This article was downloaded by: [RMIT University] On: 05 September 2013, At: 09:52 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesa20 Detoxification and Bioremediation Potential of a Pseudomonas fluorescens Isolate against the Major Indian Water Pollutants MOHD. WAJID ALI KHAN a & MASOOD AHMAD a a Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, India Published online: 22 Sep 2006. To cite this article: MOHD. WAJID ALI KHAN & MASOOD AHMAD (2006) Detoxification and Bioremediation Potential of a Pseudomonas fluorescens Isolate against the Major Indian Water Pollutants, Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 41:4, 659-674, DOI: 10.1080/10934520600575051 To link to this article: http://dx.doi.org/10.1080/10934520600575051 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Transcript
This article was downloaded by: [RMIT University]On: 05 September 2013, At: 09:52Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Environmental Scienceand Health, Part A: Toxic/HazardousSubstances and EnvironmentalEngineeringPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/lesa20
Detoxification and BioremediationPotential of a Pseudomonas fluorescensIsolate against the Major Indian WaterPollutantsMOHD. WAJID ALI KHAN a & MASOOD AHMAD aa Department of Biochemistry, Faculty of Life Sciences, AligarhMuslim University, Aligarh, IndiaPublished online: 22 Sep 2006.
To cite this article: MOHD. WAJID ALI KHAN & MASOOD AHMAD (2006) Detoxification andBioremediation Potential of a Pseudomonas fluorescens Isolate against the Major Indian WaterPollutants, Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances andEnvironmental Engineering, 41:4, 659-674, DOI: 10.1080/10934520600575051
To link to this article: http://dx.doi.org/10.1080/10934520600575051
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the“Content”) contained in the publications on our platform. However, Taylor & Francis,our agents, and our licensors make no representations or warranties whatsoever as tothe accuracy, completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Contentshould not be relied upon and should be independently verified with primary sourcesof information. Taylor and Francis shall not be liable for any losses, actions, claims,proceedings, demands, costs, expenses, damages, and other liabilities whatsoever orhowsoever caused arising directly or indirectly in connection with, in relation to or arisingout of the use of the Content.
This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Detoxification andBioremediation Potential of aPseudomonas fluorescensIsolate against the MajorIndian Water Pollutants
Mohd. Wajid Ali Khan and Masood AhmadDepartment of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University,Aligarh, India
A Pseudomonas fluorescens strain was isolated from the soil of industrial estate ofAligarh, India. This strain was resistant to some of the major Indian water pollutants,namely Cd2+, Cr6+, Cu2+, Ni2+, Pb2+, BHC, 2,4-D, mancozeb and phenols up to the lev-els occurring in the highly polluted regions. Moreover, the test strain seems to havea great potential for the detoxification of these pollutants. The decrease in toxicity asdetermined by the Allium cepa test was recorded as 62.5% for the model water contain-ing the mixture of test heavy metals, 71.9% for the pesticides, 73.2% for phenols, and58.5% for combination of all these toxicants. These values were obtained after 24 hours,exposure to the immobilized cells of the test isolate in the calcium alginate matrix atthe concentrations of these polutants supposedly present in the highly polluted watersystems in India. The efficiency of bioremediation for certain heavy metals at the sameconcentrations by means of immobilized cells of the test Pseudomonas fluorescens isolatewas estimated to be 75.9% for cadmium, 74.2% for hexavalent chromium and 61.0% forlead during the 24 hours’ treatment. In view of the preliminary work, the test isolateseems to be a good candidate for the bioremediation of water pollutants.
Key Words: Bioremediation; Detoxification; Pseudomonas fluorescens; Water pollutants.
