Journal of Hazardous Materials 283 (2015) 705–711
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Journal of Hazardous Materials
journal homepage: www.elsevier.com/locate/jhazmat
Biodegradation of tributyl phosphate, an organosphate triester, by
aerobic granular biofilms
Y.V. Nancharaiah ∗ , G. Kiran Kumar Reddy, T.V. Krishna Mohan, V.P. Venugopalan
Biofouling and Biofilm Processes Section, Water and Steam Chemistry Division, Chemistry Group, Bhabha Atomic Research Centre, Kalpakkam 603102,
Tamil Nadu, India
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• Aerobic granular biomass was cul-
•
•
•
•
tivated by feeding TBP along with
acetate.
Rapid biodegradation of TBP when
used as a co-substrate or as the sole
carbon source.
Biodegradation
of
2 mM
TBP
in 5 h with degradation rate of
0.4 mol mL−1 h−1 .
High phosphatase activity was
observed in TBP-degrading granular
biomass.
n-Butanol, hydrolyzed product of
TBP, was rapidly metabolized by aerobic granules.
a r t i c l e
i n f o
Article history:
Received 21 April 2014
Received in revised form
22 September 2014
Accepted 27 September 2014
Available online 17 October 2014
Keywords:
Aerobic granular sludge
Aerobic granules
Biodegradation
Phosphatase
Tributyl phosphate
a b s t r a c t
Tributyl phosphate (TBP) is commercially used in large volumes for reprocessing of spent nuclear fuel.
TBP is a very stable compound and persistent in natural environments and it is not removed in conventional wastewater treatment plants. In this study, cultivation of aerobic granular biofilms in a sequencing
batch reactor was investigated for efficient biodegradation of TBP. Enrichment of TBP-degrading strains
resulted in efficient degradation of TBP as sole carbon or along with acetate. Complete biodegradation of
2 mM of TBP was achieved within 5 h with a degradation rate of 0.4 mol mL−1 h−1 . TBP biodegradation
was accompanied by release of inorganic phosphate in stoichiometric amounts. n-Butanol, hydrolysed
product of TBP was rapidly biodegraded. But, dibutyl phosphate, a putative intermediate of TBP degradation was only partially degraded pointing to an alternative degradation pathway. Phosphatase activity
was 22- and 7.5-fold higher in TBP-degrading biofilms as compared to bioflocs and acetate-fed aerobic
granules. Community analysis by terminal restriction length polymorphism revealed presence of 30 different bacterial strains. Seven bacterial stains, including Sphingobium sp. a known TBP degrader were
isolated. The results show that aerobic granular biofilms are promising for treatment of TBP-bearing
wastes or ex situ bioremediation of TBP-contaminated sites.
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
∗ Corresponding author. Tel.: +91 44 27480203; fax: +91 44 27480097.
E-mail addresses: venkatany@gmail.com, yvn@igcar.gov.in (Y.V. Nancharaiah).
http://dx.doi.org/10.1016/j.jhazmat.2014.09.065
0304-3894/© 2014 Elsevier B.V. All rights reserved.
Tributyl phosphate (TBP), an organophosphate or phosphate
triester compound has a number of industrial uses. TBP is commercially used in large volumes (∼3000–5000 t/annum) as an
706
Y.V. Nancharaiah et al. / Journal of Hazardous Materials 283 (2015) 705–711
extractant for uranium and plutonium at nuclear fuel reprocessing
facilities [1]. It is also used in defoamers, plastisizers, herbicides,
and hydraulic fluids [2]. TBP bearing aqueous wastes can arise
in large amounts due to its wide-spread use 2. Often, depending
on the application, TBP wastes contain metals or radionuclides as
co-contaminants [3,4]. Wastes containing TBP with or without cocontaminants require treatment in order to prevent environmental
pollution. Moreover, TBP is a very stable compound and persistent
in natural environments such as soil and water. Consequently, TBP
contamination has been reported in indoor air from domestic and
occupational sites [5], in aquatic environments [6] and in influent
and effluent of wastewater treatment plants [5]. The TBP levels
ranged from 5.2 to 35 g L−1 in the influents of Swedish sewage
treatment plants (STP). Elevated levels of up to 52 g L−1 TBP were
observed in the influent of STP of a major airport [5], where TBP
was widely used in aircraft hydraulic fluids.
