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Activity and survival of tribromophenol-degrading bacteria in a contaminated desert soil
Soil Biology & Biochemistry 32 (2000) 1643±1650
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Activity and survival of tribromophenol-degrading bacteria in a contaminated desert soil Zeev Ronen, Luba Vasiluk, Aharon Abeliovich, Ali Nejidat* Department of Environmental Hydrology and Microbiology, The Jacob Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, Sede Boqer, Campus 84990, Israel Accepted 23 March 2000
Abstract A strain of bromophenol degrading bacteria was isolated from a contaminated desert soil. The isolate identi®ed as Achromobacter piechaudii and designated as strain TBPZ was able to metabolize both 2,4,6-tribromophenol and chlorophenols. The degradation of halophenols resulted in the stechiometric release of bromide or chloride. Growth and degradation of bromophenol were enhanced in the presence of yeast extract. To follow the survival of an introduced bacteria in the contaminated soil, TBPZ was transformed with a plasmid carrying a gene for kanamycin resistance and the lux CDABE operon from the luminescent bacteria Vibrio ®scheri under the control of a constitutive promoter producing strain TBPZ-N61. The activity of the transformed bacteria was not aected by the insertion of the plasmid. Speci®c detection of the introduced isolate in the contaminated soil samples was achieved by selection on kanamycin. Survival of the introduced bacteria, TBPZ-N61, in the contaminated soil was in¯uenced by soil moisture. Biodegradation of TBP occurred only in soil with at least 25% water content. Addition of yeast extract increased the survival and the activity of the introduced bacteria. The current study demonstrated that the limiting factors controlling pollutant degradation in a contaminated desert soil are water content, nutrient availability and the bioaugmentation of an appropriate microbial population. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Bioaugmentation; Biodegradation; Nutrient availability; Water content
1. Introduction The Negev desert occupies almost two-thirds of the land area in Israel. There is a tendency to relocate major industrial activities to this area from the more populated ones. Industrial parks in the northern Negev comprise chemical factories including Israel's largest producers of insecticides, fungicides, herbicides, ¯ame retardants and various industrial intermediates, many of which are halogenated aromatics (Belkin et al., 1994). In addition, industrial and municipal wastes from other areas of the country have been transported to the Negev to be stored and treated. As a result, * Corresponding author. Tel.: +1972-765-96-830; fax: +1972-76596-832. E-mail address: [email protected] (A. Nejidat).
contamination from hazardous wastes has occurred in several locations threatening the fragile desert ecosystem and the quality of aquifer waters (Nativ et al., 1995). Recovery of a desert ecosystem from perturbation is slow, as the main biological attenuation process of organic chemicals in the soil (mainly microbial activity) is much aected by the low soil water content (Grin, 1977). Bacterial activity occurs at water activity above 0.90 (Skujins, 1977), which happens rarely in desert soils. Thus, in order to evaluate the potential of desert soils to remediate organic compounds from this system, one must ®rst consider the eect of soil water content on the microbial activity (Abeliovich and Ronen, 1998). The winter season in the Negev desert is short and characterized by erratic and irregular rainfalls with annual rainfall in the range of 50±200 mm
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(Shem-Tov et al., 1999). Thus, water activity that is sucient to support bacterial activity in the soil occurs only during a short period of the year, although, microbial activity can be in¯uenced by daily changes of relative air humidity (Mazor et al., 1996). The current study was aimed at studying factors that may aect the biodegradation of haloaromatics in soil samples drawn from the contaminated sites in the Negev desert. Numerous studies on the biodegradation of haloaromatics and bioremediation of halophenolscontaminated soil under dierent conditions have been carried out (Haggblom and Valo, 1995). However, to the best of our knowledge, there is no information available on the fate of these compounds in desert soils. The aerobic degradation of polychlorinated phenols follows a series of hydroxylation dehalogenation reactions before ring breakdown (Fetzner and Lingens, 1994). A factor that may limit the extent of the metabolism of brominated phenols is the low carbon content of the compound. For example, in 2,4,6-tribromophenol (TBP), carbon and bromide account for 21.6% and 72% of the compound weight, respectively. As a result, the high concentration of the compound required for the supply of sucient carbon for growth may have a toxic eect. Thus, there is need of an auxiliary carbon source to support the growth and biodegradation of the target compound. In this study, we report on the isolation of a halophenol-degrading bacterium from contaminated soils in the Negev desert. To monitor its survival and activity, the TBP-degrading strain was genetically transformed to display kanamycin resistance and luminescence. The survival and activity of the engineered strain were determined in the contaminated soil samples. 2. Materials and methods 2.1. Isolation of tribromophenol-degrading bacteria Four soil samples from dierent depths (0±50 cm)
were collected from the vicinity of an industrial park in the Israeli northern Negev desert during the summer of 1993. Physical and chemical characteristics of the soil are described in Table 1. Soil samples were stored in polyethylene bags at 48C until use. Enrichment cultures (four samples) were performed in 100 ml chloride-free medium (Shochat et al., 1993) containing TBP (100 mg/1) and yeast extract (100 mg/l) and were inoculated with 10 g soil for 4 weeks. A TBP degrading bacterial strain (designated TBPZ) was isolated from the enrichments by repetitive streaking on solid medium and was used throughout this study. TBPZ was characterized based on 16S rDNA sequences (Genbank Accession No. AF237784), fatty acid pro®le and physiological tests (DSMZ, Braunschweig, Germany). For the determination of a viable microbial count, six soil samples from dierent depths were collected using a manual auger. Ten grams from each sample were suspended in 90 ml of 0.1% (w/v) pyrophosphate solution and shaken (150 rpm) for 1 h to release the bacteria (Trolldenier, 1996a). After settling, decimal dilutions (10ÿ3±10ÿ9) were prepared in a saline buer and 50 ml from each dilution were plated on PTYG medium. 2.2. Transformation of TBPZ In order to allow the speci®c detection of TBPZ strain in inoculated soil samples, it was transformed with a plasmid that provided two functions; kanamycin resistance and luminescence. Cells of the isolate TBPZ were transformed using an electroporation apparatus (BioRad Gene Pulser). Preparation of competent cells and electroporation were performed as suggested by the manufacturer's instructions for Escherichia coli. The wide range plasmid pUCD615 (Rozen et al., 1999) containing promoterless Vibrio ®scheri lux CDABE genes and a gene for kanamycin resistance was applied in the transformation of TBPZ cells. Several promoters were cloned into the BamHI site upstream of the promoterless lux CDABE operon to drive its expression. The highest luminescence, in a
Table 1 Physical and chemical properties of the soil used in this study Depth (cm)
Sand (%)
Silt (%)
Clay (%)
Water holding capacity (%)
Water extracted organic mattera (mg/kg)
pH
EC (mS)
TBP (mg/kg)
0±5 10±25 25±65 65±95
57 56 50 41
30 27 27 28
13 17 23 22
25.27 23.93 Not determined Not determined
181.67217.55b 40.67210.06 33.67211.93 40.00216.09
7.8720.12 8.2220.11 8.4920.15 8.6120.19
24.6527.2 14.7822.43 9.5023.25 8.0322.85
99.4328.15 48.20221.60 19.07213.03 9.0721.01
a b
Extracted with water (1:1) and analysed by total organic carbon analyser. The data represents the average of four dierent samples2SD.
