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Microbiological Research 163 (2008) 277—285

0944-5013/$ - sdoi:10.1016/j.

�CorrespondState of Qatar,

E-mail addr

www.elsevier.de/micres

Influence of phenolics on the sensitivity offree and immobilized bioluminescentAcinetobacter bacterium

Sahar Zakia, Desouky Abd-El-Haleema,�, Ashraf Abulhamdb,Hassan Elberyb, Gadallah AbuElreesha

aEnvironmental Biotechnology Department, Genetic Engineering and Biotechnology Research Institute, Mubarak Cityfor Scientific Research and Technology Applications, Post Code 21934, New Burg-Elarab, Alexandria, EgyptbMicrobiology Department, Faculty of Science, Alazhar University, Cairo, Egypt

Received 25 January 2006; received in revised form 5 March 2006; accepted 6 July 2006

KEYWORDSBioreporter;Phenols;Toxicity;Assessment;Immobilization

ee front matter & 2006micres.2006.07.006

ing author. Mailing addrPO Box 2713, Tel: (+97ess: abdelhaleemm@ya

SummaryIn this work, the constructed bioluminescent Acinetobacter strain DF4/PUTK2 wasemployed to assess the toxicity of phenolic compounds and the 5min EC50 valueswere calculated. The results of the DF4/PUTK2 assay were further evaluated bycomparing with the results of the Vibrio fischeri luminescence inhibition assay. Todevelop a bioassay system appropriate to be used in continuous toxicity testing,strain DF4/PUTK2 was subjected for immobilization in microtiter plates into thematrices Ca-alginate, polyacrylamide, agar and agarose. After a choice of materialswas tried, Ca-alginate was selected as the most suitable candidate material.Because, it could be stored at least 8 weeks at 4 1C, during which the ability of thebioreporter DF4/PUTK2 to detect the toxicity of phenolics was maintained. However,the stability of the bioluminescence for DF4/PUTK2 cells immobilized into agaroseand agar was significantly less than that of cells stored in alginate suspensions. Thisstudy recommended that luxCDABE-marked Acinetobacter strain DF4/PUTK2 couldbe employed to assay the ecotoxicity of environmental samples contaminated withphenols. The host strain of the bioreporter DF4/PUTK2 is Acinetobacter strain DF4. Itis known that members of the genus Acinetobacter are widespread in nature and alsoinvolved in biodegradation, leaching and removal of several organic and inorganicman-made hazardous wastes.& 2006 Elsevier GmbH. All rights reserved.

Elsevier GmbH. All rights reserved.

ess: Department of Biological Sciences, College of Arts and Sciences, University of Qatar, Doha,4) 4852702; fax: (+974) 4851584hoo.de (D. Abd-El-Haleem).

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Introduction

Phenols are distributed in various environmentalsites either as natural or man-made aromaticcompounds. Their existence as major pollutants inindustrial wastewater treatment plants, such as oilrefineries, petrochemical plants, coking plants, andphenol resin industry plants, has been well estab-lished (Rebhun and Galil, 1988; Watanabe et al.,1996). Phenol and its homologs exhibit environ-mental toxicity and are among the most frequentlyfound pollutants in rivers, industrial effluents, andlandfill runoff waters (Lee et al., 2006). They arealso problematic toxicants in waste treatmentsystems (Abd-El-Haleem et al., 2002a–c). Conse-quently, inexpensive and real-time methods fordetecting phenolic toxicity in the environment arewarranted.

