R Ettt E D I ATI 0 N Su.ntner 2 0 I 4

Enhancing Perfo rmance of

S ph i n gobo cte ri u m spi ritivo ru m in Phenanthrene Contaminated Sand

'lor-Ama ni-Fitzah Mohd-Kamit

Noor-Hana Hussain

Meizareena Binti Mizad Many biodegradatian studies have facused on survival of isolated bacteria to increase the bacte- (PAH) ria population and subseque ntly enhance the ef{iciency o{ polycyclic aramatic hydrocarbon Abdut-TaLib Suhaimi biadegradation. Hewever, there is limited research on enhancing the per{orntance o{ isolated bac' teria through reinoculat[on. Thus, this study was designed to evaluate the efects o{ reinoculation on the pertormance of Sphingobacterium spiritivorum in degradation of phenanthrene contama- performed nated sand. Fxperirnents were pertormed in three different reactors. Inoculatian was (daf 5)' The bac- once (day A) in reactor 1. ln rcactor 2, inoculation was performed twice A and day reactor teria was isalated from reactor 2 and inoculated into reactar 3. The study results show 3 (95.36 percent)' ln having the highest degradarion rate (1j.61 ng/kg/day) and percentage removal Ihus' the centrast,without reinoculation in reactor 1, 68.93 percent of phenanthrene was removed' reinocula' performance of S. spiritivoru m in phenanthrene degradation can be enhanced through tion. a2014Wiley Periodicals, lnc.

INTRODUCTION

Th" *ort diflicult issue in the application of bioremediation by inoculation of isolated some of the bacteria at contaminated sites is the survival of the bacteria. Studies show that isolatecl bacteria population clecreased shortly after soil inoculation (Nilrozik & piotrq$,ska,S"g"i, iOt O;. This may be due to the lack of certain nutrients that are needed to the isolated fbr the bactcrii growth, the environment in the containinaterl site is toxic ofthe bacteria, or the bacteriahave less potential to degrade the contarninant lfsurdval strain can be improved and increase the bacteria population, the efficiency of soil bioremediation also can be increased, and A few studies have reported on the use of hirx;arriers, such as activated carbon zeolite (Liang et al., 2009) or chitin and chitosan (Gentili et al., 2006), to convey mass nutrients to ihe bacteria for growth. Biot:arriers enhanced oxygen diffusion, nutrient Tortora et al' trarrsfern and rvater retainagc (Liang et al., 2OA\. Besides biocarriers, as nitrogen and phosphorus plant QAOT reported that the uie of bioenharlcers' such

'r0.i002lrem 119 ?jBlirYJ"J"iil?i'iil?r'[';,,* Library (wireyonrinericrary.com). DOr: z13e8

II rutiltgl!+!dr{r:, Enhancing Per{ormance o{ Sphingobacterium sp i(itivotu m

fertilizers, rvith contaminatcd soil increased the numbcr of degrarling bactcria, as compared to an unf'ertilized control soil. In addition, the bioenhancers subscquently improved the bioremediation process. In line rvith improving survival of degraders in nerv environqretrts, Karamaiidis et al. (2010i reported on a technology that provides protection for isolated bactcria using an encalrsulaticrn rnethc.rd. Sotlium alginate encapsulated Pseutlomonas aeruginosa srroin rTref u'as used to rernediate soil contaminated rvidr polycyclic aromatic hvdrocarbons (PAf ls). Holver'er,thestudvshorvedthatinoculatjonr+'ithorrvithouttheencapsulationhadno t^ significant efl'ect on I'AH biodegratlation. Reinoculation simple, I ; is Furthermore, microbiologists have becn working on improving the elficiencv of but the important effect i degrading bacteria by altering the bacteria rvith recombinant DNA technologv. The I o{ increasing the bacteria alteration is performed artiGciallv bv incorporating DNA from trvo or morc sourccs int ! & i population itself through single recombinant moiecule (Tr,rrtota er al,, 200?). Me-ver et al. (2005) incorporatcd ireinoculation of the same monooxygenase enzyme lrom Pseudomonas putjJa into Eschefichia coJi. This i strain is unknown. Inocula- xylene :tion with higher bacteria rt:combinant oxitiizcd m-nitrotoluene to the corresponding alcohols, alclehvdes, and i counts may increase the aci

