Journal of Environmental Science and Health, Part A

Toxic/Hazardous Substances and Environmental Engineering

ISSN: 1093-4529 (Print) 1532-4117 (Online) Journal homepage: http://www.tandfonline.com/loi/lesa20

Degradation of the long-resistant pharmaceutical

compounds carbamazepine and diatrizoate using
mixed microbial culture

Hunmoon Ha, Biswanath Mahanty, Soonuk Yoon & Chang-Gyun Kim

To cite this article: Hunmoon Ha, Biswanath Mahanty, Soonuk Yoon & Chang-Gyun Kim (2016):
Degradation of the long-resistant pharmaceutical compounds carbamazepine and diatrizoate
using mixed microbial culture, Journal of Environmental Science and Health, Part A, DOI:
10.1080/10934529.2015.1128712

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Published online: 11 Feb 2016.

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 JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART A
2016, VOL. 0, NO. 0, 1–5
http://dx.doi.org/10.1080/10934529.2015.1128712

Degradation of the long-resistant pharmaceutical compounds carbamazepine and

diatrizoate using mixed microbial culture
Hunmoon Ha, Biswanath Mahanty, Soonuk Yoon, and Chang-Gyun Kim
Department of Environmental Engineering, INHA University, Incheon, South Korea

ABSTRACT ARTICLE HISTORY

The microbial degradation of two recalcitrant pharmaceutical compounds, carbamazepine (CBZ) and Received 16 July 2015
diatrizoate (DTZ), was studied in laboratory batch experiments. We used a defined mixed microbial culture
KEYWORDS
comprising four distinct microbial species that were previously known to have high decomposition Carbamazepine; diatrizoate;
capacity toward recalcitrant substances. Biological decomposition in liquid phase cultures for either CBZ DOC; HPLC; mixed microbial
or DTZ, or in a combination of the two, was conducted for 12 days. DTZ and CBZ were degraded by 43.2% culture
and 60%, respectively from an initial concentration of 100 mg L¡1. When degradation was assessed using a
mixture of the two compounds, the initial degradation rates of CBZ and DTZ were lower than those
observed in the single-compound study. However, the final cumulative removal efficiency was very
Downloaded by [Inha University] at 20:52 21 February 2016

similar. The extent of dissolved organic carbon (DOC) removal was correlated with the degradation of the
pharmaceuticals.

