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Strain improvement of Pseudomonas pseudoalcaligenes MHF ENV for biodegradation of
dibutyl phosphate: Strategies and Methods
Trupti D. Chaudhari# and M.H. Fulekar#*
#
Department of Life Sciences, University of Mumbai, Santacruz, Mumbai-400 098, India.
* Current address: School of Environment Science and sustainable development, Central University of Gujarat,
Gandhinagar, Gujarat-382030, India
Corresponding author: M.H. Fulekar E-mail address: mhfulekar@yahoo.com
Tel: +91-2226528847, Fax: +91-2226526053
ABSTRACT
Bioremediation of dibutyl phosphate (DBP), an organophosphorus flame retardant and
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plasticizer has been studied using the bacterium – Pseudomonas pseudoalcaligenes MHF ENV.
Different methods have been developed for the improvement of the strain of P. pseudoalcaligenes
for bioremediation of DBP viz. adaptation and mutation. DBP degradation was noticeably
enhanced after pre-adaptation of the bacterium to 500 mg/l of DBP. Random UV mutagenesis
was induced in the culture and the mutated culture was checked for its efficacy in DBP
degradation at 500 mg/l. Different parameters like change in cell count, BOD and COD were
monitored during degradation studies. UV mutated culture was found to degrade about 90% of
500 mg/l of DBP as compared to 30% by the pre-adapted culture over a period of 6 d. Thus, the
mutated strain exhibited three-fold better DBP degradation than the adapted culture. UV
mutagenesis of P. pseudoalcaligenes contributed significantly to strain improvement with highest
DBP degradation potential at high DBP concentrations of up to 500 mg/l, and thus would be
applicable for remediation of DBP contaminated sites.
Keywords: Biodegradation, Dibutyl phosphate, Adaptation, UV mutagenesis
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1. Introduction
A variety of flame retardants and plasticizers have evolved with modern technological
advancements and rapid industrialization. Flame retardants and plasticizers are widely used all
over the world and are a part of common items found in home and the workplace including
plastics, varnishes, upholstery and textiles [3,17]. Dibutyl phosphate (DBP) is an
organophosphorus flame retardant and plasticizer widely used in the manufacture of phenol and
urea resins, as an antifoaming agent, in metal separation and extraction, polyurethane foams,
coatings, antistatic in textile industry, and thermostats provides [21]. Moreover, with the ban of
polychlorinated biphenyls and poly brominated diphenyl ethers as flame retardants, the usage of
DBP is bound to increase in coming years [21, 27]. Apart from industrial uses, DBP is the major
secondary waste resulting from the hydrolysis of tributyl phosphate (TBP) [6, 28]. Degradation of
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TBP leads to the formation of are dibutyl phosphoric (HDBP), monobutyl phosphoric (H 2 MBP)
and phosphoric (H 3 PO 4 ) acid [2, 36]. About 40-64% of the parent compound is metabolized to
DBP and that 11-21% is metabolized to monobutyl phosphate. Since these degradation products
affect the solvent property of TBP, they are discarded as `Organic Waste' [20].
The various products and processes of nuclear and chemical industries generate wastes
that contain DBP. If untreated; it leads to soil-water pollution [14]. DBP is a pollutant of
environment concern due to its wide applications, release, stability (in sunlight, neutral, alkaline
and acidic solutions) and toxicity. DBP cause irritation of the eyes, nose, throat, & lungs.
Repeated or prolonged exposure to DBP causes irritation of the skin. The LC 50 for Daphnia
magna is 210 mg/l/24 h and LC 50 for Oryzias latipes is 110 mg/l/72 h (EPA, 2001). Hepato-toxic
effects such as swelling of hepatocyte and increase in liver weight is also observed. Management
of DBP laden wastes is thus an important goal for the preservation of the public health, aquatic
animals and environment. Moreover, the European Union (EU) has classified DBP amongst the
“emerging pollutants’’ and thus needs to be treated and disposed safely [27]. Processes that are
considered globally for treatment of these wastes include incineration, chemical destruction and
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direct immobilization in suitable matrices [24]. Even though incineration of this organic waste
appears attractive, its adoption is limited since corrosive P 2 O 5 is formed during the incineration
of DBP. Biodegradation would be an easy, economical, safe and environment friendly method
[35] for treating such wastes.