Chemical degradation, incineration or immobilization methods
have been considered for treatment and disposal of TBP bearing radioactive wastes. However, these methods produce either
corrosive byproducts or secondary wastes, which require further treatment [7,8]. Biological treatment of TBP bearing wastes
is attractive because it does not generate secondary wastes and
may be less expensive. TBP can be mineralized by mixed cultures
of bacteria [9,10] or by defined bacterial cultures [1,2,9,11,12].
Pure bacterial cultures such as Acinetobacter sp., Serratia odorifera, Prodencia sp., Delftia sp., Pseudomonas pseudoalkaligenes
and Sphingobium have been reported to degrade TBP. Thomas
et al. [3] have reported TBP biodegradation by mixed bacterial
consortium. Immobilization of bacterial cultures in agarose was
reported for developing a continuous process for TBP biodegradation [9,12]. However, in majority of the studies, microbial
degradation of TBP was rather inefficient; that is, degradation was
either slow or incomplete. In addition, loss of TBP biodegradation
ability was observed during repeated sub-culturing and maintenance of the bacterial strains or the mixed cultures employed
[2–4].
The present study was undertaken with the objective of developing a treatment method for TBP prior to its environmental
discharge. Being a potential inorganic phosphate donor, TBP
could be employed for bioprecipitation of metals and radionuclides. Additional motivation for undertaking the study was that
organophosphate degradation by aerobic granular biofilms has
not been reported and TBP could be a model compound to study
organophosphate degradation by aerobic granules. Aerobic microbial granules are a novel type of microbial biomass developed in the
form of compact polymicrobial aggregates using bubble-column
sequencing batch reactors (SBR). Aerobic granular biofilm technology allows design of new generation wastewater treatment plants
with significant improvements in terms of compact reactor design,
good biomass retention, excellent biomass separation and ability
to withstand shock or toxic loadings [13,14]. Basically, bioflocs
used in conventional activated sludge plants are engineered into
dense compact aggregates possessing excellent settling properties. Aerobic microbial granules with good degradative ability have
been cultivated for biodegradation of several toxic and recalcitrant compounds through enrichment [15–18] or bioaugmentation
[19].
We have successfully cultivated aerobic granular biofilms for
efficient biodegradation of TBP using an SBR by seeding the reactor
with activated sludge flocs. Biodegradation of TBP by the aerobic granular biofilms was studied by feeding TBP along with and
without acetate. Complete and stable biodegradation of TBP was
demonstrated in the SBR during long-term operation. To the best
of our knowledge, this is the first report on successful cultivation
of aerobic granular biofilms for rapid and complete biodegradation
of an organophosphorous compound, TBP.
2. Experimental
2.1. Cultivation of aerobic granular biofilms
Aerobic granular biofilms were cultivated in a bubble-column
reactor (total reactor volume 1.6 L, working volume 1 L). The glass
column reactor height was 50 cm, with working height 30 cm and
diameter 6.5 cm and the effective height to diameter ratio was 4.6.
The reactor was inoculated with 0.2 L activated sludge collected
from the outlet of an operating domestic wastewater treatment
plant at Kalpakkam, India and operated in an SBR mode. The SBR
was fed with synthetic wastewater (SWW) prepared in deionized
water and consisted of the following (mM): sodium acetate (6.3),
TBP (0.66–2), MgSO4 ·7H2 O (0.36), KCl (0.47), NH4 Cl (3.54), K2 HPO4
(0.42), KH2 PO4 (0.21), and CaCl2 ·2H2 O (0.25). Trace elements were
supplemented by adding 1 mL of stock solution [19] to 1 L of SWW.
The cycle period consisting of the following: 10 min fill, 23 h aeration, 5 min settle, 10 min decant and 45 min idle. The effluent was
discharged from a port situated at 9 cm from reactor bottom. The
reactor was operated with 80% volumetric exchange ratio at ambient temperature (∼28 ◦ C). Aeration at a superficial air velocity of
1.2 cm s−1 was provided at the bottom of the SBR using compressed
air and a porous aquarium stone.