Introduction The frequent detection of CBZ and DTZ in the environmen-

tal matrix requires the adoption of proper remediation technol-
Managed aquifer recharge (MAR) is an accepted and effective
ogy. Physiochemical approaches such as gamma irradiation,
water resource management approach to securing water
Fenton’s treatment, and UV/persulfate to treat these pharma-
resources in the face of rising global temperatures and dis-
ceuticals have generally been effective. However, harsh treatment
rupted weather patterns. In addition to the stream water from
condition, increased expense and generation of secondary
rivers and precipitation, the effluent of sewage treatment plants
byproduct pollutants are major limitations.[15,16] Furthermore,
can also be injected into aquifers for recharge and recircula-
the implementation of conventional biological treatments in sew-
tion.[1–3] However, some pharmaceutical compounds in waste-
age and water treatment plants has not proceeded promis-
water are not completely degraded in treatment plants, which
ingly.[17–20] These pharmaceuticals can be degraded to some
necessitates quality management of effluents before utilization
extent in biological processes to generate a range of stable metab-
in MAR. These pharmaceutical compounds, used in residences
olites, but complete mineralization has not been reported.[21–25]
and hospitals, can perturb every aspect of the environment
Therefore, a feasibility study on the biodegradation of these
with widespread detrimental consequences to the ecosystem.[4]
two pharmaceutical compounds (CBZ, DTZ) in a liquid-phase
Of various pharmaceutical compounds, carbamazepine
batch experiment was undertaken using a defined mixed micro-
(CBZ) and diatrizoate (DTZ) are gaining wider attention
bial culture known to have a high decomposition capacity
because of their toxicity in aquatic environments. As an anti-
toward recalcitrant substances. The extent of microbial growth,
convulsant drug, CBZ is used in the management of epilepsy
the degradation of the pharmaceuticals, and the efficiency of
and its annual global consumption has been estimated at
DOC removal were monitored in batches containing either
1,014 tons.[5] In the long run, a significant amount of CBZ is
CBZ, DTZ, or a combination of the two.
released during manufacturing or processing in health care
facilities, leading to its high detection frequency in river and
groundwater.[6,7] The potential toxicity of CBZ in the aquatic Materials and methods
environment has also been established from recent studies.[8,9]
Chemicals
In addition to the parent molecule, different transformation
products (including acridine) generated in the water treatment CBZ (5H-dibenz[b,f]azepine-5-carboxamide, C15H12N2O,
process are also potentially toxic and carcinogenic.[10] On the MW:236.27) and sodium DTZ (3,5-diacetamido-2,4,6-triio-
other hand, DTZ is intensively used in hospitals as an X-ray dobenzoic acid sodium salt, C11H8I3N2NaO4, MW:635.90)
contrast medium. Because it is relatively hydrophobic and bio- were purchased from Sigma Aldrich Chemical (St. Louis,
logically stable (excreted without metabolism), it is frequently MO, USA). Stocks of CBZ and DTZ were prepared in
detected in sewage treatment plants and surface water.[11,12] methanol at 10 mg L¡1. Unless specified, an appropriate
This indirect discharge makes it a leading cause of emerging volume of this stock was dispensed into batch experiments
toxicity problems in the environment.[13,14] to give final concentrations of 100 mg L¡1.