The growing nuclear industry and use of DBP in chemical industries have dictated the
demand of a voracious strain capable of degrading DBP at an efficient rate. Many strain
improvement techniques have been employed for increasing the degradation yields of the
bacterial strains [41, 9, 19]. Strain improvement involves manipulating and improving microbial
strains, in order to enhance their metabolic capacities involved in contaminant degradation [25].
Moreover, other strain characteristics can also be improved such as the increased stability and
resistance to environmental factors. In the present study we investigated the potential of the
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bacterial strain P. pseudoalcaligenes MHF ENV to degrade DBP. Various strain improvement
methods have been employed to increase the degradation rate and substrate tolerance of the strain
with respect to DBP. The performance of these strains obtained is compared, and tested for their
stability with respect to DBP degradation.
2. Materials and Methods
2.1 DBP degrading bacterium
Many laboratory strains were screened for their ability to utilize DBP as sole carbon
source. Amongst them, the bacterium that exhibited highest DBP degrading potential, P.
pseudoalcaligenes MHF ENV (Gene Bank Accession No.GQ301537) was selected for further
studies. This bacterium is further designated as P. pseudoalcaligenes in this study. DBP was
added directly in the medium
2.2 Media used
Mineral salts medium (MSM) used for DBP degrading bacteria was adapted from
Thomas and Macaskie [37] and has the following composition in (g/l): CaCl2 , 0.025;
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MgSO 4 .7H 2 O, 0.2; NaCl, 0.1; (NH 4 ) 2 SO 4 , 5.0; FeSO 4 .7H 2 O, 0.015; ZnSO 4 .7H 2 O, 0.00171;
FeSO 4 , 7H 2 O, 0.0015; K 2 HPO 4 0.2, KH 2 PO 4 0.2, CoCl 2 .6H 2 O, 0.000483; CuSO 4 .5H 2 O,
0.000471; NaMoO 4 .2H 2 O, 0.000453. The carbon source in MSM was DBP. The pH was adjusted
to 7.0. Controls were DBP-free culture media.
2.3 Batch studies using wild bacteria
Cells were grown in nutrient broth (NB) and the exponential phase cells (after 24 h) were
used as inoculum for shake flask studies. After 24 h, the culture was centrifuged (10,000 x g, 4 oC
for 15 min), washed with saline and transferred to minimal medium with final optical density at
600 nm being 0.02 (40 x 105 CFU/ml) and containing 25, 50, 100 and 200 mg/l DBP as sole
carbon source. Control flasks consisted of cell free medium containing DBP. The flasks were
incubated in shaker at 130 rpm and 33 ºC for 6 d. The degradation was monitored with respect to
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the cell count and GC-MS analysis. The degradation of DBP was calculated by comparing the
area ratio with that of the standard peaks.
2.4 Adaptation of the bacterium to DBP using scale up process
The culture was grown in NB for 24 h and was then added in to an erlenmeyer flask
containing minimal media with DBP concentration of 100 mg/l and final optical density (OD) at
600 nm of 0.02. After a span of 6 d, this culture was transferred to another flask containing
minimal medium and 200 mg/l of DBP with OD at 600 nm being 0.02. This culture was
incubated on a rotatory shaker for 6 d at 33 ºC for adaptation. Likewise, after a period of 6 d, the
culture was transferred to 300 mg/l, 400 mg/l and then to 500 mg/l. This adapted culture was then
used further to study DBP degradation of 200, 300, 400 and 500 mg/l with initial OD at 600 nm
being 0.02. Biodegradation was checked by monitoring the change in cell count using spread
plate technique on nutrient agar plates. The residual DBP was monitored after every 24 h. DBP
degradation was monitored using GC-MS over a period of 6 d. Control consisted of wild cultures.
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2.5 Random UV Mutagenesis in the bacteria
UV light is stated to be mutagenic in a variety of organisms including bacteria.
Mutagenesis with ultraviolet (UV) irradiation was carried out in a UV chamber. The UV source
was a 15 W mercury vapor bulb (Model Sankyo Denki Germicidal lamp G30T8). The wild
culture, grown in NB broth (OD 600 = 1.0) was diluted with dilutions of 10-3 to 10-5 respectively.
The diluted samples were exposed to UV radiation for variable time periods (5, 10 and 15 min)
keeping the distance of the UV source fixed to 40 cm. Following mutation, cultures were plated
on minimal agar plates containing 500 mg/l of DBP. The plates were fully covered with silver foil
to avoid photo reactivation and were then incubated at 33ºC for 24 h. The colonies obtained on
minimal medium with different zone diameter were then picked individually using a toothpick
and inoculated into minimal medium containing 500 mg/l of DBP and incubated on a rotary
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shaker for a period of 6 d. DBP degradation was checked by GC-MS.
