Pentachlorophenol Degradation by Janibacter sp., a New Actinobacterium Isolated from Saline Sediment...

Research ArticlePentachlorophenol Degradation by Janibacter sp a NewActinobacterium Isolated from Saline Sediment of Arid Land

Amel Khessairi12 Imene Fhoula1 Atef Jaouani1 Yousra Turki2 Ameur Cherif3

Abdellatif Boudabous1 Abdennaceur Hassen2 and Hadda Ouzari1

1 Universite Tunis El Manar Faculte des Sciences de Tunis (FST) LR03ES03 Laboratoire de Microorganisme etBiomolecules Actives Campus Universitaire 2092 Tunis Tunisia

2 Laboratoire de Traitement et Recyclage des Eaux Centre des Recherches et Technologie des Eaux (CERTE)Technopole Borj-Cedria BP 273 8020 Soliman Tunisia

3 Universite de Manouba Institut Superieur de Biotechnologie de Sidi Thabet LR11ES31 Laboratoire de Biotechnologie etValorization des Bio-Geo Resources Biotechpole de Sidi Thabet 2020 Ariana Tunisia

Correspondence should be addressed to Hadda Ouzari imeneouzarifstrnutn

Received 1 May 2014 Accepted 17 August 2014 Published 17 September 2014

Academic Editor George Tsiamis

Copyright copy 2014 Amel Khessairi et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Many pentachlorophenol- (PCP-) contaminated environments are characterized by low or elevated temperatures acidic or alkalinepH and high salt concentrations PCP-degrading microorganisms adapted to grow and prosper in these environments play animportant role in the biological treatment of polluted extreme habitats A PCP-degrading bacteriumwas isolated and characterizedfrom arid and saline soil in southern Tunisia andwas enriched inmineral salts medium supplemented with PCP as source of carbonand energy Based on 16S rRNA coding gene sequence analysis the strain FAS23 was identified as Janibacter sp As revealed by highperformance liquid chromatography (HPLC) analysis FAS23 strain was found to be efficient for PCP removal in the presence of 1of glucoseThe conditions of growth and PCP removal by FAS23 strain were found to be optimal in neutral pH and at a temperatureof 30∘C Moreover this strain was found to be halotolerant at a range of 1ndash10 of NaCl and able to degrade PCP at a concentrationup to 300mgL while the addition of nonionic surfactant (Tween 80) enhanced the PCP removal capacity

1 Introduction

The polyorganochlorophenolic (POP) compounds have beenextensively used as wide spectrum biocides in industryand agriculture [1] The toxicity of these compounds tendsto increase according to their degree of chlorination [2]Among chlorinated phenols pentachlorophenol (PCP) hasbeen widely used as wood and leather preservative owingto its toxicity toward bacteria mould algae and fungi [3]However PCP is also toxic to all forms of life since it isan inhibitor of oxidative phosphorylation [4] The extensiveexposure to PCP could cause cancer acute pancreatitisimmunodeficiency and neurological disorders [5] Conse-quently this compound is listed among the priority pollutantsof the US Environmental Protection Agency [6] Moreover itis recalcitrant to degradation because of its stable aromaticring and high chloride contents thus persisting in the

environment [7] Although contamination of soils and waterswith chemically synthesized PCP is a serious environmentalproblem their remediation may be possible using physi-cal chemical and biological methods [8] Bioremediationrepresents a choice process thanks to its low costs andreduction of toxic residue generated in the environment Thebiodegradation of PCP has been studied in both aerobic andanaerobic systems Aerobic degradation of PCP especiallyhas been extensively studied and several bacterial isolateswere found to degrade and use PCP as a sole source ofcarbon and energyThe most studied aerobic PCP-degradingmicroorganisms included Mycobacterium chlorophenolicum[9] Alcaligenes sp [10] Rhodococcus chlorophenolicus [11]Flavobacterium [12] Novosphingobium lentum [13] and Sph-ingomonas chlorophenolica [14] Bacillus [15] Pseudomonas[16] and Acinetobacter [17] as well as some fungi speciesSaline and arid environments are found in a wide variety

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 296472 9 pageshttpdxdoiorg1011552014296472

2 BioMed Research International

of aquatic and terrestrial ecosystems A low taxonomicbiodiversity is observed in all these saline environments [18]most probably due to the high salt concentrations prevailingin these environmentsMoreover the biodegradation processis difficult to perform under saline conditions [19] Besidesthese metabolical and physiological features halophilic andhalotolerant microorganisms are known to play importantroles in transforming and degrading waste and organic pol-lutants in saline and arid environment [20] These microor-ganisms particularly actinobacteria are frequently isolatedfrom extreme environments such as Sabkha Chott andSahara which are known to have a great metabolic diversityand biotechnological potential The occurrence of actinobac-teria in saline environment and their tolerance to highsalt concentrations were thus described [21] However fewactinobacteria genera such as Arthrobacter [22] and Kocuria[23] were reported for PCP-degradation process The genusJanibacterwhich is recognized byMartin et al [24] belongs tothe family Intrasporangiaceae in the Actinomycetales orderand included five major species J limosus [24] J terrae[25] J melonis [26] J corallicola [27] and J anophelis [28]Interestingly most of these species were reported for theirability to degrade a large spectrum of aromatic andor chlori-nated compounds including polychlorinated biphenyls [29]monochlorinated dibenzo-p-dioxin [30] dibenzofuran [31]anthracene phenanthrene [32] dibenzo-p-dioxin carbazolediphenyl ether fluorene [33] and polycyclic aromatic hydro-carbons [34] However no data reporting PCP degradationby Janibacter members was described PCP and other POPcompounds shared many physical properties which limitedbiodegradation processes and one of these properties wastheir lower solubility and therefore low bioavailability to thedegrading bacteria Nevertheless the use of surfactants suchas Tween 80 has the potential to increase the biodegradationrates of hydrophobic organic compounds by increasing thetotal aqueous solubility of these pesticides [35] In this studywe evaluated for the first time the PCP removal potentialunder different physicochemical conditions by Janibacter spa halotolerant actinobacterium member isolated from aridand saline land in southern Tunisia

2 Materials and Methods

21 Chemicals and Solvents PCP (MW 26634 and gt99purity) and acetonitrile (HPLC grade) were purchased fromSigma Aldrich (USA) All other inorganic chemicals used toprepare the different media are commercially available withhighest purity and are used without further purification

22 Sample Collection and PCP-Degrading Bacterium Isola-tion The sediment samples were collected in March 2011from arid and saline ecosystems belonging to the site ldquoSebkhaEl Naouelrdquo with GPS coordinates N 34∘26101584095110158401015840 E 09∘54101584010210158401015840altitude 150 ft46m in southern Tunisia Bacterial isolationwas performed as described by Rosch et al [36] with somemodifications 10 g of soil sample was suspended in 100mL ofphosphate-buffered salt solution (137mMNaCl 27mMKCl10mMNa

