Pentachlorophenol is a wide spread pollutant with high toxicity on the biological systems, activated sludges and wastewater treatment processes. It is one of the best models for the investigation of the biodegradation of aromatic halogen-containing xenobiotics. The indicative role of dehalogenase activity (DA) for the initial detoxication of PCP has been investigated in this paper. The study is carried out with an activated sludge, originated from anaerobic PCP- detoxication process realized at step by step increase of the concentration of PCP (2 – 9,5 mg/l) in conditions in vivo and in vitro. The relationship among DA, bacteria from PCP-degrading complex, aerobic heterotrophic-, anaerobic heterotrophic bacteria as well as the changes microscopic and electron-microscopic structure of the activated sludge is followed. The obtained results confirmed the possibility DA to be used as a express initial test for the assessment of the PCP-detoxication potential of the activated sludges functioning in the presence of halogen-containing aromatic xenobiotics.

Key words: Pentachlorophenol, Dehalogenase activity (DA), Activated sludge, Biodegradation

 

INTRODUCTION

The step by step detoxication of PCP in the wastewaters is determined by its rather complicated chemical structure. The unlock of the biodegradation of PCP is initiated by the elimination of chlorine anions. This reaction is catalyzed by the dehalogenase enzymes that have three mechanisms of dehalogenation – reductive, oxidative and hydroxylation mechanism. The most important ecological role plays the reductive mechanism of chlorine elimination but other mechanisms - hydroxylation and oxidative have a supplementary significance for the detoxication of PCP (Feist and Hegeman, 1969; Topalova et al. 1995).

The chlorinated phenols are not typical for the living systems in the nature. That is why the adaptive potential of the activated sludges (AS) towards biodegradation of the chlorophenols is not revealed, especially that part of adaptation that has a enzymological base of the dehalogenase activity. The main biodegraders of PCP – bacteria possess high activity of dehalogenases. This activity can be induced by the PCP or by its low halogenated derivatives. There is a lack of data about the process of induction of DA, the mechanisms of the biosynthesis of these enzymes, as well as the possibilities of increase of initial rate of detoxication of PCP. In 1992 Mohn and Kenney proposed that the dehalogenases of microorganisms, adapted to the biodegradation of PCP are constitutive according to the their synthesis.

In the process of adaptation of AS towards PCP a lot of structural changes in all parts of AS – (bacteria, micro- and metafauna) can be decoded. These changes were – a formation of granulas, including of PCP-degrading bacteria in consortiums. The essential question is - in what way this functional AS-reconstruction influenced on the DA and on whole PCP-detoxication process? The elucidation of this question is directly connected with the management of the practical valuable bioremediation process. This study was realized to bring the light on the above described applied question.

 

MATERIALS AND METHODS

The analyzed trivial anaerobic AS was taken from the secondary setlimentors of the Sofia Waste Water Treatment Plant, Bulgaria with suspended solids (SS) 20 mg/l and then transferring in a lab scale bioreactor. An anaerobic treatment process was simulated at parameters: SS 10 mg/l, waste water with chemical oxygen demand COD 166¸182 mg/l, T° 30°C, pH 7,2¸7,5, rH (-420 mV). Step by step adaptation was realized at above mentioned parameters and addition on 2¸9,5 mg/l PCP and bioreactor functioning as the Sequencing Batch Reactor (SBR) (Ribarova et al. 2001).

Analytical procedures

PCP in watery phase and the cells were determined by method on high effectively liquid chromatography (HPLC) (Topalova et al. 1997).

Enzyme assays

Dehalogenase activity (DA) was determined simultaneously by nonadapted AS, adapted by three variants: 1) nonadapted AS (nAS); 2) in systems in vivo (AS step by step adapted at 2¸9,5 mg/l PCP); 3) systems in vitro (crude cell extract were isolated to determine of the enzyme activities of AS step by step adapted at 2¸9,5 mg/l PCP) (Feist and Hegeman, 1969; Topalova et al. 1995). DA was measured by means of analysis. Chlorine ions with ion-selective meter Jenway and was expressed in mkMol/min.mg protein. The protein content of the samples was determined by the method described by Herbert et al. (1971).