Bioluminescent bacteria were proposed by Kahruet al. (2000) as whole-cell biosensors for thetoxicity monitoring of phenolic compounds. Theadvantages of a bioluminescent bacteria-basedtoxicity assay include; a very conveniently mea-sured signal, a short exposure time, and costefficiency (Ren et al., 2003). The most thoroughlystudied luminescent bacteria-based bioassay foraquatic toxicity testing is the Microtox assaymarketed by Azur Environmental (Carlsbad, CA).The bacterium used in the Microtox assay isPhotobacterium phosphoreum, a marine bacterialstrain. Pure cultures of freeze-dried P. phosphor-eum are purchased and reconstituted as neededand toxicity tests are carried out by mixing thereconstituted cells with aquatic samples for aspecified exposure time (usually 5, 10, or 15min)and measuring the bioluminescence in an analyzerat 15 1C. Since P. phosphoreum, the bacterial strainused in the Microtox assay, is a marine bacterium, itis excessively sensitive to many toxicants comparedto other methods (Ren et al., 2003). Additionally,for the assay to work well, filtration is requiredbefore every test, and it works only in salinesolution.

In the present work a new strain of luminescentbacterium, designated DF4/PUTK2, was con-structed for measuring phenolic toxicity. The hoststrain (Acinetobacter sp. DF4) of this bioreporterhas been previously used for phenol biodegradationand detection (Abd-El-Haleem et al., 2002a, b).Therefore, toxicity assaying using strain DF4/PUTK2 might be serves as a realistic indicator ofthe environmental toxicity. Species of Acinetobac-ter have been attracting increasing attention inenvironmental applications. Some strains of thisgenus are known to be involved in biodegradationof a number of different pollutants such as biphenyl

and chlorinated biphenyl, amino acids, phenol,benzoate, crude oil, acetonitrile, and in theremoval of phosphate or heavy metals (Abd-El-Haleem, 2003).

While the high sensitivity and applicabilityof genetically modified reporter microorganismsunder controlled laboratory conditions hasbeen repeatedly demonstrated, less attention hasbeen devoted to their long-term storage anduse in an immobilized form. The successful incor-poration of such microorganisms in solid-statematrices is an important step en-route to theconversion of these bioassays into user-friendlysensing devices capable of continual monitoringor multiple use detection (Premkumar et al., 2001).Here therefore, to serve as more realisticindicator of toxicity, selection an appropriateimmobilization matrix and the developmentof a system appropriates to commercialize strainDF4/PUTK2; a range of entrapment gels wasexamined.

Materials and methods

Bioluminescent bioreporter, optimization ofgrowth and light output

The bioluminescence toxicity assays were per-formed using Acinetobacter strain DF4/PUTK2that had been genetically modified by conjugation(AbuElreesh, 2005; Abd-El-Haleem et al., 2006)to contain the plasmid PUTK2 (Burlage et al.,1990), with the Tn4431 lux transposon down-stream of a putative plasmid maintenancepromoter to produce continual visible light so-called bioluminescence. The correlationbetween cell growth and light emission of theconstructed bioreporter DF4/PUTK2 was studied bygrowing cells on an orbital shaker incubator(200 rpm) at 30 1C in LB/tetracycline medium.Subsequently, both bioluminescence and theoptical density (at 600 nm) were measured to thesame sample each 30min intervals for 24 h.Bioluminescence of the culture was measured atregular time intervals by a plate luminometer(LumiStar, Galaxy, BMG, Germany). The lumin-ometer was set at a temperature of 30 1C and theplate shaker was set to high and a 1 s durationbefore each reading, which were done automati-cally every 5min. The results were transferredto Microsoft ExcelTM and analyzed and are pre-sented as the count per second (CPS) of thebioluminescence.

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Bioluminescence toxicity assay to differenttypes of phenols in liquid cultures

A culture of strain DF4/PUTK2 was grown in LBmedium (10 Bacto-Tryptone, 5 Bacto-yeast extractand 10NaCl g/l) on an orbital shaker at 200 rpm at30 1C for 16 h. Subsequently, the cells were diluted1:10 in LB medium and incubated on an orbitalshaker at 30 1C up to achieve an optical density 0.6measured with a spectrophotometer (SpectronicGenesys 5; Spectronic Instruments, Rochester, NY)at 600 nm wavelength. After two washes in a sterileLB medium, the pellet was resuspended in 1mlsterile LB. Two milliliters of the culture were thenadded to 20ml scintillation vials containing 2.0mlMSM supplemented with phenolic-saturated MSM(Abd-El-Haleem et al., 2002b) at final concentra-tions ranging from 0 to 1000 ppm. Aliquots of 200 mlwere removed from each scintillation vial andtransferred to low-fluorescence 96-well microtiterplates (Nunc) to produce three replications of eachdilution.

