Pentachlorophenol (PCP) and its sodium salt (Na-PCP) are extremely toxic chemicals responsible for important soil and groundwater pollution, mainly caused by wastes from wood-treatment plants, because chlorinated phenols are widely used as wood preservatives. The methods most commonly used for routine analysis of pesticides such as PCP and Na-PCP are high-performance liquid chromatography (HPLC) and gas chromatography– mass spectroscopy (GC–MS). A variety of rapid biological screening tests using marine organisms, bioluminescent bacteria, and enzymes have also been reported. In this study, rapid biological screening analysis using Bacillus subtilis was developed, to assess the biodegradation of PCP and its by-products in liquid samples. An empirical model is proposed for spectrophotometric analysis of Na-PCP concentration after growth of Bacillus subtilis

Ayude,  M. A.

González, J. F.

Haure, P. M.

Murialdo, Silvia Elena

Okada, E.

Undetermined

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Bacillus subtilis as a bioindicator to estimate pentachlorophenol toxicity and concentration

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ORIGINAL PAPERBacillus subtilis as a bioindicator for estimatingpentachlorophenol toxicity and concentrationM. A. Ayude Æ E. Okada Æ J. F. González ÆP. M. Haure Æ S. E. MurialdoReceived: 30 December 2008 / Accepted: 12 February 2009! Society for Industrial Microbiology 2009Abstract Pentachlorophenol (PCP) and its sodium salt(Na-PCP) are extremely toxic chemicals responsible forimportant soil and groundwater pollution, mainly causedby wastes from wood-treatment plants, because chlorinatedphenols are widely used as wood preservatives. Themethods most commonly used for routine analysis ofpesticides such as PCP and Na-PCP are high-performanceliquid chromatography (HPLC) and gas chromatography–mass spectroscopy (GC–MS). A variety of rapid biologicalscreening tests using marine organisms, bioluminescentbacteria, and enzymes have also been reported. In thisstudy, rapid biological screening analysis using Bacillussubtilis was developed, to assess the biodegradation of PCPand its by-products in liquid samples. An empirical modelis proposed for spectrophotometric analysis of Na-PCPconcentration after growth of Bacillus subtilis.Keywords Pentachlorophenol ! Bacillus subtilis !Bioindicators ! Toxicity assessmentIntroductionSubstantial amounts of chlorinated aromatic compounds,including chlorophenols, have been discharged into theenvironment [1]. Chlorination of phenol greatly increasesthe toxicity of the resulting molecules relative to phenol,consequently pentachlorophenol (PCP and Na-PCP) is byfar the most studied because of its high toxicity. PCP andNa-PCP are extremely toxic chemicals responsible forimportant soil and groundwater pollution, principally fromwood-treatment plants, because chlorinated phenols arewidely used as wood preservatives. At least, 32 photo-lytic or biodegradation products of PCP and Na-PCPhave been identified, including: octachlorodibenzo-p-dioxin, tetrachlororesorcinol, tetrachloro, trichloro, anddichlorophenols, pentachloro, tetrachloro, trichloro, anddichloroanisoles, and tetrachlorohydroquinone [2]. Manyof these constituents are potentially toxic, mutagenic, orteratogenic [3].The most commonly employed methods for routineanalysis of pesticides such as PCP and Na-PCP arehigh-performance liquid chromatography (HPLC) andgas chromatography–mass spectroscopy (GC–MS). Thesemethods enable reliable analysis of compounds at the tracelevel but require sophisticated and expensive equipment.The most promising alternatives in environmental toxi-cology include the use of biomolecules and organisms withlimited sensitivity, for example enzymes, bacteria, fungi,and algae [4]. For example, in toxicity assays (Microtox(Azure, Bucks, UK) or ToxAlert (Merck, Darmstadt,Germany)), whole organisms are used to measure thetoxicity of water and soil samples. These systems are basedon use of the luminescent bacteria Vibrio fischeri to mea-sure the toxicity of environmental samples. Furthermore,Escherichia coli has been genetically modified with light-producing genes and, under normal conditions, give outvisible light (bioluminescence) [5, 6]. Young