Microbiological analysis

The samples nonadapted AS, adapted AS at 2 mg/l and 9,5 mg/l PCP were treated with ultrasonic desintegrator for 3x10 seconds to obtain homogenous microbial suspension necessary to determine microbial count. The amount of key to PCP-biodegradation bacteria groups - heterotrophic aerobic bacteria (AH) and heterotrophic anaerobic bacteria (AnN) were determined on a solid medium according to Kuznetzov and Dubinina (1989). The PCP-degrading bacteria were determined on a selective solid medium with PCP as a carbon and energy sources (20 mg/l) (Furukava et al. 1983).

Microscopic and electron-microscopic analysis

The characterization of changes in micro- and macrostructure of AS at the time of adaptation towards PCP-degradation accomplished by the methods of microscopic and transmission electron-microscopic (TEM) analysis (Kuznetzov and Dubinina, 1989).

 

RESULTS AND DISCUSSION

The investigation of the influence of the PCP-concentration on DA at in vitro conditions showed that at 2 and 10 mg/l PCP aAS-4 possessed 3 times higher DA in comparison of this of nAS, and 2 times higher than aAS-9,5 (fig. 1).

At PCP concentrations 5mg/l and 20 mg/l aAS-4 showed relatively 4 times larger DA than nAS and 1,5 times than aAS-9,5. The clearly expressed proportional dependence between DA of aAS-4 and concentration of PCP like a toxic substrate have been proved. At the same time it was impossible to find the connection between the concentration of PCP and DA of the nAS. This fact affords to propose that at these experimental conditions the main complex of dehalogenases has constitutive mechanism of biosynthesis. The increase of the DA of aAS-4 created the base for the proposal – at the time of AS-adaptation to the high PCP-concentration additional inductive mechanisms are included in the regulation of DA. The high and constant DA of the aAS towards 4 mg/l PCP at all investigated PCP-concentrations most probably is connected with the reconstruction of the AS. Two functional parts of AS were estimated – areas with granulas structure (pellets) and areas with floculas structure, abounded by a large amount of homogenic (free swimming) highly adapted PCP-degrading bacteria. This bifunctional structure possessed extremely high DA. It is possible to make the proposal that in the different parts of bifunctional system dehalogenases have various mechanism of the biosynthesis and regulation of their activity.

From the other side it is important to show the fact, that at aAS-4 the different mechanisms of the dehalogenase reaction in the different parts of the biological system are very well balanced and they ensured an effective dehalogenation. The decrease of DA at aAS-9,5 has serious and complicated reasons – high PCP-concentration close to the critical one, inhibitory effect of the PCP in this high concentration, accumulation of toxic metabolites, as well as hard competition for the reductive equivalents.

 

 Fig. 1 Effect of PCP-concentration on DA at incubation in vivo in the phases of adaptation

(nAS - nonadapted AS; aAS-4 - AS adapted at 4 mg/l PCP; aAS-9,5 - AS at final phase of adaptation at 9,5 mg/l PCP)

 

In fig. 2 are presented the obtained results about the influence of the PCP-concentration on the DA, measured ion conditions in vitro at aerobic incubation (fig. 2a) and at anaerobic incubation (fig. 2b). The aAS-4 possessed 40 times higher DA at aerobic incubation than DA of the nAS. At anaerobic conditions the DA of aDA-9,5 and 3¸5 times higher than DA of aAS-4 and 30¸40 times higher than the activity of nAS. As a whole the DA of the adapted and non-adapted AS, incubated at anaerobic nitrogen media for 1 hour was larger than DA of the same AS incubated at aerobic conditions for 1 hour. The comparison of the measured DA in vitro at aerobic and at anaerobic conditions showed that both types of incubation are appropriate for the measuring of the DA in vitro. There were not distinguished essential differences in the measured DA.