Along with each treatment two controls wereincorporated into each plate, the first containedonly MSM medium, while the second contained thewild-type Acinetobacter strain DF4 as a negativecontrol. Wells were covered with transparent platesealer and placed in the luminometer for lumines-cence detection at room temperature. Duration ofexposure was the same for all phenolics andconcentrations, ranging from zero (just afteraddition of the phenolics in the medium) to amaximum of 350min. All phenolics were of analy-tical grade and were used without further purifica-tion. Phenol, 4-nitrophenol, 3,5-dichlorophenol, 5-chlorophenol and chatechol were purchased fromSigma chemicals (St. Louis, USA). In all measures,the values of average and standard deviation wereobtained from triplicate measurements.

The toxicity of the toxicants was reflected by therepression of DF4/PUTK2 bioluminescence as de-fined by equation described previously by Ren andFrymier (2003). Calculated bioluminescence re-pression (BR%) was then plotted versus the con-centrations of the phenolic compounds, and theconcentrations that corresponded to 50% biolumi-nescence repression were recorded as the observedDF4/PUTK2 EC50 values.

Chemical spiking test of field water samples

In February 2006, a water sample used fortoxicity monitoring was obtained from Burg-Elarabdrinking water treatment plant. The sample wastaken just before it entered the treatment plant

(pH of the sample was around 6.9). Prior toanalysis, water sample was filtered through 0.2-mm-syringe filter to remove suspended matter andbacteria. Prior to subject the samples to toxicitybioassay, phenol concentration was determinedusing the 4-aminoantipyrene method (Eatonet al., 1995). For the bioluminescence assays,aliquots 100 ml of the filtered water sample mixedwith 100 ml of a PUTK2/DF4 cell suspension weretransferred to 96-well microtiter plates (Nunc).Subsequently, the plates were subjected for lumi-nescence detection at room temperature as de-scribed above. The water sample was spiked withdifferent concentrations of phenol adjusted to 50,100, 200, 300, 400, 500 and 800 ppm, respectively.

Immobilization experiments

Immobilization conditions of DF4/PUTK2 cellswere compared for four different matrices includ-ing calcium alginate agar/agar, agarose and poly-acrylamide in 96 well plates. Cell immobilizationwas performed into calcium alginate with slightmodification according to Abd-El-Haleem et al.(2002c). Briefly, liquid cultures were centrifuged ina 50-ml plastic centrifuge tube (2500g) at roomtemperature for 10min and the supernatant wasdiscarded. The pellet was resuspended with apreviously autoclaved solution of sodium alginatewith a final concentration of 4% (w/v) and 10% (v/v)bacterial biomass. The alginate-bacterial mixturewas added drop-wise with sterile syringe (20ml)fitted with a wide bore needle (2mm diameter)into a 96 well microtiter-plate containing (dropfor each well) 25 ml solution of calcium chloride{3% (w/v), adjusted to pH 7.0} and the entrapmentalginate gel was left for hardening overnightat 4 1C.