2HPO4 and 2mMKH

2PO4) and stirred vigorously

for 30minThe soil suspensionwas diluted and 01mL sample

was spread on the surface of yeast extract-mannitol medium(YEM) YEM medium contained the following componentsat the specified concentrations (in gL) mannitol 5 yeastextract 05 MgSO

4sdot7H2O 02 NaCl 01 K

2HPO4 05 Na

gluconate 5 agar 15 pH = 6 8 After sterilization for 20minat 120∘C 1mL of 166 CaCl

2solution was added to 1 liter

of YEMmedium (1 1000) The plates were then incubated at30∘C for 7 days Pure cultures of the isolates were obtained bystreaking a single colony on the same medium

23 16S rRNA Gene Amplification and Sequence AnalysisFor DNA extraction the FAS23 strain was grown in trypticsoy broth (TSB) containing (in gL) casein peptone 17 soyapeptone 3 glucose 25 sodium chloride 5 dipotassiumhydrogen phosphate 4 DNA extraction was performedusing CTABNaCl method as described by Wilson [37] andmodified by using lysozyme (1mgmL) for cell wall digestionThe 16S rRNA gene was amplified using universal primersSD-Bact-0008-a-S-20 (51015840-AGA GTT TGA TCC TGG CTCAG-10158403) and S-D-Bact-1495-a-A-20 (51015840-CTA CGG CTA CCTTGT TACGA-10158403) [38] PCR was performed in a final volumeof 25 120583L containing 1 120583L of the template DNA 05 120583Mof eachprimer 05 120583M of deoxynucleotide triphosphate (dNTP)25 120583L 10X PCR buffer for Taq polymerase MgCl

215mM

1UI of Taq polymerase The amplification cycle was asfollows denaturation step at 94∘C for 3min followed by 35cycles (45 sec at 94∘C 1min at 55∘C and 2min at 72∘C) plusone additional cycle at 72∘C for 7min as a final elongationstep The 16S rDNA PCR amplicons were purified withExonuclease-I and Shrimp Alkaline Phosphatase (Exo-SapFermentas Life Sciences) following the manufacturerrsquos stan-dard protocol Sequence analyses of the purified DNAs wereperformed using a Big Dye Terminator cycle sequencing kitV31 (AppliedBiosystems) and anAppliedBiosystems 3130XLCapillary DNA Sequencer machine Sequence similaritieswere found by BLAST analysis [39] using the GenBank DNAdatabases (httpwwwncbinlmnihgov) and the Riboso-mal Database Project (RDP) Phylogenetic analyses of the 16SrRNA gene sequences were conducted withMolecular Evolu-tionary Genetics Analysis (MEGA) software version 5 [40]Trees were constructed by using neighbor-joining method[41]The sequence was deposited in GenBank database underthe accession number KC959984

24 Degradation of PCP by Isolated Strain The kinetics ofthe PCP removal under different conditions were conductedin 500mL flasks sealed with cotton stoppers containing100mL of mineral salt medium (MSM) adjusted to pH69 supplemented with 1 glucose and inoculated with1 of 106 CFUmL of the strain FAS23 The MSM con-tained the following components at the specified concen-trations (in gL) KH

2PO4 08 Na

2HP4 08 MgSO

4sdot7H2O

02 CaCl2sdot2H2O 001 NH

4Cl 05 plus 1mL of trace

metal solution which includes (in mgL) FeSO4sdot7H2O 5

ZnSO4sdot7H2O 4 MnSO

4sdot4H2O 02 NiClsdot6H

2O 01 H

3BO3

015 CoCl2sdot6H2O 05 ZnCl

2025 and EDTA 25 PCP

was added to the medium after autoclaving [19] Whennecessary solid MSM plates were prepared by adding 15 gLbacteriological grade agar The inoculum was prepared as

BioMed Research International 3

Janibacter melonis (JX865444) Janibacter marinus (AY533561) Janibacter terrae (KC469957) Janibacter hoylei (FR749912) Janibacter sanguinis (JX435047)

Janibacter sp FAS23 Janibacter sp ATCC 33790 (GU933618)

Janibacter limosus (KC469951)

Arthrobacter chlorophenolicus (EU102284)

9976

82100

58

5240

001

Janibacter corallicola (NR 041558)

Terrabacter tumescens (NR 044984)Terracoccus luteus (NR 026412)

Kocuria rhizophila (NR 026452)

Figure 1The phylogenetic position of Janibacter sp strain in relation to somemembers of actinobacteria (genus of Janibacter SphingomonasTerrabacter Terracoccus Kocuria and Arthrobacter) based on 16S rRNA gene Bootstrap values for a total of 1000 replicates are shown at thenodes of the tree The scale bar corresponds to 005 units of the number of base substitutions per site changes per nucleotide

follows overnight culture was centrifuged and the pellet wasrinsed twice with fresh MSM PCP removal was monitoredduring 144 h of incubation by varying different parameters(i) initial pH (ii) initial PCP concentrations 20 50 100200 and 300mgL corresponding to 0075mM 019mM037 075mM and 114mM respectively (iii) temperatureof incubation 25 30 and 37∘C (iv) NaCl concentrations10 gL 30 gL 60 gL and 100 gL (v) the addition of nonionicsurfactant Tween 80 (40mgL) Bacterial cell growth wasevaluated by measuring the optical density at 600 nm usingUV-VIS spectrophotometer (Spectro UVS-2700 Dual BeamLabomed Inc) every 24 h of the incubation Three controlswere used PCP-free MSM uninoculated PCP containingMSM and PCP containing MSM inoculated with heatedinactivated cells The cell suspension was centrifuged (5min8000 rpm) and the supernatant was filtered through 022 120583mfilters [16] Samples of 100 120583L were applied to C18 reversephase column (LiChrospher 100 RP-18 endcapped column250mm times 46mm id and particle size of 5 120583m) at a flowrate of 1mLminminus1 The retained molecules were eluted over35min using the following gradient 1 (vv) phosphoricacid in water for 4min followed by an increase to 100(vv) acetonitrile within 21min which was kept constant for5min and then decreased back to initial concentration andkept constant for another 5min PCP was quantified usingexternal standards method Percent removal was estimatedusing the following formula removal () = area minus areaarea[42]

25 Statistical Analysis Data were subjected to analysis ofvariance using SPSS software (version 140) and the meandifferences were compared by Student-Newman-Keuls com-parison test A 119875 value of less than 005 was consideredstatistically significant (test at119875 lt 005)Three replicates wereprepared for each treatment