efliciency remains unknown, In this study, Sphingobactelum sPiritot'orum, isolated from i municipal sludge, rvas used as the PAH-degracling microorganisms rvhile phenanthrene served as tlie PAH surrogate. The primarv aim of this study is to enhance dre performance o[ 5. sPtuiLivorum i\ pLenanthrene degradation through reinoculation. Reinoculation rvith a sirnilar bacterir count was evaluate

METHODOLOGY

Materials

All chcmicals uscd for cxtraction, prcparation of minimal mcdia, and bacteria culturc rvere of analvtical gradc and supplied bv Merck, Germany. The phenanthrcne standard for the gas ghromatograph,v mass slJcctrometer (GCMS) analyscs was also obtained li'on-r Merck, Germany. Ultra-pure lvater (UPW) used in this studv was Produced from Alga Pureiab Ultra (18.2 MCl, United Kingdom).

120 Remediation DOI: 10.1002/rem @2014 Wiley Periodicals, lnc

1t.,1. REilI E D IATTO N Sumnil 201 4

Sample Preparation

Preparation of Contaminated Sand

Silica sand rvas samplc

Preparation of Minimal Media

IMinimal media solution was prepared try dissolving 8."5 g NarHPOa, 3.0 g KHrPOn, 0.5 g NaCl, 1.0 g NHuCl, 0.5 g MgSO*.7H2O, 0.0i+7 g CaClr, 0.000.+ g CuSOa, 0.001 g Kl, 0.004 g MnSOn.l IrO, 0.004 g ZnSO*, 0.005 g H,IJO' and 0.002 g FeCl, in 1L UPW The preparecl minimal nredia rvere autoclaved (Hiravarna, HVE-50, Japan) for 20 rninutes at 121 "C.

Preparation of Bacteria Strain

The strain, S. sprritovorum, w"as isolated from municipal sludgc by Othman et al. (2009) preserved at 80'C in microbeads (nricrobankrr{, Round Rock, TX)- Thc strain u'as revived by transferring a ferr of the frozen beacls into universal bottlcs containing 20 mL o of Nutrient Broth that q'as incubated at 30 C for thre e da1's. A series of dilution streaking was performed and the strain *'as sulrcultured three timer to attain the active bacterium befbre it rvas used in biodegradation studies.

Growth Curve Test

The plate count nrethod rvas used to establish the gowth curve after the bacteria u.as inoculated in contaminated sand. 'l'his test lvas conducted in reactor t and reactor 2. The bacteria number in the sand sarnples rvere quantified by mixing 1 g of sand w-ith 9 mL of sterile phosphate l:ruffered sa]ine and homogenized at high speed for I minute using a vortex mixer. Successir.e I / I 0 dilutions were made bv adding i mL of the sand suspension to 9 mL of phosphate butl-ered saline. An aliquot (0.1 mL) of each dilution lr'as oC transferrecl to nutrient lgar on a Petri dish- The tlishes rvere incubated at 30 at an invcrtcd position. After 4 days, the numbers of bacterial cokrnies tere counted using a platc countcr. Platcs with different dilutions were prepared and t}ose with colnnies in the rangc of 30 to 300 rt'crc uscd to cstimatc the numbcr of bacteria. Thig number of colr:nies was then multiplicd by thc dilution f-actor to find tlc total numbcr of bacteria per 1 g nf the sand. The numbers of colonies are expressed as colony-forming units pcr gram of sand (CFU/g). All tests were conciucted in *iplicate.