Entrapment of cells into polyacrylamide wasperformed by re-suspending 100mg of washed cellsin 1ml of physiological saline solution at 0 1C with90mg acrylamide, and 10mg N,N0-methylenebisa-crylamide. The suspension was then saturated withnitrogen. Aliquot of 150 ml was pipetted into eachwell of a microtiter plate. While, the polymeriza-tion was initiated by adding 1 ml of 10% (w/v)dimethylaminopro-pionitrile and 0.1 mg potassiumpersulfate. Agar and agarose entrapment wasperformed by dissolving 200mg agar or agarose in10.0ml physiological saline at 100 1C, and thencooled to 50 1C. Subsequently, the agar or agarosesolution was mixed with the cell suspensionprepared as described above with polyacrylamideentrapment. Then the matrices with the immobi-lized organisms poured in a microtiter 96 well

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plate, 150 ml/well. The 96 well plates were allowedto stand as described above to ensure that theimmobilization matrices solidified, fitted with aplastic cover to prevent evaporation from theimmobilization matrix, stored at 4 1C until re-quired. Bioluminescence was monitored for bothfree and immobilized cells.

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Results

Optimization of growth and light output

Figure 1 shows the results of a bioluminescentbacteria culture to determine the time course ofluminescence and absorbance. The optical densityvalue is correlated to the bacteria number andindicates bacterial growth; the bioluminescence iscorrelated to the bacteria number and to lumines-cent efficiency. As shown in Fig. 1, the lumines-cence peak corresponds to bacterial growth and inthe early logarithmic phase (after �30min incuba-tion) the maximum luminescent resulted value wasrecorded. At this growth point, the optical densityvalue of the strain DF4/PUTK2 at 600 nm (OD600 nm)reached 0.6. Therefore, in order to obtain thehighest luminescence during assays, cultures wereroutinely grown to OD600 nm ¼ 0.6–0.7.

Specific responses of the strain DF4/PUTK2to phenolics in liquid cultures

The influence of exposure period of strain DF4/PUTK2 for detecting phenolics toxicity was eval-uated using a constant phenolic concentration(100 ppm each) at 5min intervals assay for

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350min. As shown in Fig. 2, an immediate inhibi-tion of the bioluminescence was occurred with 3,5-dichlorophenol, 5-chlorophenol, 4-nitrophenol cha-techol and phenol, respectively. However, reduc-tion of the bioluminescence values was showedwith phenol in the first 50min exposure thenincreased dramatically to reach values approxi-mate to the control.

The bioluminescence courses at the shortestexposure time for the bioassay (5min), underinfluence of various phenolic concentrations arepresented in Fig. 3. These experiments wereperformed for both free and immobilized cells atthe same time and in the same plates. As shown inFig. 3, 3,5-dichlorophenol, 5-chlorophenol, 4-ni-trophenol, chatechol and phenol, respectively,showed significant reduction (Po0:01, t-test) ofthe bioluminescence values. The data showed alsothat bioluminescence values of all phenolics wereincreased by increasing the concentrations. Thecalculated DF4/PUTK2 5min EC50 values of the fivephenolic compounds are given in Table 1. For thepurpose of comparison, the 5min Microtox EC50

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Table 1. EC50 values {5min data (ppm)} of phenolic compounds of free and immobilized PUTK2/DF4 cells comparedwith Microtox assay

Compound Free-cells of Observed PUTK2/DF4 cells immobilized into

Observed PUTK2/DF4 Microtoxa SHK1a Ca-alginate Polyacrylamide Agar Agarose

Phenol 170 18 482 340 351 773 3484-nitrophenol 65 6.4 43 136 295 185 1375-chlorophenol 10.8 3.2 20 132 151 183 1333,5-dichlorophenol 8.1 7.3 41 168 182 238 172Catechol 136 — — 235 240 308 279

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Influence of phenolics on the sensitivity of free and immobilized bioluminescent A. bacterium 281

values and the strain Shk1 EC50 values are alsoshown. A comparison of the DF4/PUTK2, Shk1 andMicrotox EC50 values shows that DF4/PUTK2 ismore sensitive than Shk1, while it was slightly lesssensitive than Microtox to all phenolic compoundstested.

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Figure 4. Bioluminescent inhibition percentages ofstrain DF4/PUTK2 during toxicity monitoring of phenolspiked drinking water and municipal wastewater.

