Microb Ecol (1994) 28:53-65

MICROBIAL ECOLOGY © 1994Springer-VerlagNewYorkInc.

Degradation by and Toxicity to Bacteria of Chlorinated Phenols and Benzenes, and Hexachlorocyclohexane Isomers E. Lang,* H. Viedt Institut ffir Mikrobiologie, Technische Universit~itBraunschweig, Konstantin-Uhde-Str. 4, 38106 Braunschweig, Germany Received: 11 May 1993; Revised: 14 February 1994

Abstract. Mixed cultures degrading chlorinated benzenes, chlorinated phenols, or hexachlorocyclohexane (HCH) as the sole source of carbon and energy were obtained by enrichment from contaminated soil samples. Cultures which metabolized 3-chlorophenol (3-CP), 2,3-dichlorophenol (2,3-DCP), or 2,6dichlorophenol (2,6-DCP) were able to utilize several other chlorinated compounds as substrates, whereas cultures enriched with 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB), ~-HCH, or -/-HCH did not metabolize most of the other chlorinated congeners tested. Chloride release and growth rates with all four chlorinated phenols decreased with increasing initial substrate concentrations within the range of 30-250 txmol liter -1. Maximum chloride release was 3.8 mg liter- 1 corresponding to 35 p~mol liter- 1 trichlorophenol within 7 weeks. In contrast, the rate of metabolism of the nonphenolic compounds 1,2,4,5-TeCB, a-HCH, or ~/-HCH increased with increasing substrate concentrations. Initial concentrations of 750 txmol liter -~ ~-HCH or 1,2,4,5-TeCB were completely dechlorinated within 2 weeks. Because aqueous solubility and bioavailability of the chlorophenolic compounds is much higher than that of the nonphenolic compounds, it is suggested that the high bioavailability of the chlorophenolic compounds is the reason for the high toxicity of these substrates to the degrading cultures. In contrast, the low aqueous solubilities of the chlorinated benzenes and HCH-isomers caused consistently low concentrations in the medium, which were high enough to induce degradation but too low to damage the bacterial cells. Introduction Degradation of chlorinated hydrocarbons (CHCs), e.g., chlorinated benzenes and phenols, or hexachlorocyclohexane isomers (HCH), by mixed populations and individual bacterial strains is well documented. Efforts have been made to investi-

*Present address." Institut flit Bodenbiologie, FAL, Bundesallee 50, 38116 Braunschweig, Germany Correspondence to: E. Lang

54

E. Lang, H. Viedt

gate the aerobic and anaerobic pathways of microbial CHC metabolism, and to describe the intermediates and enzymes involved. Meanwhile, at least one metabolic pathway for almost all the cited compounds has been elucidated [22,24]. Less attention has been given to the influence of substrate concentration on degradation. Chlorinated benzenes, HCH-isomers, and highly chlorinated phenols are hydrophobic substances with water solubilities of less than 150 mg liter -a . At sites contaminated with CHCs, they can be adsorbed to the soil matrix, especially if organic matter is present [6,20]. Low solubility and adsorption can prevent an even distribution of the CHCs within the soil, and the spatial distribution of the contaminants in soil may vary over several orders of magnitude on a meter scale as well as on a millimeter scale (microenvironments). The in situ bioconversion of CHCs is strongly influenced by this concentration pattern, because it determines the availability and toxicity of the compounds to the degrading microflora. In this study, the aerobic dechlorination of several CHCs by mixed bacterial cultures enriched from a contaminated site was investigated. Soil samples were taken from an industrial land site polluted with a mixture of the CHCs mentioned above. The CHCs were metabolized aerobically in soil and ground water samples of this site, but no dechlorination was found under anaerobic conditions [16]. We wanted to determine which compounds could be metabolized by the microfora of this site, how fast the compounds were dechlorinated, which similar CHC could be attacked by a culture adapted to one particular CHC, and especially, how CHC concentrations influenced their own bioconversion.