INTRODUCTION
Pseudomonas fluorescens belongs to a group of common non-pathogenic sapro-phytes that colonize soil, water and plant surface environments. Physiological
Received August 11, 2005.Address correspondence to Masood Ahmad, Department of Biochemistry, Faculty of LifeSciences, Aligarh Muslim University, Aligarh 202002, India; E-mail: smasood [email protected]
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and genetic features of Pseudomonas make them a promising agent for utiliza-tion in biotechnology, agriculture and environmental bioremediation applica-tions. The ability to biodegrade hazardous chemical wastes is an interestingfeature of Pseudomonas fluorescens.[1,2] The efficient remediation of xenobioticpollutants by microbial communities,[3] which may otherwise threaten publichealth, is known as bioremediation. Heavy metals, pesticides, as well as phe-nols all pose a severe threat to living systems. They can react with proteins,nucleic acids and phospholipids, and thus arrest cellular proliferation.[4]
Heavy metals are ubiquitous and persistent environmental pollutants thatare introduced in the environment through anthropogenic activities, such asmining and smelting, as well as through other sources of industrial waste.[5]
Various species of Pseudomonas have developed the ability to tolerate millimo-lar amounts of heavy metals.[1]
Pesticides can enter crop residues, municipal sludges, farm manures andsoils from industrial usage, military actions, home and agriculture.[6] Insectsand weed control measures in farming may also lead to accumulation of pesti-cides in soils, in which decomposition of these materials may occur slowly.[7,8]
Pesticides such as DDT, aldrin, BHC, heptachlor, 2,4-D, mancozeb, etc. are quitestable and persist in the environment for prolonged periods.[9] They have a ten-dency to accumulate in the fatty tissues when the food containing these residuesis ingested.[10] Moreover, the pesticides are highly toxic compounds that causea wide variety of effects, ranging from immunological disorders to adverse ef-fects on the liver, kidney, thyroid and lungs, and are frequently accompaniedby porphyria.[11]
Phenolic compounds are common pollutants in many industrialwastewaters.[12] These pollutants can enter the environment from severalsources, including the partial degradation of phenoxy herbicides,[13] the useof wood preservatives[14] and the generation of wastes in petroleum-relatedindustries.[15] Phenols are also quite toxic and exhibit varying degrees oftoxicity.[16] The present paper deals with the study on the bioremediation anddetoxification of some of the major pollutants in Indian water bodies by meansof a Pseudomonas fluorescens strain isolated from a highly polluted site.
MATERIAL AND METHODS
MediaMineral salt medium used to grow the pure culture was prepared in deion-
ized water and consisted of the following ingredients (per liter): 10 g of peptone,10 g of tryptone, 1.5 g of MgSO4, 1.5 g of K2HPO4, and 10 mL glycerol. Nutrientagar plates were prepared for colony counts that contained nutrient broth withagar-agar at 7 g/L.
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Pseudomonas Use Against Indian Water Pollutants 661
ChemicalsPeptones, tryptone, agar-agar, were purchased from Hi-Media, India. BHC
and 2,4-D were obtained from Sigma Aldrich Co. Cadmium chloride, calciumchloride, lead acetate, potassium chromate, and Millipore filter (0.45 µm) wereobtained from SRL, India. Copper sulphate, nickel sulphate, magnesium sul-phate, dipotassium hydrogen phosphate, glacial acetic acid, catechol, cresol,phenol and resorcinol were from Qualigens India Ltd. Mancozeb was a giftfrom Dr. B.S. Parmar, AIRI, New Delhi.
Isolation of Pseudomonas fluorescens Strainfrom the Polluted SoilThe soil was collected from an industrial area of Aligarh, India. First, 25 g
soil sample was suspended in 100 mL distilled water and allowed to settleafter vigorous shaking. The enrichment technique was employed for the iso-lation of the test strain.[17] The colonies appeared on the Pseudomonas agarsupplemented with test toxicants taken separately and were further checkedfor growth on the increasing amount of toxicants taken in combination. A sin-gle clone viz Pseudomonas fluorescens WM1 exhibiting the best growth wasselected for further studies.