Chemical spiking test of field water sample

To show the possibility of using the strain DF4/PUTK2 to monitor phenolics in real ecosystems,drinking water spiked with different concentrationsof phenol were tested. No phenol was detected inthe sample when assayed colorimetrically with4-aminoantipyrene (detection limit ¼ 1 ppb). Asshown in Fig. 4 the unspiked drinking water samplewas considered as non-toxic (BR% ¼ 0.0). However,450% bioluminescence inhibition was observed atphenol concentration 300 ppm (BR% ¼ 54.73) ex-hibiting EC50 value of 333 ppm. In contrast, thebioluminescence was markedly depressed (450%inhibition) at concentrations 400 ppm, exhibiting BIvalue of �87%. It was also observed that increasingphenol concentration increased the biolumines-cence inhibition values up to phenol concentration500 ppm (BR%X99%).

Immobilization experiments

Cells of strain DF4/PUTK2 were immobilized incalcium alginate, agarose, agar/agar and polyacry-lamide. Phenolics were added to DF4/PUTK2 cellsin microtiter plates at different concentrationsranging from 50 to 1000 ppm in a 5min assay. Asshown in Figs. 5(A)–(C), with all phenolics theimmobilized cells in the four matrices showed lowbioluminescence inhibition values compared withthat of free cells. These results indicate thatprotection of cells caused by the immobilization.Phenol and catechol gave an induction case atconcentration 50 and 100 ppm with all carriers

tested. Out of the EC50 values presented in Table 1,it was noted that the DF4/PUTK2 strain gave muchmore sensitive responses when immobilized withinCa-alginate beads, while it was less sensitive whenagar, agarose and polyacrylamide were used as thematrix.

Stability of the response

The sensing ability of strain DF4/PUTK2 to aphenolic compound during long-term storage wasexamined. After removing the plates from 4 1Cstorage, they were allowed to warm for 30min at30 1C. A 100 ml sample of 5-chlorophenol stocksolution was injected into each well, and lumines-cence was measured at room temperature.5-chlorophenol (50 ppm) was selected as sampletoxic chemical to demonstrate the dose response ofthe bacteria to toxicity, toxic mode of action andthe high throughput character of this system. Theability of the strain to sense toxicity was main-tained for 8 weeks.

As shown in Fig. 6A, the maximum biolumines-cent levels of the control in the first 3 weeks were

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Figure 5. Influence of phenolic compounds at different concentration (50–1000 ppm) on the sensitivity of theimmobilized DF4/PUTK2 cells into Ca-alginate (A), polyacrylamide (B), agar (C) and agarose (D), respectively.

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significantly higher than the rest of storage period(Po0:05, t-test). All matrices showed similarcharacteristics in this period, except that thebioluminescence signal was higher with Ca-algi-nate. Though we did not test cell viability duringlong-term assay, bioluminescent level changes ofthe control wells can explain the cell viability. Themaximum bioluminescent levels of the control inthe last week were significantly lower (Po0:01, t-test) than in the first one.

As shown in Fig. 6B, no observed losses inthe ability of the strain DF4/PUTK2 to sense thetoxicity of pentachlorophenol up to the end of thestorage period, especially with Ca-alginate gel.Bioluminescence inhibition values of Ca-alginateimmobilized cells remains unchanged regardless ofthe reduction of bioluminescence production in thecontrol cells (prior to the addition of pentachlor-ophenol). The BI% in the first 3 weeks with Ca-alginate immobilized cells were ranged from 95% to93%. In the rest of the storage period (4–8 weeks),BI% were ranged from 72% to 68%, respectively,exhibiting lose of the bioreporter sensibility of

�20%. It is also observed that these sensibilityvalues was in the range to that obtained with thefree and freshly prepared DF4/PUTK2 cell suspen-sion at the same pentachlorophenol concentration(50 ppm, BI% ¼ 89.58).