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pure culture, was obtained with a promoter isolated from the bacterium Nitrosomonas europaea (Nejidat et al., 1996). The bacterial strain bearing the resulting plasmid (pUCD615 with active lux CDABE operon) was designated TBPZ-N61. 2.3. Soil inoculation Bacterial cells (TBPZ-N61) were cultured in a PTYG medium (containing in 1 l: peptone 0.5 g, tryptone 0.5 g, yeast extract 1 g, glucose 1 g, MgSO47H2O 0.6 g and CaCl2 0.07 g). Cells were concentrated from a 500 ml culture, the pellet washed twice with 50 ml saline solution (0.85% NaCl) and resuspended in a saline solution to a concentration of 109 cells/ml. Soil inoculation for TBP degradation was performed as follows: 2 g of dry soil were weighed into 20 ml vials and the following components were added: yeast extract (50 mg/g), TBP (50 mg/g), 100 ml of bacterial suspension (108 cells) and distilled sterile water was added to complete the desired soil water content (10%, 25%, or 50%) taking into account the water volume of the other additives. A non-inoculated soil sample was used as a control (®nal water content of 50%). Microbial counts were done as previously described (Trolldenier, 1996a). To each vial (2 g soil freshly collected), 8 ml of 0.1% (w/v) pyrophosphate solution was added and the suspension was shaken (150 rpm) for 1 h to release the bacteria. After settling, decimal dilutions (10ÿ3±10ÿ9) were prepared in saline buer and 50 ml from each dilution were plated on PTYG medium supplemented with 50 mg/ml kanamycin and solidi®ed with 15 g/l agar (Difco). Randomly selected colonies were tested for luminescence and TBP dehalogenation in order to con®rm the identity of the bacterial colonies. Bacterial count was calculated based on the ®rst lower dilution, where separate colonies can be easily distinguished and counted. For every time point in each experiment, two vials were sacri®ced and duplicate plates were cultured for each dilution. Aliquots of 1 ml from the ®rst dilution (10ÿ1) were centrifuged (5 min in a microfuge) and then used for TBP determination. As a possible substitute for the viable colony counts, we attempted to use the luminescence capacity of strain TBPZ-N61 to determine its number in the soil samples. Thus, a most-probable-number (MPN) method for the detection and enumeration of the bioluminescent TBPZ-N61 was tested. From the ®rst soil dilution, 20 ml was used to prepare the decimal dilutions in 96 wells microtiter plates (Dynatech, V, USA) containing 180 ml of PTYG medium. Light production was measured for over 9 h with 5 wells for each dilution. Luminescence was measured in a microtiter plate luminometer (Anthos Labtech, Salzburg, Austria, model Lucy 1). Luminescence values are pre-
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sented in the instrument's arbitrary relative light units (RLU) and positive score was given to wells emitting 100% more light than background (50±80 RLU). Microbial count was then calculated from the MPN tables (Trolldenier, 1996b) and compared with the viable colony counts. Experiments were repeated at least three times. 2.4. Analytical assays TBP concentrations were measured with reverse phase HPLC (Kontron Milan). The compound was separated on a 25-cm long LC-18 column (Supelco, Bellefonte, PA). The mobile phase was a mixture of methanol with 1% acetic acid (phase A) and ammonium acetate (18 mM) buer with 1% acetic acid (phase B). TBP was separated by a gradient run that started at 60% phase A and was then increased linearly to 100% over 13 min. The ¯ow rate of the mobile phase was 1.5 ml/min. TBP was detected at 290 nm and quanti®ed by using an external standard. Bromide was determined by a colorimetric method (Shochat et al., 1993). 3. Results 3.1. Characterization and identi®cation of strain TBPZ A gram-negative bacterial strain, designated as TBPZ and capable of degrading TBP, was isolated from the enrichment cultures. Partial sequences (468 bp) of 16S rDNA showed 98% identity to Achromobacter piechaudii. Fatty acid pro®le (data not shown) and physiological tests (Table 2) con®rmed the 16S rDNA sequence data. TBPZ was able to metabolize 2,4,6-tribromophenol (TBP) and trichlorophenols (TCP) and could also utiTable 2 Physiological properties of Achromobacter piechaudii, strain TBPZ Shape of cells
Rod
Utilization of:
Width (mm) Length Gram reaction Lysis by 3% KOH Autotrophic growth Denitri®cation Motility Oxidase Catalase Aminopeptidase (cerny) ADH Urease Hydrolysis of gelatin
0.5±0.7 1.5±2.5 ÿ + ÿ ÿ + + + + ÿ + ÿ
Glucose Phenylacetate Citrate Malate Arabinose Adipate Isovalerate Itaconate Gluconate OF tests: Glucose, aerobic Glucose, anaerobic Xylose, aerobic Xylose, anaerobic
ÿ + + + ÿ + + + ÿ ÿ ÿ ÿ ÿ
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lize phenol and catachol, but was not able to metabolize mono- or di-bromophenol isomers (data not shown). Dehalogenation of the halophenols by TBPZ resulted in a nearly stechiometric release of the halogen ion into the culture medium (Fig. 1) and we were not able to detect any accumulation of partially dehalogenated intermediates. The maximum concentrations of TBP and TCP tested for dehalogenation were 1.25 mM (Fig. 1). On an average, dehalogenation of 1 mmol of TBP or TCP resulted in the release of 2.44 and 2.52 mmol bromide and chloride, respectively, indicating that some halogenated organic material remained in the medium. Growth of strain TBPZ on TBP alone was slow, as was the dehalogenation process (Fig. 2). However, growth and dehalogenation were enhanced by the addition of yeast extract (Fig. 2). The growth of the strain TBPZ was also aected by the salinity of the culture medium. Growth rate of TBPZ showed almost constant increase between 0% and 3% NaCl. At 0% NaCl, on PTYG medium, growth rate was 0.22 hÿ1 which then increased to 0.32 hÿ1 at 3% NaCl but declined to 0.05 hÿ1 at 4% NaCl.
phase were observed (Fig. 3) possibly due to the additional expression of kanamycin transforming enzyme and the lux operon. No growth of the wild type was observed on kanamycin.