ToxicantsThe present work was initiated with the aim of combating the menace
of water pollution by heavy metals, pesticides and phenols in Indian waters.Thus, these pollutants were taken alone or in combinations in different exper-iments. The individual pollutants were selected and taken at the maximumconcentrations where they have been reported in the highly polluted regions ofIndia.[18,19] The concentrations of various heavy metals considered as 1 × in thisstudy were as follows: Cd2+12 ppm, Cr6+10 ppm, Cu2+ 735 ppm, Ni2+ 300 ppm,and Pb2+ 194 ppm. Accordingly, the half-dilution and double concentration ofthe above metals were regarded as 0.5× and 2×, respectively.
Similarly, 1× concentration of pesticides based on their concentration in thehighly polluted regions was taken as 500 ppb benzene hexachloride (BHC), 78ppb 2,4-dichlorophenoxy acetic acid (2,4-D), and 312 ppm manganese ethylenebis dithiocarbamate (mancozeb), and the 50% and 200% concentrations wereaccordingly prepared. Although the phenols were not detected in the samplescollected from Aligarh. However, the concentration in the highly polluted watersystems in India due to the contamination of the effluents of chemical industrieswere obtained from the literature and were arbitrarily taken to be 1 mM ofindividual phenols i.e., 94 ppm of phenol, 108 ppm cresol, 110 ppm catecholand 110 ppm resorcinol, as 1× for this study.
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Determination of Growth Rate of the Isolate under StressTo check the growth pattern of resistant strain under stress, a freshly grown
overnight culture was prepared. Then, 10 mL of liquid Pseudomonas brothwas taken in the culture flasks and 0.3 mL of pure culture was added to 3×concentrations of heavy metals alone (Cd2+, Cr6+, Cu2+, Ni2+ and Pb4+), 3xconcentrations of pesticides alone (BHC, 2,4-D and mancozeb), 3× phenols alone(catechol, cresol, phenol and resorcinol) as well as 1× and 3× test heavy metals,pesticides and phenols in combination. All the flasks were incubated at 37◦Cand absorbance was measured at 540 nm after 4, 8, 12, 24 and 48 hours. Aseparate flask containing the pure culture without any toxicant was also runwhich served as the negative control.
Cell Viability of the Pseudomonas fluorescens WM1 under StressAn aliquot (0.2 mL) of the overnight culture of the test isolate was added in
10 mL Pseudomonas broth and allowed to grow at 37◦C till the O.D at 540 nmwas doubled. The cells were then harvested by centrifugation at 5000 rpm for5 minutes. After discarding the supernatant, the cell pellet was suspended in10 mL of sterile normal saline supplemented with various concentrations oftest toxicants. The cells were incubated for 6 hours at 37◦C. Serial dilution ofsample was done up to 105-fold and from that diluted solution 0.1 mL dilutedsample was taken for spreading on the plates. The plates were incubated overnight at 37◦C and number of colonies was counted.[20]
Entrapment of the Test Isolate in the Alginate Gel BeadsThe entrapment of cells was carried out according to the method of Eikmeier
and Rehm.[21] Alginate (Manugel DJX) was obtained from alginate industries,Hamburg, FRG. 0.1 mL of cell suspension was mixed with 0.9 mL of 2% sodiumalginate solution, maintained at room temperature. The slurry was extrudeddrop wise through syringe into 250 mL of 0.8 M calcium chloride solution.The beads formed were removed and washed and then incubated with varyingconcentrations of toxicants overnight at 37◦C. Subsequently the beads wereremoved out of toxicant solutions that were then tested for the extent of biore-mediation/detoxification.