Discussion

Among many toxicity assays, bioluminescenceassays are advantageous because they offer a quickresponse, usually on the order of minutes. Inaddition to the naturally occurring marine luminousbacterium Vibrio fischeri, which is widely used inaquatic toxicity tests (Jennings et al., 2001),constitutive luminescent genetically modified mi-croorganisms (GEMs) have also been created inrecent years (Gu and Gil, 2001; Kelly et al., 1999;Kim et al., 2003; Ren and Frymier, 2003; Ren andFrymier, 2005; Wiles et al., 2003). Out of thesestudies, it shown that each species and testprocedure has its own sensitivity pattern to

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Figure 6. The bioluminescence production and reductionlevels produced by immobilized strain DF4/PUTK2 duringa storage period for 8 weeks; (A) control, wherein nopentachlorophenol was added in the plates, (B) toxicityresponse to 50 ppm pentachlorophenol.

Influence of phenolics on the sensitivity of free and immobilized bioluminescent A. bacterium 283

toxicants. Recently, in this regard we (Abd-El-Haleem et al., 2006) described the use of aluxCDABE-marked Acinetobacter bacterium DF4/PUTK2 to assay the ecotoxicity of wastewater andeffluent samples contaminated with heavy metals.This strain was sensitive to Zn, Cd, Fe, Co, Cr andCu in order of decreasing sensitivity. The samepattern of sensitivity was observed when severalcontaminated water and wastewater effluentswere assayed.

However, more research was needed to evaluateits response to different types of toxicants.Phenolic compounds, as sewage components, arehigh on the list of organic pollutants with harmfulimpact on water systems (Kudryasheva et al.,2002). Therefore, the present study was conductedto employ strain DF4/PUTK2 to assess phenolicstoxicity. It is known that the number of phenoliccompounds currently used for industrial and agri-cultural purposes is very large and continues toincrease, and it is difficult to test the toxicities ofall of them. For these reasons, the compounds

involved in this study were limited to phenoliccompounds because they usually exhibit toxicity bythe polar narcosis mechanism (Verharr et al.,1992). Studies in the literature have shown thattoxicologically meaningful QSARs are developed forcompounds having the same toxic mechanism(Cronin and Dearden, 1995; Schultz et al., 1996).Our results proved that at lower concentration,3,5-dichlorophenol was the most effective biolumi-nescence inhibitor followed by 5-chlorophenol,4-nitrophenol, catechol and phenol respectively.Thus, in fact confirms the hypotheses that theincrease in chlorine number increases membranedamage (Bragadin and Dell’Antone, 1996; Benkoet al., 2006). Previously, Kim et al. (2003) reportedthat the E. coli strain GC2 was sensitive only to 2,4-chlorophenol and 2-chlorophenol among six pheno-lic species tested.

The EC50 values for five phenolic compoundswere determined using a DF4/PUTK2-based assay.DF4/PUK2 was less sensitive for all tested phenolicsthan Photobacterium phosphoreum, the bacteriumused in the Microtox assay. However, DF4/PUTK2was more sensitive than the strain Pseudomonasfluorescens Shk1 (Ren and Frymier, 2003), the firstluminescent GEM described by Kelly et al. (1999)for assessing wastewater toxicity. It is known thatthe utility of using native strains from such highlypolluted systems to provide microorganisms withhigh level tolerance in that they can be deployed incompartments upstream of the most active biolo-gical treatment systems, to provide robust, earlywarning systems of acute toxicity which could leadto process failure (Ren and Frymier, 2005).