';

a O20'l4Wiley Per;odicals, lnc. Remediation DOI: :0.1002/rem 121 ': .i

i. Enhancing Per{orrnance o{ Sphingobacteritnt spiritivorun

Exhibit 1 . Detail of the experimental setup

Biodegradation Study

Batch experiments tvere conduded in a 250 mL Erlenmeyer flask as ihe reactor. The reactor contained 20 g autoclaved sand, 100 ppm phenanthrene, 5,6 mL minimal mediar &d | .+ mL bacterial inoculums. 5.6 mL of minimal meclia constituted 40 p€rcent of thc total volume. This volume was selected based on the quantity of inorganic nutrient required for bioremediation of PAHs in soil (Li et al., 2008). A total t .4 mL of bacterial inoculum occupied l0 percent ofthe total volume and this quantity was selected based on an optimization study conducted by Othman et al' (2009). The bacteria inoculated into the samples rvere collected at the nriddle of the exponential growth phase based on established growth curves, that is, on day 4. This e1periment was repeated three tirnes and the average bacteria concentration on day 4 was 'I'hen, 2 . I X. | .2 X 1 07 cells,/g soil- the strain was inoculated into reactor I and reactor For reaclor 3, the bacteria strain was isolated fi'om reactor 2 on day 9 and inoc.ulated with almost a similar number as reactor I and 2. The method of isolation was arlopted from Othnt;ur et al. (201O). tletails of the experimental variations studies for all reactors are shown in Exhibit 1. .l5O After inoculation, all flasks were shaken in an incubator shaker at rpm in tle dark oC. at 30 Sterile water wa* srrpplied at 2 percent remaining weight cvery day. All samples were analyzed in triplicate" Control samples consisted of 20 g autoclavcd sand, 100 ppm phenanthrene, and 5.5 ml. minimal media without bactcrial inoculums. The plate count method was perfbrmed on the control samplcs and no colony w-as found.

Extraction and Analysis

For sample analysis, Sffi mg of the contaminatcd sand sample was dissolved in 25 mL of n-hexane and acetone 7:3 (v/v). Thc cxtractions l'r'ere performed using the Pressurized microwave-assisted extraction svstcm (MAE) Multiwave 3000 (Rotor 8XFt00 SOLV and solvcnt safety systemi Graz, Austria). Ali samples that were placed in the MAE were extractcd for 40 minutcs under a pressure of 10 bars, When the extraction Period was complctcd, thc cguipment was allowed to cool down to room temperature (20 minutes). Subsequentl,v, the samples were filtered with a Whatman fiber filter with a pore size of I I ;rm and stored in a 25 mL universal bottle. The samples were concentrated by means of a rotary eyaporator to 1 mL. The extraction method had been pretested for recoverY efficiency for phenanthrene and demonstrated an efficiency of 99 percent. Phenanthrene concentrations \ rere analyzed using a gas chromatography mass spectrometer (Perkin Elmer Clarus 600; Shelton, CT), equipped with Elite Column 5MS of 30 m long X 0.25 mm internal dimension X 0.25 ;.tm thickness, The injector was

oeriodicals. Remed;aiion DOr: 10.1002/rer O2014 Wile/ ln:

,.::G . :... :

, REMIEDIATION Swnrcr 2014

1.8+10 aReactor I trReactor2

ab! l.E+08 E

l.E+41

qt

€, l.E+{6 g F

l.E+05

0t 2 t 4 5, 6 7 8 9 l0 1l Day

Exhibit 2. Growth curve of Sphingobacterium spiritovorum in reactor 1 and 2

operated at 250 'C in the splitless mode with a 3-minute splitless period. Helium lvas used as the carrier gas u'ith 1 ml,/min constant flow rate. The column temperature \{.as oC oC initially set at 50 for 1 minute, increased to 250 at a rate of 2SoClmin, kept constant at I minute, and held constant until the end of the 22-minute total run time,