2.2. Biodegradation of tributyl phosphate and its intermediates
Biodegradation of TBP and its intermediates was studied in 1 L
volume SBRs. TBP was fed into the reactor as co-substrate along
with acetate. The concentration of TBP at the beginning of the
cycle period was varied from 0.66 mM to 2 mM. Biodegradation
of putative intermediates of TBP such as dibutyl phosphate (DBP)
and n-butanol was also studied under similar experimental conditions. Dissolved oxygen (DO) pattern in the SBR was monitored
online during the aeration period of SBR cycle, in order to understand substrate utilization profiles. Liquid samples were collected
at regular time intervals during and at the end of SBR cycle period
for monitoring substrate removal pattern. The samples were centrifuged at 10,000 rpm for 5 min to remove suspended cells and the
supernatants were analyzed for total organic carbon (TOC), acetate,
TBP, DBP and inorganic phosphate. TBP degradation rate was calculated by determining TBP consumption in unit time for 2 mM initial
concentration [1].
2.3. Phosphatase activity
Phosphatase activity of three different biomass samples (activated sludge, acetate-fed granular sludge, and TBP-fed granular
sludge) was determined using a commercial EnzChek phosphatase
assay kit (Invitrogen, USA) according to Kiran Kumar Reddy et al.
[20]. The biomass samples were homogenized by vortexing for
5 min along with glass beads. The homogenate was centrifuged at
10,000 rpm for 5 min and the cell pellet was washed two times
with phosphate buffered saline. The pellet was resuspended in
lysis buffer and subjected to sonication for 2 min with 10 s ON and
10 s OFF mode. The cell lysate was centrifuged at 10,500 rpm for
15 min, and the supernatant was used as the source of enzyme
and incubated with 6,8-difluoro-4-methylumbelliferyl phosphate
(DiFMUP) substrate for 30 min at room temperature. The fluorescence of DiFMU released was measured using a fluorescence
microplate reader (BioTek, USA). The phosphatase activity was
expressed as M of DiFMU released min−1 g−1 of protein.
2.4. Microscopy of aerobic granular biofilms
Morphology of the granules was documented with an Olympus DP70 camera connected to a SMZ1000 stereo-zoom microscope
Y.V. Nancharaiah et al. / Journal of Hazardous Materials 283 (2015) 705–711
707
(Nikon, Japan). Average size and circularity of the individual granules were determined by using the freeware ImageJ v1.43, as
described earlier [15,21]. Microstructure of the aerobic microbial
granules was determined by confocal laser scanning microscopy
(CLSM). For CLSM imaging, the microbial granules were stained
with LIVE/DEAD BacLight bacterial viability kit (Molecular Probes,
USA). Imaging was performed using a CLSM model TCS SP2 AOBS
equipped with an inverted microscope DMIRE2 (Leica Microsystems, Germany), according to published protocols [22].
2.5. Microbial community of TBP-degrading granular biofilms
The microbial community of TBP-degrading granules was
determined using cultivation dependent isolation and cultivation
independent terminal restriction fragment length polymorphism
(t-RFLP) analysis. The detailed description of bacterial isolation,
characterization and t-RFLP analysis are included in supplementary
material.
2.6. Organophosphate analysis
Residual TBP present in the samples was extracted into organic
phase using n-hexane. n-Hexane was removed completely by
evaporation at room temperature. The TBP residue was dissolved
in known volume of acetonitrile and determined using Dionex
UltiMate 3000 HPLC-refractive index detector system (Thermo Scientific, USA). The HPLC equipped with a 5 m ODS Hypersil C18
column was operated in an isocratic mode using 100% acetonitrile
as mobile phase at a flow rate of 1 mL min−1 . Known concentration
of TBP standard prepared in acetonitrile was used for computing
concentration of TBP in unknown samples. DBP was monitored
using ICS2100 ion chromatograph (Dionex, USA) equipped with
AS18 column, an automatic sampler and a conductivity detector,
according to Dodi and Verda [23]. Potassium hydroxide (25 mM)
prepared in double distilled water was used as the eluent at a flow
rate of 1 mL min−1 . Liquid samples were filtered through 0.45 m
cellulose acetate membrane and used for DBP analysis. For routine
monitoring of organophosphate removal, TOC was measured.
