International Biodeterioration & Biodegradation 52 (2003) 261–267www.elsevier.com/locate/ibiod

Biodegradation and detoxi!cation of phenolic compounds by pure andmixed indigenous cultures in aerobic reactors

A. Gallegoa, M.S. Fortunatoa, J. Fogliab, S. Rossia, V. Geminia, L. Gomezb, C.E. Gomezb,L.E. Higab, S.E. Korola ;∗

aChair of Hygiene, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Junin 956 4o Piso, 1113, Buenos Aires, ArgentinabNational Institute of Water, C.C. No. 7 (1802) Aeropuerto Internacional de Ezeiza, Buenos Aires, Argentina

Received 10 April 2002; received in revised form 12 May 2003; accepted 10 July 2003

Abstract

Degradation and detoxi!cation of a mixture of persistent compounds (2-chlorophenol, phenol and m-cresol) were studied by usingpure and mixed indigenous cultures in aerobic reactors. Biodegradation assays were performed in batch and continuous 9ow reactors.Biodegradation was evaluated by determining total phenols, ultraviolet spectrophotometry and chemical oxygen demand (COD). Microbialgrowth was measured by the plate count method. Scanning electronic microscopy was employed to observe the microbial community inthe reactor. Detoxi!cation was evaluated by using Daphnia magna toxicity tests. Individual compounds were degraded by pure bacteriacultures within 27 h. The mixture of 2-clorophenol (100 mg l−1), phenol (50 mg l−1) and m-cresol (50 mg l−1) was degraded by mixedbacteria cultures under batch conditions within 36 h: 99.8% of total phenols and 92.5% of COD were removed; under continuous9ow conditions 99.8% of total phenols and 94.9% of COD were removed. Mineralization of phenolic compounds was assessed by gaschromatography performed at the end of the batch assays and in the eAuent of the continuous-9ow reactor. Toxicity was not detected inthe eAuent of the continuous-9ow reactor.? 2003 Elsevier Ltd. All rights reserved.

Keywords: 2-Chlorophenol; Phenol; m-Cresol; Biodegradation; Detoxi!cation; Aerobic reactors

1. Introduction

Wastewater from industries rarely contains a single pol-lutant as chemicals manufacture involves multiple unitoperations and processes. The multi-substrate nature of anindustrial eAuent is characterized by the presence of a vari-ety of compounds in varying concentrations (Godbole andChakrabarti, 1991). 2-Chlorophenol, phenol and m-cresolcompounds, for instance, are released into the environmentby the industrial eAuents of petrochemical, textile, pharma-ceutical, chemical plants, and the like. These compoundsare toxic and persistent: they accumulate in the environ-ment and usually aEect the performance of industrial aswell as urban treatment plants. Phenolic compounds areserious river water pollutants in Argentina, where industrialeAuents are frequently discharged into streams after being

∗ Corresponding author.E-mail address: sekorol@Eyb.uba.ar (S.E. Korol).

slightly treated or untreated (AGOSBA-OSN-SIHN, 1994;IEIMA, 1990). The development of improved technologiescapable of degrading persistent and recalcitrant compoundsthen becomes necessary.Microbial degradation is a useful strategy to eliminate

these compounds and detoxify wastewaters and polluted en-vironments (Puhakka et al., 1995; Morgan and Watkinson,1989). Several bacterial strains belonging to a varietyof genera degrade phenolic compounds (HIaggblom andValo, 1995; Chitra et al., 1995; Shimp and Pfaender, 1987;Gurujeyalakshmi and Oriel, 1989). Despite the availabilityof biochemical information on the catabolism of pheno-lic compounds, there is a lack of information regardingthe design and operation of treatment facilities. The pur-pose of this investigation has been to study biodegradationand detoxi!cation of a mixture of persistent compounds:2-chlorophenol, phenol, m-cresol, by pure and mixedindigenous cultures in both batch and continuous 9ow9uidized-bed aerobic reactors.

0964-8305/$ - see front matter ? 2003 Elsevier Ltd. All rights reserved.doi:10.1016/j.ibiod.2003.07.001

262 A. Gallego et al. / International Biodeterioration & Biodegradation 52 (2003) 261–267

2. Materials and methods

2.1. Selection and identi9cation of the bacterialcommunity

Enrichment was performed by a periodic subculturing ofsamples from a polluted Buenos Aires river in synthetic min-imal medium (Korol et al., 1989) containing 2-chlorophenol,phenol and m-cresol (100, 50 and 50 mg l−1, respectively).

To isolate individual bacterial strains from the mixtureof persistent compounds, we used streaking in tryptone soyagar medium supplemented with 20 mg l−1 of each phe-nolic compound. Phenolic compounds thus seve the pur-pose of maintaining a selective pressure on bacterial strainsand avoiding the loss of genes responsible for compoundscatabolism.Isolated pure cultures were examined for reaction to Gram

staining and identi!ed using the API system (Bio MLerieux,L’ LEtoile, France).