et al. [4]investigated the effect of PCP on two isozymes of lactatedehydrogenase (LDH) and found that detection limits canM. A. Ayude (&) ! P. M. HaureDiv. Catálisis, INTEMA, Facultad de Ingenierı́a,Universidad Nacional de Mar del Plata (UNMdP),Mar del Plata, Argentinae-mail: mayude@fi.mdp.edu.arE. Okada ! J. F. González ! S. E. Murialdo (&)Grupo de Ingenierı́a Bioquı́mica (GIB),Departamento de Ingenierı́a Qca, Facultad de Ingenierı́a,UNMdP, Mar del Plata, Argentinae-mail: silviem@fi.mdp.edu.ar123J Ind Microbiol BiotechnolDOI 10.1007/s10295-009-0550-ybe significantly reduced by modification of enzyme inhi-bition assays with a non-optimum cofactor.Repetto et al. [7] studied the specific effect of PCP usingdifferent ecotoxicological model systems, includingimmobilization of Daphnia magna, bioluminiscence inhi-bition in the bacterium Vibrio fischeri, growth inhibition ofthe alga Chlorella vulgaris, and micronuclei induction inthe plant Allium cepa. PCP was very toxic to all biota andcells; micronuclei induction in Allium cepa was the mostsensitive.The evolution of toxicity and biodegradability of liquidstreams from oxidation treatments should be evaluated, inorder to effectively assess the impact of discharge ofxenobiotics on the biota of the receiving water bodies. Inthis sense, bioassays provide an alternative because theyconstitute a measurement of environmentally relevanttoxicity. In this study, a method for rapid biologicalscreening analysis was developed to assess the biodegra-dation of Na-PCP, and the presence of toxic by-products inliquid samples. An empirical model is proposed for simplespectrophotometric (A600) estimation of Na-PCP concen-tration after growth of Bacillus subtilis.Materials and methodsChemicals and reagentsPentachlorophenol (99% pure) was obtained from SigmaChemical (St Louis, MO, USA). The salt sodium penta-chlorophenate (Na-PCP) was used in all assays. Allchemicals used were of the highest purity availablecommercially.PCP mineralizationMixed culture [8] able to degrade Na-PCP, was inocu-lated in mineral salts (MS) solution (in grams per litre:NaNO3 2.25, K2HPO4 2.83, KH2PO4 0.507, MgSO4.7H2O 0.45, FeSO4 0.135, and CaCl2 0.45) plus Na-PCP asthe only source of carbon and energy (NaPCP–MSmedium). Details on culture conditions and media aregiven in Murialdo et al. [8]. Acclimated cells in NaPCP–MS liquid medium containing 25–500 mg/l Na-PCP wereused as the inoculum for all Na-PCP degradation andtoxicity experiments. Batch culture experiments werecarried out in 250 ml Erlenmeyer flasks filled with 50 mlNaPCP–MS medium, and attached to a shaker (120 rpm)at 25"C.Liquid samples were periodically withdrawn and filter-sterilized (pore size 0.22 lm) for further analysis. Na-PCPcontent was determined with a Shimadzu UV–visiblespectrophotometer by comparison with a standard curve(Na-PCPAbs320 = 1 corresponds to 50 mg/l). The flaskswere kept at 25"C in the dark to avoid photo-decomposi-tion of Na-PCP. In each experiment a flask with noinoculum was run as control for Na-PCP volatilisation [9].Toxicity bioassaysA suspension of the bacterium Bacillus subtilis ATCC6051 sensitive to the bactericidal action of PCP and manyof its metabolites [10] was used. Samples taken periodi-cally from batch Na-PCP-biodegradation experiments werefilter-sterilized (pore size 0.22 lm). Different dilutions, inthe range 1/2 to 1/128, were made in tryptone broth (TB),and these were then inoculated with B. subtilis (10% v/v)previously grown in TB overnight. After incubation timesof 48 h at 120 rpm and 25"C, the growth of B. subtilis wasassessed by A600. The results were compared with theoutput of B. subtilis ATCC 6051 exposed to differentdilutions of Na-PCP in TB. The concentrations of Na-PCPwere determined by HPLC (Shimadzu LC-10AD). Parallelto this assay, the last sample depleted of Na-PCP was alsoanalyzed by HPLC in order to confirm the disappearance ofNa-PCP and its by-products.Additional experiments were performed to quantify andcorrelate the bacterial growth with A600. Bacteria weregrown in an Erlenmeyer flask with 0.5 mg dehydrated LBmedium powder per millilitre at 25"C. After 24 h ofincubation the cells were collected at 14,000 rpm. Thecells were re-suspended in 2 ml phosphate