3. Analytical procedures
DO concentration during the aeration period was monitored
online for determining substrate utilization patterns. DO was measured using a luminescent dissolved oxygen (LDO) sensor probe
connected to a portable multi-parameter device (Hach, USA). The
DO measurement accuracy of the LDO probe was ±0.2 mg L−1 . Liquid samples collected during and at the end of SBR cycle period
were centrifuged at 10,000 rpm for removing suspended cells. TOC
was determined using a Shimadzu TOC-VCSH analyzer, after suitable dilution. Inorganic phosphate was measured using ascorbic
acid method according to standard methods [24]. Other parameters such as mixed liquor suspended solids, biomass density and
dry weight of biomass were measured according to standard methods [24]. Total protein was quantified fluorimetrically using Qubit
protein assay kit and Qubit 2.0 fluorometer (Invitrogen, USA).
4. Results and discussion
4.1. Granule formation
A column reactor was inoculated with bioflocs, fed with TBP
along with acetate and operated in SBR mode for one year to
enrich and select for TBP utilizing microbial granules. Over a period
of time, gradual evolution of bioflocs to compact granules was
observed (Fig. 1). It was evident that the microbial granules were
Fig. 1. (A) Morphology of TBP-degrading aerobic granular biofilms. Scale
bar = 0.5 cm. (B) A 3-D projection of single microbial granule obtained using Imaris
software. Granules were stained with BacLightTM live/dead kit and imaged using
CLSM.
highly compact, dense and exhibited excellent settling abilities.
The biomass density of TBP degrading granules was 91 g L−1 . The
average size of the granules stabilized at about 1.2 ± 0.9 mm (Fig.
S1). The microbial granules were completely devoid of flocculent
biomass (Fig. 1a). The granules were spherical in shape with an
aspect ratio of 0.78 ± 0.2. The size distribution of TBP-fed aerobic
granules was more homogenous as compared to aerobic granules
of an operating laboratory scale SBR that was fed with acetate alone
(data not shown). It is evident that the average size and size distribution were influenced by the presence of TBP. Individual microbial
granules were composed of compact aggregates and majority of the
cells in them could be stained with a green fluorescent nucleic acid
stain, SYTO 9 but not with propidium iodide, indicating the viability
of bacteria (Fig. 1b). The granules developed under these conditions
were stable during several months of reactor operation and storage at room temperature or at 4 ◦ C. Community analysis by t-RFLP
revealed presence of 30 different ribotypes in the TBP-degrading
granular biofilms (Fig. 2). However, only 7 morphologically distinct
bacterial strains were obtained on agar plate supplemented with
TBP and acetate. The bacterial strains isolated from TBP-degrading
aerobic granules were identified based on 16S rRNA gene sequencing (Table 1). The total number of culturable strains obtained by
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Fig. 2. The t-RFLP profile of TBP-degrading aerobic granular biofilms. The blue peaks represent the FAM labeled terminal restriction fragments while the orange peaks
represent the internal size standard. (For interpretation of the references to color in this text, the reader is referred to the web version of the article.)
isolation corresponded to 23% of strains showed by the t-RFLP technique.
4.2. Biodegradation of tributylphosphate
Biodegradation of TBP was evident from (1) a complete match
between TBP/TOC removal and decrease in DO during aeration
(Figs. 3–5), (2) release of inorganic phosphate during TBP removal
(Fig. 6), and (3) stable TBP/TOC removal patterns in multiple SBR
cycles. DO and chemical measurements under different feeding
conditions (acetate, TBP, acetate + TBP) revealed preferential utilization of acetate over TBP. A two-step dip in dissolved oxygen
(DO) profile was observed when TBP was fed along with acetate
(Fig. 3), while a single dip DO profile was observed when acetate or
TBP were fed as sole carbon sources (Figs. 4 and 5). The chemical
analysis data showed that dip 1 and dip 2 of the two-step DO profile
were related to acetate and TBP oxidation, respectively. We have
consistently observed rapid drop in DO as soon as the carbon source
was added. However, the extent of DO decrease was greater with
acetate addition as compared to TBP. We presume that it is associated with the rate of oxidation and utilization of oxygen. Acetate,
being a labile substrate, is completely utilized within the first one
hour of feeding. Preferential utilization of acetate over TBP presented in this study is similar to earlier studies, wherein glucose
was preferentially utilized prior to TBP degradation by Klebsiella
pneumonia S3 [25] and S. odorifera [2]. However, presence of glucose
inhibited growth and TBP utilization by Sphingobium sp. [1].
Removal of about 2 mM of TBP at different degradation rates by
bacterial cultures and mixed consortium has been reported earlier,
with complete degradation happening within a few days to weeks
[3,26,27]. Recently, very efficient degradation of TBP by bacterial
Table 1
Different bacterial strains obtained from TBP degrading granular biofilms by the
spread plating technique and identified by 16S rRNA gene sequencing.