3.2. Transformation of strain TBPZ Transformation of strain TBPZ with the plasmid pUCD615, in order to confer kanamycin resistance, did not signi®cantly aect its growth and dehalogenation activity when compared to the wild-type strain. However, in the presence of kanamycin (50 mg/ml), a longer lag phase and slower growth rate during the log
Fig. 1. Bromide and chloride released by Achromobacter piechaudii TBPZ at dierent concentrations of 2,4,6-TBP and 2,4,6-TCP. Cultures were incubated for 1 week in a mineral medium with the addition of 100 mg/l yeast extract before analysis. Symbols: (--) chloride, and (-.-) bromide release. The results are the mean of three similar experiments.
Fig. 2. Eect of yeast extract concentrations on the growth of Achromobacter piechaudii TBPZ (A), TBP biodegradation (B), and bromide release (C). Symbols: (-.-) 0, (--) 50, (-W-) 100, (-q-) 200 mg/l yeast extract. The results are the mean of two similar experiments 2 SD.
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Fig. 4. Distribution of bacterial counts (A) and soil water content (B) in the contaminated soil pro®le. Microbial count was performed with (--) or without (-W-) addition of 3% NaCl into the culture. The results are the mean of three replicates for each depth2SD.
3.3. Activity and survival of the transformed strain in soil samples
Fig. 3. Biodegradation of TBP (A), growth (B) and bromide release (C) by Achromobacter piechaudii TBPZ and Achromobacter piechaudii TBPZ-N61 in the absence and presence of kanamycin (50 mg/ml). Symbols: (-.-) Achromobacter piechaudii TBPZ with kanamycin, (--) Achromobacter piechaudii TBPZ without kanamycin, (-q-) Achromobacter piechaudii TBPZ-N61 with kanamycin, (-W-) Achromobacter piechaudii TBPZ-N61 without kanamycin. The results are the mean of three experiments2SD.
Before inoculation of the contaminated soil samples, the distribution of microbial populations and moisture content were determined from dierent depths at the contaminated site. The results indicate that both, bacterial count and water content were signi®cantly increased in deeper samples (Fig. 4). Including 3% of NaCl (equivalent to the estimated salt content of the soil, Table 1) in the growth medium signi®cantly increased the viable counts. Plotting of bacterial counts against water content values (data not shown) indicates a signi®cant correlation R 0:86 and 0.93 with and without NaCl, respectively) between the two variables. The strain TBPZ was isolated from the homogenized soil columns between 0 and 50 cm depth where water content was between 2% and 10% and total microbial count was 5 103 ±1:5 105 /g soil. TBP concentration ranges between 100 mg/kg in the
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surface and 19 mg/kg at 45 cm below surface (Table 1). No kanamycin resistant bacteria were detected in the soil samples. In the following experiments, the activity and survival of the transformant (TBPZ-N61) were tested in soil samples. Signi®cant degradation of TBP and survival of the introduced bacteria were not observed in samples without the addition of yeast extract (data not shown). Thus, yeast extract was included in all experiments. Initially we compared the MPN-lux method for determining the number of bacteria in the soil to the spread plate method. When adding 2:2 108 cells/ g soil, based upon the spread plate method, 2:7 107 cell/g were recovered. This reduction in the viable-cells number was re¯ected in the luminescence measurements, where luminescence produced by 10ÿ3 dilution of bacteria after mixing with soil samples was 10 times lower than that produced by the corresponding dilution of the culture (Fig. 5). Based on the MPN-lux method, the calculation indicated that only 2:7 106 cell/g soil were recovered at zero time after inoculation with 2:2 108 cells/g soil compared to 2:7 107 cells/g soil obtained with the spread plate method. Thus, the spread plate method was chosen for the next set of experiments. However, three criteria were used for the speci®c detection of the transformant in the soil samples, kanamycin resistance, luminescence and dehalogenation of TBP. The eect of water content on the survival of the transformant and degradation of TBP in soil samples was studied. The results indicate that at 25% and 50% water content, TBP degradation was rapid (Fig. 6(A)), whereas there was almost no change in its concentration in soil samples with only 10% water content.
Fig. 5. Kinetics of light production by Achromobacter piechaudii TBPZ-N61 inoculated into medium (-.-) and soil (-W-). Suspensions of Achromobacter piechaudii TBPZ-N61 were added into soil and after mixing, the cells were recovered as described in Section 2. The light produced by the 10ÿ3 dilution of the culture or recovered suspension was measured over 9 h.
Adding another dose of TBP (50 mg/g soil) after 17 days to the inoculated soil resulted in its rapid degradation in samples including 25 and 50% water content. Under a controled treatment (50% water content without inoculation), biodegradation of TBP was very slow. In parallel, colony count, in the 25% and 50% water content treatments, showed an initial decline in the cell number that stabilized at about 5 106 CFU/g soil (Fig. 6(B)). Subsequently, microbial counts con-
Fig. 6. Eect of water content on biodegradation of TBP (A) and survival of Achromobacter piechaudii TBPZ-N61 (B) in contaminated soil samples. (-W-) Control (non-inoculated soil with 50% water content), (-q-) inoculated 2 108 cells/g) 10% water content, (-R-) inoculated 2 108 cells/g) 25% water and (--) inoculated 2 108 cells/g) 50% water content. After 17 days (arrow) a second dose of TBP (50 mg/g soil) was added to the 25 and 50% water content treatments. The results are representative experiment of three similar ones.