Materials and Methods

Enrichment and Culturing of CHC Degrading Bacteria Mineral medium (MM) free of chlorides [7] was added to 250-ml Erlenmeyer flasks (with gas-tight screw caps) supplied with the respective chlorinated organic compound as the sole source of carbon and energy. The medium contained (NH4)2SO4, 0.5 g; MgSO 4 - 7H20, 0.2 g; Ca(NO3)2, 0.05 g; Na2HPO4, 2.44 g; KH2PO4,1.52 g; 3 ml vitamin solution and 10 ml trace element solution per liter of distilled water and had a pH of 6.9. The vitamin solution consisted of 0.2 mg biotin, 2 mg nicotinic acid, 1 mg thiamine, 1 mgp-aminobenzoic acid, 0.5 mg pantothenic acid, 5 mg pyridoxamine, and 2 mg cyanocobalamine per 100 ml of distilled water. The chlorophenols were dissolved in a NaOH solution (0.01 mol liter-l), filter sterilized, and added to the autoclaved mineral medium. 1,2,4,5TeCB, a-HCH, or ",/-HCH were dissolved in acetone. Aliquots of these stock solutions were pipetted into empty, sterile culture flasks. As the solvent vaporized at room temperature (about 30 min), the substrates left a thin layer of crystals at the bottom and wall of each flask. Following the addition of 30 ml of sterile medium, a part of the nonphenolic CHCs remained undissolved as crystals, i.e., the actual solution concentration was lower than nominal. Mixed cultures of CHC degraders were obtained using soil samples from an industrial plant site (northern Germany) which had been polluted with a mixture of mono- through hexachlorinated benzenes and phenols, and with hexachlorocyclohexane isomers (HCH) over a period of about 40 years. The concentrations of the xenobiotics in soil samples varied widely from 200 up to 3300 mg kg- ~ EOX (extractable organic halogens). For enrichment cultures, 250-ml Erlenmeyer flasks supplied with 30 ml MM and a CHC were inoculated with 1 ml of a soil suspension. This suspension consisted of 10 g soil in 95 ml of a sodium pyrophosphate solution (2 g liter- 1) to support dispersion of cells from the soil matrix. The suspension was mixed end over end at 100 rpm for 1 h. Other cultures were inoculated with 1 rrd of a preculture. All cultures were incubated at 20°C. Incubation times are reported with the results.

Degradation by Bacteria of Chlorinated Hydrocarbons

55

Microscopic Cell Counts Bacterial densities of the cultures were counted microscopically in a bacterial counting chamber.

Chloride Concentration in Culture Solutions After separating the cells from the culture solutions by centrifugation at 4,000 g, inorganic chloride content of the supernatants was measured turbidimetricallyas AgC1. Silver nitrate solution was made in the following manner: 4.25 g AgNO3, 1 ml HNO3 (65%), and 0.05 g NaC1were dissolved in 100 ml of distilled water. The solution was heated to 80°C while stirring until a greyish precipitate formed. The solution was filtered through medium-fast filter paper. One milliliter HNO3 (2 mol liter 1), and 0.5 ml silver nitrate solution were added to 10 ml of sample. After mixing and incubating the samples for 5-10 min in the dark, the optical density was measured at 365 nm. Test concentrations ranged from 0.1 to 10 mg liter-1 chloride.

Analysis of Chlorinated Phenol Concentration Concentrations of chlorinated phenols in the cultures were analyzed by gas chromatography. For extraction of the chlorophenols from the cultures, 1 volume n-pentane per 5 volumes of medium was injected directly to the cultivation flasks. The chlorinated phenols were extracted by shaking end over end for 1 h. The phases were separated by freezing the flasks over night. Pentane samples of 1 p~lwere injected into a gas chromatograph (Perkin Elmer 8500) equipped with a flame ionization detector and fitted with a Perkin Elmer PVMS SE 54 column (50 m, diameter 0.32 mm, particles 1 ixm). Analysis conditions were as follows: hydrogen gas at 1.5 ml min 1, injector and detector temperatures 240°C and 300°C, respectively, oven temperature increasing from 70°C to 240°C.

