies grew around the 120 h sample well. This result
cannot be used as the sole electron donor and carbon source for cell further confirms that strain JL9 was able to co-metabolize SMX with

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D.h. Liang and Y. Hu Bioresource Technology 293 (2019) 122061

Fig. 5. Proposed reaction processes of SMX biodegradation and nitrogen conversion with different carbon and nitrogen resources. (Stape 1: Baterial growth; Stape 2:
Nitrogen conversion; Stape 3: SMX biodegradation).

Table 1
Carbon balance analysis during nitrogen dependence experiment.
Carbon balance

Initial carbon concentration (mg/L) Final carbon concentration (mg/L) Inorganic-C

Biomass-C Organic-C TC Biomass-C Organic-C TOC
10.86 ± 0.25 475.89 ± 2.76 481.75 ± 2.01 68.76 ± 0.59 242.9 ± 1.89 311.66 ± 1.48 35.30%

Table 2
Nitrogen balance analysis during carbon dependence experiment.
Nitrogen balance

Nitrogen concentration (mg/L)

Initial SMX as sole carbon source Final concentration Loss-N

TN TN Biomass-N NO3− NH4+ NO2− Organic-N TN
110.62 ± 0.67 108.23 ± 0.54 18.78 ± 0.26 8.78 ± 0.26 10.12 ± 0.24 0.08 ± 0.02 2.92 ± 0.06 40.68 ± 0.68 69.94 ± 1.41

sodium acetate in aerobic conditions and led to a loss of sample bio- biotoxicity of samples decreases with increasing SMX biodegradation
toxicity with increasing reaction time. time.
The toxicities of intermediate compounds were also measured
during nitrogen dependence experiments. Fig. 6(b) shows that the SMX 4. Conclusions
concentration gradually decreased from 53.38 mg/L to 19.70 mg/L and
intermediate compound concentrations gradually increased when SMX This research explored effects of different carbon and nitrogen
was the sole nitrogen source. Meanwhile, the PI of E. coli by samples sources on the nitrogen and carbon balance. Results showed that the
gradually decreased from 44.79% at 0 h to 12.64% after 120 h. How- strain JL9 can utilize SMX as its sole nitrogen source and sodium acetate
ever, even 120 h samples showed biotoxicity still beyond toxic inhibi- as a co-metabolic carbon source for growth and SMX biodegradation. In
tion region boundaries. This experimental data implies that although no addition, nitrogen and carbon balance experiments indicated that ap-
additional nitrogen source was added, biotoxicity was gradually re- proximately 51.91% and 63.22% of carbon and nitrogen were lost as
duced with increasing SMX biodegradation time. However, the bio- gaseous forms. Furthermore, solution toxicity gradually decreased with
toxicity of the samples cannot be reduced to non-toxic without of other SMX biodegradation. This study can thus serve as an important source
nitrogen source, according to growth of bacteria was control by the for SMX biodegradation and biotoxicity reduction in pharmaceutical
nitrogen sources. The agar-well diffusion method was conducted to wastewater.
further evaluate sample toxicity when SMX was the sole nitrogen
source. A clear inhibition zone appeared around the initial 50 mg/L Acknowledgements
SMX sample and the inhibition zones disappeared around samples after
48 h and 120 h of treatment. The experiment further confirms that the The authors gratefully thank the financial support provided by the

7
D.h. Liang and Y. Hu Bioresource Technology 293 (2019) 122061

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