Isolation of sodium dodecyl sulfate degrading strains from a ...

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Isolation of sodium dodecyl sulfate degrading strains from a ...

Journal of Cell and Molecular Biology 8(2): 103-111, 2010 Research Article

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

Isolation of sodium dodecyl sulfate degrading strains from a detergent

polluted pond situated in Varanasi city, India

Venkatesh CHATURVEDI * and Ashok KUMAR

School of Biotechnology, Banaras Hindu University, Varanasi, India.

(*author for correspondence; venkybt123@gmail.com)

Received: 08 September 2010; Accepted: 21 December 2010

Abstract

Sodium Dodecyl Sulfate (SDS) is one of the most widely used anionic detergent in households and in industry.

After use it is discharged in large amounts in water systems like ponds and rivers etc. It is now well established

that SDS is toxic to the health and survival of aquatic organisms. So, in the present study, we have made an

attempt to isolate and identify SDS degrading bacteria from detergent contaminated ponds situated in Varanasi

city, India. Using enrichment technique in minimal medium containing SDS as a sole carbon source, initially

six SDS degrading isolates where recovered. However, only two isolates where found to be efficient degraders

of SDS. These isolates where identified as P. alcaligenes and P. mendocina respectively. These isolates can be

exploited for bioremediation of this detergent from water systems.

Keywords: Biodegradation, SDS, Pseudomonas, zymography, alkyl sulfatase.

Sodyum dodesil sülfat yıkan suşların Varanasi şehri, Hindistan’da deterjan kirliliği olan göletten

izolasyonu

Özet

Sodyum dodesil sülfat (SDS) ev ve endüstride en yaygın kullanılan deterjanlardan bir tanesidir. Kullanımından

sonra büyük miktarlarda gölet ve nehirler gibi su sistemlerine atılmaktadır.. SDS’nin su oganizmalarının

sağlığına ve yaşamasına toksik olduğu iyice ortaya çıkmıştır. Bu sebeple, bu çalışmada, Hindistan’ın Veranasi

şehrinde bulunan deterjan ile kontamine olmuş göletlerden SDS yıkan bakterileri tespit ve izole etmek için bir

girişim yaptık. Karbon kaynağı olarak sadece SDS içeren minimal ortamda zenginleştirme tekniği kullanarak

ilk olarak altı SDS yıkan izolatlar bulundu. Ancak, sadece iki izolatın SDS’yi verimli olarak yıktığı saptandı.

Bu izolatlar sırası ile P. alcaligenes ve P. mendocina olarak tespit edildi. Bu izolatlardan su sistemlerinden bu

deterjanın biyoremediasyonu için faydalanılabilinir.

Anahtar sözcükler: Biyoyıkım, SDS, Pseudomonas, zimografi, alkil sulfataz


104 Venkatesh CHATURVEDI and Ashok KUMAR

Introduction

In India, ponds are often situated in vicinity of

temples and are used for bathing purposes for people

who visit the temples for worshipping. Some ponds

are also used for washing of clothes by local washer

men. Hence, these ponds are contaminated with high

amounts of detergents. Varanasi is an ancient holy

city, situated near the banks of river Ganges. This

city is often named as city of temples. In this city,

daily a number of visitors come for worshipping

purposes. In the vicinity of each temple, a pond is

situated which is meant for bathing purpose for

visitors. So, as a result of extensive use, the ponds

have become contaminated with detergents.

SDS is widely used anionic detergent in

household products such as, toothpaste’s, shampoos,

shaving foams, bubble baths, and cosmetics (Dhuiib

et al., 2003). In industry it is used as leather

softening agent, wool cleaning agent, in paper

industry as penetrant, flocculating agent, de-inking

agent, in building construction as additive of

concrete, oil well fire fighting, fire fighting devices,

engine degreasers, floor cleaners, and car wash

soaps etc. SDS can enhance absorption of chemicals

through skin, gastrointestinal mucosa, and other

mucous membranes. Importantly it is also used in

trans- epidermal, nasal and ocular drug delivery

systems, to enhance the intestinal absorption of

poorly absorbed drugs and it is also now widely used

in biochemical research involving electrophoresis

(Karsa, 1992).