Chemicals The HCH isomers were 99% grade gas chromatography standards from Riedel-de-Haen (Seelze, Germany), 3-chlorophenol (3-CP) and 2,6-dichlorophenol (2,6-DCP) (98% and 99%) were bought from Aldrich Chemicals (Steinheim, Germany), 2,3-dichlorophenol (2,3-DCP) and 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB) (>98%) from Fluka (Ulm, Germany), and n-pentane from Merck (Darmstadt, Germany).

Results Mixed bacterial cultures were enriched with suspensions of soil samples severely c o n t a m i n a t e d with chlorinated benzenes and phenols and HCHs. These cultures utilizes one of the CHCs m e n t i o n e d in Table 1 as the sole source of carbon and energy u n d e r aerobic conditions. In all cases, an increase of optical density due to bacterial growth was a c c o m p a n i e d by a release of inorganic chloride into the culture solution as a result of C H C metabolism. Controls supplied with sterilized soil suspensions did not show any increase of chloride concentration during incubation. Bacteria seeded to m i n e r a l m e d i u m without C H C did not exhibit any growth. Cell counts in these controls r e m a i n e d constant at 3 × 106 per ml over 3 weeks. Figure 1 shows chloride release during growth on o~-HCH, T - H C H , or 1,2,4,5TeCB as carbon source. The bacteria degraded 750 Ixmol liter -1 ~ - H C H or

56

E. Lang, H. Viedt

Table 1. Substrates in enrichment cultures of soil samples from a CHCpolluted site Substrate for enrichment

Growth with

ct-HCH "r-HCH

r-HCH c~-HCH

Dechlorinationin No growth or % of enrichment~ dechlorinationwith 52 100

1,2,4,5-TeCB

{3-HCH 8-HCH

1,2,4,5-TeCB

~-HCHb "r-HCHb

49 44

3-CP

2,3-DCP 2,6-DCP 3-CP 2,6-DCP 3-CP 2,3-DCP a-HCH 1,4-DCB

13 27 100 26 34 20 20 86

2,3-DCP 2,6-DCP

1,2,3,4-ToCB 1,4-DCB 1,2,3-TCB 1,2,4-TCB 1,3,5-TCB 1,2,3,4-TeCB

~Dechlorination: production of inorganic chloride within 6 weeks from the CHC supplied as the sole source of carbon and energywas measured. Dechlorination is expressed as the chloridereleased with the replacement substrate as compared to the enrichment culture (dechlorination determined in the enrichment culture is set to 100%). Dechlorination detection limit: 3 mg liter-1 chloride bGrowth only after adaption in substrate mix of 250 p.mol liter 1 of each c~-HCH,"r-I-ICH,and 1,2,4,5-TeCB

1,2,4,5-TeCB completely within 2 weeks. The total amount of organically bound chlorine, 160 mg liter -1 and 106 mg liter -1, respectively, was metabolized. "r-HCH was dechlorinated to 54% within this time. The growth and transformation rate on 2,6-DCP or 2,4,5-TCP was much slower than on nonphenolic compounds. Even after 4 or 7 weeks, the cultures had dechlorinated only 32% of the 30 txmol liter -1 2,6-DCP or 72% of the 50 Ixmol liter -1 2,4,5-TCP present in the medium. For cultures with 2,6-DCP as the substrate, it was shown by gas chromatographic analysis that chloride release was accompanied by a corresponding decrease of 2,6-DCP (Table 2). Growth rates were highest during the second and third week with 2,6-DCP, and during the first week with 2,4,5-TCP as substrate (Fig. 2). Release of chloride ions into the medium continued during the whole incubation period and did not incease with decreasing growth rate. 2,4,5-TCP was degraded to the highest extent of all phenolic compounds (Table 2). Each mixed bacterial culture growing on a chlorinated hydrocarbon was tested for its capability to degrade CHCs different from the compound it was enriched from. The culture enriched with a - H C H could dechlorinate and grow on ~-HCH and vice versa. No degradation was found with [3- or 8-HCH or 1,2,4,5-TeCB (Table 1). The 1,2,4,5-TeCB-enriched culture did not grow on any other di-, tri-, or