2.6 Batch studies using mutated strain
The isolate no. 7 that depicted the maximum DBP degradation was selected for further
studies. This mutated strain was used (final OD at 600 nm 0.02) to check DBP degradation
potential at 500 mg/l. The bacterial growth was monitored by studying the increase in CFU/ml on
nutrient agar plates after every 24 h. A similar set up was arranged using adapted and wild strain
to compare the efficacies of degradation using wild, adapted and the mutated strain. Various
parameters like BOD, COD were checked during degradation.
2.7 Stability of the mutated strain
The mutated strain used for DBP degradation at 500 mg/l was harvested (10,000 x g, 4 ºC
for 10min) and used again for biodegradation of DBP to check its efficiency and stability. The
strain was used repeatedly for DBP degradation in shake flask cultures. Degradation was checked
by GC-MS after 6 d and the culture was used again for a fresh cycle of DBP degradation.
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2.8 Sample preparation for DBP assay
Samples were extracted by liquid-liquid extraction with CCl 4 as a solvent (extraction
efficiency of 94%). The organic fraction obtained was passed through anhydrous sodium sulphate
powder to remove the aqueous phase traces if any. This organic extract (2µl) was injected in GCMS for measurement of the residual TBP concentration.
2.9 Analysis of DBP
DBP degradation was confirmed by an integrated GC-MS (Model No- GC 1800A,
Hewlett Packard co., Palo Alto, Calif.) equipped with a HP-5 column (30m; 0.25mm inner
diameter) and an electron ionization detector. The carrier gas was He (0.7 ml/min) and other
parameters were adapted from Kawagoshi et al. [11] used for analysis of different
organophosphorus esters. The DBP degradation was defined by comparing the analyzed product
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peak area and standard DBP peak areas. The compound was confirmed as di-butyl phosphate
using the MS library.
2.10 Sample preparation for SEM
Bacterial cells were grown in DBP containing 500 mg/l for 6 d and were then harvested
by centrifugation. The cells were washed in phosphate buffer and fixed in phosphate buffer
containing 1% glutaraldehyde. The fixed bacteria were rinsed in phosphate buffer, post fixed by
incubation in 1% osmium tetraoxide for 24 h at room temperature, rinsed in phosphate buffer,
dehydrated in a graded series of alcohol solutions, critical point dried as described by Vaca et al.
[38], mounted on the stub, sputter coated with gold and observed under scanning electron
microscope (Model 435 VP Leo Electron Microcscopy Ltd, Cambridge, UK).
3. Results and discussion
3.1 DBP utilization by P. pseudoalcaligenes MHF ENV
The potential of the bacterial strain P. pseudoalcaligenes to degrade DBP was
investigated in shake flask cultures. The bacterium was exposed to selected concentrations of
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DBP in the range of 25-200 mg/l of DBP. The change in cell count was monitored every 24 h.
The inoculated culture controls lacking DBP showed no growth. Analysis of the experimental
flasks showed that there was an increase in CFU/ml till the concentration of 100 mg/l (Fig.1). The
increase in cell number as reflected by increase in CFU/ml indicates that DBP supports biomass
growth and served as sole carbon source. However, at DBP concentration of 200 mg/l the culture
depicted a lag phase of one day suggesting that the culture requires some acclimatization period
at higher DBP concentrations.
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Fig. 1. Growth of P. pseudoalcaligenes when exposed to various initial DBP concentrations over
a period of 6 d.
Fig. 2. Degradation of DBP at various initial concentrations over a period of 6 d.
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The samples were also analyzed for residual DBP in the spent medium using GC-MS.
GC-MS analysis of cell free control flasks showed no DBP degradation and no other compound
was found in the medium that could serve as carbon source. Complete degradation of 25 and 50
mg/l was achieved after 2 d and 3 d respectively. Whereas; for 100 mg/l of DBP complete
degradation was observed after a period of 5 d. At the highest concentration of 200 mg/l of DBP,
the bacterium exhibited slower degradation with only 15 % in a time span of 6 d. It is known that,
a feasible remedial technology requires microorganisms being capable of quick adaptation at high
contaminant concentrations and efficient use of pollutants of interest in a reasonable period of
time [30]. Thus, our study further aimed at strain improvement with respect to better degradation
rate at high DBP concentration.
