4-Chlorophenol degradation by a bacterial consortium: development of a granular activated carbon bio®lm reactor

A bacterial consortium that can degrade chloroand nitrophenols has been isolated from the rhizosphere of Phragmitis communis. Degradation of 4-chlorophenol (4-CP) by a consortium attached to granular activated carbon (GAC) in a bio®lm reactor was evaluated during both open and closed modes of operation. During the operation of the bio®lm reactor, 4-CP was not detected in the column e‚uent, being either adsorbed to the GAC or biodegraded by the consortium. When 4-CP at 100 mg l was fed to the column in open mode operation (20 mg g GAC total supply), up to 27% was immediately available for biodegradation, the rest being adsorbed to the GAC. Biodegradation continued after the system was returned to closed mode operation, indicating that GAC bound 4-CP became available to the consortium. Bio®lm batch cultures supplied with 10±216 mg 4-CP g GAC suggested that a residual fraction of GAC-bound 4-CP was biologically unavailable. The consortium was able to metabolise 4-CP after perturbations by the addition of chromium (Cr VI) at 1±5 mg l and nitrate at concentrations up to 400 mg l. The development of the bio®lm structure was analysed by scanning electron microscopy and confocal laser scanning microscopy (CLSM) techniques. CLSM revealed a heterogeneous structure with a network of channels throughout the bio®lm, partially occupied by microbial exopolymer structures. Introduction A number of chlorinated aromatic compounds have been designated priority environmental pollutants by the US EPA and the EEC. Some are used extensively in several industries, including pulp and paper manufacture (Keith and Telliard 1979; Mason 1991). This has led to their widespread release into the environment, affecting both soil and groundwater. The degradation of these compounds under both aerobic and anaerobic conditions has been reported (Haggblom 1992; Puhakka et al. 1995; Sanford and Tiedje 1997) and degradation pathways have been elucidated (Reineke and Knackmuss 1989; Hollander et al. 1997). In recent years, the potential of bio®lm reactors to remediate contaminated groundwater and toxic liquid e‚uents has been recognised. Bio®lm reactors are advantageous in that the biomass is active even at very low concentrations of target organic chemicals, they are less sensitive to the presence of toxic and inhibitory materials, and are more resistant to shock loading than dispersed growth systems (Lee et al. 1994; Fauzi 1995; Shi et al. 1995). The microbial degradation of aromatic compounds adsorbed onto powdered activated carbon (PAC) has been shown to be e€ective at concentrations which caused a dramatic inhibition of degradation in suspended or sand-attached cultures (AbuSalah et al. 1996). Granular activated carbon (GAC) is a good bacterial immobilisation matrix as it is very adsorptive and has a very high surfaceto-volume ratio, due to its large number of internal pores and rough surface texture (Weber et al. 1979; Christiansen and Characklis 1990). Immobilised bacterial GAC reactors have been assessed for the microbiological clean-up of water contaminated with BTX (Xing and Hickey 1994), halogenated aliphatics (Fauzi 1995), toluene (Shi et al. 1995) and chlorobenzene (Klecka et al. 1996). Klecka et al. (1996) used a GAC ̄uidized-bed reactor for the degradation of chlorobenzene present in an in̄uent stream at 125±145 mg l, with more than 99% M. Caldeira á M. F. Carvalho á I. Vasconcelos P. M. L. Castro (&) Escola Superior de Biotecnologia, Universidade Cato lica Portuguesa, Rua Dr. Anto nio Bernardino de Almeida, P-4200 Porto, Portugal e-mail: [email protected] Tel.: +351-2-5580042 Fax: +351-2-5090351 S. C. Heald á A. T. Bull Research School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK eciency. Adsorption e€ects were also noticed and the adsorbed chlorobenzene was available for later biodegradation. Fauzi (1995) developed a GAC reactor to degrade very low concentrations of dichloropropanol (lg l range) in a continuous ̄ow system, and the operation was stable and e€ective. Treatment of groundwater contaminated with toluene at 2.7 mg l in a GAC ̄uidized-bed bioreactor was achieved with 99.4% eciency during steady-state operation, while during shock loading the GAC served to adsorb the compound for subsequent degradation (Shi et al. 1995). Both bio®lm composition and activity are important parameters for the successful operation of ®xed ®lm processes for water treatment (Moller et al. 1996). However, experimental information on bio®lm microstructure has been limited until recently because of the lack of analytical techniques for observing ®xed bacteria in situ. Techniques for bio®lm analysis were reviewed by Costerton et al. (1995). Scanning electron microscopy (SEM) provides information on the number and distribution of bacteria within the bio®lm and on exopolymer structures (EPS). But advances in non-destructive methods, principally confocal scanning laser microscopy (CLSM), provide a more detailed insight into the structural organisation of bio®lms and allow the quanti®cation of bio®lm constituents (Caldwell et al. 1992; Fauzi 1995; Massol-Deya et al. 1995; Moller et al. 1996). We have isolated chloroand nitro-aromatic degrading bacterial consortia from the rhizosphere of Phragmitis communis colonising an industrial site contaminated with chloroorganics, heavy metals, nitrates and sulphates. The primary objective of our work was to establish a GAC bio®lm reactor for the degradation of the aromatic pollutants. Here we report the eciency of the bioreactor to degrade 4-chlorophenol (4-CP) during closed mode (recycle system) and open mode of operation; and the e€ects of hexavalent chromium (CrVI) and nitrate perturbations on consortium activity. The development and structure of the bio®lm community was followed using microscopic techniques. Materials and methods Isolation of bacterial consortia Rhizosphere samples (roots and associated soil) of the reed P. communis, collected from a chemically contaminated industrial site in Northern Portugal (Estarreja), were used to inoculate batch ̄asks containing 50 ml minimal salts medium (per litre: Na2HPO4 á 2H2O 2.7 g; KH2PO4 1.4 g; (NH4)2SO4 0.5 g; MgSO4 á 7H2O 0.2 g; yeast extract 0.02 g and 10 ml trace elements solution of the following ®nal composition per litre: Na2EDTA2H2O, 12.0 g; NaOH, 2.0 g; MnSO4 á 4H2O, 0.4 g; ZnSO4 á 7H2O, 0.4 g; H2SO4, 0.5 ml; Na2SO4, 10.0 g; Na2MoO4 á 2H2O, 0.1 g; FeSO4 á 7H2O, 2.0 g; CuSO4 á 5H2O, 0.1 g; CaCl2 1.0 g). Aromatic compounds were supplied as the sole carbon and energy sources, at concentrations of 50±100 mg l. Cultures were incubated aerobically at 25 °C at 200 rpm and the optical density was monitored at 600 nm. Cultures (5 ml) were transferred to 50 ml identical fresh medium at weekly intervals, at which time aliquots (0.1 ml) were plated onto minimal agar plates containing the relevant substrate. Enrichments were established using 4-CP, 4-nitrophenol (4-NP), nitrobenzene, chlorobenzene and various mixtures of these compounds.