In this regard, the parent strain of the biorepor-ter DF4/PUTK2 is belonging to the genus Acineto-bacter. This genus is environmentally relevant andwidespread in nature, and can be obtained fromwater, soil, living organisms and even from humanskins. They also involved in biodegradation, leach-ing and removal of several organic and inorganicman-made hazardous wastes (Abd-El-Haleem,2003). In the evaluation of drinking water sampleusing phenol as the sole toxicant, the bioreporterstrain DF4/PUTK2 displayed luminescence responseto increasing phenol concentrations up to 400 ppm,which allowed it to be used in highly toxic systems.This is in direct contrast with the response of themarine bacterium V. fischeri which exhibitedpreviously (Wiles et al., 2003) a limited sensingrange in response to wastewater samples, andnegligible luminescence output was observedwhere phenolic concentrations exceeded150–200 ppm.

For the development of a biosensor or a probe forpollution monitoring, immobilization of whole cells

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on or in solid supports has several advantages overfree cells, including the potential for stabilizingbiological activity. Whole cell immobilization isconvenient, has a short preparation time and isrelatively economical. Here, we show from theEC50 values presented in Table 1 and the dataillustrated in Fig. 5 that immobilized DF4/PUTK2cells are protected from the toxicity and were lesssensitive to the majority of the phenolic com-pounds tested in compare to the free cells.However, DF4/PUTK2 cells stored in Ca-alginateshowed the highest sensitivity in compare to othertested entrapment gels. With regard to theobservations, Heipieper et al. (1991) supposedthat, phenols as membrane-active compounds atsublethal concentrations are less inhibitory toimmobilized microorganisms than they are to freemicroorganisms. In addition, our previous studies(Abd-El-Haleem et al., 2002c) demonstrated thatthe application of immobilized Acinetobacter cellsin wastewater treatment offered the possibility ofdegrading higher concentrations of phenol(500 ppm) than can be achieved with free cells.

Here a long-term experiment was performed todetermine how immobilizing materials and storageconditions affected bioluminescence stability.Figure 6 shows results obtained following storageover 8 weeks at 4 1C for DF4/PUTK2 cells gelled inimmobilizing materials Ca-alginate, agar, agaroseand polyacrylamide. Among the four examinedentrapment gels, Ca-alginate was chosen as themost suitable candidate material in the presentcontext. Because, it could be stored at least 8weeks at 4 1C, during which the ability of thebioreporter DF4/PUTK2 to detect the toxicity of 5-chlorophenol was maintained. Thus, indicating thatthe presence of alginate did not impair cellmetabolism and, indeed, assisted its maintenance.In addition, over the first 3 weeks of storage, cellsgelled in polyacrylamide showed a similar patternof bioluminescence decline but stability declinedthereafter. In contrast to this finding, previously,Chun et al. (1996) reported that cells of thePhotobacterium phosphoreum (Microtox bacter-ium) were directly died when stored in polyacryla-mide gel. Our results further showed that thestability of bioluminescence for DF4/PUTK2 cellsimmobilized into agarose and agar was significantlyless than that of cells stored in alginate suspen-sions. This suggesting that either the gellingmaterials or the immobilization processes hadharmful effects on the bacteria.

In summary, the results reported in this paperindicate that DF4/PUTK2 can be successfullyimmobilized and maintained in Ca-alginate, andthat immobilized cells can be used to detect the

presence of phenolic pollutants. However, ourresults show that a DF4/PUTK2-based toxicity-monitoring assay would be more appropriate forthe influent toxicity monitoring, and compared tothe Microtox assay such a system would reduce thefrequency that an incoming wastewater is diverted.Therefore, currently we are conducting field-scaleexperiments in an industrial wastewater treatmentplant. The field-scale experiments will enable us toevaluate the performance of the DF4/PUTK2-basedassay for mixtures of toxicants.

Acknowledgements

This work was supported completely by GeneticEngineering and Biotechnology Research Institute,Mubarak City, for scientific research and technologyapplications, Alexandria, Egypt. Great thanks toboth Prof. Dr. Gay Sayler and Prof. Dr. Alice Laytonat Center for Environmental Biotechnology, Ten-nessee University, Knoxville, USA, who kindlyprovided us the plasmid PUTK2.

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