Degradation by Bacteria of Chlorinated Hydrocarbons

57

chloride (mg I"1) 175

150

- 8 - alpha-HCH

125

~

- ~ - 1,2,4,5-TeCB

gamma-HCH

IO0

75

5O

25

i

0

3

6

9

chloride i n p u t w i t h T e C B ..........

chloride input with HCH

12

days

15 Fig. 1. Release of ionic chloride during growth of three enrichment cultures with 750 p~mol liter- 1 1,2,4,5-tetrachlorobenzene, tx-HCH, or "r-HCH as the sole source of carbon and energy.

tetrachlorinated benzene tested. Growth on HCH was also very weak when this culture was transferred directly from 1,2,4,5-TeCB to HCH. However, after growth with a mixture of 1,2,4,5-TeCB, a-HCH, and "r-HCH, the bacteria were able to utilize HCH as the sole source of carbon and dechlorinated almost half of the HCH present in the cultures. The slow-growing chlorophenol cultures were able to degrade, at least partially, every replacement substrate tested. Not only other chlorinated phenols, but also a-HCH, and 1,4-DCB were utilized by the 2,6-DCPenriched culture (Table 1) and supported growth of about 1.4 × 108 cells m1-1 (data not shown). The effect of CHC concentration on chloride production and cell growth was tested with seven enrichment cultures already adapted to growth with this CHC as a substrate. Only a small portion of the substrate was metabolized to inorganic chloride with 30 p~mol liter -1 of oL- or "r-HCH or 1,2,4,5-TeCB. Increasing the initial concentration of these nonphenolic substrates resulted in an increase of chloride production. The final absolute chloride concentration (Table 2) as well as the percentage of these CHCs dechlorinated increased drastically (Fig. 3) with increasing initial CHC concentration. Increasing the concentration of the chlorophenolic substrates resulted in an opposite effect. The 3-MCP and 2,3-DCP cultures released only slightly more chloride with 150 Ixmol liter-1 than with 50 txmol liter-1 (Table 2). Increasing the 2,6-DCP concentration from 100 to 250 txmol liter-a decreased chloride production from 0.96 to 0.14 mg liter -1. At a concentration of 50 ~xmol liter -1 2,4,5TCP, 38 mg liter - t chloride was released. But at a concentration of 100 txmol liter -1, only 0.72 mg liter-1 was mineralized. If the extent of CHC metabolism is expressed as the percentage of organically bound chlorine mineralized, transformation of the chlorinated phenols was also low at higher, compared to lower, CHC concentrations (Fig. 3). Cell growth corresponded with substrate degradation. 2,6-DCP cell counts increased from 6.3 x 10 6 to 1 x 108 cells m 1 - 1 in the culture with 30 t~mol liter -1 2,6-DCP (Fig. 2). At a concentration of 250 p~mol liter -1 on the same substrate,

58

E. Lang, H. Viedt

Table 2. Dechlorination of CHCs in mixed cultures with varying substrate concentrations after 7 weeks of incubation Initial concentration

Substrate 3-MCP 2,3-DCP 2,6-DCP

2,4,5-TCP

1,2,4,5-TeCB

c~-HCH

-r-HCH

of substrate (Ixmol liter- 1)

of substrate-bound chlorine (mg liter- 1)

Production of chloride ions (rag liter- 1)

Dechlorination (%)

50 150 50 150 30 100 250 50 100 150 30 150 750 30 150 750 30 150

1.77 5.32 3.55 10.6 2.12 7.09 17.8 5.32 10.6 15.9 4.3 21.2 106.3 6.4 31.9 159 6.4 31.9

0.27 0.43 0.31 0.35 0.67 0.96 0.14 3.8 0.72 0.54 0 2.2 31.5 1.4 18.1 123 2.5 18.3