buffer. Serialdilutions were made and A600 was determined using aShimadzu UV–visible spectrophotometer. Each dilutionwas surface plated on 0.5 g dehydrated LB medium, and1.5 g of agar per 100 ml. The plates were incubated at25"C, and the colonies were counted after 48 h andexpressed as colony-forming units per milliliter (CFU/ml).Dry weight was determined by weighing the cells afterdrying in an oven at 105"C for 24 h. Figure 1 shows thecorrelation of CFU/ml and dry weight as a function ofA600.0.0E+005.0E+081.0E+091.5E+092.0E+092.5E+093.0E+090 0.2 0.4 0.6 0.8 1A60000.10.20.30.40.50.6CFU/mlDry weight (g/ml)Fig. 1 Colony-forming units per millilitre (filled diamonds) and dryweight (open diamonds) as a function of A600 for B. subtilis growthJ Ind Microbiol Biotechnol123Statistical analysisAll yield measurements were performed three times induplicate. Statistically significant differences between A600measurements were evaluated by use of Excel software.The significance levels are quoted at the 95% confidencelevel (P B 0.05).Results and discussionEstimation of Na-PCP using B. subtilis as a bioindicatorControl assayNa-PCP toxicity to Bacillus subtilis was investigated usinga known concentration of Na-PCP in a control assay.Results are presented in Fig. 2. Cell growth was apparentwhen the absorbance at 600 nm was greater than 0.1. Thus,B. subtilis was not able to grow when Na-PCP concentra-tion was above 1.5 mg/l, and maximum growth wasachieved without Na-PCP.The dependence on Na-PCP concentration of B. subtilisgrowth for Na-PCP concentrations below 1.5 mg/l wasinvestigated thoroughly. Data were correlated using thenonlinear regression package MathCad (genfit function) as:PCP "mg=l# $ 0:4036A600% 0:3321 "1#This method is suitable for predicting responses forA600[ 0.1. The maximum growth of B. subtilis during48 h of incubation corresponds to an absorbance (A600) ofapproximately 1. Figure 2 shows that fitting is moreaccurate for A600[ 0.2, which is for Na-PCP contentbelow 1 mg/l. Therefore, monitoring the growth ofB. subtilis by use of A600 enables estimation of Na-PCPconcentration.Na-PCP mineralisation: toxicity screeningNa-PCP mineralisation experiments, using mixed culture[8], were performed in a batch reactor. Samples wereperiodically withdrawn and centrifuged. Na-PCP concen-tration was determined by A320. Then, samples were filtersterilized (pore size 0.22 lm) and diluted (1/32) in TBbefore inoculating B. Subtilis in order to analyze thedependence of the growth of B. subtilis in media previouslytreated with mixed culture. Results are presented in Fig. 3.These outcomes effectively demonstrate that the media,after being treated with mixed culture, does not containintermediate products toxic to B. subtilis. This was alsoconfirmed by parallel HPLC assays (data not shown).For the first samples taken from the batch reactor (0 and6.33 h), 1/32 dilution is not enough to allow B. subtilisgrowth (Fig. 3), because Na-PCP concentration is greaterthan 1.5 mg/l. Consequently, in the whole set of samples,different dilutions in TB from 1/2 to 1/128 were made forall the samples taken in biodegradation experiments. Eachdilution was then inoculated with a B. subtilis suspensionof known concentration. This procedure is useful todetermine in which range the dilution should be made toenable B. subtilis growth. Na-PCP concentration wasdetermined by A320. Results for a typical experiment areshown in Table 1. When A600 & 0.1 after 48 h of incu-bation at 25"C, Na-PCP concentration was estimated usingEq. (1). Thus, the estimated Na-PCP concentration in theoriginal sample was obtained. For the whole testing data-set, the mean relative error was 17.3%.ConclusionsThe toxicity of chlorophenols varies between differentcongeners and bacteria. In our results we found that theminimum concentration of Na-PCP, which completelyinhibited growth of B. subtilis ATCC 6051, was above00.20.40.60.810 5 10 15 20 25A600Na-PCP (mg/l)Fig. 2 Control assay: filled diamonds, experimental data; solid line,genfit fitting; dashed line, A600 = 0.1 (the dashed line represents thevalue above which the growth of B. subtilis can be observedspectrophotometrically)012340 5 10 15 20time (h)00.20.40.60.81A600Na-PCP (mg/l)Fig. 3 Effect of residual PCP