Allium cepa TestAllium cepa test for the model water at the given concentrations of toxi-
cants was carried out according to the method of Fiskesjo.[22] Small red onionsof equal size were taken for this experiment. Using a small sharp knife, theyellowish brown outer scales and the brownish bottom plates were removedcarefully, leaving the ring primordial intact. Test tubes in triplicate were filled
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Pseudomonas Use Against Indian Water Pollutants 663
with different solutions of heavy metal salts, pesticides and phenols obtainedbefore and after passing through immobilized WM1 cell system. Aqua guardwater was used as solvent control. One onion was placed on the top of each tubewith root primordial downward touching the liquid samples. At every 12 hours,fresh liquid samples were flooded to ensure contact with the onion bulbs. Thetreatment was continued for 5 days in the dark. After the treatment was over,the onions were taken out and the average length of the roots (an average of 10roots) was measured for each concentration. Inhibition in the growth of Alliumcepa roots was considered as an index of toxicity.
Analysis of Heavy MetalsHeavy metals were analyzed in the test sample before and after treatment
with the immobilized cells by means of GBC932+ atomic absorption spectropho-tometer. The standards were from SRL, India and the solutions were preparedin double distilled water.
RESULTS AND DISCUSSION
Environmental pollution is one of the most formidable problems of the moderntime especially the water pollution in India has assumed an alarming dimen-sion due to the dangerously high levels of pesticides, heavy metals and otherorganic pollutants. Aligarh city houses several small electroplating and lockmanufacturing industries. The effluents supposedly containing the heavy met-als from the industrial waste, pesticides from agricultural run off, and phenolsfrom certain chemical industries gain entry into the waterways including thegroundwater system as well as in the cultivable land. Thus, there is a need todevelop a technique of bioremediation of these major pollutants from the pol-lutant water to make it potable. The present study is a preliminary attempttowards that direction.
Pseudomonas fluorescens is a non-pathogenic soil bacterium, especiallyequipped with the machinery for biotransformation of xenobiotics.[23−25] Amongthe natural population of microflora existing in the heavily polluted soil, wesucceeded in isolating a strain of Pseudomonas flurorescens, which seems tobe quite resistant and is apparently useful for bioremediation of heavy metals,pesticides and phenols commonly present in Indian water system.
Figure 1 shows the growth pattern of the test isolate in the presence ofvarious concentrations of the said water pollutants. The test strain regainedroughly similar rate of growth with and without toxicants following a variableperiod of lag phase. This similarity of slopes is indicative of the fact of acquiringcomplete resistance after the dose-dependent preparatory periods. However, thetime lag in the presence of test chemicals could also be attributed to the killingeffect of these toxicants on the test strain. Another experiment was, therefore,
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Figure 1: Growth pattern of isolate Pseudomonas fluorescens strain in the presence ofvarious toxicants.
performed to rule out the possibility of significant irreversible damage to thecells of the test isolate (Table 1).
The hazardous nature of pollutants can only be assessed and evaluated bymeans of toxicity bioassays.[26] Hence our primary objective was to estimate thereduction in toxicity of the model contaminated water sample following treat-ment with immobilized WM1 strain regardless of whether biodegradation of theorganic toxicants had occurred or not. This had become pertinent in view of thesignificant reduction in the phenolic contents in case of green olives wastewatertreated with P. ostreatus strain without a significant reduction in phytotoxicity.Similarly in case of GOW treated with purified laccase from Polyporous pensi-tius there was no reduction in the phytotoxicity concomitant with remediation ofphenolics.[27] Recently Aggelis et al.[28] have also demonstrated that in all cases
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Pseudomonas Use Against Indian Water Pollutants 665
Table 1: Survival of the test Pseudomonas fluorescens strain following exposure tovarying concentrations of toxicants for 6 hours.
