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Journal of Environmental Quality 32:55-62 (2003)
© 2003 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America

TECHNICAL REPORTS
Bioremediation and Biodegradation

The Potential of Rhizosphere Microbes Isolated from a Constructed Wetland to Biomethylate Selenium

H. A. Azaizeh*,a, N. Salhanib, Z. Sebesvarib and H. Emonsb

a Research and Development Center, The Galilee Society (affiliated with Haifa University), P.O. Box 437, Shefa-Amr 20200, Israel
b Institute of Phytospheric Research, Research Center Juelich, 52425 Juelich, Germany

* Corresponding author (hazaizi{at}gal-soc.org)

Received for publication February 12, 2002.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The potential of rhizosphere microbes isolated from common reed [Phragmites australis (Cav.) Trin. ex Steud] plants grown in a subsurface-flow constructed wetland to biomethylate selenate or selenite was studied in liquid cultures under controlled conditions. Total mean percentages of volatilized Se from half-strength Hoagland culture solutions (low C content) supplemented with selenate or selenite and inoculated with cultured rhizosphere microbes after 15 d of incubation were 7.9 and 49.1%, respectively. There was a relative best fit (r = 0.87) between total number of rhizosphere and cultured microbes and the percentage of volatilized Se in Hoagland solution after 15 d of incubation. However, when the same microbes were cultured in tryptic soybean broth (TSB) medium (high C content), the percentages of volatilized Se from selenate and selenite were 1.3 and 1.9%, respectively. The volatilization percentages of Se from selenate or selenite in culture solutions inoculated with rhizosphere suspension instead of cultured rhizosphere microbes were very low (1.2–3.0%) in both cultivation media. In all experiments, selenite was volatilized significantly (p < 0.05) in higher amounts by cultured rhizosphere microbes after 15 d of incubation compared with selenate. Dissolved biomethylated dimethylselenide (DMSe) in water samples taken from the subsurface-flow bed was determined by purging with helium. The DMSe in water samples was indirectly detected up to 2.4 µg Se L-1, which indicates that part of the produced DMSe was dissolved in the matrix before being released into the atmosphere. Our results show that rhizosphere microbes isolated from common reed plants have a high potential of Se biomethylation and volatilization from selenate and selenite.

Abbreviations: AMVF, aquatic microcosm vegetation facility • CFU, colony forming units • DMDSe, dimethyldiselenide • DMSe, dimethylselenide • HG–AAS, hydride generation–atomic absorption spectrometry • ICP–MS, inductively coupled plasma–mass spectrometry • TSB, tryptic soybean broth


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SELENIUM (Se) is an essential micronutrient and has important benefits for animal and human nutrition; however, Se at high dosages may be toxic (Wilber, 1980; Von Vleet and Ferrans, 1992). Selenium is a metalloid that exists in five oxidation states where the Se redox speciation depends on redox potential and pH. Selenate is the major species in aerobic and neutral to alkaline environments, whereas selenite and elemental Se dominate in anaerobic environments (Lauchli, 1993). Selenium also exists in volatile forms such as dimethylselenide (DMSe), dimethyldiselenide (DMDSe), and probably dimethyl selenone, dimethyl selenylsulfide, and methaneselenol (Karlson, 1994; Fan et al., 1997). The oxidized forms of Se, selenate and selenite, are highly soluble in water and therefore bioavailable and potentially toxic. The biomethylation of inorganic Se by microbes, plants, and animals is an important pathway by which toxic Se compounds are transformed into relatively nontoxic volatile forms such as DMSe and DMDSe (Frankenberger and Karlson, 1994; Terry et al., 2000).