concentration on B. subtilis growth inculture media (with PCP-SM) where mixed culture has previouslybeen grown: filled diamonds, Na-PCP (m/l); open squares, A600J Ind Microbiol Biotechnol1231.5 mg/l. It is clear that B. subtilis is sensitive, specific, anduseful for detecting low levels of Na-PCP concentration.The bioassay is simple, inexpensive, and fast, and does notrequire complex sample-preparation procedures or sophis-ticated analytical instrumentation. An empirical modelenables estimation of Na-PCP concentration within 17.3%error. This toxicity test may therefore enable accurateassessment of the efficacy of bioremediation of contami-nated soils and groundwater, especially for compoundssuch as Na-PCP, for which many toxic degradationby-products may exist. Several bacteria degrade PCP andNa-PCP but there are various unknown toxic metabolicintermediates which could be detected by sensitive bacteriasuch as B. subtilis.Furthermore, this work contributes an effective tool thatcan be used in situations in which technology presentstemporary constraints. Although this research used a spec-trophotometer, it avoids the use of UV light, which isnecessary to detect Na-PCP. Moreover, the use of a spec-trophotometer can be replaced when using the direct methodof microorganism counting (CFU) or dry weight (g/ml).Acknowledgments The authors would like to thank Dr. ClaudiaStuddert and Dr. Celina Martone for their support, CONICET forassisting with fellowships, SECyT for financial aid, and INTEMA,CONICET, and Universidad Nacional de Mar del Plata for facilitatingthe development of this research.References1. Steinle P, Stucki G, Stettler R, Hanselmann KW (1998) Aerobicmineralisation of 2,6-dichlorophenol by Ralstonia sp. strain RK1.Appl Environ Microbiol 64:2566–25712. Middaugh DP, Resnick SM, Lantz SE, Heard CS, Mueller JG(1993) Toxicological assessment of biodegraded pentachloro-phenol: microtox and fish embryos. Arch Environ ContamToxicol 24:165–1723. Lu PRL, Metcalf E, Cole LK (1977) The environmental fate of14C-pentachlorophenol in laboratory model ecosystems. In: RaoKR (ed) Pentachlorophenol: chemistry, pharmacology, andenvironmental toxicology. Plenum, New York, pp 53–634. Young J, Dowman AA, Cowell DC (1999) The detection ofpentachlorophenol by its inhibitory effectiveness on lactatedehydrogenase of rabbit muscle and Bovine Heart. J PestBiochem Physiol 64:1–8. doi:10.1006/pest.1999.24045. Redshaw N, Dickson SJ, Ambrose V, Horswell J (2007) A pre-liminary investigation into the use of biosensors to screenstomach contents for selected poisons and drugs. Forensic Sci Int172:106–1116. Rattray EAS, Prosser JI, Killham K, Glover LA (1990) Lumi-nescence-based nonextractive technique for in situ detection ofEscherichia coli in soil. Appl Environ Microbiol 56:3368–33747. Repetto G, Jos A, Hazen MJ, Molero ML, del Peso A, SalgueroM, del Castillo P, Rodriguez-Vicente MC, Repetto M (2001) Atest battery for the ecotoxicological evaluation of Pentachloro-phenol. Toxicol In Vitro 15:503–509. doi:10.1016/S0887-2333(01)00055-88. Murialdo SE, Fenoglio R, Haure PM, Gonzalez JF (2003) Deg-radation of phenol and chlorophenols by mixed and pure cultures.Water SA 29:457–4639. Radehaus RM, Schmidt SK (1992) Characterization of a novelPseudomonas sp. that mineralizes high concentrations of penta-chlorophenol. Appl Environ Microbiol 58:2879–288510. Ruckdeschel G, Renner G, Schwarz K (1987) Effects of penta-chlorophenol and some of its known and possible metabolites ondifferent species of bacteria. Appl Environ Microbiol 53(11):2689–2692Table 1 Results for a typical PCP biodegradation experimentTime (h) PCP in originalsample (mg/l)aSampledilutionPCP in dilutedsample (mg/l)aA600 (dilutedsample)bEstimated PCP in dilutedsample (Eq. 1) (mg/l)Estimated PCP inoriginal sample (mg/l)0 110 1/128 0.86 0.3704 0.76 96.96 71 1/64 1.11 0.3585 0.79 50.87.3 43.5 1/32 1.36 0.215 1.54 49.413.5 11 1/8 1.37 0.2095 1.59 12.7PCP estimation using B. subtilisa Measured by A320b Measured after 48 h of incubationJ Ind Microbiol Biotechnol123

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Bacillus subtilis as a bioindicator to estimate pentachlorophenol toxicity and concentration

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