S. no. Experimental conditions No. of colonies/mL∗
∗Each experiment was performed twice and the plates for each dose were run in triplicate.Values are mean ± SD.Heavy metals—Cd2+, Cu2+, Cr6+, Ni2+ and Pb2+.Pesticides—BHC, 2,4-D and mancozeb.Phenols—catechol, cresol, phenol and resorcinol.
of olive oil mill waste treatment systems employed by them the decrease intoxicity was never proportional to the removal of phenolics. It is in this contextthat we had selected the Allium cepa test, which is one of the best indicators ofphytotoxicity of chemicals and has been validated by several workers for testingchemical pollutants posing environmental hazards.[29−31] This test has manyattractive features including its simplicity, sensitivity, reproducibility and costeffectiveness.[32−34]
The test Pseudomonas fluorescens WM1 strain was immobilized in the cal-cium alginate gel matrix in view of the reports in the literature that immobilizedsystem has several advantages over the free cell system.[35] The detoxificationefficiency of the test isolate was estimated in term of the residual toxicity ofmodel water containing commonly occurring heavy metals, pesticides and phe-nols alone and in combination by means of Allium cepa test. Tables 2–4 showthe data of Allium cepa root inhibition assay of the model water before and afterpassing through the calcium alginate beads carrying the test Pseudomonas flu-orescens strain. The maximum and minimum efficiency of metal detoxificationby immobilized cell system of WM1 was found to be 75% and 50.8%, respectively(Table 2).
The pre-and post-treatment toxicity of model water containing the test pes-ticides as determined by Allium cepa test in the immobilized WM1 cell systemis presented in Table 3. The maximum and minimum efficiencies of pesticidesdetoxification by the immobilized cell system were estimated to be 78.1% and65.6%, respectively (Table 3). Table 4 shows the data of the toxicity of aquaguard water supplemented with varying concentration of phenols before andafter passing through the test cell immobilized system. The efficiencies of detox-ification of phenols as determined by the Allium cepa test, after treatment withthe immobilized cells were recorded to be 76.5%, 68.0% and 64.1%, respectivelyat 0.5x, 1x and 2x phenol concentrations.
Extent of detoxification brought about by combination of toxicants is shownin Table 5. The efficiency of detoxification of combined toxicants was recordedto be 76.4%, 58.5% and 40.6% for 0.5×, 1× and 2× toxicant concentration,
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Tab
le2:
Eva
lua
tion
of
the
toxi
city
of
he
avy
me
tals∗
of
the
wa
ter
sam
ple
sb
efo
rea
nd
aft
er
tre
atm
en
tw
ithth
eto
lera
nt
isola
teim
mo
bili
zed
inth
ea
lgin
ate
ge
l.
Co
nce
ntra
tion
ofh
eavy
me
tals
0.5×
1×∗∗
2×N
oto
xic
ant
s(−
vec
ont
rol)
Exp
erim
ent
al
Ave
rag
e%
Ave
rag
e%
Ave
rag
e%
Ave
rag
ec
ond
itio
nsle
ngth
(cm
)In
hib
itio
n∗∗∗
leng
th(c
m)
Inhi
biti
on
leng
th(c
m)
Inhi
biti
on
leng
th(c
m)
Un
tre
ate
dm
od
elw
ate
rc
on
tain
ing
he
avy
me
tals
mix
ture
(+ve
co
ntr
ol)
0.7
±0.
0389
0.3
±0.
0295
.3N
og
row
th10
06.
4±
0.07
Mo
de
lwa
ter
tre
ate
dw
ithim
mo
bili
zed
ce
llso
fP
seu
do
mo
na
sflu
ore
sce
ns
isola
te
5.6
±0.
1414
4.4
±0.
1432
.83.
2±
0.42
49.2
6.4
±0.
14
Effic
ien
cy
of
me
tal
de
toxi
fica
tion
inim
mo
bili
zed
ce
llsy
ste
m∗∗
∗∗
75.0
%62
.5%
50.8
%—
Va
lue
sa
rem
ea
ns
±SD
.∗ H
ea
vym
eta
ls:a
mix
ture
of
Cd
2++
Cr6+
+P
b2+
∗∗1×
he
avy
me
tals
:C
d2+
(124
pp
m)
+C
r6+(1
0p
pm
)+
Pb
2+(1
98.1
8p
pm
).∗∗
∗ In
hib
itio
nw
ithre
spe
ct
to−v
ec
on
tro
li.e
.w
itho
ut
toxi
ca
nts
.∗∗
∗∗Ef
ficie
nc
yo
fd
eto
xific
atio
n=
pe
rce
nt
inh
ibiti
on
inu
ntr
ea
ted
—p
erc
en
tin
hib
itio
nin
tre
ate
dsa
mp
les
at
the
toxi
ca
nts
co
nc
en
tra
tion
s.