CONTACT Chang-Gyun Kim cgk@inha.ac.kr Department of Environmental Engineering, INHA University, 253 Yonghyundong, Incheon 402–751, South Korea.
© 2016 Taylor & Francis Group, LLC
 2 H. HA ET AL.
Mixed microbial culture and their identification
Mixed microbial culture originally isolated from oil-contami-
nated soil in Ulsan, Korea, was purchased from E. Corp., Ltd.
The purity and composition of the mixed culture were verified
using 16s rDNA sequence analysis. About 20 mL of microbial
suspension grown overnight was centrifuged (Hanil, HA-1000-
3) at 3000 £ g for 20 min. Total genomic DNA was extracted
by bead beating using FastPrep® instrument (Q-Biogene, Carls-
bad, CA, USA) for 5 seconds and FastDNA spin kit (Bio101
system, Q-Biogene). Extracted genomic DNAs were amplified
in PCR using two universal primers: 27F (50 AGA GTT TGA
TCM TGG CTC AG30 ) and 1492R (50 TAC GCY TAC CTT
GTT ACG ACT T30 ).
Amplification was performed by initial denaturation at 95 C
for 5 min, 35 cycles of denaturation (95 C for 1 min), annealing
at 54 C for 1 min, extension at 72 C for 1 min and then final
extension at 72 C for 10 min in a thermal cycler (Techgene,
Irvind, TX, USA). The PCR products were purified by using
Wizard® SV Gel and PCR Clean-Up System (Promega, Fitch-
Downloaded by [Inha University] at 20:52 21 February 2016
burg, WI, USA). The purified PCR products were ligated to a
pHEM-T easy vector (Promega) and then transfected into E.
coli XL1-Blue host cells. The recombinant cells, screened in LB
broths containing ampiciline, X-Gal and IPTG, were collected
by using the Wizard® Plus Minipreps DNA Purification System
(Promega). The 16S rDNA gene in the plasmids was sequenced
using the 3100 Capillary DNA sequencer (Applied Biosystems,
Foster City, CA, USA). Nucleotide sequence alignments were
performed and analyzed using BLAST with National Center for
Biotechnology Information (NCBI; http://www.lncbi.nkm.nih.
gov/) database.
Media and culture conditions
An equal volume mixture of LB medium and Dominic-Gra-
ham’s culture medium was used in the batch biodegradation
study. The LB medium (per liter of solution) contained 10 g
tryptone, 5 g yeast extract, and 8 g NaCl. Dominic-Graham’s
culture medium was composed of 0.15 g MgSO4¢7H2O, 1.5 g
KH2PO4, 0.5 g (NH)2SO4, 0.5 g NaCl, 3.5 g K2HPO4
and 1 mL of trace elements per liter of solution. The trace
element solution contained 0.1 g CuSO4¢5H2O, 0.3 g
MnSO4¢4H2O, 0.2 g ZnSO4¢7H2O, 2 g NaHCO3¢10H2O, 0.5 g
CoCl2¢6H2O, 0.05 g CaCl2¢2H2O, 0.5 g FeSO4¢7H2O and
0.02 g (NH4)6Mo7O24¢4H2O per liter.[26] The medium was
autoclaved at 120 C for 30 min. The mixed microbial culture
was inoculated in 200 mL of medium and then incubated
under aerobic condition at 30 C, 160 rpm. Under the given
condition, the culture was grown until the optical density of
the suspension (at 600 nm) reached about 0.8. Subsequently,
the liquid phase biodegradation experiment was conducted
as described next.
Liquid phase batch biodegradation experiments
To evaluate the feasibility of CBZ and DTZ biodegradation,
liquid phase batch biodegradation experiments were con-
ducted in a set of 300-mL triangular flasks with 100 mL of
LB media. A pre-incubated mixed microbial culture was
added at 2% v/v. Subsequently, CBZ and DTZ were added
at 100 mg L¡1, and the flasks were then sealed with silicone
bio-stoppers (Shin-Etsu, Japan, T-42) and incubated at
30 C. A control with heat-killed microbes (at 120 C for
30 min) was conducted to quantify the abiotic loss of the
target compounds.
Analytical procedures
Analysis of target compounds
The concentrations of CBZ and DTZ from the liquid phase
batch test were determined through solid phase extraction and
HPLC analysis (Agilent Technology 1200 Series, Wilmington,
DE, USA).[27] Samples were first centrifuged (Hanil Science
HA-1000-3) at 3,500 rpm for 15 min, and then the supernatant
was successively filtered through both glass fiber and 0.45 mm
membrane filters. The processed filtrate was extracted by solid-
phase extraction using HLB (OasisTM , 60 mg, 3 mL) and
ENVC (Waters®, 50 mg, 3 mL) cartridges. Each cartridge was
installed on a vacuum manifold and pre-conditioned with
3 mL of methanol and 3 mL of deionized water. Samples, which
had been previously adjusted to pH 2.7 by 3.5 M H2SO4, were
passed through the cartridge at a rate of 15 mL min¡1. The car-
tridge was washed two times with 3 mL of deionized water, and
dried under vacuum for 10 min. Finally, CBZ and DTZ were
eluted with 3 £ 3 mL of methanol dripping with vacuum. The
eluted CBZ and DTZ fractions were pooled and concentrated
(to »1 mL) in a nitrogen gas-aided evaporation system (Turbo-
Vap system, Biotage, Uppsala, Sweden).
HPLC analysis of the sample was carried out on a Zorbax
Eclipse XDB-C18 column (4.8 £ 150 mm, 5 mm, Agilent Tech-
nology) with UV-detection at 258 nm. A binary gradient con-
sisting of 0.1% v/v formic acid and methanol at a flow rate of
1 mL min¡1 was adopted as follows: 5% methanol held for
3.5 min, increased linearly to 50% for 5 min and held for
2 min, then increased linearly to 60% and 80% every 2 min and
held for 2 min and finally stepped to 100% for 16 min. The col-
umn was washed with 100% methanol to remove any residue
between sample runs.
Microbial biomass
Microbial growth in the experimental sets was measured using
optical density at 600 nm in a UV-Vis spectrophotometer (HP-
Agilent 8453). This indirect method is superior to dry cell
weight measurement, which can have substantial error in small
sample volumes.
Dissolved organic carbon (DOC) content
Changes in dissolved organic carbon (DOC) due to pharma-
ceutical compound degradation or microorganism growth were
measured using a TOC analysis instrument (Sievers TOC 900,
GE Analytical Instruments, Boulder, CO, USA).
Results and discussion
Identity of the mix culture
Among the 14 sequences, 10 (M1, M3, M4, M6, M8, M9, M10,
M11, M13 and M14) were identified by BLAST and included in