RESULTS AND DISCUSSION

Grawth Curve

The strain grew rapidly after inoculation until dav 1 rvith the grow.th rate of 3.9 X 10" CFU /g/day as shown in Exhibit 2 . After day I , tle bacteria concentration in reactor 1 decreased throughout the experiment- For rcacJor 2, after reinoculation on day 5, the bacteria concentration increased until day 5 but decreased again rrntil day 10. In reactor 2, the growth c'urve can be divided irto two phascs, tlrat is, phase l: Ilnm da1- 0 until day 4 and phase 2: fiom day 4 until day 10. Betueen thcsc trvo phases, the growth of the strain rvas similar with the maximum bacteria concentration of 3 x I 08 CFU /g. Exhibit 2 shows the reduction of bacteria counts after inoculation into contaminatecl sand in both reactors. These results indicate that the bacteria were less adaptable to the new cnvironment, which lead to mortality. This low survival may be due to low bioavailability ofphenanthrene. Results hom this study are contrasted from the studv reported by Othman ct al. (2009) where the bacteria showed encouraging growth rates in minirnal media. Minimal media can be categorized as homogenous medium. There is unlirnited bacteria access to phenanthrene in a homogenous medium. In heterogeneous media, such as contaminated sand, dre phenanthrene is not homogeneously distributed and may be located in small pores or absorbed inside organic particles, which are

Q?Q14 \Nilex Pe

l:i.ii;lti.!#;iif2:t:li:::.lx$ir/: Erhancing Performsnce of Sphingobacterium spiritivorur,

F a 'lE E 1,! 1.F.06 t L.i t6 I .g .l' EE l.E{7 ; g!t : ::E i-Uoi o l' . Reattor I E .i( t ,r! o0 6r It t.E{8 l oReactor 2 &D€ :.:

j LE-09 ,

: l.E-10 . Day I I

t. - ... -. -. . . .. * -. ,, *. , ,., " -. -... - -,. " ", ",. -. -, _i Exhibit 3, Degradation rate per colony in reactor 1 and 2

irraccessible to bacteria (Johnsen et al., 2005). In ad&tion, minimal media n'hich is a liquid, mixes well and, thus, increases the bioavailability of pheaanthrene to bacteria. Whereas, this is not the case for contaminated sand. Consequentlv, the physical separation n betu'een phenanthrene and the sand results in low bioavailability. Cao et al. (2009) also mentioned that the degradation process may tre retarded if there is an insufficient amount ?. ofone ofthese important elements (i.e., phenanthrene as electron clonor, oxygen as tl eleclron acceptor, or bacteria as ). In this degradation process, clxygen lvas not sl limitecl, as thc covcr frrr thc flasks consistcd of stcrile cotton and the flasks were shaken L :. throughout dre experiment. Comparing this studv to onc bv Kumar ct al. (2009), which was conductrld undcr anacrobic conditions and provided a degradation proces$ driven by thc unlimitcd availability of sulfide as the electron acceptor, resulted in similar reductions

ofbacteria counts. P sa Potential ol a Single Colony b' ci The potential fbr one colony can tre evaluated by

124 Remediation DOI: 10.1002/rem 02014 Wiley Periodicals, Inc REMEDIATI ON Summer 2Ol4

100 r n . I Reactof I 90 1 : sReflctor 2 : E0i 2* inoculation o0 Reactor 3 T ^ b 70

cg 60 g 50 + E 6 40" f+tr I 30 i+ll i++ I t m, o l0 iI

3456 89 10 Day * Data shown are nrean of thre+ replicates; error bors rcpresent statdard dcviarion

Exhibit 4. Phenanthrene degradation by Sphingobacterium spiritovarum in reactor 1, 2, and 3

In addition, the bacteria may be utilizing other carbon sourccs containerl in the nutrient broth. Agathos and Reineke (2002) reported on degradation of'PAHs in soils, where bacteria rrere able to utilize other carbon-containing compounds instead of only the targeted PA[.Is. If other carbon compountls are relatively easier to utilize as compared to the targeted PAH, then the potential of the bacteria to degrade PAHs may be reduccd. As shown in Exhibit 2, the bacteri4 nurnber decreased alier inoculation and reinoculation. However, the degradation activitv o[ the co]onv in degrading phenanthrene had slowly increased for reactor I. On the other hand, the degradation rate per colony in reactor 2 rapiclly increased until the last day of the experinrent. The increment of the degradation rate per colony could be due to surr"ival of high potential bacteria (bacteria rvith more adaptabilitv under nonindigenous conditions). The samples wcrc inoculated with a high bacteria concentration. However, the reduction of bacteria concentration occurred due to low bioavailability, as discussed abuve. Under tlris condition, the lo*'potcntial bacteria may not survive due to lesser adaptability to a new environment and low bioarailability, resrrlting in fatalitv. Thus, the high potential bacteria have agreater ability to survive compared to low potential bacteria. ln addition, the high potential bacteria may access and break the carbon choin of phenanthrene in thc sand media. The high potential bacteria show'ed a higher degradation rate per colony'.