666
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Tab
le3:
Eva
lua
tion
of
the
toxi
city
of
pe
stic
ide
s∗o
fth
ew
ate
rsa
mp
les
be
fore
an
da
fte
rtr
ea
tme
nt
with
the
tole
ran
tiso
late
imm
ob
ilize
din
the
alg
ina
teg
el.
Co
nce
ntra
tion
ofp
est
icid
es
0.5×
1×∗∗
2×N
oto
xic
ant
s(−
vec
ont
rol)
Exp
erim
ent
al
Ave
rag
e%
Ave
rag
e%
Ave
rag
e%
Ave
rag
ec
ond
itio
nsle
ngth
(cm
)In
hib
itio
n∗∗∗
leng
th(c
m)
Inhi
biti
on
leng
th(c
m)
Inhi
biti
on
leng
th(c
m)
Un
tre
ate
dm
od
elw
ate
rc
on
tain
ing
pe
stic
ide
sm
ixtu
re(+
vec
on
tro
l)
0.8
±0.
0787
.50.
6±
0.04
90.6
No
gro
wth
100
6.4
±0.
14
Mo
de
lwa
ter
tre
ate
dw
ithim
mo
bili
zed
ce
llso
fP
seu
do
mo
na
sflu
ore
sce
ns
isola
te
5.8
±0.
149.
45.
2±
0.07
18.7
4.6
±0.
1434
.46.
4±
0.07
Effic
ien
cy
of
me
tal
de
toxi
fica
tion
inim
mo
bili
zed
ce
llsy
ste
m∗∗
∗∗
78.1
%71
.9%
65.6
%—
Va
lue
sa
rem
ea
ns
±SD
.∗ P
est
icid
es:
am
ixtu
reo
fBH
C+
2,4-
D+
ma
nc
oze
b.
∗∗1×
Pe
stic
ide
s:
BHC
(500
pp
b)
+2,
4-D
(78
pp
b)
+m
an
co
zeb
(312
pp
b).
∗∗∗ I
nh
ibiti
on
with
resp
ec
tto
co
ntr
oli
.e.,
with
ou
tto
xic
an
ts.
∗∗∗∗
Effic
ien
cy
of
de
toxi
fica
tion
=p
erc
en
tin
hib
itio
nin
un
tre
ate
d—
pe
rce
nt
inh
ibiti
on
intr
ea
ted
sam
ple
sa
tth
esa
me
toxi
ca
nts
co
nc
en
tra
tion
s.
667
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Tab
le4:
Eva
lua
tion
of
the
toxi
city
of
ph
en
ols∗
of
the
wa
ter
sam
ple
sb
efo
rea
nd
aft
er
tre
atm
en
tw
ithth
eto
lera
nt
isola
teim
mo
bili
zed
inth
ea
lgin
ate
ge
l.
Co
nc
en
tra
tion
of
ph
en
ols
0.5×
1×∗∗
2×N
oto
xic
ant
s(−
vec
ont
rol)
Exp
erim
ent
al
Ave
rag
e%
Ave
rag
e%
Ave
rag
e%
Ave
rag
ec
ond
itio
nsle
ngth
(cm
)In
hib
itio
n∗∗∗
leng
th(c
m)
Inhi
biti
on
leng
th(c
m)
Inhi
biti
on
leng
th(c
m)
Un
tre
ate
dm
od
elw
ate
rc
on
tain
ing
ph
en
ols
(+ve
co
ntr
ol)
0.8
±0.