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tinuously declined, and after 30 days the microbial count was minimal. Addition of TBP at this time showed no degradation (data not shown). At 10% water content, the microbial population declined rapidly to below 100 CFU/g after 13 days. In the control treatment, no appearance of kanamycin resistance colonies was observed. 4. Discussion An Achromobacter piechaudii strain (TBPZ) capable of degrading TBP was isolated from a contaminated desert soil, although gram-negative bacteria is usually not abundant in soil from the Negev desert (Dobrovol'skaya et al., 1996). However, its survival and biodegradative activity occurred only at water content levels which are not typical to desert soil (Fig. 6). Achromobacter sp. were previously used for the remediation of soils contaminated with polycyclic aromatic hydrocarbons (Cutright and Lee, 1994, 1995). The biodegradation pathway of halophenols involves a series of oxidative dehalogenation reactions. For example, Azotobacter GPl degrades 2,4,6-trichlorophenol to 2,6dichlorohydroquinone (Li et al., 1991). At this time, we do not know if the degradation of TBP by Achromobacter piechaudii TBPZ follows a similar route. Addition of yeast extract was necessary to obtain an enhanced growth of TBPZ and dehalogenation (Fig. 2). Furthermore, the addition of yeast extract was necessary for the survival and activity of the transformed strain in the contaminated soil samples (Fig. 6). It is possible that yeast extract provides necessary nutrients including an additional carbon source for growth, since TBP contains little assimilable carbon. However, the eect of yeast extract on biodegradation of haloaromatics was attributed to its protective eect that may reduce the toxic eect of the degradable compounds (Fava et al., 1995; Armenante et al., 1995; Kafkewitz et al., 1996). Thus, the possibility that yeast extract may have multiple eects cannot be ruled out. The transformation of strain TBP with a plasmid did not have a signi®cant eect on its basic characteristics (Fig. 3). Therefore, the transformant (TBPZN61) was used as an indication for activity and survival of Achromobacter piechaudii TBPZ in the soil. The application of the MPN-lux method (Flemming et al., 1994; Arias et al., 1996) for detecting the bacteria in the soil was hampered by the low light output produced by Achromobacter piechaudii TBPZ-N61 (Fig. 5). Thus, the speci®c detection of TBPZ-N61 was done by selection on kanamycin and the luminescence capacity was used as supporting criterion. At 50% water content, the survival of the inoculated bacteria and TBP degradation rates were not signi®cantly dierent from those at 25% water content.
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When a second dose of TBP was added to the soil samples containing 25% or 50% water content, TBP degradation was fast, even 15 days after inoculation. However, no signi®cant TBP degradation was observed when additional dose was added 15 days later when bacterial counts recovered were low (data not shown). This indicates a correlation between the recovered bacterial counts and activity and supports the possibility that the reduction in bacterial counts after mixing with soil samples is due to the loss of viability rather than adsorption or low recovery. Bacterial survival in the soil is determined by biotic factors such as perdition and competition or abiotic factors such as water tension, organic and inorganic nutrients, pH, temperature, and toxic chemicals (van Veen et al., 1997). The results reported here demonstrate that contaminated soil in the Negev desert can be inoculated with bacteria that are able to degrade haloaromatics. Water content and the availability of nutrients have a major eect on the survival and activity of the introduced bacteria. Acknowledgements This study was funded in part by the Israeli Ministry of the Environment. References Abeliovich, A., Ronen, Z., 1998. Pollution and bioremediation of soils in arid zone. In: Sikdar, S.K., Irvine, R.L. (Eds.), Biodegradation Technology Development. Techonomic Publishing, Lancaster, pp. 111±121. Arias, R.S., Mochizuki, G., Fukui, R., Alvarez, A.M., 1996. Most probable number method to enumerate a bioluminescent Xanthomonas campestris pv. Campestris in soil. Soil Biology and Biochemistry 28, 1725±1728. Armenante, P.M., Fava, F., Kafkewitz, D., 1995. Eect of yeast extract on growth kinetics during aerobic biodegradation of chlorobenzoic acids. Biotechnology and Bioengineering 47, 227± 233. Belkin, S., Abeliovich, A., Brenner, A., Lebel, A., 1994. Wastewater treatment in the desert. Industrial Wastewater 2, 34±39. Cutright, T.J., Lee, S.G., 1994. Remediation of PAH-contaminated soil using Achromobacter sp. Energy Sources 16, 279±287. Cutright, T.J., Lee, S.G., 1995. In-situ soil remediation Ð bacteria or fungi. Energy Sources 17, 413±419. Dobrovol'skaya, T.G., Chernov, I.Y., Zenova, G.M., 1996. Vertical structure of bacterial complexes in the Negev desert (Israel). Mikrobiologiya (in Russian) 65, 282±287. Fava, F., Armenante, P.M., Kafkewitz, D., Marchetti, L., 1995. In¯uence of organic and inorganic growth supplements on the aerobic biodegradation of chlorobenzoic acids. Applied Microbiology and Biotechnology 43, 171±177. Fetzner, S., Lingens, F., 1994. Bacterial dehalogenases: biochemistry, genetics and biotechnological applications. Microbiological Reviews 58, 641±685. Flemming, C.A., Lee, H., Trevors, J.T., 1994. Bioluminescent mostprobable-number method to enumerate lux-marked Pseudomonas
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aeruginosa UG2Lr in soil. Applied and Environmental Microbiology 60, 3458±3461. Grin, D.M., 1977. Water and microbial stress. Advances in Microbial Ecology 5, 91±136. Haggblom, M.M., Valo, R.J., 1995. Bioremediation of chlorophenol wastes. In: Young, L.Y., Cerniglia, C.E. (Eds.), Microbial Transformation and Degradation of Toxic Organic Chemicals. Wiley-Liss, New York, pp. 389±434. Kafkewitz, D., Fava, F., Armenante, P.M., 1996. Eect of vitamins on the aerobic degradation of 2-chlorophenol, 4-chlorophenol, and 4-chlorobiphenyl. Applied Microbiology and Biotechnology 46, 414±421. Li, D.Y., Ebersacher, J., Wagner, B., Kuntzer, J., Lingens, F., 1991. Degradation of 2,4,6-Trichlorophenol by Azotobacter sp. strain GP1. Applied and Environmental Microbiology 57, 1920±1928. Mazor, G., Kidron, G.J., Vonshak, A., Abeliovich, A., 1996. The role of cyanobacterial exopolysaccharides in structuring desert microbial crusts. FEMS Microbiology Ecology 21, 121±130. Nativ, R., Adar, E., Daham, O., Geyh, M., 1995. Water recharge and solute transport through the vadose zone of fractured chalk under desert conditions. Water Resources Research 31, 253±261. Nejidat, A., Spector, O., Abeliovich, A., 1996. Isolation and characterization of a functional promoter from Nitrosomonas europaea. FEMS Microbiological Letters 137, 9±12.
Rozen, Y., Nejidat, A., Gartemann, K.-H., Belkin, S., 1999. Speci®c detection of p-Chlorobenzoic acid by Escherichia coli bearing a plasmid-borne fcbA'::lyx fusion. Chemosphere 38, 633±641. Shem-Tov, S., Zaady, E., Groman, P.M., Gutterman, Y., 1999. Soil carbon content along a rainfall gradient and inhibition of germination: a potential mechanism for regulating distribution of Plantago coronopus. Soil Biology and Biochemistry 31, 1209± 1217. Shochat, E., Hermoni, I., Cohen, Z., Abeliovich, A., Belkin, S., 1993. Bromoalkane-degrading Pseudomonas strains. Applied and Environmental Microbiology 59, 1403±1409. Skujins, J., 1977. Microbial ecology of desert soils. Advances in Microbial Ecology 7, 49±91. Trolldenier, G., 1996a. Plate count technique. In: Schinner, E., Ohlinger, R., Kandeler, E., Margesine, R. (Eds.), Methods in Soil Biology. Springer-Verlag, Berlin, Heidelberg, pp. 20±26. Trolldenier, G., 1996b. Physiological groups by most probable number method. In: Schinner, E., Ohlinger, R., Kandeler, E., Margesine, R. (Eds.), Methods in Soil Biology. Springer-Verlag, Berlin, Heidelberg, pp. 26±28. van Veen, J.A., van Overbeek, L.S., van Elsas, J.D., 1997. Fate and activity of microorganisms introduced into soil. Microbiological and Molecular Biology Review 61, 121±135.