Isolate ID
Closest match
% Similarity
Accession number
T1
T2
Agromyces sp. QDZ-A
Shinella yambaruensis
strain: NBRC 102122
Alicycliphilus denitrificans
Stenotrophomonas
acidaminiphila strain
SR50-5
Agrobacterium tumefaciens
strain AMHSOL 286
Sphingobium fuliginis strain
HC3
Cupriavidus sp. USMAA2-4
100
100
HQ713375.1
AB681707.1
100
99
NR074585.1
KF279369.1
100
KF914408.1
100
KC747727.1
100
KF460029.1
T3
T5
T6
T7
T8
Fig. 3. Dissolved oxygen (A), tributyl phosphate and TOC (B) profiles during
sequencing batch reactor (SBR) cycle period. The SBR was fed with 2 mM of tributyl
phosphate along with acetate.
Y.V. Nancharaiah et al. / Journal of Hazardous Materials 283 (2015) 705–711
Fig. 4. Dissolved oxygen (A), acetate and TOC (B) profiles during SBR cycle period.
The SBR was fed with acetate as sole carbon source.
strains was reported [1,11]. Efficient and complete degradation of
2 mM of TBP with a degradation rate of 0.25 mol mL−1 h−1 was
achieved using Sphingobium sp. in 8 h. This strain appears to be far
more superior to those used in earlier studies on TBP biodegradation. In contrast, we have achieved complete degradation of 2 mM
of TBP in 5 h with a degradation rate of 0.4 mol mL−1 h−1 . Moreover, degradation of TBP was demonstrated during multiple cycles
709
Fig. 6. (A) Release of inorganic phosphate (PO4 -P) during microbial degradation of
1 mM of tributyl phosphate. (B) mM of inorganic phosphate (PO4 -P) released during
biodegradation of different initial concentrations of tributyl phosphate. The data is
the average of two cycles. Error bars represent standard deviation.
of reactor operation, indicating the stability and suitability of the
system for treating TBP containing wastewaters.
TBP removal was accompanied by release of its hydrolysed
product, orthophosphate, into the medium in stoichiometric manner (Fig. 6). Release of inorganic phosphate into the medium has
been observed in various TBP-degrading mixed cultures or defined
bacterial cultures [1,3,25]. In an earlier study, the released phosphate has been used for removal of uranium from aqueous solution
through bioprecipitation [9]. In the present study, inorganic phosphate is not removed during the process and must be handled
separately. Although significant removal of phosphate has been
reported by aerobic granular biofilms [28], such removal was not
observed in the present case because the differences in cultivation conditions and feed composition. In case of TBP, hydrolytic
action of phosphatases results in the release of inorganic phosphate. Phosphatases are known to hydrolyse organophosphate
compounds into inorganic phosphate and the corresponding alcohol. The phosphatase activity was almost 22- and 7.5-fold higher
in TBP-degrading granules as compared to activated sludge and
acetate-fed aerobic granules, respectively (Fig. 7). The involvement
of phosphatases in the hydrolysis of TBP has been implicated for
microbial degradation of TBP and its intermediates [2,25,29]. Consequently, approximately 4- to 5-fold increases in phosphatase
activity was observed in TBP degrading bacterial cells upon exposure to TBP [25]. The phosphatase activity of TBP degrading granules
was 3.18 ± 0.7 M min−1 mg−1 protein and was much higher when
compared to other studies on phosphatase activity of defined TBP
degrading bacterial cultures [2,25]. It is likely that the SBR operation strategy and repeated exposure of microorganisms to TBP
during long-term reactor operation could have acted as a selection
pressure for high phosphatase activity.
4.3. Pathway of microbial degradation of tributyl phosphate
Fig. 5. Dissolved oxygen (A) and TOC (B) profiles during single SBR cycle period. The
SBR was fed with 2 mM of tributyl phosphate as the sole carbon source.