2.2. Chemicals

The 2-chlorophenol, phenol and m-cresol used were ofchromatographic grade and purchased from Merck (Darm-stadt, Germany). All the other chemicals were of analyticalreagent grade and purchased from Mallinckrodt ChemicalCo. (St. Louis, USA), and Merck (Darmstadt, Germany).Phenolic compounds solutions were prepared aseptically bydissolving the necessary amount in sterile 0:1 N NaOH.

2.3. Biodegradation test in batch reactor

Assays of biodegradation were !rst carried out by usingbacterial isolates, pre-exposed to each of the phenolic com-pounds. They were then carried out by using mixed bacteria,adapted by inoculation in synthetic minimal medium, sup-plemented with the three phenolic compounds they use astheir carbon sources, and incubated in a rotatory shaker at28◦C for 48 h (stock culture).All batch experiments were performed in a New

Brunswick Multigen TA microfermentor aerobically op-erated, at 28◦C with an eEective volume of 1250 ml.The system was inoculated with either 5 ml of a suspen-sion of any of the isolated bacteria or 5 ml of the stockculture according to the assay (!nal cell concentration:106 cell ml−1).

During incubation, 10-ml samples were removed fromthe system at appropriate intervals in order to determine theamount of remaining phenolic compounds and to evaluatemicrobial growth.

2.4. Biodegradation test in ;uidized-bed reactor

Biodegradation assays in continuous 9ow were performedin an aerobic 9uidized-bed reactor with recycling (eEective

Effluent

Granularactivatedcarbon

Liquidrecycle

Recirculationpump

Influent

Air

Scale

10 cm

Fig. 1. Experimental aerobic 9uidized-bed reactor.

volume 6 l) !lled with granular activated carbon (GAC)CARVEGAT, type ACCF (FACASA). The reactor, whichwas described in a previous work (Gallego et al., 2001),utilizes a Plexiglas column 152 cm long× 9:2 cm wide (in-ternal diameter), and is shown in Fig. 1.Before loading, GAC was sieved using 16 and 20 ASTM

sieves. The reactor was operated under environmental con-ditions (without sterility), at room temperature (between14:8◦C and 31:4◦C), the pH being 7.6 and with a 24-h hy-draulic retention time. Working conditions are shown inTable 1.The reactor was continuously fed with synthetic waste

liquid prepared by using 2-chlorophenol (100 mg l−1), phe-nol (50 mg l−1) and m-cresol (50 mg l−1), and a farmingfertilizer (BASF) free from chloride, ratio of N:P:K 10:2:6.Non-sterilized groundwater used for drinking was employedas dilution water. At startup, the reactor was inoculated with

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Table 1Fluidized-bed reactor operating conditions

Parameter Range Average

Volume (l) — 6Hydraulic retention time (h) — 24Temperature (◦C) 14.8–31.4 23.1pH 7.3–8.1 7.6In9uent 9owrate (l day−1) 3.9–7.6 5.5Recycle 9owrate (l min−1) — 4

approximately 1 l of the bacterial community inoculum ob-tained in the batch assays. The reactor was kept in operationfor 6 months.Scanning electronic microscopy (PSEM 500) was em-

ployed in order to observe the microbial community in thereactor.

2.5. Analytical methods and control parameters

To determine the amount of remaining phenolic com-pounds, bacterial cells were separated by centrifugation,and the !ltered supernatant 9uid was submitted to spec-trophotometrical analysis (Metrolab UV 1700 spectropho-tometer), the absorbance was measured at 292 nm; andtotal phenols and chemical oxygen demand (COD) weremeasured during feeding and in the eAuent according toAPHA (1998).To ensure the complete degradation of the compounds un-

der study, selected samples from the reactors were also an-alyzed by gas chromatography. A TRIO-2 VG Masslab gaschromatograph-mass spectrometer, furnished with a capil-lary column (CP-SIL 5 CB, 50 m × 0:25 mm), was usedwith He as carrier gas and a temperature gradient of 60–290◦C increasing at 10◦C min−1.Determination of cell viability was performed by spread-

ing sample dilutions on the surface of nutrient agar plates(APHA, 1998).

2.6. Toxicity test

Bioassays of acute toxicity were performed to evaluatedetoxi!cation using Daphnia magna according to ISO 6341(E) (1989). Toxicity was expressed as eEective concentra-tion 50 (EC50) concentration/dilution, which produces theimmobility of 50% of the organisms of the population testedafter a period of exposure of 24 or 48 h.Before assays were performed, the organisms sensitivity

was evaluated; potassium dichromate was used as the ref-erence toxic compound. General conditions of exposure areshown in Table 2. In bacterial community biodegradationassays, toxicity was evaluated in samples taken both at thebeginning and at the end of the batch processes and in thein9uent and eAuent of the 9uidized-bed reactor.