3 Results

31 Isolation Identification of FAS23 Strain and 16S rDNASequence Based Phylogenetic Analysis The bacterial strainFAS23 was isolated from the saline and arid sediment

The morphological aspect of FAS23 strain culture on theisolation medium YEM showed opaque pale cream andconvex colonies with glistening surface Cells were Gram-positive rod-shaped and positive for catalase and oxidasetests No growth under anaerobic conditions and no sporeformation were recorded The optimal growth conditions ofFAS23 strain were pH of 70ndash85 and a temperature rangeof 28ndash30∘C The strain was able to grow at a range ofsalt concentrations from 1 to 100 gL of NaCl 16S rDNAsequencing and phylogenetic analysis allowed the assignmentof FAS23 strain to Janibacter sp (Figure 1)

32 The Optimum Growth Conditions of Janibacter sp StrainThe effect of physiological and biochemical variations (glu-cose supplement temperature pH PCP concentration andpresence of biosurfactant) on bacterial growth of Janibactersp FAS23 and PCP removal was studied

321 Effect of Glucose on the Growth of Janibacter sp and PCPRemoval The effect of glucose as cosubstrate on the growthof Janibacter sp strain and PCP removal was studied inMSMThe result showed that the growth of the strain was possibleonly after the addition of glucose (Figure 2(a)) As well thePCP was efficiently removed in the presence of glucose and7184 of PCP was degraded within 24 hours and more than90 after 72 h (Figure 2(b)) The obtained results indicatedthe phenomenon of cometabolism in which microorganismsdo not obtain energy from the transformation reaction theyrather require another substrate for growth [43]

322 Effect of pH and Temperature on the Growth of theStrain and PCP Removal The effect of pH variations (4069 and 90) on the growth and PCP removal was assessed(Figure 3) At both pH 40 and 90 a low rate of growth andPCP removal was observed after 24 and 48 h of incubationHowever after 144 h of incubation the rate of PCP removalhas reached values of 4480 and 7022 at pH 40 and pH90 respectivelyThe optimumgrowth and PCP removal werehowever observed at pH69 as we noted a significant removalof PCP of 7184 8447 and 9906 after 24 48 and 144 hof incubation respectivelyThe strain FAS23 was able to grow


Incubation period (h)

0002040608101214161820

0 24 48 72 96 120 144

Glucose

PCPPCP ndash Glucose

OD

at600

nm

(a)

0

20

40

60

80

100

0 24 48 72 96 120 144Incubation period (h)

Resid

ual P

CP (

)

PCPPCP + Glucose

(b)

Figure 2 The growth (a) and the PCP removal (b) in the presence and in deficiency of the supplementary carbon source (glucose 1) byJanibacter sp FAS23 Error bars represent the standard deviation

00

02

04

06

08

10

12

14

16

18

20

0 24 48 72 96 120 144Incubation periods (h)

pH 4pH 69-PCP

pH 69

pH 9

OD

at600

nm

pH 4-PCPpH 9-PCP

(a)

0102030405060708090

100

0 24 48 72 96 120 144


pH 4pH 69pH 9

Resid

ual P

CP (

)

(b)

Figure 3 (a) Growth of Janibacter sp at different pH of culture medium pH 40 pH 69 and pH 90 with 20mgL of PCP at 30∘C (b) Effectof different pH on the PCP removal efficiency by Janibacter sp Error bars represent the standard deviation

in the temperature range of 25ndash37∘C with an optimum at30∘C At 25 and 37∘C the growth of the bacterial strain aswell as PCP removal was affected (Figure 4) However at25∘C the strain showed a better growth and PCP removalcompared to temperature of 37∘C Likewise the PCP removalwas optimal at 30∘C reaching 7184 and 9906 after 24 hand 144 h of incubation respectively

323 Effect of PCP Amount on the Growth and PCP Removalby Janibacter Variation of PCP amount in the mediumshowed that the growth of the strain as well as PCP removaldecreased with the increase of PCP concentration (Figure 5)At low concentrations (20 and 50mgL) the bacterial strainwas able to remove the majority of PCP after 72 h of

incubation time Up to 100mgL 50 of PCP could beremoved after 72 h of incubation However with higherconcentrations (200 and 300mgL) equivalent level of PCPremoval could be reached if the incubation time is extended

324 Effect of Various NaCl Concentrations on the PCPRemoval The strain was tested for its ability to remove PCP(20mgL) at different NaCl concentrations (0 1 36 and 10) The best rate of growth and PCP removalwas recorded at 1 of NaCl (more than 92 after 144 hof incubation) The growth and thus the capacity of PCPremoval were inhibited when the concentration of sodiumchloride was increased (Figure 6) At 3 NaCl the PCPremoval was 72 after 144 h of incubation When the NaCl



OD

at600

nm

00

02

04

06

08

10

12

14

16

18

20

37∘C

30∘C

25∘C

37∘C-PCP

25∘C-PCP

30∘C-PCP

(a)

0

10

20

30

40

50

60

70

80

90

100


Resid

ual P

CP (

)37

∘C30

∘C25

∘C

(b)

Figure 4 (a) Growth of Janibacter sp at different temperatures in presence of 20mgL of PCP and at pH 69 (b) Effect of temperature changeson the PCP removal efficiency by Janibacter sp Error bars represent the standard deviation


02050

100200300

OD

at600

nm

00

02

04

06

08

10

12

14

16

18

20

(a)

0

10

20

30

40

50

60

70

80

90

100


2050100

200300

Resid

ual P

CP (

)

(b)

Figure 5 (a) Growth of Janibacter sp in the presence of 0 20 50 100 200 and 300mgL of PCP (b) Effect of different PCP concentrationson the PCP removal by Janibacter sp Error bars represent the standard deviation

concentration was increased to 6 and 10 PCP removalfalls to 4653 and 1762 respectively

325 Effect of Nonionic Surfactant Tween 80 on the Biodegra-dation of PCP In this study the nonionic surfactant Tween80was found to enhance the growth and PCP-biodegradationprocess (Figure 7) Interestingly removal of high amount

of PCP (300mgmL) was improved by 30 after 72 h ofincubation compared to the control (Figure 7(b))

4 Discussion

The strain FAS23 isolated from saline sediment collectedfrom Tunisian arid ecosystems was identified as an


0 24 48 72 96 120 144Incubation period (hours)

013

610

OD

at600

nm

0002040608101214161820

(a)

0102030405060708090

100


013

610

Resid

ual P

CP (

)

(b)