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Activity and survival of tribromophenol-degrading bacteria in a contaminated desert soil Zeev Ronen, Luba Vasiluk, Aharon Abeliovich, Ali Nejidat* Department of Environmental Hydrology and Microbiology, The Jacob Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, Sede Boqer, Campus 84990, Israel Accepted 23 March 2000
Abstract A strain of bromophenol degrading bacteria was isolated from a contaminated desert soil. The isolate identi®ed as Achromobacter piechaudii and designated as strain TBPZ was able to metabolize both 2,4,6-tribromophenol and chlorophenols. The degradation of halophenols resulted in the stechiometric release of bromide or chloride. Growth and degradation of bromophenol were enhanced in the presence of yeast extract. To follow the survival of an introduced bacteria in the contaminated soil, TBPZ was transformed with a plasmid carrying a gene for kanamycin resistance and the lux CDABE operon from the luminescent bacteria Vibrio ®scheri under the control of a constitutive promoter producing strain TBPZ-N61. The activity of the transformed bacteria was not aected by the insertion of the plasmid. Speci®c detection of the introduced isolate in the contaminated soil samples was achieved by selection on kanamycin. Survival of the introduced bacteria, TBPZ-N61, in the contaminated soil was in¯uenced by soil moisture. Biodegradation of TBP occurred only in soil with at least 25% water content. Addition of yeast extract increased the survival and the activity of the introduced bacteria. The current study demonstrated that the limiting factors controlling pollutant degradation in a contaminated desert soil are water content, nutrient availability and the bioaugmentation of an appropriate microbial population. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Bioaugmentation; Biodegradation; Nutrient availability; Water content
1. Introduction The Negev desert occupies almost two-thirds of the land area in Israel. There is a tendency to relocate major industrial activities to this area from the more populated ones. Industrial parks in the northern Negev comprise chemical factories including Israel's largest producers of insecticides, fungicides, herbicides, ¯ame retardants and various industrial intermediates, many of which are halogenated aromatics (Belkin et al., 1994). In addition, industrial and municipal wastes from other areas of the country have been transported to the Negev to be stored and treated. As a result, * Corresponding author. Tel.: +1972-765-96-830; fax: +1972-76596-832. E-mail address: [email protected] (A. Nejidat).
contamination from hazardous wastes has occurred in several locations threatening the fragile desert ecosystem and the quality of aquifer waters (Nativ et al., 1995). Recovery of a desert ecosystem from perturbation is slow, as the main biological attenuation process of organic chemicals in the soil (mainly microbial activity) is much aected by the low soil water content (Grin, 1977). Bacterial activity occurs at water activity above 0.90 (Skujins, 1977), which happens rarely in desert soils. Thus, in order to evaluate the potential of desert soils to remediate organic compounds from this system, one must ®rst consider the eect of soil water content on the microbial activity (Abeliovich and Ronen, 1998). The winter season in the Negev desert is short and characterized by erratic and irregular rainfalls with annual rainfall in the range of 50±200 mm
0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 8 0 - 8
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(Shem-Tov et al., 1999). Thus, water activity that is sucient to support bacterial activity in the soil occurs only during a short period of the year, although, microbial activity can be in¯uenced by daily changes of relative air humidity (Mazor et al., 1996). The current study was aimed at studying factors that may aect the biodegradation of haloaromatics in soil samples drawn from the contaminated sites in the Negev desert. Numerous studies on the biodegradation of haloaromatics and bioremediation of halophenolscontaminated soil under dierent conditions have been carried out (Haggblom and Valo, 1995). However, to the best of our knowledge, there is no information available on the fate of these compounds in desert soils. The aerobic degradation of polychlorinated phenols follows a series of hydroxylation dehalogenation reactions before ring breakdown (Fetzner and Lingens, 1994). A factor that may limit the extent of the metabolism of brominated phenols is the low carbon content of the compound. For example, in 2,4,6-tribromophenol (TBP), carbon and bromide account for 21.6% and 72% of the compound weight, respectively. As a result, the high concentration of the compound required for the supply of sucient carbon for growth may have a toxic eect. Thus, there is need of an auxiliary carbon source to support the growth and biodegradation of the target compound. In this study, we report on the isolation of a halophenol-degrading bacterium from contaminated soils in the Negev desert. To monitor its survival and activity, the TBP-degrading strain was genetically transformed to display kanamycin resistance and luminescence. The survival and activity of the engineered strain were determined in the contaminated soil samples. 2. Materials and methods 2.1. Isolation of tribromophenol-degrading bacteria Four soil samples from dierent depths (0±50 cm)
were collected from the vicinity of an industrial park in the Israeli northern Negev desert during the summer of 1993. Physical and chemical characteristics of the soil are described in Table 1. Soil samples were stored in polyethylene bags at 48C until use. Enrichment cultures (four samples) were performed in 100 ml chloride-free medium (Shochat et al., 1993) containing TBP (100 mg/1) and yeast extract (100 mg/l) and were inoculated with 10 g soil for 4 weeks. A TBP degrading bacterial strain (designated TBPZ) was isolated from the enrichments by repetitive streaking on solid medium and was used throughout this study. TBPZ was characterized based on 16S rDNA sequences (Genbank Accession No. AF237784), fatty acid pro®le and physiological tests (DSMZ, Braunschweig, Germany). For the determination of a viable microbial count, six soil samples from dierent depths were collected using a manual auger. Ten grams from each sample were suspended in 90 ml of 0.1% (w/v) pyrophosphate solution and shaken (150 rpm) for 1 h to release the bacteria (Trolldenier, 1996a). After settling, decimal dilutions (10ÿ3±10ÿ9) were prepared in a saline buer and 50 ml from each dilution were plated on PTYG medium. 2.2. Transformation of TBPZ In order to allow the speci®c detection of TBPZ strain in inoculated soil samples, it was transformed with a plasmid that provided two functions; kanamycin resistance and luminescence. Cells of the isolate TBPZ were transformed using an electroporation apparatus (BioRad Gene Pulser). Preparation of competent cells and electroporation were performed as suggested by the manufacturer's instructions for Escherichia coli. The wide range plasmid pUCD615 (Rozen et al., 1999) containing promoterless Vibrio ®scheri lux CDABE genes and a gene for kanamycin resistance was applied in the transformation of TBPZ cells. Several promoters were cloned into the BamHI site upstream of the promoterless lux CDABE operon to drive its expression. The highest luminescence, in a
Table 1 Physical and chemical properties of the soil used in this study Depth (cm)
Sand (%)
Silt (%)
Clay (%)
Water holding capacity (%)
Water extracted organic mattera (mg/kg)
pH
EC (mS)
TBP (mg/kg)
0±5 10±25 25±65 65±95
57 56 50 41
30 27 27 28
13 17 23 22
25.27 23.93 Not determined Not determined
181.67217.55b 40.67210.06 33.67211.93 40.00216.09
7.8720.12 8.2220.11 8.4920.15 8.6120.19
24.6527.2 14.7822.43 9.5023.25 8.0322.85
99.4328.15 48.20221.60 19.07213.03 9.0721.01
a b
Extracted with water (1:1) and analysed by total organic carbon analyser. The data represents the average of four dierent samples2SD.