15 8 9 3 32 13 1 72 7 3 0 10 30 22 57 77 39 57

Concentration of 2,6-DCP measured

2,6-DCP

initial (ixmol liter- 1) 29 104 250

degradeda (p~mol liter- 1) 5.8 7 1.8

Corresponding production of chloride ionsb (mg liter- 1) 0.4 0.52 0.13

The amount of DCP degraded was calculated from the differences in DCP concentration (determined after incubation by gas chromatography) with an inoculated culture and a sterile control bCalculated from the DCP decrease measured, assuming that the DCP was dechlorinated totally a

o n l y 1.9 x 106 cells were p r o d u c e d . W i t h 50 p~mol l i t e r - 1 2 , 4 , 5 - T C P , the bacteria g r e w to a density o f 2.3 x 109 iill - 1 within 5 weeks, whereas only 2 x 108 cells m l - 1 were o b t a i n e d with 150 Ixmol l i t e r - 1

Discussion T h e c a p a b i l i t y o f the m i x e d cultures to degrade C H C s other than those they were enriched b y was restricted for the nonphenolic c o m p o u n d s : o n l y a - and "r-HCH could substitute each other as the sole carbon source. The culture g r o w i n g with 1,2,4,5-TeCB was not able to utilize any o f the l o w e r chlorinated b e n z e n e s tested. N o such specialization was o b s e r v e d with two pure cultures o f 1 , 2 , 4 - T C B - or 1 , 2 , 4 , 5 - T e C B - d e g r a d i n g Pseudomonas sp. strains [26]. T h e y also t r a n s f o r m all d i c h l o r o b e n z e n e s even though the m e t a b o l i c p a t h w a y for T e C B does not include

59

Degradation by Bacteria of Chlorinated Hydrocarbons DCP 3 0

50

TOP

chloride ling/L}

log of cell counts per rnl

log of cell counts per

10

chloride [rag/L]

ml

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Fig. 2.

Cell growth and chloride production by a 2,4,5-trichlorophenol enrichment culture and by a 2,6-dichlorophenol enrichment culture during growth with chlorinated phenols as sole carbon source. Initial substrate concentrations: TCP 50, 50 txmol liter -1 2,4,5-trichlorophenol; DCP 30, 30 p~mol liter -L 2,6-dichlorophenol; DCP 100, 100 p,mol liter -1 2,6-dichlorophenol; DCP 250, 250 txmol liter 1 2,6-dichlorophenol.

these compounds [26]. The adaptation of our 1,2,4,5-TeCB culture to HCH by temporary incubation with both substrates showed that the cultures harbored the genetic resources for the uptake and metabolism of different CHCs. Nevertheless the cultures needed prolonged incubation times under growing conditions to adapt to the new substrate either by changing in strain composition or by expressing the appropriate enzyme system. The substrate spectrum of the chlorophenolic compound-degrading cultures was much wider. All the mono- or dichlorophenols tested were utilized by all three cultures to some extent. Even o~- and "r-HCH were dechlorinated. Pure cultures metabolizing chlorinated phenols also show wide substrate spectra for chlorinated phenols [1,17]. Nevertheless, 2,4,6-TCP-degrading Azotobacter sp. GP1 does not mineralize 3-CP [17], and 4-CP-grown cells of Pseudomonas sp. B 13 or Alcaligenes eutrophus do not cometabolize 2,6-DCP [15] as did our 3-CP culture. The aqueous solubility of substrates is thought to correspond to some degree with bioavailability [ 12]. In this study, CHC degradation rates were inversely related to the water solubility of the substrates, suggesting that factors other than availability

60

E. Lang, H. Viedt gamrna-HCH

aipha-HCH

m

il_

2,3-DCP

50

1,2,4,5-TeCB

I

2,6-DCP

1 150

3O

L 100

2,4,a-TCP

250

initial concentration of chlorinated hydrocarbon (pMol

v

50

100

150

I~)

Fig. 3. Effect of increasing initial concentrations of chlorinated hydrocarbons on mineralization of the compounds by mixed cultures. Chloride production from the chlorinated hydrocarbons as the sole source of carbon after 4 weeks (HCH and TeCB) and 7 weeks (DCP and TCP) of incubation at 22°C, respectively.