Occurrence of SDS in environment arises mainly

from its presence in complex domestic and

industrial effluents as well as its release directly

from some applications (e.g., oil dispersants and

pesticides) (Fendinger et al., 1994). It has been

reported that SDS is toxic and affects survival of

aquatic animals such as fishes, microbes like yeasts

and bacteria (Singer and Tjeerdema, 1992; Martinez

and Munoz, 2007; Sandbacka et al., 2000). It is also

toxic to mammals like mice and humans but to a

lesser extent. It was felt that bioremediation of this

detergent is a necessity. So, screening was done for

bacteria capable of degrading this detergent and

utilizing it as a carbon source. Members of the

family Pseudomonas were found capable of

degrading SDS and utilizing it as a carbon source

(Harada, 1964). The pathway of SDS

biodegradation is also well documented (Thomas

and White, 1989). In the present study, we have

made an attempt to isolate and identify SDS

degrading bacteria from some ponds situated in

Varanasi city India. We have selected two ponds,

which were used by washer men and local

inhabitants for washing and bathing purposes. SDS

degrading bacteria were isolated from pond water

from two selected ponds using enrichment technique

in PBM containing SDS as a sole carbon source. A

total of six SDS degrading bacteria were isolated

from two ponds. Initial characterization of these

isolates was done using standard biochemical tests.

SDS biodegradation by these isolates was tested in

liquid PBM supplemented with SDS as a source of

carbon. Time course study of SDS biodegradation

was studied by monitoring disappearance of SDS

with time. Specific activity of alkyl sulfatase, the

key enzyme involved in the pathway was estimated

in all the isolates. Further multiplicity alkyl

sulfatases were studied by NATIVE PAGE

zymography (Payne et al., 1974).

Materials and methods

Chemicals and culture media

Sodium dodecyl sulfate (98.5% purity) was

purchased from Sigma- Aldrich, Inc, st. Louis, MO,

USA. Other chemicals used in this study were of the

highest grade available. The Phosphate Buffered

Medium (PBM) contained (g/L): K 2HPO 4 1.0,

KH 2PO 4 1.0, NH 4Cl 1.0, MgSO 4.7H 2O 0.20, NaCl

0.5, CaCl 2 0.02, pH 7.5. The medium also contained

trace elements (1 ml of stock) having (g/L):

FeCl 3·6H 2O 0.24, CoCl 2·6H 2O 0.04, CuSO 4·5H 2O

0.06, MnCl 2·4H 2O 0.03, ZnSO 4·7H 2O 0.31,

Na 2MoO 4·2H 2O 0.03. After autoclaving, SDS

(1g/L) was added as the sole carbon source.

Luria-Bertani (LB) medium contained (g/L):

tryptone 10.0, NaCl 5.0, yeast extracts 5.0, pH 7.0.

Isolation of SDS degrading bacteria

Water samples (20 ml) from four corners of a pond


were collected in sterilized screw-capped culture

tubes. Water of this pond was heavily used for

house hold purposes including cleaning and washing

of clothes. For the isolation and enrichment of SDS

degrading bacteria, 0.1 ml of pond water was added

to 100 ml sterilized PBM supplemented with SDS (1

g/L) in a culture flask and incubated at 37 °C in a

shaker (150 rpm). After 3-4 days of growth, the

culture (1 ml) was transferred to fresh PBM

supplemented with SDS. Repeated sub-culturing (at

least 3-4 times) resulted in the enrichment of

putative SDS degrading bacteria. Spreading on solid

agar-agar PBM resulted in the formation of minute

colonies and based on colony morphology different

SDS degrading bacteria were isolated.

Kinetics of SDS biodegradation

For the study of kinetics of SDS degradation, all the

test isolates were grown overnight in LB medium

.The cultures were centrifuged at 8000 rpm for 5 min

at room temperature, washed with PBM and then

OD at 600 nm of each isolate was adjusted to

approximately 10.0 with PBM, 0.1 ml of the culture

was used to inoculate 100 ml of PBM containing

SDS (1g/L). The cultures were incubated at 30°C

with shaking at 120 rpm. At regular intervals,

samples were removed and assayed for residual SDS

by stains all method as described above. Culture OD

was also recorded at indicated time interval.