The chemical degradation of TBP has been proposed to proceed
through three sequential hydrolysis steps. The intermediates of
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Y.V. Nancharaiah et al. / Journal of Hazardous Materials 283 (2015) 705–711
Fig. 7. Phosphatase activity in bioflocs, acetate utilizing and tributyl phosphate
degrading granular biofilms. Bioflocs represent activated sludge from an operating
sewage treatment plant. Acetate-granules and TBP-granules were collected from
laboratory reactors fed with acetate and acetate plus TBP, respectively.
chemical degradation of TBP are dibutyl phosphate (DBP) and
monobutyl phosphate (MBP) and the final products are n-butanol
(3 mol/mol TBP) and inorganic phosphate (1 mol/mol TBP) (Fig. 8A).
Although there is not much information on the metabolic pathway of TBP degradation, microbial degradation of TBP has been
proposed to occur through a series of reactions similar to that
of chemical degradation [9]. Microbial degradation of TBP is
thought to be mediated by phosphoesterases (i.e. tri-, di-, monophopshoesterases). In order to understand degradation pathway,
we have tried to monitor the intermediates (i.e. DBP and n-butanol)
of TBP degradation. However, release of DBP and n-butanol could
not be detected in the present study. Biodegradation of intermediates has been shown in Figs. S2–S5. Surprisingly, degradation of
1 mM of DBP was rather slow and incomplete (Figs. S3 and S5).
Berne et al. [2] have also found that a TBP degrading bacterium, S.
odorifera was unable to utilize DBP. However, we have observed
partial degradation of DBP by the aerobic granular biofilms. In contrast to DBP, n-butanol degradation was very rapid, almost similar
to that of acetate (Figs. S3 and S4). In fact, the degradation of 3 mM
of n-butanol was complete during the first 1 h of aeration period.
Based on the data, the following observations could be made:
(1) preferential utilization of acetate over TBP; (2) slow and incomplete degradation of DBP, the putative intermediate of TBP, when
fed separately; and (3) no preferential utilization of acetate over
n-butanol, a hydrolysed product of TBP. Based on the available
experimental data, a pathway for TBP degradation by aerobic
granular biomass is proposed (Fig. 8B). The microbial degradation
of TBP could be divided into hydrolysis and degradation steps.
It appears that hydrolysis is an important step which is mediated by the phosphoesterases and limits the overall degradation
rate. Formation of intermediates such DBP and MBP also depends
on the enzymatic machinery involved and their substrate specificity. In the present case, TBP degradation does not appear to
proceed through DBP formation. We were unable to detect nbutanol because it was probably consumed as soon as it is produced.
In fact, the degradation of n-butanol was almost similar to that of
acetate. Further studies on the organisms and the enzymes involved
are needed to provide definite evidence for the TBP degradation
pathway of aerobic granular biofilms. Nevertheless, the process
described here based on aerobic granular biofilm technology is
quite promising for developing a treatment method for TBP bearing wastes or for ex situ bioremediation of sites contaminated with
TBP.
Fig. 8. Proposed pathway of TBP biodegradation by bacterial cultures (A) and aerobic granular biofilms (B). Sequential hydrolysis of TBP involves intermediates such as DBP
and MBP. Final products of TBP hydrolysis are n-butanol and inorganic phosphate. TBP, tributyl phosphate; DBP, dibutyl phosphate; MBP, monobutyl phosphate.
Y.V. Nancharaiah et al. / Journal of Hazardous Materials 283 (2015) 705–711
5. Conclusions
This study reports cultivation of compact and dense aerobic
granular biofilms capable of TBP biodegradation in a sequencing
batch reactor by feeding TBP along with acetate. Biodegradation
of TBP was accompanied by a stoichiometric release of inorganic
phosphate into the medium. Complete biodegradation of 2 mM
of TBP was achieved within 5 h using cultivated aerobic granular
biofilms. It was observed that n-butanol, the hydrolyzed product of
TBP, was rapidly biodegraded, while, dibutyl phosphate, a putative
intermediate of TBP biodegradation, was only partially degraded,
pointing to an alternate degradation pathway. High phosphatase
activity was observed in TBP degrading aerobic granular biofilms,
as compared bioflocs and acetate fed aerobic granules, indicating involvement of enzyme activity in biodegradation. Community
analysis by t-RFLP revealed the presence of 30 unique ribotypes
in the TBP degrading microbial consortium. Seven bacterial stains,
including Sphingobium sp. a known TBP degrader, were isolated.
This study indicates that aerobic granular biofilm sequencing batch
reactors are suitable for treatment of TBP bearing waste streams.
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.jhazmat.
2014.09.065.
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