Figure 6 (a) Growth of Janibacter sp in MS medium supplemented with different concentrations of NaCl 0 1 3 6 and 10 in thepresence of 20mgL of PCP at 30∘C (b) Effect of NaCl on the PCP removal efficiency by Janibacter sp Error bars represent the standarddeviation


20-T8020

300-T80300

OD

at600

nm

0002040608101214161820

(a)

0102030405060708090

100


20-T8020

300-T80300

Resid

ual P

CP (

)

(b)

Figure 7 (a)Growth of Janibacter sp inMSmedium supplementedwith nonionic surfactant Tween 80 (40mgL) containing 20 and 300mgLof PCP (b) Effect of nonionic surfactant Tween 80 on the PCP removal efficiency by Janibacter sp Error bars represent the standard deviation

actinobacterium belonging to the genus Janibacter sp withrespect to morphological and biochemical tests and 16SrRNA gene sequence Despite their known high potential inrecalcitrant compounds biodegradation [29 33] bacteria ofthe genus Janibacter described in this study are reportedfor the first time for their ability to degrade PCP Inbiodegradation process glucose is commonly used as anadditional source of carbon and energy and is the mostmetabolizable sugar which supported a maximum growth[44] In this context our results are in agreement with thoseof Singh et al [45] and Singh et al [15] who reported theenhancement of bacterial growth and PCP-degradationprocess using MSM supplemented with 1 of glucose [43]This effect can be explained by the connection of the two

substrates metabolism In fact the NADH provided byglucose metabolism may increase the biomass and thusincrease the total activity for PCP metabolizing [46]

In the present study PCP removal is affected by pHvariation As it was reported by Premalatha and Rajakumar[43] Wolski et al [47] Barbeau et al [48] and Edgehill[22] for Arthrobacter and different Pseudomonas species theneutral pH was found to be optimal for PCP degradationHowever for other bacterial species such as Sphingomonaschlorophenolica PCP degradation was more important at pH92 [49]

Temperature is another important environmental factorthat can influence the rate of pollutants degradation [48]The optimal temperature for the PCP removal was recorded


at 30∘C but lower temperatures (25∘C) allowed significantremoval than the upper values These results were in accor-dance with those of Wittmann et al [9] and Crawfordand Mohn [50] Overall deviation in pH and tempera-ture from the optimum results in alteration of microbialgrowth and metabolism as well as the pollutants properties[51 52]

The effect of different concentrations of PCP on growth ofthe strain proved that the PCP removal was more efficient atlow concentrations (20mgL) This result was coherent withdata of Webb et al [53] reporting that all strains tested wereable to degrade up to 90 of the PCP when the concentrationwas 10mgL Moreover as it was revealed by Karn et al[16] the ability of PCP removal of Janibacter sp decreaseswhen PCP concentration was increased Furthermore wefound that the removal ability by Janibacter sp has reached40 after 144 h of incubation when PCP concentration of300mgL was used These results are in accordance withthose of Chandra et al [54] for Bacillus cereus strain andmay suggest that these bacteria may tolerate and remove highconcentrations of PCP if we increase the incubation time Onthe contrary Kao et al [55] reported that no PCP removal wasdetected with PCP concentrations of 320mgL even after 20days of incubation

As for bioremediation the strain should possess not onlythe high removal efficiency for the target compounds butalso the strong abilities of adapting some conditions suchas pH temperature and salinity fluctuations In this studyit was shown that Janibacter sp was able to remove PCPeven with salinity fluctuations (less than 10) These resultswere in accordance with those of Gayathri and Vasudevan[56] suggesting that the reduction in phenolic componentsremoval efficiency above 10 NaCl may be due to increasein salinity These results indicated that Janibacter sp strainhas an inherent flexibility to adapt to salinity fluctua-tions

The use of surfactants has the potential to increase thebiodegradation rate of hydrophobic organic compounds incontaminated environments Nonionic surfactants are usu-ally used in the bioavailability studies due to their relativelylow toxicity compared to ionic surfactants [57]The enhancedbiodegradation in the micelles solution can be attributableto the increased solubility dissolution and bioavailabilityof compound to bacteria [58] and the surfactant enhancedsubstrate transport through the microbial cell wall [59] Theeffects of the surfactants on PCP removal have been invari-ably attributed to the increased solubility and dissolution ofPCP or enhancement of mass transport in the presence ofsurfactants In this context at high concentration of PCP(300mgL) Tween 80 increases the removal rate of PCPwhenthe Tween 80 concentration is 40mgL The enhancement ofPCP removal was slightly detected when the concentrationof PCP is 20mgL These results can be confirmed with thestudy of Cort et al [60] when the biodegradation rate ofPCP was enhanced for the concentration of PCP at 140 and220mgL but it was inhibited for the concentration of PCPat 100 and 50mgL Consequently successful integration ofPCP and Tween 80 degradation was achieved by Janibactersp strain

5 Conclusion

In this study a novel efficient PCP-degrading actinobac-terium (Janibacter sp) was isolated from saline soil of aridland and investigated for its physiological characteristicsJanibacter was able to remove high concentration of PCP andto tolerate fluctuation of NaCl This removal potential wasmoreover accelerated by the addition of Tween 80This studysuggested that strain Janibacter sp could be widely used forPCP bioremediation of polluted aridextreme environments

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was financially supported by the NATO ProjectSFP (ESPMDFFP981674) 0073 ldquoPreventive and RemediationStrategies for Continuous Elimination of Poly-ChlorinatedPhenols from Forest Soils and Groundwaterrdquo It was in partfurther supported by the European Union in the ambit ofthe Project ULIXES (EuropeanCommunityrsquos Seventh Frame-work Programme KBBE-2010-4 under Grant Agreement no266473)

References

[1] A Vallecillo P A Garcia-Encina and M Pena ldquoAnaerobicbiodegradability and toxicity of chlorophenolsrdquo Water Scienceand Technology vol 40 no 8 pp 161ndash168 1999

[2] S Fetzner and F Lingens ldquoBacterial dehalogenases biochem-istry genetics and biotechnological applicationsrdquoMicrobiolog-ical Reviews vol 58 no 4 pp 641ndash685 1994

[3] C M Kao C T Chai J K Liu T Y Yeh K F Chen and SC Chen ldquoEvaluation of natural and enhanced PCP biodegrada-tion at a former pesticidemanufacturing plantrdquoWater Researchvol 38 no 3 pp 663ndash672 2004

[4] D-S Shen X-W Liu and H-J Feng ldquoEffect of easily degrad-able substrate on anaerobic degradation of pentachlorophenolin an upflow anaerobic sludge blanket (UASB) reactorrdquo Journalof Hazardous Materials vol 119 no 1ndash3 pp 239ndash243 2005