0787
.50.
5±
0.04
92.2
No
gro
wth
100
6.4
±0.
28
Mo
de
lwa
ter
tre
ate
dw
ithim
mo
bili
zed
ce
llso
fP
seu
do
mo
na
sflu
ore
sce
ns
isola
te
5.9
±0.
289.
45.
1±
0.14
19.0
4.2
±0.
1433
.36.
4±
0.14
Effic
ien
cy
of
me
tal
de
toxi
fica
tion
inim
mo
bili
zed
ce
llsy
ste
m∗∗
∗∗
81.1
%73
.2%
66.7
%—
Va
lue
sa
rem
ea
ns
±SD
.∗ P
he
no
ls:a
mix
ture
of
ca
tec
ho
l+c
reso
l+p
he
no
l+re
sorc
ino
l.∗∗
1×p
he
no
ls:c
ath
ec
ho
l(11
0p
pm
)+
cre
sol(
108
pp
m)
+p
he
no
l(94
pp
m)
+re
sorc
ino
l(11
0).
∗∗∗ I
nh
ibiti
on
with
resp
ec
tto
co
ntr
oli
.e.,
with
ou
tto
xic
an
ts.
∗∗∗∗
Effic
ien
cy
of
de
toxi
fica
tion
=p
erc
en
tin
hib
itio
nin
un
tre
ate
d—
pe
rce
nt
inh
ibiti
on
intr
ea
ted
sam
ple
sa
tth
esa
me
toxi
ca
nts
co
nc
en
tra
tion
s.
668
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Tab
le5:
Eva
lua
tion
of
the
toxi
city
of
the
ma
jor
toxi
ca
nts
∗in
co
mb
ina
tion
be
fore
an
da
fte
rtr
ea
tme
nt
with
the
tole
ran
tiso
late
WM
1im
mo
bili
zed
inth
ea
lgin
ate
ge
l.
Co
nce
ntra
tion
oft
oxi
ca
nts
0.5×
1×∗∗
2×Ex
pe
rime
nta
lA
vera
ge
%A
vera
ge
%A
vera
ge
%
No
toxi
ca
nts
(−ve
co
ntro
l)A
vera
ge
co
nditi
ons
leng
th(c
m)
Inhi
biti
on∗∗
∗le
ngth
(cm
)In
hib
itio
nle
ngth
(cm
)In
hib
itio
nle
ngth
(cm
)
Un
tre
ate
dm
od
elw
ate
rc
on
tain
ing
he
avy
me
tals
mix
ture
(+ve
co
ntr
ol)
0.5
±0.
0792
.00.
2±
0.07
96.0
No
gro
wth
100
6.4
±0.
28
Mo
de
lwa
ter
tre
ate
dw
ithim
mo
bili
zed
ce
llso
fP
seu
do
mo
na
sflu
ore
sce
ns
isola
te
5.4
±0.
2815
.64.
0±
0.28
37.5
2.6
±0.
4259
.46.
4±
0.14
Effic
ien
cy
of
me
tal
de
toxi
fica
tion
inim
mo
bili
zed
ce
llsy
ste
m∗∗
∗∗
76.4
%58
.5%
40.6
%—
Va
lue
sa
rem
ea
ns
±SD
.∗ T
oxi
ca
nts
:a
mix
ture
of
he
avy
me
tals
+p
est
icid
es
+p
he
no
ls.∗∗
Inh
ibiti
on
with
resp
ec
tto
co
ntr
oli
.e.,
with
ou
tto
xic
an
ts.
∗∗∗ P
erc
en
td
eto
xific
atio
n=
pe
rce
nt
inh
ibiti
on
inu
ntr
ea
ted
—p
erc
en
tin
hib
itio
nin
tre
ate
dsa
mp
les
at
the
toxi
ca
nts
co
nc
en
tra
tion
s.