Degradation Rate and Percentage Removal ln reactor 1, phenantlrrene degradation can be divided into two ghases, that is, phase l: rapid degradation within five days with a degradation rate of 12.46 mg/kg/day tbllowed by phase 2: slow degradation rvith a rate of 0.74 mglkg/day as shorvn in Exhitrit 4. In enhancing the slow degradation observed in phase 2, reactor 2 was reinoculated with similar bacteria on day 5. In reactor 2, the degradation rate for the first 5 davs was

O20l4WileyPeriodicals, Inc. Remedration DOI: 10.100?/rerr Enhancing Performance oI Sphingabacteriun spiritivorum

llcthcd ot :glrtion of lerch6 U!dt00 lartmam ot Baatarb m. rnhrnencr* Erei!ffi| 3 Mth .crturted ce6on:4E.Bg pscem PAHg liMrrler: 2l ;oil 17.49 qsceil {ctfurtad carbon: l0ro celht-l EtiYaled B.b@ Mtfio{t divat€d €rbflr 3t.{ s@nt PAIG

Mth bixaraicr: 60 gcem hw|@1t4 tin|m.l liHnier: chitin 3l l0 Frroat tol crllrgl Flt md chitolan Mth@t bim.rier:30 Dsent lrydrctbont

lddfior of or$rtc nltrotcn: 55 9crcnt cnrd, lil Dmdld riNohorer: 141 ;oil Mthdt otianic nitrogen: 50.1 frud€ o 1,5 p€rcent ldellsg'' rrt nknluogetr F€ftst

.drc: 20 days nculatio with or w{thst effigill.tiol Incaptulatoni f,ithod h|d E 58nific.nt cfr€ct on PAH 5l Soll ) percefi {A bdim iBioate '79 percc$ ol 16 PNls rcwved In 791 doyt. l€irullalion: Mthoqt rclnfi ulatl,on: 68.93 wnt 'lrh rtsdv ioil !5.4f, perccnt ldclks-1 ms 5trein i/ith rain€ulauofi : 95i36 serent

Exhibit 5. Enhancement method in increasino of slte bio.emediation efficiency

observed to be quite similar to reactor I with a degradation rate of 12.31 rng/kg/day. After reinoculation on day 5, tie degradation rate was found to be 4.49 mg/kg/day, u'hich was much higher than that observed in reactor 1 . In evaluating the high potential bacteria, as previous\ discussed, bacteria in reactor 2 rvas isolated and inoculated in reactor 3. In reactor 3, the degradation rate was determined to be I 3.61 mg/kglday and 4,66 ng/ kg/ dxy in phase I and phase 2, respectively. As a whole, the degradation rate in phase 2 was lower than phase 1. As duration increased, concentrations of phenanthrene were reduced. Thus, it decreased the carbon sources to be degraded by the bacteria (Boopathy, 2000). The percentage removals for reactor 1, 2, efv|3 wcrc dctcrmincd to bc 58.93 persent, 86.75 percent, and 95,36 percent, respectively. Reactor 3 showed the highest degradation compared to reactor 1 and 2. Thus, ttre bacteria produced from the reinoculation process is the best phenanthrene degrader in contaminated sand. Exhibit 5 shows the enhancement methods of increasing bacteria performance in site bioremediation. Escalation of percentage removal from this study was slightly lower than t}rat demonstrated in a study conducted by Gentili et al. (2005). Hou'wer, the escalatiqn was higher t-han in other studies .orrdu.t"i by Liang et al. (2009) and liah and Ukpe (1992). Ontheotherhand, Karamalidisetal. 12010) showednosignificanteffectof the encapsulation methorl in PAH biodegradation. The study conducted by Gentili et al. (2006) showed the highest escalation of percentage removal. The studl was conducted in a liquid culture, that is, minimal salt medium. Thus, there was a higher bioavailability qf contauilnant$ corlpared to the soil rnedium (Liang et al., 2009). However, a study using a similar method of enhancement (Liang et al., 2009), that is, biocarrier, only showed I 1.49 percent of escalation of percentage reuroval. The results from Uang et al. show that biocarrier rnay notbe sufficient in soil medium. Utilizatiorr ofactivated carbon as a biocarrier by Liang et al. (2009) shorved the highest increases in bacteria concentration, that is, 1010 cells/g. However, this study only