Z. Ronen et al. / Soil Biology & Biochemistry 32 (2000) 1643±1650
pure culture, was obtained with a promoter isolated from the bacterium Nitrosomonas europaea (Nejidat et al., 1996). The bacterial strain bearing the resulting plasmid (pUCD615 with active lux CDABE operon) was designated TBPZ-N61. 2.3. Soil inoculation Bacterial cells (TBPZ-N61) were cultured in a PTYG medium (containing in 1 l: peptone 0.5 g, tryptone 0.5 g, yeast extract 1 g, glucose 1 g, MgSO47H2O 0.6 g and CaCl2 0.07 g). Cells were concentrated from a 500 ml culture, the pellet washed twice with 50 ml saline solution (0.85% NaCl) and resuspended in a saline solution to a concentration of 109 cells/ml. Soil inoculation for TBP degradation was performed as follows: 2 g of dry soil were weighed into 20 ml vials and the following components were added: yeast extract (50 mg/g), TBP (50 mg/g), 100 ml of bacterial suspension (108 cells) and distilled sterile water was added to complete the desired soil water content (10%, 25%, or 50%) taking into account the water volume of the other additives. A non-inoculated soil sample was used as a control (®nal water content of 50%). Microbial counts were done as previously described (Trolldenier, 1996a). To each vial (2 g soil freshly collected), 8 ml of 0.1% (w/v) pyrophosphate solution was added and the suspension was shaken (150 rpm) for 1 h to release the bacteria. After settling, decimal dilutions (10ÿ3±10ÿ9) were prepared in saline buer and 50 ml from each dilution were plated on PTYG medium supplemented with 50 mg/ml kanamycin and solidi®ed with 15 g/l agar (Difco). Randomly selected colonies were tested for luminescence and TBP dehalogenation in order to con®rm the identity of the bacterial colonies. Bacterial count was calculated based on the ®rst lower dilution, where separate colonies can be easily distinguished and counted. For every time point in each experiment, two vials were sacri®ced and duplicate plates were cultured for each dilution. Aliquots of 1 ml from the ®rst dilution (10ÿ1) were centrifuged (5 min in a microfuge) and then used for TBP determination. As a possible substitute for the viable colony counts, we attempted to use the luminescence capacity of strain TBPZ-N61 to determine its number in the soil samples. Thus, a most-probable-number (MPN) method for the detection and enumeration of the bioluminescent TBPZ-N61 was tested. From the ®rst soil dilution, 20 ml was used to prepare the decimal dilutions in 96 wells microtiter plates (Dynatech, V, USA) containing 180 ml of PTYG medium. Light production was measured for over 9 h with 5 wells for each dilution. Luminescence was measured in a microtiter plate luminometer (Anthos Labtech, Salzburg, Austria, model Lucy 1). Luminescence values are pre-
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sented in the instrument's arbitrary relative light units (RLU) and positive score was given to wells emitting 100% more light than background (50±80 RLU). Microbial count was then calculated from the MPN tables (Trolldenier, 1996b) and compared with the viable colony counts. Experiments were repeated at least three times. 2.4. Analytical assays TBP concentrations were measured with reverse phase HPLC (Kontron Milan). The compound was separated on a 25-cm long LC-18 column (Supelco, Bellefonte, PA). The mobile phase was a mixture of methanol with 1% acetic acid (phase A) and ammonium acetate (18 mM) buer with 1% acetic acid (phase B). TBP was separated by a gradient run that started at 60% phase A and was then increased linearly to 100% over 13 min. The ¯ow rate of the mobile phase was 1.5 ml/min. TBP was detected at 290 nm and quanti®ed by using an external standard. Bromide was determined by a colorimetric method (Shochat et al., 1993). 3. Results 3.1. Characterization and identi®cation of strain TBPZ A gram-negative bacterial strain, designated as TBPZ and capable of degrading TBP, was isolated from the enrichment cultures. Partial sequences (468 bp) of 16S rDNA showed 98% identity to Achromobacter piechaudii. Fatty acid pro®le (data not shown) and physiological tests (Table 2) con®rmed the 16S rDNA sequence data. TBPZ was able to metabolize 2,4,6-tribromophenol (TBP) and trichlorophenols (TCP) and could also utiTable 2 Physiological properties of Achromobacter piechaudii, strain TBPZ Shape of cells
Rod
Utilization of:
Width (mm) Length Gram reaction Lysis by 3% KOH Autotrophic growth Denitri®cation Motility Oxidase Catalase Aminopeptidase (cerny) ADH Urease Hydrolysis of gelatin
0.5±0.7 1.5±2.5 ÿ + ÿ ÿ + + + + ÿ + ÿ
Glucose Phenylacetate Citrate Malate Arabinose Adipate Isovalerate Itaconate Gluconate OF tests: Glucose, aerobic Glucose, anaerobic Xylose, aerobic Xylose, anaerobic
ÿ + + + ÿ + + + ÿ ÿ ÿ ÿ ÿ
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lize phenol and catachol, but was not able to metabolize mono- or di-bromophenol isomers (data not shown). Dehalogenation of the halophenols by TBPZ resulted in a nearly stechiometric release of the halogen ion into the culture medium (Fig. 1) and we were not able to detect any accumulation of partially dehalogenated intermediates. The maximum concentrations of TBP and TCP tested for dehalogenation were 1.25 mM (Fig. 1). On an average, dehalogenation of 1 mmol of TBP or TCP resulted in the release of 2.44 and 2.52 mmol bromide and chloride, respectively, indicating that some halogenated organic material remained in the medium. Growth of strain TBPZ on TBP alone was slow, as was the dehalogenation process (Fig. 2). However, growth and dehalogenation were enhanced by the addition of yeast extract (Fig. 2). The growth of the strain TBPZ was also aected by the salinity of the culture medium. Growth rate of TBPZ showed almost constant increase between 0% and 3% NaCl. At 0% NaCl, on PTYG medium, growth rate was 0.22 hÿ1 which then increased to 0.32 hÿ1 at 3% NaCl but declined to 0.05 hÿ1 at 4% NaCl.
phase were observed (Fig. 3) possibly due to the additional expression of kanamycin transforming enzyme and the lux operon. No growth of the wild type was observed on kanamycin.