Concentration of SDS was measured by stains All

method (Rusconi et al., 2001)

Preparation of crude cell extracts

Preparation of crude cell extract was done according

to the method of Ellis et al. (2002). Bacterial cells

were grown in PBM containing SDS (1g/L) as a sole

carbon source. Cells were harvested in mid

logarithmic phase by centrifugation at 10,000 rpm

for 20 min at 4°C in a Sorvall RC-5B superspeed

refrigerated centrifuge (Sorvall Instruments,

DuPont, Wilmington, DE, USA). Cell pellets were

washed twice and resuspended in 10 mM Tris-HCl

(pH 7.5). The cells were ruptured by sonication for a

total duration of 3 min, consisting of intermittent

sonication for 30 s on and 30 s off, operated at level

Isolation of sodium dodecyl sulfate degrading strains 105

5 and a 25% duty cycle in a Branson Sonifier 450

(Branson Ultrasonics Corp., Danbury, CT, USA).

To minimize heat inactivation of enzymes, samples

were sonicated in eppendorf tubes kept on ice. Cell

debris was removed by centrifugation at 12,000 rpm

for 10 min at 4°C. The cell extracts were stored at

-20°C prior to assay for alkyl sulfatase activity.

Alkyl sulfatase assay

Alkyl sulfatase activity in crude cell extracts was

estimated as per the method of Ellis et al. (2002).

Briefly, crude cell extract (10 to 50 μl containing

approximately 40 μg protein) was incubated at 30oC

in 1 ml 10 mM Tris-HCl (pH 7.5) containing SDS at

a final concentration of 50 μg /ml. Samples (100 μl)

were removed at fixed intervals and assayed for

residual SDS by using the MBAS method. One unit

of enzyme activity was defined as the amount of

enzyme which converted 1 μmol of SDS per minute

under the assay conditions.

NATIVE PAGE zymography for Alkyl sulfatase

With a view to determine the multiplicity of alkyl

sulfatase activity in cell extracts, native

polyacrylamide gel electrophoresis ( NATIVE

PAGE) for identification of alkyl sulfatase was

carried out as per the method of Payne et al. (1974).

Briefly, native PAGE was carried out with crude cell

extract (containing150 µg of protein) in 6%

polyacrylamide gel prepared in 0.378 M

Tris-glycine buffer (pH 8.3). The electrophoresis

was carried out at 100 V and 4ºC. Extruded gel was

incubated in developing solution containing 20 mM

SDS in 0.1 M Tris-Cl (pH 7.5) at 30ºC. Location of

active alkyl sulfatase was revealed by the formation

of white band of insoluble alcohol droplets. The

bands of alkyl sulfatases were designated on the

basis of their respective Rf values. Rf value was

calculated by measuring the ratio of distance

migrated by alkyl sulfatase band and distance

migrated by tracking dye in native polyacrylamide

gel.


106 Venkatesh CHATURVEDI and Ashok KUMAR

Preparation of PCR products for Sequencing and

Identification

16S rDNA (1.5 kb) was amplified by universal

primer (Schmalenberger et al., 2001) was purified

by Invitrogen kit( Invitrogen Corpn, CA, USA)

following the instructions of manufacturer. The

PCR product was eluted in Milli Q water and purity

and concentration of DNA was checked by reading

OD at 230, 260 and 280 nm. The sequencing PCR

reaction mix (DNA sequencing kit, Applied

Biosystes, Foster city, CA, USA) included 8.0 μL

Bigdye Terminator V3.0 cycle sequencing ready

reaction mixture with ApliTaq, 10ng PCR product

DNA, 3.2 pmole forward primer and deionised

water was added to achieve total volume of 20 μl.

The thermal cycle conditions were set as; initial

denaturation at 98oC for 1 min, 25 cycles of

denaturation at 96oC for 10 s, annealing at 55oC for

5s and extension at 60oC for 4 in and hold at 4oC

until the purification step. Ethanol precipitation was

done for the removal of unused BigDye Terminator

present in the reaction mixture. After drying the

samples, 20 μl of TSR (template suppression

reagent) was added, mixed well and heated for 2 min

at 95oC. Subsequently, the samples were chilled on

ice and after vortexing thoroughly, centrifuged

briefly in a microcentrifuge. Samples were hold in

ice until loading. Sequencing was performed in an

ABI-PRISM 310 Genetic Analyzer (Applied

Biosystems). PCR and direct sequencing were

performed at least twice to determine and confirm

the DNA sequences for each isolate. All the

sequences were matched against nucleotide

sequences present in Gen Bank using BLASTn

program (Altschul et al., 1990).