determined how well the individual CHCs were mineralized. We propose that substrate toxicity was responsible for the observed inverse relationship between CHC concentration and metabolism of phenolic and of nonphenolic chlorinated compounds. The toxicity of the phenolic compounds to the degrading enrichment cultures corresponds to the inhibiting effect of chlorinated phenols on the growth on yeast extract of the microflora from soil samples of the same site [16]. At concentrations of 30-750 ~mol liter-1, only the two chlorinated phenols tested, 2,4,5-TCP and PCP, decreased cell growth more than 50% compared to growth without CHC. No such growth inhibition occurred with ten chlorinated benzenes or HCH isomers tested [ 16]. The CHC-degrading cultures were not more resistant to CHCs than the overall microflora of the site. Several studies describe the toxic effects of chlorinated phenols on bacterial respiration or growth with an easily degradable substrate. The least toxic dichlorophenol, 2,6-DCP, for instance, gave IC5o-values (the concentration that inhibits

Degradation by Bacteria of Chlorinated Hydrocarbons

61

activity to 50%) of 400-3,380 Ixmol liter-1 [5,18]. Minimal inhibiting concentrations (MICs) for 2,6-DCP were determined to be as high as 6, 130 ~mol liter -1 [8, 14]. These inhibiting concentrations are higher than those found in our experiments for degrading bacteria where 250 Ixmol liter-1 2,6-DCP inhibited growth. Kiyohara et al. [14] found that bacteria never challenged by CHC can be more resistant to chlorophenols than chlorophenol degraders. To our knowledge, comparable toxicity tests for chlorobenzenes do not exist. Table 3 compiles CHC degradation studies considering the effect of CHC concentration on growth and degradation. The minimal concentrations inhibiting growth with chlorinated benzenes vary from 30 mg kg -1 soil up to 1,260 mg liter-1. Chlorinated phenols reduce their own degradation rates from a lower limit of 2-260 mg liter -1. A fundamental difference in the toxicities of phenolic and nonphenolic CHCs cannot be derived from this synoptic list of degradation studies of single CHCs. Some of the authors observed adverse effects of chlorinated benzenes on the survival of cells, on the removal of CHC from soil, and on their own metabolism, as was observed for chlorinated phenols. Increasing absolute degradation rates with increasing concentrations have been cited for chlorophenols and for chlorobenzenes. Therefore, the pronounced contrary effect of increasing concentrations of chlorinated phenols and nonphenolic compounds in our enrichment cultures has to be emphasized. HCH isomers and 1,2,4,5-TeCB were degraded most effectively at 750 txmol liter-1 (Fig. 1), a concentration 3 times higher than the concentration that caused 90% inhibition to growth with chlorophenolic compounds. Fritz et al. [11] describe two mechanisms that might be responsible for the sensitivity of cells to CHCs: (1) the highly lipophilic character of the compounds, causing adverse effects on the cell envelope, and (2) enzyme toxicity of degradation intermediates as found by Reineke and Knackmuss [23]. Both mechanisms apply equally to phenolic and nonphenolic compounds. Lipophilicity is measured as octanol:water-partition coefficient (log Pow) [5]. The log Pow values of dichlorinated phenols and of dichlorinated benzenes are similar, as are those of o~-HCH and trichlorophenols [5,28]. Critical key metabolites for both substance classes are the chlorinated catechols [22,23]. Thus, these two points cannot solely account for the fact that only chlorinated phenols showed a toxic effect in our experiments. Whether toxicity was caused exclusively by the substrate itself or by some (even more) toxic metabolites produced during cultivation cannot be determined from our experiments. This question should be the subject of further investigations. While chlorinated benzenes and HCH isomers dissolved in water at concentrations of 150 mg liter -1 (1,2-DCB) or less, more than 1,000 mg liter -1 of di- and trichlorophenols are water soluble. Hence the dissolved concentrations of the nonphenolic compounds during our experiments were generally much lower than nominal (aqueous solubility of HCH is 10 mg liter- 1, lowest concentration tested was 30 txmol liter-1 corresponding to 8.73 mg liter-1), whereas the chlorophenols were dissolved totally. Assuming that only the dissolved concentration is the effective one, the results of our experiments were influenced by the bioavailability of the CHCs. However, we propose that the dominant character of bioavailability was not the availability as a substrate but its availability as a toxic agent. On the other hand, it must be emphasized that "concentrations" of nonphenolic CHCs well above saturation had a marked positive effect on degradation rates. This phenome-