Table 2Conditions of exposure of the acute toxicity test carried out with Daphniamagna

Exposure type Static without water exchangeTemperature 20:0± 0:5Light No illuminationDilution water Reconstituted destilled waterpH 7:8± 0:2Hardness 250± 25 mg CaCO3 l−1

Dissolved oxygen 80% of saturationConductivity 170 �S cm−1

Size of the container 20 ml (test tubes)Volume of the sample 10 mlAge of the organisms 6–24 hNumber of organisms/test 5Number of replicates 4Total number of test 20organismsFeeding No feedingAeration No aerationTest duration 24–48 hMeasure eEect InmobilizationEnd point EC50 24=48 h, Spearman–Karber

method

3. Results and discussion

3.1. Selection and identi9cation of bacterial community

Bacterial community consists of three pre-dominantgram-negative, non-fermentative strains AG 21, AG 22,and AG 23 found in the enrichment culture from a pollutedBuenos Aires river. AG 21 and AG 23 were identi!ed asbelonging to the genus Alcaligenes. AG21 was capable ofdegrading 2-chlorophenol. AG 23 was capable of degradingm-cresol. Phenol was used as the only carbon source byboth AG 22 and AG 23. The former (AG 22) was identi!edas belonging to the genus Acinetobacter.

3.2. Biodegradation test in batch reactor

Batch experiments were performed in a microfermentorto avoid sudden variations in oxygen, agitation, pH, andtemperature during growth period.Fig. 2 shows growth kinetics and individual substrate re-

moval by pure and mixed bacteria. Alcaligenes (AG21)metabolizes 99.5% of 2-chlorophenol (100 mg l−1) within27 h with a speci!c growth rate (�) of 0:19 h−1, (Fig. 2A).Fig. 2B shows the exponential growth of Acinetobactersp in the presence of phenol (50 mg l−1) with a speci!cgrowth rate (�) of 0:36 h−1, biodegrading 99.5% of the sub-strate within 11 h while Alcaligenes (AG23) requires 20 hto degrade 99.5% of phenol. Fig. 2C shows that AG23 de-grades m-cresol within 8 h, with a speci!c growth rate (�)of 0:48 h−1. In mixtures of bacteria, the rates of degrada-tion of each compound were similar to those observed inisolated bacteria, which demonstrates that the presence of

264 A. Gallego et al. / International Biodeterioration & Biodegradation 52 (2003) 261–267

1006

7

8

5 10 15 2000

20

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% R

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hen

ol

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FU

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Time (h)

9 100

1006

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5 10 15 20 250

0

20

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60

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% R

emai

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-Ch

loro

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ml -1

Time (h)

Time (h)

1006

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emai

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g m

-Cre

sol

Lo

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0 C

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100

0 2 4 6 8 10

(A) (B)

(C)

Fig. 2. Batch reactor: (A) Biodegradation of 2-chlorophenol (100 mg l−1) by Alcaligenes sp. (AG 21) and bacterial community with an initial numberof cells: 106 cells ml−1. Growth kinetics of Alcaligenes sp. (AG21) (− −� − −); bacterial community (− − • − −). Remaining 2-chlorophenolin minimal medium: Alcaligenes sp. (AG 21) (— —); bacterial community (—◦—). (B) Biodegradation of phenol (50 mg l−1) by Alcaligenes sp.(AG23), Acinetobacter sp. (AG 22) and bacterial community with an initial number of cells: 106 cells ml−1. Growth kinetics of Alcaligenes sp. (AG 23)(− −4− −); Acinetobacter sp. (AG 22) (− −�− −); bacterial community (− −•− −). Remaining phenol in minimal medium: Alcaligenes sp. (AG23) (—�—); Acinetobacter sp. (AG 22) (− − �− −); bacterial community (—◦—). (C) Biodegradation of m-cresol (50 mg l−1) by Alcaligenessp. (AG 23) and bacterial community with an initial number of cells: 106 cells ml−1. Growth kinetics of Alcaligenes sp. (AG 23) (− − 4 − −);bacterial community (− − • − −). Remaining m-cresol in minimal medium: Alcaligenes sp. (AG 23) (—�—); bacterial community (—◦—).

the other microorganisms does not aEect the process of in-dividual degradation.Assays using a mixture of 2-chlorophenol 100 mg l−1,

phenol 50 mg l−1,m-cresol 50 mg l−1 prove that these com-pounds are degraded by the bacterial community within 38 h(Fig. 3). The results of COD and total phenols were 92.5%and 99.8%, respectively (Table 3).