Results

Alkyl sulfatase assay

SDS degrading bacteria were isolated from two

ponds situated in Jawahar Nagar and Pisach Mochan

in Varanasi City, India. These ponds were selected

because they were used extensively by local people

and washer men for bathing purposes and for

washing of clothes. The pond water was analyzed

for presence of anionic detergents. The pond water

was collected and concentration of anionic detergent

was measured using MBAS method (Hayashi,

1975). The pond water from the two Jawahar Nagar

and Pisach Mochan showed high amount of anionic

detergent i.e.0.07 and 0.074 mg/L respectively.

Using enrichment technique, SDS degrading

bacteria were isolated from these pond water

samples. Briefly, 1 mL of pond water from each

pond was inoculated in 100 mL PBM containing

SDS (1g/L) as a sole carbon source. The flasks were

incubated in a shaker set at 100 rpm and at 37oC.

After 2-3 days, when prominent growth was

observed. 1ml culture was transferred to a fresh

flask containing 100ml PBM containing SDS. This

process was repeated thrice. Finally, the culture was

serially diluted in fresh PBM and 100 µL was spread

on PBM agar plates containing SDS (1g/L). The

plates were kept in a BOD incubator set at 37oC for

2-3 days until colonies appeared. Isolates were

selected on the basis of having distinct morphology,

color, colony shape, appearance, pigment

production. The isolates were streaked to purity on

PBM agar plates containing SDS. These isolates

were maintained on these plates. A total of 6

different types on isolates were recovered from 2

ponds. These isolates recovered from Jawahar Nagar

pond was designated as JN1, JN2 and JN3

respectively. The isolates recovered from Pisach

Mochan pond was designated as PM1, PM2 and

PM3 respectively. These isolates were different

from each other on the basis of colony morphology,

appearance, color, pigment production etc. These

isolates where maintained on PBM agar plates

containing SDS (1g/L) as a sole carbon source.

Study of SDS degradation

Degradation potential of SDS by these isolates was

studied by there growth in PBM containing SDS as

a sole carbon source. Each isolate was grown for

overnight in LB medium. After proper growth, the

cells where harvested by centrifugation. The cells

where washed twice and resuspended in PBM.

These cells where inoculated in 250 ml flasks

containing100 ml PBM supplemented with SDS

(1g/L).The initial absorbance OD 600nm of each


isolate was set close to 0.025. After fixed interval of

time, the concentration of residual SDS was

estimated by stains all method and cell growth was

monitored by taking absorbance at 600 nm. All the

isolates where found to degrade and metabolize SDS

as a carbon source. However, the rates of

degradation of SDS were different. Only two

isolates namely, JN1 and PM2 were found to be

efficient degraders of SDS. Other isolates showed

slow and incomplete degradation of SDS. When

Isolation of sodium dodecyl sulfate degrading strains 107

strain JN1 was grown in PBM in presence of SDS

(1g/L) complete degradation of SDS was observed

in 20h with concomitant increase in the culture OD

indicating utilization of this detergent as a carbon

source. Similarly when strain PM2 was grown in

similar conditions, complete degradation of SDS

was observed in about 16h (Figure 1). Strain PM2

was observed to be a better degrader of SDS as

compared to strain JN1.

Figure 1. Time course study of SDS biodegradation. (A) JN1, (B) PM2.


108 Venkatesh CHATURVEDI and Ashok KUMAR

Table 1. Alkyl sulfatase activity ,specific activity and Relative migration (Rf value) in SDS degrading strains.

Alkyl sulfatase specific activity

Strain Alkyl sulfatase activity (U/ml) Rf value

(U/ml/mg protein)

JN1

PM2

Alkyl sulfatase activity

The alkyl sulfatase activity in crude cell extracts was

monitored, when these isolates where in exponential

phase of growth with SDS. As evident from Table 1,

strain JN1 showed alkyl sulfatase activity i.e. 2.05

U/ml and specific activity of 0.103 U/ml/mg protein.