62

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non, also reported by Bachmann et al. [2] and Stanlake and Finn [30], requires further elucidation. Our results imply that bacterial growth and metabolism can be inhibited at in situ sites (macro- or micro-) with phenolic CHC concentrations of about 30 txmol liter -1 or higher in the soil solution. At sites contaminated with a mixture of nonphenolic and phenolic CHCs, the dissolved concentration of the phenolic compounds can be critical for the biodegradation of all CHCs. Bioremediation plans for such sites have to take into account these toxic effects. Besides the usual monitoring of the contaminant concentration as a bulk parameter, e.g., as AOX or EOX (adsorbable or extractable organic halogens), the concentration of the phenolic fraction must be measured and controlled during bioremediation.

Acknowledgments. We thank Anja Rhoda and Stefan Schieseck for excellent technical assistance. The project was financially supported by Dekonta GmbH, Mainz.

References 1. Apalajahti, JHA, Salkinoja-Salonen MS (1986) Degradation of polychlorinated phenols by Rhodococcus chlorophenolicus. Appl Microbiol Biotechnol 25:62-67 2. Bachmann A, de Briun W, Jumelet JC, Rijnaarts HNN, Zehnder AJ (1988) Aerobic biomineralization of alpha-hexachlorocyclohexane in contaminated soil. Appl Environ Microbiol 54:548554 3. Bachmann A, Walet P, Wijnen P, de Bruin W, Huntjens JLM, Roelofse (1988) Biodgradation of alpha- and beta-hexachlorocyclohexane in a soil slurry under different redox conditions. Appl Environ Microbiol 54:143-149 4. Balajee S, Mahadevan A (1990) Utilization of chloroaromatic substances by Azotobacter chroococcum. Syst Appl Microbiol 13:194-198 5. Beltrame P, Beltrame PL, Carniti P (1984) Inhibiting action of chloro- and nitro-phenols on biodegradation of phenol: a structure-toxicity relationship. Chemosphere 13:3-9 6. Blackburn JW (1987) Prediction of organic chemical fates in biological treatment systems. Environ Progress 6:217-223 7. Brunner W, Staub D, Leisinger T (1980) Bacterial degradation of dichloromethane. Appl Environ Microbio140:950-958 8. Chu J, Kirsch EJ (1973) Utilization of halophenols by a pentachlorophenol metabolizing bacterium. Dev Ind Microbiol 14:264-273 9. Crawford RL, Mohn WW (1985) Microbiological removal of pentachlorphenol from soil using a Flavobacterium. Enzyme Microb Technol 7:617-620 10. de Bont JAM, Vorage MJAW, Hartmans S, van den Tweel WJ (1986) Microbial degradation of 1,3-dichlorobenzene. Appl Environ Microbiol 52:677--680 11. Fritz H, Reineke W, Schmidt E (1992) Toxicity of chlorobenzene on Pseudomonas sp. strain RHO 1, a chlorobenzene-degradingstrain. Biodegradation 2:165-170 12. Goldstein RB, Mallory LM, Alexander M (1985) Reasons for possible failure of inoculation to enhance biodegradation. Appl Environ Microbiol 50:977-983 13. Haider K, Jagnow G, Kohnen R, Lim SU (1974) Abbau chlorierter Benzole, Phenole und Cyclohexan-Derivatedurch Benzol und Phenol verwertende Bodenbakterienunter aeroben Bedingungen. Arch Microbiol 96:183-200 14. Kiyohara H, Hatta T, Ogawa Y, Kakuda T, Yokoyama H, Takizawa N (1992) Isolation of Pseudomonas pickettii strains that degrade 2,4,6-trichlorophenol and their dechlorination of chlorophenols. Appl Environ Microbiol 58:1276-1283 15. Knackmuss H-J, Hellwig M (1978) Utilization and cooxidation of chlorinated phenols by Pseudomonas sp. B 13. Arch Microbiol 117:1-7

Degradation by Bacteria of Chlorinated Hydrocarbons

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