3.3. Biodegradation test in ;uidized-bed reactor

The bacterial community assays performed in the9uidized-bed reactor under operating conditions demon-strate that a removal of total phenols and COD of 99.8%and 94.9%, respectively, is achieved, and a total reductionin toxicity is observed (EC50 24 h expressed in % v/vnon-detectable) (Table 3).

The role of biological processes in phenolic compoundstotal degradation has to be accepted. We have to take intoaccount that the reactor was continuously fed with phenoliccompounds during a 6-month period. GAC ought to havereached a saturation point as an adsorbent, and consequentlya phenolic compounds increase in the eAuent of the reac-tor should have been observed. This was not the case: onthe contrary, the removal of phenolic compounds remainedconstant at 99.8%. This fact can only be explained by thepresence of microorganisms.Fig. 4 shows the scanning electron microscopy of carbon

surface before and after inoculation.Though there was an important presence of other envi-

ronmental bacteria within the system, batch conditions lab-oratory testing we performed showed that these bacteria cannot degrade phenolic compounds.

A. Gallego et al. / International Biodeterioration & Biodegradation 52 (2003) 261–267 265

0 5 10 15 20 25 30 35 40 45

Time (h)

0

10

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80

90

100

% R

emai

nin

g p

hen

ols

9

8

7

6

Lo

g 1

0 C

FU

ml -

1

Fig. 3. Batch reactor: Biodegradation of 2-chlorophenol, phenol and m-cresol by the bacterial community with an initial number of cells: 106 cells ml−1.Growth kinetics (− − • − −); remaining phenols in minimal medium (—◦—).

Table 3Biodegradation of 2-chlorophenol, phenol and m-cresol by the bacterial community in batch and continuous reactorsa

Parameter Batch reactor Fluidized-bed reactor

Initial Final % Removal In9uent EAuent % Removal

Total phenols(mg l−1) 210 0.5 99.8 197 0.4 99.8Chemical oxygen demand(mg l−1) 400 30 92.5 389.9 19.9 94.9EC50

b 24 h(% v/v) 0.7 40.6 — 0.7 ND —

aAverage result.bEEective concentration: concentration/dilution that produces the immobility of 50% of the organisms of the population. ND: Not detected.

Fig. 4. SEM-photomicrograph of the carbon surface of 9uidized-bed reactor: (a) before inoculation; (b) after inoculation.

266 A. Gallego et al. / International Biodeterioration & Biodegradation 52 (2003) 261–267

Fig. 5. Batch reactor. GC-MS chromatograms 2-chlorophenol, phenol and m-cresol: (A) initial; (B) !nal.

Fig. 6. Fluidized-bed reactor. GC-MS chromatograms 2-chlorophenol, phenol and m-cresol: (A) in9uent; (B) eAuent.

When the 9uidized-bed reactor was functioning, we ob-served the presence of some algae (Chlorphyta and diatoms,among the latter,Navicula sp. andPinularia sp., especially);rotifers, nematodes, amoebas and ciliates were also found.It is noteworthy that the biomass of degrading micro-

organisms in the reactor remained approximately constant inthe aqueous phase and it developed only from initial inocula;the reinoculation was not necessary.Complete degradation of phenolic compounds and ab-

sence of metabolites (Figs. 5 and 6) were proved by gaschromatography performed at the end of the batch assaysand in the eAuent of the continuous-9ow reactor.

3.4. Toxicity test

High average levels of toxicity expressed by EC50 24 hof 0.7% v/v were found in the feeding of the 9uidized-bedreactor as well as in the initial condition of the batchreactor.Despite high percentages of removal of mixed phenolic

compounds, the samples taken from the batch reactor at theend of each assay turned out to be toxic (EC50 24 h of 40.6%v/v) according to EPA (1991). However, toxicity was not

detected in the eAuent of the continuous reactor (Table 3).This absence of toxicity may be explained by the presence ofGAC and the heterogeneity of the organisms in equilibriumsuch as algae, rotifers, and ciliates.The results of the continuous-9ow reactor assays are sig-

ni!cant, taking into account that the Argentine National Acton Hazardous Wastes (Act 24051/92) and its regulatingDecree (Decree 830/93), de!ne toxicity as an essential char-acteristic which identi!es hazardous waste.We hope that these !ndings will facilitate the treatment of

industrial eAuents and the bioremediation of environmentspolluted by phenolic compounds.

Acknowledgements

We thank the University of Buenos Aires for the grantgiven for this study, supported by the UBACYT Program—Projects AB024-B014.We also thank the Instituto Nacional del Agua (INA)

(National Institute of Water) for allowing us to carry outthis study and make it public.

A. Gallego et al. / International Biodeterioration & Biodegradation 52 (2003) 261–267 267

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