3.2 Effect of adaptation on DBP degradation
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The biodegradation of pollutants may be affected by a variety of factors, including
adaptation of the microbial community to a particular chemical. Adaptation may be operationally
defined as an increase in the ability of a microbial community to degrade a chemical after
prolonged exposure to the compound [31]. Thus, in order to enhance the degradative potential of
the wild-type bacterial culture to degrade high concentration of DBP, the bacterium was adapted
to 500 mg/l of DBP. Many degradation studies have reported that, an increase in the rate at which
microorganism transforms the contaminant provides evidence that adaptation has occurred and
bioremediation is working [34]. In accordance to these findings, increased DBP removal of 200
mg/l from 15% to 96% in 6 d was observed with the culture pre-adapted to DBP. Our findings
show that, pre-adaptation of P. pseudoalcaligenes to DBP increased the DBP degradation rate
and also enabled DBP degradation at higher concentrations. These findings are in accordance
with biodegradation studies of compounds like benzene [42] and phenol [8] where adaptation of
the microbial culture to the substrate increased the substrate tolerance and thereby increased the
contaminant removal rate of the bacterium. Further adaptation to the same concentration did not
increase the degradation rate. The ability of the adapted culture to degrade higher DBP
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concentration viz. 300, 400 and 500 mg/l of DBP was also checked. The change in CFU/ml
during DBP degradation by this adapted culture is shown in Fig. 3. Adaptation may be the result
of several alterations in the structure and function of microorganisms. These include induction or
de-repression of enzymes, genetic change, and increase in the number of degrading organisms
[23]. In accordance to this, increase in cell number at 500 mg/l of DBP when compared to the
wild strain was due to adaptation. It was observed that, at 500 mg/l of DBP the culture exhibited
an extended lag phase for first 2d. This suggests that at higher DBP concentration of 500 mg/l,
the adapted culture requires some time probably for acclimatization or for enzyme synthesis.
Moreover, subculture to the same concentration (500 mg/l) decreased the lag phase by only 4 h
with negligible DBP degradation in first 2d. Adaptation studies infer that, at higher DBP
concentration viz. 500 mg/l adaptation is relatively slower and require longer degradation time.
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GC-MS analysis of the spent medium showed that, the adapted culture depicted DBP
removal of 78, 65, and 30% at 300, 400 and 500 mg/l of DBP respectively in a time span of 6 d
(Fig. 4).
Fig. 3. Growth pattern of pre- adapted P. pseudoalcaligenes at various DBP concentrations over a
period of 6 d.
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Fig. 4. DBP degradation by pre- adapted P. pseudoalcaligenes at various DBP concentrations
after a period of 6 days at 33ºC.
The DBP utilization of the adapted culture are significantly higher from those obtained
with inoculums not acclimated to DBP. From this, we can conclude that the adaptation of the
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bacteria to DBP is necessary for the survival of the biodegradation of the substrate in a medium
with higher concentration of DBP. In other words, adaptation contributed to the strain
improvement with respect to increased substrate tolerance, enabled DBP degradation at high
concentration of up to 500 mg/l and enhanced the DBP degradation rate as compared to the wild
type.
3.3 UV mutagenesis for strain improvement:
The induction of mutation by physical and chemical agents has been extensively used in
strain improvement programmes in bacteria. Mutagenic agents are numerous but selection of
mutagen in this study is based on safety of the mutagen, since most are highly toxigenic;
simplicity of technique and availability of the mutagen. Random UV mutations were induced in
P. pseudoalcaligenes for improving the potential of the strain with respect to degradation of DBP
at high concentrations. Amongst the various mutants obtained after UV exposure, seven positive
mutants were selected on the basis of DBP degradation (Table.1)
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Table 1 Results of UV mutagenesis of P. pseudoalcaligenes and DBP degradation yield by the
mutated strains after a period of 6 d.
Dilution
Exposure
DBP
Exposure time=10
DBP
Exposure
time=5 min
yield
min
yield
time=15 min
(%)
P. pseudoalcaligenes
10-3
10-4
(%)
Isolate 1
42
Isolate 4
76
----
Isolate 2
82
Isolate 5
85
----
Isolate 3
26
----
Isolate 6
71
Isolate 7
---90
----
3.4 Comparison of DBP degradation by adapted and mutated strain
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The mutant strain (Isolate 7) that depicted maximum DBP degradation yield was selected
for further studies. To check the potential of the mutated strain, it was exposed to 500 mg/l of
DBP and the increase in cell count was checked with respect to time. Results showed that
mutated strain could utilize 500 mg/l of DBP with better growth rate than the adapted culture as
depicted by the exponential growth during first two days that resulted in increased biomass. The
acclimatization period of 2 d required by the adapted strain was overruled by the mutant strain as
observed in Fig. 5.