This was low as compared to strain PM2 which

showed an enzyme activity of 2.4 U/ml and specific

activity of 0.124 U/ml/mg protein as compared to

strain PM2 (Table 1). The results were in

accordance with the kinetics of SDS degradation.

Where strain JN1 showed lower rates of SDS

degradation, as compared to strain PM2.

Native PAGE Zymography

Native PAGE Zymography of alkyl sulfatase in

crude cell extracts was performed. Briefly, the cells

where grown in PBM supplemented with SDS

(1g/L). When the cells were in exponential phase of

growth, the cells were harvested and analyzed for

the presence of alkyl sulfatase by Native PAGE

Zymography. It was observed that both the strains

harbored different types of alkyl sulfatases as

A B

2.04

Figure 2. Native PAGE Zymography of alkyl

sulfatase. (A) JN1, (B) PM2.

2.4

0.103

0.124

observed by calculating the migration of enzyme

when compared to the migration of dye front (Figure

2). For each enzyme the Rf value was calculated by

dividing the distance migrated by the enzyme by the

distance migrated by the dye. Strain JN1 showed a

Rf value of 0.57 and strain PM2 showed Rf value of

0.36 (Table 1). It was clearly evident that these two

strains harbored alkyl sulfatase of different

multiplicity.

Identification of isolates

The two strains where identified by 16S rDNA

sequencing, which is a widely used method for the

identification of isolates. The 1.5 kb 16S rDNA gene

was PCR amplified using universal primers and was

sequenced by forward primer. The sequence was

matched with NCBI database. The sequence of JN1

showed 99% similarity to Pseudomonas alcaligenes

and strain PM2 showed 98% similarity of

Pseudomonas mendocina. So, these strains where

designated as P. alcaligenes strain JN1 (Accession

no. HM756168) and P. mendocina strain PM2

(Accession no. HM627521) respectively. This is the

first report of isolation of SDS degrading bacteria

belonging to P. alcaligenes and P. mendocina.