126 Renediatior, DOlr 10.1(E2./rern 02014 Wilev Periooicals, lnc. REMEDIATION Su.nrcr 2014

showed a | | .49 percent €scalation of'percentage removal, which is lower than Gentili et al. (2005) and this study. In addition, the study used more than one species of bacteria, resulting in the idghest bacteria concentration. In this study, the bacteria concentration increased to 106 cells,/g after refuroculation, which is the sarne order ot'magnitude as the study conducted by Gentili et al. (2006) and Ijah and Ukpe (1992). However, this study showed the highest escalation of phenanthrene removal of the three studies.

CONCLUSION

The performance of S. spirftivorun can be enhanced through reinoculation with a percentage removal of 95.35 percent. On the other hand, without reinoculation, 68.93 percent of phenautlrrene was removed. It was also tbund that the strain ca&ot survive in 'sand. It was observed that t}e potential of a single cok-rnv increased after reinoculation. This result shows the early indication of survival on high potential basteria. Even though the increased bacteria concettrations are of the same order magnitude as prev'ious studies, the escalation of percentage removal from this study (26.43 pereent) was higher. Elucidating the mechanism of high potential bacteria in PAH degradation will require further research.

ACKNOWLEDGMENTS

Financial support lrorn Ge l{inistry of Higher Education under a Longlferm Research Grant Scheme and tlle Universiti Teknologi Mara under an Excellent Fund Scheme is gratefullv acknorvledged. Assistance fionr the malagenrent of the Marvar Wastewater Treatment Plant in providing raw wastelvater samples is greatly appreciated.

REFERENCES

Agathos, S. N., & Reineke, W (2002). Focus on biotechnology. Biotechnology for the environment: Strategy and fundamentals (Vol. 34,). Dordrecht, l,letherlands: Kluwer Academic Publisher.

Boopathy, R. (2000), Factors limrling bioremediation technologies. Bioresource Technology, 74(1r, 63-67.

Cao, 8., Nagarajan, K., & Loh, K. (2009). Biodegradation of aromatic compounds: Current gtatus and opportunities for biomolecular approaches. Applied Microbilogy and Biotechnology,85Q1,2A7-228.

Gent;li, A. R., Cubitto. M. A., Ferero, M., & Rodriguez, M. 5- (2006). Bjoremediation of crude oil polluted seawater by a hydrocarbon-degrading bacterial strain immobilized on chitin and chitosan flakes.

I nternation al B iodeterioration & B iodeg radatio n, 57 (4), 222*228. l.iah, U. J. J., & Ukpe, L. l. (1992), Biodegradation of crudo oil by Eacillus strains 28A and 618 isolated irom oil spilled soil. Management, 12(1), 55-60.

Johnsen, A. R., Wick, L. Y., & Harms, H. {2005). Principles oi microbial PAHdegradation in soil. Environmental Pollution, I 33(1), 71 -84.

Karamalidis, A. K., Evangelou, A. C., Karabika, E., Koukkou, A. 1.. Drainas, C., & Voudrias, E. A. (2O1O). Laboratory scale bioremediation of petroleum-contaminated soil by indigenous microorganisms and added Pseudomonas aeruginasa strain spet. Bioresource Technology, 101(16), 6545+552.

rO20l4Wiiey Periodlca s, nc. Rsmediation DOI: 10,1O0Zrem 127 Enh;ncing Per{orm6nqs e1 5thingobacteri u m spiritivo r u m

Kumar, M., Wu, P C., Tsai, J, C., & Un, J. G. (2009). Biodegradation of soil-applied polycyclic aromatic hydrocarbons by sulfate reducing bacterial consortium. Journal of Environmental Science and Health,

Part A, 44(1 ), 1 2*20.