Selenium volatilization through the methylation process is thought to be a protective mechanism used by microorganisms to avoid toxicity in seleniferous environments (Frankenberger and Karlson, 1994). This process permanently removes Se from contaminated soils and waters under aerobic conditions, and minimizes its entry into the food chain. Rhizosphere and bulk soil microbes isolated from roots of plants grown in a surface-flow constructed wetland were effective in Se volatilization (Azaizeh et al., 1997). This constructed wetland has been shown to efficiently remove Se from contaminated wastewater: 89% of the Se entering the constructed wetland as selenite-contaminated oil refinery effluent was removed (Hansen et al., 1998). The biological volatilization (plants and microbes) may have accounted for as much as 10 to 30% of the Se removed. The remainder of Se was accumulated in plant tissues and the immobilized forms were sedimented in the wetland. These authors in addition to others (Allen, 1991; Zayed and Terry, 1994; Azaizeh et al., 1997) concluded that biological Se volatilization is a significant pathway of Se removal in a surface-flow constructed wetland and in plant roots. The rhizosphere is a site of increased microbial activity that may enhance accumulation, transformation, degradation, and biomethylation of Se and other trace elements (Allen, 1991; Anderson et al., 1993; Azaizeh et al., 1997). Plants provide fixed C and other nutrients to the microbes in the rhizosphere: this occurs through root exudation (Azaizeh et al., 1995) and plant decomposition. The maintenance of oxygen at the root respiratory sites depends on development of open lacunar space in the stems, roots, and rhizomes. This gaseous space acts as an oxygen conduit from the photosynthetic shoot tissue to the subsurface tissues, where aerobic processes maintain normal root absorptive functions for nutrient uptake (Bedford et al., 1991). It is also believed that an external aerobic microzone is established around parts of the growing roots of wetland plants (Armstrong and Armstrong, 1990). Therefore, wetland plants may provide the rhizosphere microbes with a C source, suitable pH, and oxygen, which are required for microbial growth. Selenium volatilization is an attractive method of removing Se from contaminated wastewater with wetland plants because it minimizes the entry of Se into the food chain. This is because biomethylation takes place mainly in the region of roots so that most of the Se volatilized escapes directly into the atmosphere (Azaizeh et al., 1997; Terry et al., 2000).

Dimethylselenide has an approximate vapor pressure of 32 kPa at 35°C and a solubility of 24.4 g L-1 in water (Chasteen, 1998). Therefore, the stability of the DMSe followed by its emission potential into the air is affected by the water content of the matrix where the DMSe is produced. In aquatic systems and moist terrestrial environments, such as moist soil, DMSe may be partially dissolved into water or trapped in the water and soil–sediment phase before its emission into the air.

A subsurface-flow constructed wetland (aquatic microcosm vegetation facility, AMVF) cultivated with common reed was used for a few years to study the potential of this system in phytoremediation of Se and other trace elements (Salhani and Stengel, 2001; Shardendu et al., 2002). In this AMVF system common reed plants were grown in the gravel and supplied continuously for few weeks with 20 µg Se L-1 as selenate. The results showed that less than 20% of the supplied Se was taken up by the cultivated plants and the rest apparently was biomethylated, absorbed to the gravel, or sedimented in the organic matter.

The main objectives of the present work were to evaluate the potential of rhizosphere microbes isolated from roots of AMVF plants on Se biomethylation and the fate of DMSe in water flown through the system. To investigate these objectives, rhizosphere microorganisms were isolated from common reed plants grown in the AMVF and supplied continuously with selenate. The isolated rhizosphere microorganisms were used to inoculate culture solutions supplemented with either selenate or selenite. Water samples were also taken from six different sampling points along the AMVF bed for Se and DMSe determination (Fig. 1) .



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  		Fig. 1. Diagram of the aquatic microcosm vegetation facility (AMVF system) showing plant vegetation technique, six sampling points (Inlet, 1, 2, 3, 4, and Outlet), and Se supplementation device.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study Site
Plant specimens and rhizosphere organic matter samples used to isolate the microbes were collected from an established outdoor subsurface-flow constructed wetland, the aquatic microcosm vegetation facility (AMVF) (Fig. 1). Each AMVF bed (7.0 m long, 0.65 m wide, and 0.4 m deep with a total surface area of 4.55 m2) was cultivated since 1980 with common reed. Each bed was filled with gravel 3 to 8 mm in diameter and contained four sampling points (1, 2, 3, 4) in addition to the inlet and the outlet. Six sampling points were used to take water samples throughout the experiment. Selenium was supplemented continuously as sodium salt (Na2SeO4) at a final concentration of 100 µg Se L-1 in the inlet of the beds. Each bed received Se mixed with tap water at a rate of 15 L h-1 with membrane pumps (Type R 409 1-50 E pump; Seybert & Rahier, Immenhausen, Germany). These pumps are displacement pumps where the water supply takes place through a shape-changeable elastic membrane and they guarantee a continuous and constant supply of water for many years.