3.2. Transformation of strain TBPZ Transformation of strain TBPZ with the plasmid pUCD615, in order to confer kanamycin resistance, did not signi®cantly aect its growth and dehalogenation activity when compared to the wild-type strain. However, in the presence of kanamycin (50 mg/ml), a longer lag phase and slower growth rate during the log
Fig. 1. Bromide and chloride released by Achromobacter piechaudii TBPZ at dierent concentrations of 2,4,6-TBP and 2,4,6-TCP. Cultures were incubated for 1 week in a mineral medium with the addition of 100 mg/l yeast extract before analysis. Symbols: (--) chloride, and (-.-) bromide release. The results are the mean of three similar experiments.
Fig. 2. Eect of yeast extract concentrations on the growth of Achromobacter piechaudii TBPZ (A), TBP biodegradation (B), and bromide release (C). Symbols: (-.-) 0, (--) 50, (-W-) 100, (-q-) 200 mg/l yeast extract. The results are the mean of two similar experiments 2 SD.
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Fig. 4. Distribution of bacterial counts (A) and soil water content (B) in the contaminated soil pro®le. Microbial count was performed with (--) or without (-W-) addition of 3% NaCl into the culture. The results are the mean of three replicates for each depth2SD.
3.3. Activity and survival of the transformed strain in soil samples
Fig. 3. Biodegradation of TBP (A), growth (B) and bromide release (C) by Achromobacter piechaudii TBPZ and Achromobacter piechaudii TBPZ-N61 in the absence and presence of kanamycin (50 mg/ml). Symbols: (-.-) Achromobacter piechaudii TBPZ with kanamycin, (--) Achromobacter piechaudii TBPZ without kanamycin, (-q-) Achromobacter piechaudii TBPZ-N61 with kanamycin, (-W-) Achromobacter piechaudii TBPZ-N61 without kanamycin. The results are the mean of three experiments2SD.
Before inoculation of the contaminated soil samples, the distribution of microbial populations and moisture content were determined from dierent depths at the contaminated site. The results indicate that both, bacterial count and water content were signi®cantly increased in deeper samples (Fig. 4). Including 3% of NaCl (equivalent to the estimated salt content of the soil, Table 1) in the growth medium signi®cantly increased the viable counts. Plotting of bacterial counts against water content values (data not shown) indicates a signi®cant correlation R 0:86 and 0.93 with and without NaCl, respectively) between the two variables. The strain TBPZ was isolated from the homogenized soil columns between 0 and 50 cm depth where water content was between 2% and 10% and total microbial count was 5 103 ±1:5 105 /g soil. TBP concentration ranges between 100 mg/kg in the
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surface and 19 mg/kg at 45 cm below surface (Table 1). No kanamycin resistant bacteria were detected in the soil samples. In the following experiments, the activity and survival of the transformant (TBPZ-N61) were tested in soil samples. Signi®cant degradation of TBP and survival of the introduced bacteria were not observed in samples without the addition of yeast extract (data not shown). Thus, yeast extract was included in all experiments. Initially we compared the MPN-lux method for determining the number of bacteria in the soil to the spread plate method. When adding 2:2 108 cells/ g soil, based upon the spread plate method, 2:7 107 cell/g were recovered. This reduction in the viable-cells number was re¯ected in the luminescence measurements, where luminescence produced by 10ÿ3 dilution of bacteria after mixing with soil samples was 10 times lower than that produced by the corresponding dilution of the culture (Fig. 5). Based on the MPN-lux method, the calculation indicated that only 2:7 106 cell/g soil were recovered at zero time after inoculation with 2:2 108 cells/g soil compared to 2:7 107 cells/g soil obtained with the spread plate method. Thus, the spread plate method was chosen for the next set of experiments. However, three criteria were used for the speci®c detection of the transformant in the soil samples, kanamycin resistance, luminescence and dehalogenation of TBP. The eect of water content on the survival of the transformant and degradation of TBP in soil samples was studied. The results indicate that at 25% and 50% water content, TBP degradation was rapid (Fig. 6(A)), whereas there was almost no change in its concentration in soil samples with only 10% water content.
Fig. 5. Kinetics of light production by Achromobacter piechaudii TBPZ-N61 inoculated into medium (-.-) and soil (-W-). Suspensions of Achromobacter piechaudii TBPZ-N61 were added into soil and after mixing, the cells were recovered as described in Section 2. The light produced by the 10ÿ3 dilution of the culture or recovered suspension was measured over 9 h.
Adding another dose of TBP (50 mg/g soil) after 17 days to the inoculated soil resulted in its rapid degradation in samples including 25 and 50% water content. Under a controled treatment (50% water content without inoculation), biodegradation of TBP was very slow. In parallel, colony count, in the 25% and 50% water content treatments, showed an initial decline in the cell number that stabilized at about 5 106 CFU/g soil (Fig. 6(B)). Subsequently, microbial counts con-
Fig. 6. Eect of water content on biodegradation of TBP (A) and survival of Achromobacter piechaudii TBPZ-N61 (B) in contaminated soil samples. (-W-) Control (non-inoculated soil with 50% water content), (-q-) inoculated 2 108 cells/g) 10% water content, (-R-) inoculated 2 108 cells/g) 25% water and (--) inoculated 2 108 cells/g) 50% water content. After 17 days (arrow) a second dose of TBP (50 mg/g soil) was added to the 25 and 50% water content treatments. The results are representative experiment of three similar ones.