Li, X. J., Li, P. J., Lin, X., Zhang, C. G., Li, O., & Gong, Z. O. (2008). Biodegradation of aged polycyclic

aromatic hydrocarbons {PAHs) by microbial consortia in soil and slurry phases. Journal o{ Hazandous

Marerials, 1 50(1), 21*26.

Liang, y T., Zhang, X., Dai, D. J", & Li, G, H. (2009), Porous biocarner-enhanced biodegradation o{ crude oil

contaminated soil. I nternational Biodeterioration & Biodegradalion, 63(1 ), 8G-87'

Meyer, D., Withok, 8., & Schmid, A. (2005). Suitability of recombinant Esherichia coli and Pseudomonas putida strain for selective biotrans{ormation o{ m-nitrotoluene by xylene monooxygenase. Applied and

Fnvironmental Microbiology. 7 1 (1 11, 66244632. ':jj;:, .:i*r Mrozik,.4., & Piotrowska-Seget, Z. (2010). Bioaugmentation as a st|.ategy for cleaning up o{ soils contaminated with aromatic compounds. Microbiological Research, 165(5), 363-375. i

Othman, N., Huqsain, N. H., Abd-Karim, A. T, & Abdul-Talib, S. (2009) lsolation and optimization o{ naphthalene degradative bacteria. The 1st International Seminar on Sustainable In{rastructure and Built $ Environment in Developing Countries, November 2-3, Bandung ilndonesia). 'ii Othmen, N,, Hussain, N. H-, & Abdul-Talib, S. {2010). lsolation and optimization of phenanthrene degradative bacteria from nrunicipal sludge for PAHs bioremediation' 3RD Southeast Asian Natural Resources and f{ Environn'iental Management, August 3-5, Kota Kinabalu, Sabah (Malaysia)-

Tonora, G. J., Funke, B. R., & Case, C. L. (2007). Microbiology: An introduction, fth ed. San Francisco, CA: li: Pearson Benlamin Cummings. -,i'a

Nor-Amani-Filzah Mohd-Kamil, MEnq, is a PhD student at Universiti Teknologi Mara, Shah Alam, Malaysia, Her research includes soil bioremediation and wastewater treatment.5he received her MEng in envi- ronment management and BEng in civil engineering from Universiti Teknologi Malaysia, Johor. Malaysia.

Noor-Hana Hussain, PhD, is a senior lectufer at the faculty of applied scienct, Universiti Teknologi Mara, Shah Alam, MalaVsia, Her research interests include the inducible stress responses in Gram negative bacteria, bacterial plant pathogens, antibiotic resistance, and quorum sensing.She is currently $/orking on four projects funded by the Ministry of Higher Education. Dr. Hussain received her BSc in biological sciences from University of [ssex, UK, and her FhD in microbiology from Univenity Coilege London' UK.

facuity science, technology, and human development, i Meizareena Binti Mizad is a lecturer at the of Universiti Tun Hussein Qnn Malaysia, Johor, Malavsia, She received her Bachelor of Human Sciences {English j language & literature) from Intemational lslamic university Malaysia, selangol Malaysia.

,:i Suhaimi Abdul-Talib is a professor 0f civil engineering at Universiti Teknologi Mara, Shah Alam, Malavsia. = His research interests include environmental management, polluti0n {ontrol, water and wastewater engineering' I bioremediation, management of water supply, and wastewater services. He is currently working 0n ten projects funded by thr Ministry of Higher Education and other agencies. Dr. Talib received his BE (Hons) civil from Univer- ; := sitv of Melbourne, Australia, his MSc in water ft environmental management {Distinction) from Loughborough a: University, UK, and his PhD in environmental engineering from Universiti Teknologi Malaysia, Johor, Malaysia'

2eriodicals, 128 Remediatiao DC)t: 10JAOZ./rerr @2014Wiley Inc