[5] K Sai K S Kang A Hirose R Hasegawa J E Trosko and TInoue ldquoInhibition of apoptosis by pentachlorophenol in v-myc-transfected rat liver epithelial cells relation to down-regulationof gap junctional intercellular communicationrdquo Cancer Lettersvol 173 no 2 pp 163ndash174 2001

[6] EPA ldquoFinal determination and indent to cancel and denyapplications for registrations of pesticide products contain-ing pentachlorophenol (including but not limited to its saltsand esters) for non-wood usesrdquo US Environmental ProtectionAgency Federal Register vol 52 pp 2282ndash2293 1987

[7] S D Copley ldquoEvolution of ametabolic pathway for degradationof a toxic xenobiotic the patchwork approachrdquo Trends inBiochemical Sciences vol 25 no 6 pp 261ndash265 2000

[8] D Errampalli J T Trevors H Lee et al ldquoBioremediation aperspectiverdquo Journal of Soil Contamination vol 6 no 3 pp207ndash218 1997


[9] C Wittmann A P Zeng and W D Deckwer ldquoPhysiologicalcharacterization and cultivation strategies of the pentachloro-phenol-degrading bacteria Sphingomonas chlorophenolicaRA2 and Mycobacterium chlorophenolicum PCP-1rdquo Journalof Industrial Microbiology and Biotechnology vol 21 no 6 pp315ndash321 1998

[10] I SThakur R Sharma S Shukla andAHAhmed ldquoEnumera-tion and enrichment of pentachlorophenol degrading bacterialof pulp and paper miller in the chemostatrdquo in Proceedings ofthe AMI Conference vol 159 pp 25ndash27 Jaipur India November2000

[11] J H A Apajalahti P Karpanoja and M S Salkinoja-SalonenldquoRhodococcus chlorophenolicus sp nov a chlorophenol-mine-ralizing actinomyceterdquo International Journal of Systematic Bac-teriology vol 36 no 2 pp 246ndash251 1986

[12] J F Gonzalez and W S Hu ldquoEffect of glutamate on the degra-dation of pentachlorophenol by Flavobacterium sprdquo AppliedMicrobiology and Biotechnology vol 35 no 1 pp 100ndash110 1991

[13] R I Dams G I Paton and K Killham ldquoRhizoremediationof pentachlorophenol by Sphingobium chlorophenolicumATCC39723rdquo Chemosphere vol 68 no 5 pp 864ndash870 2007

[14] C-F Yang C-M Lee and C-C Wang ldquoIsolation and physi-ological characterization of the pentachlorophenol degradingbacterium Sphingomonas chlorophenolicardquo Chemosphere vol62 no 5 pp 709ndash714 2006

[15] S Singh B B Singh R Chandra D K Patel and V RaildquoSynergistic biodegradation of pentachlorophenol by Bacilluscereus (DQ002384) Serratia marcescens (AY927692) and Serra-tia marcescens (DQ002385)rdquoWorld Journal of Microbiology andBiotechnology vol 25 no 10 pp 1821ndash1828 2009

[16] S K Karn S K Chakrabarty and M S Reddy ldquoPen-tachlorophenol degradation by Pseudomonas stutzeri CL7 inthe secondary sludge of pulp and paper millrdquo Journal ofEnvironmental Sciences vol 22 no 10 pp 1608ndash1612 2010

[17] A Sharma I S Thakur and P Dureja ldquoEnrichment isola-tion and characterization of pentachlorophenol degrading bac-teriumAcinetobacter sp ISTPCP-3 from effluent discharge siterdquoBiodegradation vol 20 no 5 pp 643ndash650 2009

[18] M Kamekura ldquoDiversity of extremely halophilic bacteriardquoExtremophiles vol 2 no 3 pp 289ndash295 1998

[19] R Margesin and F Schinner ldquoPotential of halotolerant andhalophilic microorganisms for biotechnologyrdquo Extremophilesvol 5 no 2 pp 73ndash83 2001

[20] W D Grant R T Gemmell and T J McGenity ldquoHalophilesrdquoin Extremophiles Microbial Life in Extreme Environments KHorikoshi and W D Grant Eds pp 93ndash132 Wiley-Liss NewYork NY USA 1998

[21] C K Okoro R Brown A L Jones et al ldquoDiversity of culturableactinomycetes in hyper-arid soils of the AtacamaDesert ChilerdquoAntonie van Leeuwenhoek vol 95 no 2 pp 121ndash133 2009

[22] R U Edgehill ldquoPentachlorophenol removal from slightly acidicmineral salts commercial sand and clay soil by recoveredArthrobacter strain ATCC 33790rdquo Applied Microbiology andBiotechnology vol 41 no 1 pp 142ndash148 1994

[23] S K Karn S K Chakrabarti andM S Reddy ldquoDegradation ofpentachlorophenol by Kocuria sp CL2 isolated from secondarysludge of pulp and paper millrdquo Biodegradation vol 22 no 1 pp63ndash69 2011

[24] K Martin P Schumann F A Rainey B Schuetze and I GrothldquoJanibacter limosus gen nov sp nov a new actinomycetewith meso- diaminopimelic acid in the cell wallrdquo International

Journal of Systematic Bacteriology vol 47 no 2 pp 529ndash5341997

[25] J-H Yoon K-C Lee S-S Kang Y-H Kho K-H Kang andY-H Park ldquoJanibacter terrae sp nov a bacterium isolated fromsoil around a wastewater treatment plantrdquo International Journalof Systematic and Evolutionary Microbiology vol 50 no 5 pp1821ndash1827 2000

[26] J-H Yoon H B Lee S-H Yeo and J-E Choi ldquoJanibacter mel-onis sp nov isolated from abnormally spoiled oriental melonin Koreardquo International Journal of Systematic and EvolutionaryMicrobiology vol 54 no 6 pp 1975ndash1980 2004

[27] A Kageyama Y Takahashi M Yasumoto-Hirose H KasaiY Shizuri and S Omura ldquoJanibacter corallicola sp nov iso-lated from coral in Palaurdquo Journal of General and AppliedMicrobiology vol 53 no 3 pp 185ndash189 2007

[28] P Kampfer O Terenius J M Lindh and I Faye ldquoJanibacteranophelis sp nov isolated from the midgut of Anophelesarabiensisrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 56 no 2 pp 389ndash392 2006

[29] I Sierra J L Valera M L Marina and F Laborda ldquoStudyof the biodegradation process of polychlorinated biphenyls inliquid medium and soil by a new isolated aerobic bacterium(Janibacter sp)rdquo Chemosphere vol 53 no 6 pp 609ndash618 2003