Discussion

Due to their amphiphilic properties, long-chain

aliphatic sulfate esters such as sodium dodecyl

sulfate (SDS) are widely used as components of

surfactant formula¬tions and are consequently

discharged into wastewa¬ter. It is realized that rapid

removal of surfactants from the environ¬ment will

make their application safer and widespread (Zeng

et al., 2007). In the present study, we have made an

attempt to isolate SDS degrading bacteria from

detergent polluted pond situated in Varanasi city,

India. These ponds where used extensively for

washing of clothes and also for bathing purposes. In

0.57

0.36


this study, we have screened two ponds situated in

different parts of Varanasi city. Amount of anionic

detergent present in pond water was evaluated by

MBAS method. It is well established method for

quantitative estimation of total anionic detergent

from water samples. As expected, the amount of

anionic detergent in pond water was at higher level

in all the ponds. This was due to extensive use of

detergents and also due to the fact that ponds

constitute a closed system where there is point of

entry of pollutants but there is no exit. So, these

chemicals tend to accumulate with time. Also,

seasonal factors like rainfall and drought drastically

effect the concentration of chemicals in the pond

water. This system is very much different from

rivers, which is an open system. The chemicals are

entering but eventually they get distributed with

flowing water. Direct measurements of sewage

treatment plant influents, indicated that

concentrations of anionic surfactants in waste water

are typically in the1-10 p.p.m. range (Fendinger et

al., 1994). Although waste water treatment plants

reduce this concentration markedly (typically to the

1-10 p.p.b. range), direct discharge of untreated

waste water to rivers, and direct entry of surfactants

to ground water, e.g. via agrochemical sprays, can

lead to significant local concentrations in surface

and groundwater. Similar concentrations have been

detected in the UK, i.e. 4-24 p.p.m. in raw sewage

and 0-14.1 p.p.m. in river waters. Levels of 1-25 mg

surfactant per kg of sediment have also been

recorded (Marchesi et al., 1994). However, there is

no data available regarding concentration of anionic

detergents in pond water. Using enrichment

technique in minimal medium, we were able to

isolate 6 SDS degrading bacteria from two detergent

contaminated ponds. In every pond, there was

presence of SDS degrading bacteria indicating that

this detergent is easily biodegradable. The kinetics

of degradation of SDS by these isolates was studied

by monitoring disappearance of SDS with time and

also by measuring the growth of the isolate. It was

observed that these isolates showed varying rates of

SDS biodegradation. Only two isolates namely JN1

and PM2 showed appreciable level of

biodegradation. In strain PM2 where the rate of

Isolation of sodium dodecyl sulfate degrading strains 109

biodegradation was higher, took 16 h to completely

degrade SDS and in other where the rate was lower

took 20 h to completely degrade SDS. Using

microorganisms to degrade surfactants is one

promising method (Zeng et al., 2007). A range of

bacterial strains able to degrade aliphatic sulfate

esters have been isolated from contaminated sources

such as sewage sludge on the basis of their ability to

utilize aliphatic sulfate esters as carbon sources for

growth. Abboud et al. (2007) have isolated two

facultative anaerobes Acinetobacter calcoaceticus

and Pantoea agglomerans from wastewater. It was

observed that mixed consortia of these isolates could

degrade 4000 ppm SDS within 120 h of incubation.

Hosseini, et al. (2007) have isolated two isolates

belonging to Pseudomonas betelli and

Acinetobacter johnsoni. The Acinetobacter johnsoni

was able to decrease SDS level in the growth media

from an original of 522 mg/L to the extent of 93.6%

within 5 days; whereas, the Pseudomonas betelli did

so to the extent of 84.6%. However, following 10

days of incubation, Pseudomonas betelli showed

greater degradation, (97.2%) potential relative to the

Acinetobacter johnsoni, (96.4%). The highest peak

of SDS degradation occurred during the logarithmic

phase of bacterial growth. Shukor et al. (2009) have

isolated a strain belonging to Klebsiella oxytoca

from SDS polluted water samples from Malaysia.

This isolate was able to degrade approximately 80%

of 2.0 g/l SDS after 4 days of incubation and 100%

SDS in 10 days of inoculation concomitant with

increase in cellular growth. Here, our isolates

appears to be more efficient than theses isolates

since the rate of degradation of SDS is more in our

isolates. In most cases, degradation of alkyl sulfate

esters was found to be initiated by alkyl sulfatase

enzymes that catalyze hydrolytic cleavage of the

ester bond to liberate inorganic sulfate. The resulting

parent alcohol is further degraded (Dodgson et al.,

1982) or incorporated into cellular lipids (Thomas

and White, 1989). The genus Pseudomonas is

ubiquitous and well known for versatility in the

biodegradation of industrial compounds. We have

also estimated alkyl sulfatase activity in these

isolates, when cells where present in exponential

phase of growth with SDS as a carbon source. The


110 Venkatesh CHATURVEDI and Ashok KUMAR

highest alkyl sulfatase activity was observed in

strain PM2 and in strain JN2 lower activity was

observed, this was in accordance with its SDS

degrading ability. Also the enzyme alkyl sulfatase

was also different in two strains as evidenced by

different migration of these enzymes on

polyacrylamide gels. The bacteria involved in alkyl

sulfate ester metabolism are often capable of

producing a multiplicity of alkyl sulphatases.

Pseudomonas C12B can produce five such enzymes

(Dodgson et al., 1974), Pseudomonas putida FLA

six (Lillis et al., 1983) and Pseudomonas DESI four

(Hales, 1981). These isolates where identified based

on 16S rDNA sequencing. These isolates where

identified as P. alcaligenes strain JN1 (Accession

no. HM756168) and P. mendocina strain PM2

(Accession no. HM627521) respectively. This is the

first report of isolation of these bacteria capable of

degrading SDS. Other workers have reported the

role of P. aeruginosa, P. putida in SDS

biodegradation. The alkyl sulfatases present in these

isolates appear to be novel. So, a detailed study

regarding these enzymes is required to ascertain the

role of these isolates in SDS degradation.

In conclusion, it appears that these two strains are

better adapted for rapid removal of SDS as

compared to other strains isolated by different

workers. So, these strains can be efficiently utilized

for removal of detergents from detergent

contaminated water systems.

Acknowledgements

V.C. is grateful to Council for Scientific and

Industrial Research (CSIR), New Delhi, India for

the award of Junior Research Fellowship. This study

was partly supported by a research grant sanctioned

to A.K. by the Department of Biotechnology, Govt.

of India, New Delhi (BT/PR1239/AGR./02/065/98).

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