Downloaded by [Inha University] at 20:52 21 February 2016
Figure 1. Phylogenetic tree of the 16S rDNA gene sequences obtained from mixed
cultures. Sequences detected in clones are referred to as M1-M14. The tree was
constructed using sequences of comparable regions of the 16S rDNA gene sequen-
ces available in the NCBI database.
the phylogenetic tree, as shown in Figure 1. The other four
clone sequences had too many ambiguous positions to be iden-
tified. Moreover, repetition of same 16s rDNA sequence in
multiple clones could have been a possibility. As can be seen,
M3, M4 and M8 belonged to Acinetobacter sp. US1 (with
homology of >99%). M6 was identical with Bacillus halodurans
(with 100% identity). M10 had a 98% homology with Pseudo-
monas putida. These microbes are known for their capability to
degrade PAHs, oil compounds, and resin.[28] Acinetobacter spp.
has often been enriched from water with a high level of phar-
maceutical compounds such as hospital and pharmaceutical
Figure 2. Residual concentrations of CBZ and DTZ in experimental and abiotic con-
trol sets during 12 days of incubation.
JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART A 3
plant effluents.[29] Recently, Pseudomonas sp. isolated from
activated sludge in a municipal wastewater treatment plant has
been shown to effectively remove CBZ.[26]
Removal of CBZ and DTZ in batch experiments
The biodegradation of CBZ in liquid phase experiments is
shown in Figure 2. The CBZ concentration slowly decreased to
80.44 mg L¡1 during the initial 2 days, then rapidly dropped to
40 mg L¡1 after 4 days, before reaching a final residual concen-
tration of 2.8 mg L¡1 at the end of 12 days. The final residual
concentration of CBZ was 63.5 mg L¡1 in the abiotic control,
which suggests that approximately 60% of the CBZ removal
could be attributed to microbial metabolism. However, the
solid–water distribution coefficient of CBZ was negligible and
the adsorption process alone cannot explain the abiotic loss.[30]
In addition, specific ionic interaction may not be relevant for
non-ionized CBZ at the ambient neutral pH. We anticipate
that the loss of CBZ in the control experiments cannot simply
be extrapolated into the biotic experimental sets for any neces-
sary corrections.
The extent of CBZ biodegradation in this study was more
effective than that previously reported. For example, Gauthier
et al.[31] have reported only 15% of CBZ degradation, from an
initial concentration of 9.5 ppm, with Rhodococcus rhodochrous
after 28 days of aerobic incubation. Using Pseudomonas sp.
CBZ-4, Li et al.[26] have observed an average CBZ removal rate
of 46.6% after 144 for CBZ concentrations ranging from 10 to
160 mg L¡1.
With DTZ, the final residual concentrations were 36.8 mg
L¡1 and 80 mg L¡1 in the experimental and abiotic control sets,
respectively (Fig. 2). The extent of DTZ removal (43.2%) was
lower than that of CBZ at the equivalent incubation period.
This difference was tentatively attributed to the lower aqueous
solubility of DTZ, which may have reduced its bioavailability
for microbial degradation. While monitoring the biodegrad-
ability and toxicity of DTZ in laboratory-scale sewage treat-
ment plants, Haiß and K€ ummerer[21] reported only minor loss
of DTZ (12–17% of the initial concentration) up to the 16th
day, which was biotransformed but not fully mineralized after
Figure 3. Residual concentrations of CBZ and DTZ when the two compounds were
simultaneously added into liquid phase batch experiments.
 4 H. HA ET AL.
Figure 4. Biomass concentrations (OD600) of mixed microbial culture in batch
experiments with or without pharmaceuticals (CBZ or DTZ).
an extended time of 23 days. The improved and faster biodeg-
Downloaded by [Inha University] at 20:52 21 February 2016