[30] S Iwai A Yamazoe R Takahashi F Kurisu and O YagildquoDegradation of monochlorinated dibeno -p-Dioxins by Jani-bacter sp strain YA isolated from river sedimentrdquo CurrentMicrobiology vol 51 no 5 pp 353ndash358 2005

[31] S Jin T Zhu X Xu andYXu ldquoBiodegradation of dibenzofuranby Janibacter terrae strain XJ-1rdquo Current Microbiology vol 53no 1 pp 30ndash36 2006

[32] A Yamazoe O Yagi andH Oyaizu ldquoDegradation of polycyclicaromatic hydrocarbons by a newly isolated dibenzofuran-utilizing Janibacter sp strain YY-1rdquo Applied Microbiology andBiotechnology vol 65 no 2 pp 211ndash218 2004

[33] AYamazoeOYagi andHOyaizu ldquoBiotransformation of fluo-rene diphenyl ether dibenzo-p-dioxin and carbazole by Jani-bacter sprdquo Biotechnology Letters vol 26 no 6 pp 479ndash4862004

[34] G-Y Zhang J-Y Ling H-B Sun J Luo Y-Y Fan and Z-JCui ldquoIsolation and characterization of a newly isolated poly-cyclic aromatic hydrocarbons-degrading Janibacter anophelisstrain JY11rdquo Journal of HazardousMaterials vol 172 no 2-3 pp580ndash586 2009

[35] D A Edwards R G Luthy and Z Liu ldquoSolubilization of poly-cyclic aromatic hydrocarbons in micellar nonionic surfactantsolutionsrdquo Envirenmental Science and Technology vol 25 no 1pp 127ndash133 1991

[36] C Rosch AMergel andH Bothe ldquoBiodiversity of denitrifyinganddinitrogen-fixing bacteria in an acid forest soilrdquoApplied andEnvironmental Microbiology vol 68 no 8 pp 3818ndash3829 2002

[37] K Wilson ldquoPreparation of genomic DNA from bacteriardquo inCurrent Protocols in Molecular Biology F M Ausubel R BrentR E Kingston et al Eds pp 241ndash245 1987

[38] D Daffonchio S Borin G Frova P L Manachini and CSorlini ldquoPCR fingerprinting of whole genomes the spacersbetween the 16s and 23S rRNA genes and of intergenic tRNAgene regions reveal a different intraspecific genomic variabilityof Bacillus cereus and Bacillus licheniformisrdquo International Jour-nal of Systematic Bacteriology vol 48 no 1 pp 107ndash116 1998

[39] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990


[40] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[41] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquoMolecular biol-ogy and evolution vol 4 no 4 pp 406ndash425 1987

[42] I SThakur ldquoStructural and functional characterization of a sta-ble 4-chlorosalicylic-acid-degrading bacterial community in achemostatrdquo World Journal of Microbiology and Biotechnologyvol 11 no 6 pp 643ndash645 1995

[43] A Premalatha andG S Rajakumar ldquoPentachlorophenol degra-dation by Pseudomonas aeruginosardquo World Journal of Microbi-ology and Biotechnology vol 10 no 3 pp 334ndash337 1994

[44] M Tripathi and S KGarg ldquoStudies on selection of efficient bac-terial strain simultaneously tolerant to hexavalent chromiumand pentachlorophenol isolated from treated tannery effluentrdquoResearch Journal of Microbiology vol 5 no 8 pp 707ndash716 2010

[45] S Singh R Chandra D K Patel and V Rai ldquoIsolationand characterization of novel Serratia marcescens (AY927692)for pentachlorophenol degradation from pulp and paper millwasterdquoWorld Journal ofMicrobiology and Biotechnology vol 23no 12 pp 1747ndash1754 2007

[46] M Cai and L Xun ldquoOrganization and regulation of pentachlo-rophenol-degrading genes in Sphingobium chlorophenolicumATCC 39723rdquo Journal of Bacteriology vol 184 no 17 pp 4672ndash4680 2002

[47] E A Wolski S E Murialdo and J F Gonzalez ldquoEffect ofpH and inoculum size on pentachlorophenol degradation byPseudomonas sprdquoWater SA vol 32 no 1 pp 93ndash98 2006

[48] C Barbeau L Deschenes D Karamanev Y Comeau and RSamson ldquoBioremediation of pentachlorophenol-contaminatedsoil by bioaugmentation using activated soilrdquo Applied Microbi-ology and Biotechnology vol 48 no 6 pp 745ndash752 1997

[49] C-F Yang C-M Lee and C-C Wang ldquoDegradation ofchlorophenols using pentachlorophenol-degrading bacteriaSphingomonas chlorophenolica in a batch reactorrdquo CurrentMicrobiology vol 51 no 3 pp 156ndash160 2005

[50] R L Crawford and W W Mohn ldquoMicrobiological removal ofpentachlorophenol from soil using a Flavobacteriumrdquo Enzymeand Microbial Technology vol 7 no 12 pp 617ndash620 1985

[51] J T Trevors ldquoEffect of temperature on the degradation ofpentachlorophenol by Pseudomonas speciesrdquoChemosphere vol11 no 4 pp 471ndash475 1982

[52] M A Providenti H Lee and J T Trevors ldquoSelected factorslimiting the microbial degradation of recalcitrant compoundsrdquoJournal of Industrial Microbiology vol 12 no 6 pp 379ndash3951993

[53] M D Webb G Ewbank J Perkins and A J McCarthyldquoMetabolism of pentachlorophenol by Saccharomonosporaviridis strains isolated from mushroom compostrdquo Soil Biologyand Biochemistry vol 33 no 14 pp 1903ndash1914 2001

[54] R Chandra A Ghosh R K Jain and S Singh ldquoIsolation andcharacterization of two potential pentachlorophenol degradingaerobic bacteria from pulp paper effluent sludgerdquo The Journalof General and Applied Microbiology vol 52 no 2 pp 125ndash1302006

[55] C M Kao J K Liu Y L Chen C T Chai and S C ChenldquoFactors affecting the biodegradation of PCP by Pseudomonasmendocina NSYSUrdquo Journal of Hazardous Materials vol 124no 1ndash3 pp 68ndash73 2005

[56] K V Gayathri and N Vasudevan ldquoEnrichment of phenoldegrading moderately halophilic bacterial consortium fromsaline environmentrdquo Journal of Bioremediation amp Biodegrada-tion vol 2011 article 104 2010

[57] D H Yeh K D Pennell and S G Pavlostathis ldquoToxicityand biodegradability screening of nonionic surfactants usingsediment-derived methanogenic consortiardquo Water Science andTechnology vol 38 no 7 pp 55ndash62 1998