An increase in DBP degradation yield from 30% for the adapted strain to 90% with the
mutant strain was achieved in a time span of 6 d. The residual DBP in the medium using adapted,
mutated and wild strain is shown in Fig. 6 (a). The degradation rate for the adapted and the
mutated strain at 500 mg/l were 25 mg/l/d and 75 mg/l/h respectively. Mutations were found to
have enhanced the DBP degrading ability by three fold than the adapted strain. Thus, in the
present study, mutations were found to have contributed significantly in strain improvement with
respect to DBP tolerance at high concentrations and increased degradation rate. Our findings are
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in accordance with than of Chen et al. [5], Malkawi et al. [16] and Sekar et al. [29] who have
reported that, mutations may have a positive change in the DNA base pairs that finally results in
better degradation rates and high contaminant tolerance.
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Fig. 5. Growth pattern of wild, adapted and mutated culture of P. pseudoalcaligenes at various
DBP concentrations over a period of 6 d.
The change in COD provides useful insights into the mechanism of biodegradation and
was thus also studied during degradation process. It was found that the COD values decreased
over time indicating the degradation of DBP in the medium with increasing incubation period.
The change in residual DBP concentration in spent medium and COD during bioremediation at
500 mg/l was studied and is shown in Fig. 6 a,b.
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(a)
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(b)
Fig. 6. Change in residual DBP (a) and COD (b) during DBP degradation at 500 mg/l over a
period of 6 days uisng wild, adapted and mutated strain.
3.5 Stability of the mutated strain:
UV mutated strains are sometimes known to be unstable since there is a probability of
these mutated strains to revert back. Biodegradation studies focussed on strain improvement are
valid only if the strain exhibits stability with respect to degradation. Thus, though efficient and
effective in their degradation ability the strain should be checked for their stability [30].
Therefore, the mutated strain obtained in our study was checked for its stability with respect to
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DBP degradation. Results show that the culture depicted stable degradation of about 90 ± 10% for
500 mg/l of DBP for over ten cycles as shown in Fig. 7
Fig. 7 Graph depicting stability profile of mutated strain with respect to DBP degradation (a)
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during its reuse for 10 cycles. The culture was harvested and reused ten successive times for DBP
degradation.
3.6 Biochemical and morphological characteristics of mutated strain:
Mutated strains are known to exhibit change in their biochemical properties and
physiological structure [39]. The mutated culture was found to depict different biochemical
properties as compared to the wild culture. The mutated bacteria was able to utilize sugars like
cellobiose and mellibiose that was otherwise not utilized by the wild culture.
SEM analysis of the adapted and mutated strain shows that the adapted cells were
irregular in margin and smaller in size than the mutated strain. The smaller size of the bacterium
in adapted culture may be due to DBP toxicity. Another morphological change as reflected by the
flocculation of cells was also observed. Flocculation of cells has been widely observed
phenomenon in bacterial cultures at high concentration of contaminants [7]. In contrary to this,
the mutated strain was observed mainly as individual cells that infers minimum DBP toxicity to
this strain (Fig. 8 a,b). Flocculation has been found to lower the degradation rate due to anoxic
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environmnet inside the floc and also due to substrate diffusion inhibition. Due to formation of
floc, the substrate cannot diffuse inside the floc thereby renedering it inaccessible to the cells
inside the floc. Thus the formation of floccules could be one of the reasons for slow degradation
rates in adapted culture. On the contrary, individual cells in the mutated strain would overrules
the substrate inavailibilty or anoxic environment and may lead to higher degradation rate.
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(a)
(b)
Fig. 8 SEM image of the adapted (a) and mutant strain (b) of P. pseudoalcalignes taken at 10,000
X when exposed to 500mg/l of DBP.
Conclusion
Strain improvement methods were employed in the present study to enhance DBP
degradation by P. pseudoalcaligenes. Research showed that, DBP degrading efficiency of P.
pseudoalcaligenes was enhanced by adaptation and random UV mutagenesis. UV mutated strain
depicted the highest DBP degradation potential as compared to the other strains. The studies
infer that the mutant strain is a promising bioresourse for remediation of DBP contaminated
sites.The study would also provide insights into the strain improvement studies of other
organophosphorus compounds.
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