Z. Ronen et al. / Soil Biology & Biochemistry 32 (2000) 1643±1650
tinuously declined, and after 30 days the microbial count was minimal. Addition of TBP at this time showed no degradation (data not shown). At 10% water content, the microbial population declined rapidly to below 100 CFU/g after 13 days. In the control treatment, no appearance of kanamycin resistance colonies was observed. 4. Discussion An Achromobacter piechaudii strain (TBPZ) capable of degrading TBP was isolated from a contaminated desert soil, although gram-negative bacteria is usually not abundant in soil from the Negev desert (Dobrovol'skaya et al., 1996). However, its survival and biodegradative activity occurred only at water content levels which are not typical to desert soil (Fig. 6). Achromobacter sp. were previously used for the remediation of soils contaminated with polycyclic aromatic hydrocarbons (Cutright and Lee, 1994, 1995). The biodegradation pathway of halophenols involves a series of oxidative dehalogenation reactions. For example, Azotobacter GPl degrades 2,4,6-trichlorophenol to 2,6dichlorohydroquinone (Li et al., 1991). At this time, we do not know if the degradation of TBP by Achromobacter piechaudii TBPZ follows a similar route. Addition of yeast extract was necessary to obtain an enhanced growth of TBPZ and dehalogenation (Fig. 2). Furthermore, the addition of yeast extract was necessary for the survival and activity of the transformed strain in the contaminated soil samples (Fig. 6). It is possible that yeast extract provides necessary nutrients including an additional carbon source for growth, since TBP contains little assimilable carbon. However, the eect of yeast extract on biodegradation of haloaromatics was attributed to its protective eect that may reduce the toxic eect of the degradable compounds (Fava et al., 1995; Armenante et al., 1995; Kafkewitz et al., 1996). Thus, the possibility that yeast extract may have multiple eects cannot be ruled out. The transformation of strain TBP with a plasmid did not have a signi®cant eect on its basic characteristics (Fig. 3). Therefore, the transformant (TBPZN61) was used as an indication for activity and survival of Achromobacter piechaudii TBPZ in the soil. The application of the MPN-lux method (Flemming et al., 1994; Arias et al., 1996) for detecting the bacteria in the soil was hampered by the low light output produced by Achromobacter piechaudii TBPZ-N61 (Fig. 5). Thus, the speci®c detection of TBPZ-N61 was done by selection on kanamycin and the luminescence capacity was used as supporting criterion. At 50% water content, the survival of the inoculated bacteria and TBP degradation rates were not signi®cantly dierent from those at 25% water content.
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When a second dose of TBP was added to the soil samples containing 25% or 50% water content, TBP degradation was fast, even 15 days after inoculation. However, no signi®cant TBP degradation was observed when additional dose was added 15 days later when bacterial counts recovered were low (data not shown). This indicates a correlation between the recovered bacterial counts and activity and supports the possibility that the reduction in bacterial counts after mixing with soil samples is due to the loss of viability rather than adsorption or low recovery. Bacterial survival in the soil is determined by biotic factors such as perdition and competition or abiotic factors such as water tension, organic and inorganic nutrients, pH, temperature, and toxic chemicals (van Veen et al., 1997). The results reported here demonstrate that contaminated soil in the Negev desert can be inoculated with bacteria that are able to degrade haloaromatics. Water content and the availability of nutrients have a major eect on the survival and activity of the introduced bacteria. Acknowledgements This study was funded in part by the Israeli Ministry of the Environment. References Abeliovich, A., Ronen, Z., 1998. Pollution and bioremediation of soils in arid zone. In: Sikdar, S.K., Irvine, R.L. (Eds.), Biodegradation Technology Development. Techonomic Publishing, Lancaster, pp. 111±121. Arias, R.S., Mochizuki, G., Fukui, R., Alvarez, A.M., 1996. Most probable number method to enumerate a bioluminescent Xanthomonas campestris pv. Campestris in soil. Soil Biology and Biochemistry 28, 1725±1728. Armenante, P.M., Fava, F., Kafkewitz, D., 1995. Eect of yeast extract on growth kinetics during aerobic biodegradation of chlorobenzoic acids. Biotechnology and Bioengineering 47, 227± 233. Belkin, S., Abeliovich, A., Brenner, A., Lebel, A., 1994. Wastewater treatment in the desert. Industrial Wastewater 2, 34±39. Cutright, T.J., Lee, S.G., 1994. Remediation of PAH-contaminated soil using Achromobacter sp. Energy Sources 16, 279±287. Cutright, T.J., Lee, S.G., 1995. In-situ soil remediation Ð bacteria or fungi. Energy Sources 17, 413±419. Dobrovol'skaya, T.G., Chernov, I.Y., Zenova, G.M., 1996. Vertical structure of bacterial complexes in the Negev desert (Israel). Mikrobiologiya (in Russian) 65, 282±287. Fava, F., Armenante, P.M., Kafkewitz, D., Marchetti, L., 1995. In¯uence of organic and inorganic growth supplements on the aerobic biodegradation of chlorobenzoic acids. Applied Microbiology and Biotechnology 43, 171±177. Fetzner, S., Lingens, F., 1994. Bacterial dehalogenases: biochemistry, genetics and biotechnological applications. Microbiological Reviews 58, 641±685. Flemming, C.A., Lee, H., Trevors, J.T., 1994. Bioluminescent mostprobable-number method to enumerate lux-marked Pseudomonas
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aeruginosa UG2Lr in soil. Applied and Environmental Microbiology 60, 3458±3461. Grin, D.M., 1977. Water and microbial stress. Advances in Microbial Ecology 5, 91±136. Haggblom, M.M., Valo, R.J., 1995. Bioremediation of chlorophenol wastes. In: Young, L.Y., Cerniglia, C.E. (Eds.), Microbial Transformation and Degradation of Toxic Organic Chemicals. Wiley-Liss, New York, pp. 389±434. Kafkewitz, D., Fava, F., Armenante, P.M., 1996. Eect of vitamins on the aerobic degradation of 2-chlorophenol, 4-chlorophenol, and 4-chlorobiphenyl. Applied Microbiology and Biotechnology 46, 414±421. Li, D.Y., Ebersacher, J., Wagner, B., Kuntzer, J., Lingens, F., 1991. Degradation of 2,4,6-Trichlorophenol by Azotobacter sp. strain GP1. Applied and Environmental Microbiology 57, 1920±1928. Mazor, G., Kidron, G.J., Vonshak, A., Abeliovich, A., 1996. The role of cyanobacterial exopolysaccharides in structuring desert microbial crusts. FEMS Microbiology Ecology 21, 121±130. Nativ, R., Adar, E., Daham, O., Geyh, M., 1995. Water recharge and solute transport through the vadose zone of fractured chalk under desert conditions. Water Resources Research 31, 253±261. Nejidat, A., Spector, O., Abeliovich, A., 1996. Isolation and characterization of a functional promoter from Nitrosomonas europaea. FEMS Microbiological Letters 137, 9±12.
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