[58] F Volkering A M Breure J G van Andel and W H Rul-kens ldquoInfluence of nonionic surfactants on bioavailability andbiodegradation of polycyclic aromatic hydrocarbonsrdquo Appliedand Environmental Microbiology vol 61 no 5 pp 1699ndash17051995

[59] W H Noordman J H J Wachter G J de Boer and D BJanssen ldquoThe enhancement by surfactants of hexadecane degra-dation by Pseudomonas aeruginosa varies with substrate avail-abilityrdquo Journal of Biotechnology vol 94 no 2 pp 195ndash212 2002

[60] T L CortM-S Song andA R Bielefeldt ldquoNonionic surfactanteffects on pentachlorophenol biodegradationrdquo Water Researchvol 36 no 5 pp 1253ndash1261 2002


of aquatic and terrestrial ecosystems A low taxonomicbiodiversity is observed in all these saline environments [18]most probably due to the high salt concentrations prevailingin these environmentsMoreover the biodegradation processis difficult to perform under saline conditions [19] Besidesthese metabolical and physiological features halophilic andhalotolerant microorganisms are known to play importantroles in transforming and degrading waste and organic pol-lutants in saline and arid environment [20] These microor-ganisms particularly actinobacteria are frequently isolatedfrom extreme environments such as Sabkha Chott andSahara which are known to have a great metabolic diversityand biotechnological potential The occurrence of actinobac-teria in saline environment and their tolerance to highsalt concentrations were thus described [21] However fewactinobacteria genera such as Arthrobacter [22] and Kocuria[23] were reported for PCP-degradation process The genusJanibacterwhich is recognized byMartin et al [24] belongs tothe family Intrasporangiaceae in the Actinomycetales orderand included five major species J limosus [24] J terrae[25] J melonis [26] J corallicola [27] and J anophelis [28]Interestingly most of these species were reported for theirability to degrade a large spectrum of aromatic andor chlori-nated compounds including polychlorinated biphenyls [29]monochlorinated dibenzo-p-dioxin [30] dibenzofuran [31]anthracene phenanthrene [32] dibenzo-p-dioxin carbazolediphenyl ether fluorene [33] and polycyclic aromatic hydro-carbons [34] However no data reporting PCP degradationby Janibacter members was described PCP and other POPcompounds shared many physical properties which limitedbiodegradation processes and one of these properties wastheir lower solubility and therefore low bioavailability to thedegrading bacteria Nevertheless the use of surfactants suchas Tween 80 has the potential to increase the biodegradationrates of hydrophobic organic compounds by increasing thetotal aqueous solubility of these pesticides [35] In this studywe evaluated for the first time the PCP removal potentialunder different physicochemical conditions by Janibacter spa halotolerant actinobacterium member isolated from aridand saline land in southern Tunisia

2 Materials and Methods

21 Chemicals and Solvents PCP (MW 26634 and gt99purity) and acetonitrile (HPLC grade) were purchased fromSigma Aldrich (USA) All other inorganic chemicals used toprepare the different media are commercially available withhighest purity and are used without further purification

22 Sample Collection and PCP-Degrading Bacterium Isola-tion The sediment samples were collected in March 2011from arid and saline ecosystems belonging to the site ldquoSebkhaEl Naouelrdquo with GPS coordinates N 34∘26101584095110158401015840 E 09∘54101584010210158401015840altitude 150 ft46m in southern Tunisia Bacterial isolationwas performed as described by Rosch et al [36] with somemodifications 10 g of soil sample was suspended in 100mL ofphosphate-buffered salt solution (137mMNaCl 27mMKCl10mMNa

2HPO4 and 2mMKH

2PO4) and stirred vigorously

for 30minThe soil suspensionwas diluted and 01mL sample

was spread on the surface of yeast extract-mannitol medium(YEM) YEM medium contained the following componentsat the specified concentrations (in gL) mannitol 5 yeastextract 05 MgSO

4sdot7H2O 02 NaCl 01 K

2HPO4 05 Na

gluconate 5 agar 15 pH = 6 8 After sterilization for 20minat 120∘C 1mL of 166 CaCl

2solution was added to 1 liter

of YEMmedium (1 1000) The plates were then incubated at30∘C for 7 days Pure cultures of the isolates were obtained bystreaking a single colony on the same medium

23 16S rRNA Gene Amplification and Sequence AnalysisFor DNA extraction the FAS23 strain was grown in trypticsoy broth (TSB) containing (in gL) casein peptone 17 soyapeptone 3 glucose 25 sodium chloride 5 dipotassiumhydrogen phosphate 4 DNA extraction was performedusing CTABNaCl method as described by Wilson [37] andmodified by using lysozyme (1mgmL) for cell wall digestionThe 16S rRNA gene was amplified using universal primersSD-Bact-0008-a-S-20 (51015840-AGA GTT TGA TCC TGG CTCAG-10158403) and S-D-Bact-1495-a-A-20 (51015840-CTA CGG CTA CCTTGT TACGA-10158403) [38] PCR was performed in a final volumeof 25 120583L containing 1 120583L of the template DNA 05 120583Mof eachprimer 05 120583M of deoxynucleotide triphosphate (dNTP)25 120583L 10X PCR buffer for Taq polymerase MgCl

215mM

1UI of Taq polymerase The amplification cycle was asfollows denaturation step at 94∘C for 3min followed by 35cycles (45 sec at 94∘C 1min at 55∘C and 2min at 72∘C) plusone additional cycle at 72∘C for 7min as a final elongationstep The 16S rDNA PCR amplicons were purified withExonuclease-I and Shrimp Alkaline Phosphatase (Exo-SapFermentas Life Sciences) following the manufacturerrsquos stan-dard protocol Sequence analyses of the purified DNAs wereperformed using a Big Dye Terminator cycle sequencing kitV31 (AppliedBiosystems) and anAppliedBiosystems 3130XLCapillary DNA Sequencer machine Sequence similaritieswere found by BLAST analysis [39] using the GenBank DNAdatabases (httpwwwncbinlmnihgov) and the Riboso-mal Database Project (RDP) Phylogenetic analyses of the 16SrRNA gene sequences were conducted withMolecular Evolu-tionary Genetics Analysis (MEGA) software version 5 [40]Trees were constructed by using neighbor-joining method[41]The sequence was deposited in GenBank database underthe accession number KC959984

24 Degradation of PCP by Isolated Strain The kinetics ofthe PCP removal under different conditions were conductedin 500mL flasks sealed with cotton stoppers containing100mL of mineral salt medium (MSM) adjusted to pH69 supplemented with 1 glucose and inoculated with1 of 106 CFUmL of the strain FAS23 The MSM con-tained the following components at the specified concen-trations (in gL) KH