radation of DTZ reported herein may be attributed to better
adoption of the mixed culture toward the test compound.
When CBZ and DTZ were simultaneously added in combi-
nation, their initial removal rate was lower than that observed
when the compounds were added individually (Fig. 3). How-
ever, the final extent of removal for CBZ (57.11%) or DTZ
(41.40%) was not different compared to the sets with individual
pharmaceutical compounds, i.e., the degradation rate of CBZ
or DTZ was not influenced by the presence of the other. A sim-
ilar observation has also been reported by Monteiro and Box-
all,[32] who found that the degradation behaviour for a mixture
of fluoxetine and CBZ in agricultural soil was similar to that
observed in the single compound. This phenomenon was tenta-
tively attributed to the involvement of different sets of enzyme
systems in degradation of the two compounds.
Influence of CBZ and DTZ on the growth of mixed
microbial culture
The presence of pharmaceutical compounds (CBZ and/or
DTZ) can influence the growth of microorganisms in a batch
Figure 5. Variation in biomass concentration (OD600) in a batch system using
mixed-microbe culture with simultaneous injection of CBZ and DTZ.
Figure 6. DOC concentration variation according to the addition of either CBZ or
DTZ for 12 days.
experiment, as shown in Figure 4. In a negative control without
CBZ, the concentration of mixed microorganisms continued to
increase before reaching a near constant microbial concentra-
tion (optical density of 0.9 at 600 nm) after 2 days. In experi-
mental sets receiving CBZ, the culture optical density during
the initial 4 days was somewhat lower than the control but
thereafter had a similar trend to that of the control. It was con-
sidered that the growth of mixed microbial culture was not sig-
nificantly influenced at the tested CBZ concentration. In the
presence of DTZ, a lower microbial growth rate was apparent
compared with the unamended control. However, the mainte-
nance of a steady microbial concentration after 4 days implied
a nominal influence of DTZ.
The growth of microbial culture in medium with the com-
bined addition of CBZ and DTZ was similar to that when either
CBZ or DTZ was individually added (Fig. 5). This suggests that
DTZ influenced the initial growth of the mixed culture, but no
interaction between the two pharmaceuticals significantly
altered the microbial growth pattern.
Variation of DOC in batch experiments
The DOC variation during CBZ or DTZ degradation is shown
in Figure 6 In batch experiments, the change in DOC concen-
trations, i.e., from 5,840 to 3,428 mg L¡1, was proportional to
the reduction in CBZ concentration. Likewise, in the case of
DTZ addition, the DOC concentration decreased from 5,960 to
3,128 mg L¡1 in 12 days. This implies that the two pharmaceu-
tical compounds were biologically degraded by mixed microbes
into CO2 and H2O.
Conclusion
The feasibility of using a batch biodegradation process to reme-
diate CBZ and DTZ was demonstrated. A defined mixed micro-
bial culture comprising four distinct microbial species
successfully degraded CBZ and DTZ up to 60% and 40%,
respectively, after 12 days in batch experiments. The addition
of pharmaceutical compounds influenced the initial microbial
growth, but the growth profiles subsequently became very simi-
lar. The proportional change in DOC concentration with a

varying concentration of CBZ and DTZ suggests that the phar-
maceutical compounds had been mineralized.
Funding
This work was supported by a grant from “The GAIA Project” funded by
the Korea Ministry of Environment (2014000550004) and partially by the
“Kor-Indo Savi program” of the National Research Foundation of Korea
(NRF) (NRF-2013K2A1B9065411).
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