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670 Khan and Ahmad
respectively. The atomic absorption spectrophotometer analysis on the removalof heavy metals from the water by means of immobilized Pseudomonas fluo-rescens system within a short exposure of 24 hours (Table 6), was also suggestiveof the fact that this strain has a remarkable potential for the bioremediationpurpose in view of the 75.9% removal of cadmium, 74.2% removal of chromiumV1 and 61.0% removal of lead under our experimental conditions.
The Allium cepa test is a valid indicator of toxicity in the water system.[22,36]
It is clear from Tables 2–5 that the immobilized cell system was quite suitablefor the detoxification of major water pollutants of India. This is in view of theremarkably high detoxification efficiency obtained during a short span, of 24-hour treatment, up to the extent of 50.8% for heavy metals, 65.6% for pesticides,66.7% for phenols and 40.6% for combination of all these toxicants at the con-centrations doubled (2×) to that supposed to be present in the highly pollutedregion of India.
It is interesting to note that our isolate, Pseudomonas fluorescens WM1was not only capable of bioremediating the cationic species of heavy metalsviz. Cd2+ or Pb2+, it could also efficiently remove the anionic species of hex-avalent chromium in the form of chromate (Table 6). Hexavalent chromiumis enormously toxic species of heavy metals.[37,38] Moreover, bioremediationand detoxification of cationic metal species simply involve the biosorptionmechanism,[39,40] such a mechanism would not operate in case of chromaterather would require either the internalization of CrO4
2+ exploiting theSO4
2+ influx machinery[41] or any other mechanism to reduce the hexavalentchromium to trivalent species.[42] It is, therefore evident from our data thatthe test isolate is equipped with at least two systems of metal bioremedia-tion. Similarly, the detoxification of organic pollutants of diverse nature (viz.
Table 6: Efficiency of bioremediation of heavy metals by means of immobilizedcells of Pseudomonas fluorescens.
Concentration of individual heavymetals (ppm)
S. no Experimental conditions Cd2+ Cr6+ Pb2+
Untreated contaminated1 water containing the heavy 701 ± 9.7 9.8 ± 0.16 187 ± 3.8
metals as determined byAAS∗Concentration of heavy
2 metals in the water treated 169 ± 5.6 2.5 ± 0.35 72 ± 4.2with immobilized cells asdetermined by AAS∗
3 Percent bioremediation∗∗ 75.9 74.2 61.0
Values are the means of three experiments ± SD.∗AAS—Atomic absorption spectrophotometer.∗∗Percent bioremediation= Toxicant concentration before treatment − concentration after treatment
Concentration before treatment × 100.
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Pseudomonas Use Against Indian Water Pollutants 671
BHC, 2,4-D, mancozeb and four types of phenols) would also essentially re-quire multiple enzymatic machinery.[43] In view of the complexity of resistancemechanisms, a switch over from favorable environment to an unfavorable onewould be reflected by a time lag. Such a lag is also suggestive of the fact of theinducible nature of some of the enzymatic mechanisms.[44]
In view of the prevailing conditions of water pollution in India, there is aneed for developing a simple, efficient and effective system to cope with themenace of environmental pollution. Our work is a preliminary step in directionof removing pollutants from the drinking water by means of a widely prevalent,non-pathogenic and fast-growing microbe.
CONCLUSION
This study shows that the test Pseudomonas fluorescens isolate can toleratenot only the heavy metals, pesticides or phenols alone but also the combinationof these toxicants. The most important point to note is that, Pseudomonas flu-orescens WM1 strain immobilized in calcium alginate beads could be a bettersystem not only for bioremediation of heavy metals, pesticides and phenoliccompounds but would also be equally efficient for their detoxification. Indeedfurther studies have to be performed to elucidate the mechanisms of bioreme-diation and detoxification processes operating in this system.
ACKNOWLEDGMENTS
The authors are thankful to the university authorities for providing the essen-tial facilities for carrying out this research.
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