2PO4 08 Na

2HP4 08 MgSO

4sdot7H2O

02 CaCl2sdot2H2O 001 NH

4Cl 05 plus 1mL of trace

metal solution which includes (in mgL) FeSO4sdot7H2O 5

ZnSO4sdot7H2O 4 MnSO

4sdot4H2O 02 NiClsdot6H

2O 01 H

3BO3

015 CoCl2sdot6H2O 05 ZnCl

2025 and EDTA 25 PCP

was added to the medium after autoclaving [19] Whennecessary solid MSM plates were prepared by adding 15 gLbacteriological grade agar The inoculum was prepared as






9976

82100

58

5240

001







3 Results








0002040608101214161820

0 24 48 72 96 120 144

Glucose


OD

at600

nm

(a)

0

20

40

60

80

100


Resid

ual P

CP (

)

PCPPCP + Glucose

(b)


00

02

04

06

08

10

12

14

16

18

20


pH 4pH 69-PCP

pH 69

pH 9

OD

at600

nm

pH 4-PCPpH 9-PCP

(a)

0102030405060708090

100

0 24 48 72 96 120 144


pH 4pH 69pH 9

Resid

ual P

CP (

)

(b)








OD

at600

nm

00

02

04

06

08

10

12

14

16

18

20

37∘C

30∘C

25∘C

37∘C-PCP

25∘C-PCP

30∘C-PCP

(a)

0

10

20

30

40

50

60

70

80

90

100


Resid

ual P

CP (

)37

∘C30

∘C25

∘C

(b)



02050

100200300

OD

at600

nm

00

02

04

06

08

10

12

14

16

18

20

(a)

0

10

20

30

40

50

60

70

80

90

100


2050100

200300

Resid

ual P

CP (

)

(b)





4 Discussion




013

610

OD

at600

nm

0002040608101214161820

(a)

0102030405060708090

100


013

610

Resid

ual P

CP (

)

(b)



20-T8020

300-T80300

OD

at600

nm

0002040608101214161820

(a)

0102030405060708090

100


20-T8020

300-T80300

Resid

ual P

CP (

)

(b)











5 Conclusion




Acknowledgments


References





































































9976

82100

58

5240

001







3 Results








0002040608101214161820

0 24 48 72 96 120 144

Glucose


OD

at600

nm

(a)

0

20

40

60

80

100


Resid

ual P

CP (

)

PCPPCP + Glucose

(b)


00

02

04

06

08

10

12

14

16

18

20


pH 4pH 69-PCP

pH 69

pH 9

OD

at600

nm

pH 4-PCPpH 9-PCP

(a)

0102030405060708090

100

0 24 48 72 96 120 144


pH 4pH 69pH 9

Resid

ual P

CP (

)

(b)








OD

at600

nm

00

02

04

06

08

10

12

14

16

18

20

37∘C

30∘C

25∘C

37∘C-PCP

25∘C-PCP

30∘C-PCP

(a)

0

10

20

30

40

50

60

70

80

90

100


Resid

ual P

CP (

)37

∘C30

∘C25

∘C

(b)



02050

100200300

OD

at600

nm

00

02

04

06

08

10

12

14

16

18

20

(a)

0

10

20

30

40

50

60

70

80

90

100


2050100

200300

Resid

ual P

CP (

)

(b)





4 Discussion




013

610

OD

at600

nm

0002040608101214161820

(a)

0102030405060708090

100


013

610

Resid

ual P

CP (

)

(b)



20-T8020

300-T80300

OD

at600

nm

0002040608101214161820

(a)

0102030405060708090

100


20-T8020

300-T80300

Resid

ual P

CP (

)

(b)











5 Conclusion




Acknowledgments


References


































































0002040608101214161820

0 24 48 72 96 120 144

Glucose


OD

at600

nm

(a)

0

20

40

60

80

100


Resid

ual P

CP (

)

PCPPCP + Glucose

(b)


00

02

04

06

08

10

12

14

16

18

20


pH 4pH 69-PCP

pH 69

pH 9

OD

at600

nm

pH 4-PCPpH 9-PCP

(a)

0102030405060708090

100

0 24 48 72 96 120 144


pH 4pH 69pH 9

Resid

ual P

CP (

)

(b)








OD

at600

nm

00

02

04

06

08

10

12

14

16

18

20

37∘C

30∘C

25∘C

37∘C-PCP

25∘C-PCP

30∘C-PCP

(a)

0

10

20

30

40

50

60

70

80

90

100


Resid

ual P

CP (

)37

∘C30

∘C25

∘C

(b)



02050

100200300

OD

at600

nm

00

02

04

06

08

10

12

14

16

18

20

(a)

0

10

20

30

40

50

60

70

80

90

100


2050100

200300

Resid

ual P

CP (

)

(b)





4 Discussion




013

610

OD

at600

nm

0002040608101214161820

(a)

0102030405060708090

100


013

610

Resid

ual P

CP (

)

(b)



20-T8020

300-T80300

OD

at600

nm

0002040608101214161820

(a)

0102030405060708090

100


20-T8020

300-T80300

Resid

ual P

CP (

)

(b)











5 Conclusion




Acknowledgments


References


































































OD

at600

nm

00

02

04

06

08

10

12

14

16

18

20

37∘C

30∘C

25∘C

37∘C-PCP

25∘C-PCP

30∘C-PCP

(a)

0

10

20

30

40

50

60

70

80

90

100


Resid

ual P

CP (

)37

∘C30

∘C25

∘C

(b)



02050

100200300

OD

at600

nm

00

02

04

06

08

10

12

14

16

18

20

(a)

0

10

20

30

40

50

60

70

80

90

100


2050100

200300

Resid

ual P

CP (

)

(b)





4 Discussion




013

610

OD

at600

nm

0002040608101214161820

(a)

0102030405060708090

100


013

610

Resid

ual P

CP (

)

(b)



20-T8020

300-T80300

OD

at600

nm

0002040608101214161820

(a)

0102030405060708090

100


20-T8020

300-T80300

Resid

ual P

CP (

)

(b)











5 Conclusion




Acknowledgments


References


































































013

610

OD

at600

nm

0002040608101214161820

(a)

0102030405060708090

100


013

610

Resid

ual P

CP (

)

(b)



20-T8020

300-T80300

OD

at600

nm

0002040608101214161820

(a)

0102030405060708090

100


20-T8020

300-T80300

Resid

ual P

CP (

)

(b)











5 Conclusion




Acknowledgments


References





































































5 Conclusion




Acknowledgments


References
































































Pentachlorophenol Degradation by Janibacter sp., a New Actinobacterium Isolated from Saline Sediment...

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Transcript of Pentachlorophenol Degradation by Janibacter sp., a New Actinobacterium Isolated from Saline Sediment...