Isolation and Enumeration of Rhizosphere Microbes
The rhizosphere microbes were isolated from fresh root specimens of common reed plants collected close to the inlet of the AMVF, which were supplemented continuously for 2 mo with 100 µg Se L-1 (Fig. 1, Point 1). Root sections (15 g fresh weight to obtain about 1.0 g dry organic matter) were randomly collected and loosely adhering wet organic matter and gravel were gently removed. Roots were rinsed in a beaker containing 150 mL sterile 0.1% agar solution. The beaker was magnetically stirred for 15 min to obtain a homogeneous rhizosphere suspension. The dry weight of the organic matter was determined by drying 25 mL of suspension at 90°C for 48 h. Total cultured bacteria in the suspension were enumerated by the stepwise dilution of 1 mL in 10-fold sterile 0.1% agar solution prepared with tap water. Appropriate dilutions ranging between 10-3 and 10-8 (1 mL) were mixed with sterile full strength tryptic soybean agar (TSA) (Merck, Darmstadt, Germany) to enumerate total bacteria. Petri dishes were incubated at 26°C for 7 d.

Carbon, pH, and Selenium Contents of Rhizosphere Suspensions
Total soluble carbon content of rhizosphere suspension (inoculum) used in the experiments was determined with a multiphase C analyzer (RC-412; LECO, St. Joseph, MI). The pH value of the rhizosphere suspension was also determined. To determine total Se in the rhizosphere suspension, the different Se species were converted to selenite. Each filtered (0.45 µm) sample (4.0 mL) was mixed with 1.0 mL of 2.0% ammonium persulfate, boiled for 15 min at 97°C, and cooled. Then, 5.0 mL concentrated HCl were added, and the solution was boiled again for 20 min at 97°C, cooled, and analyzed. Total Se in culture suspension was determined with hydride generation–atomic absorption spectrometry (HG–AAS) (AAS 4100, FIAS 400, and AS 90 autosampler; PerkinElmer, Wellesley, MA). Selenite was used as a standard. The detection limit of this HG–AAS procedure was 0.2 µg L-1 Se and the reproducibility was better than 2% for Se concentrations above 0.5 µg L-1. This analytical method had not only been internally validated, but also by successful participation at international proficiency testing exercises, such as IMEP (Institute of Reference Materials and Measurements, http://www.irmm.jrc.be), and external evaluation during the laboratory accreditation according to European Normal Standard EN 45001.

Cultured Rhizosphere Microbes
To obtain culturable rhizosphere microbes, 5 mL rhizosphere suspension was inoculated into 100 mL sterile full strength tryptic soybean broth (TSB) medium in 250-mL Erlenmeyer flasks. The flasks were incubated in the dark on a shaker at 150 rpm at 26°C for 6 d before being used as inoculum.

Potential of Rhizosphere Microbes Cultured in Tryptic Soybean Broth to Biomethylate Selenium
Rhizosphere microbes were cultivated in 500-mL cylindrical bottles (177 mm high and 41 mm in diameter) equipped with inlet and outlet tubes (Schott Duran, Mainz, Germany). The bottles contained 200 mL of sterile full strength TSB adjusted to pH 7.0 and supplemented with either 20 µM sodium selenate or sodium selenite. The culture solutions were aerated as follows. The inlet was inserted inside the cultivation solution to provide air bubbles (aeration) into the culture; the outlet was in the gaseous phase to sweep out produced DMSe. The rate of the airflow was adjusted to 150 mL h-1 with a peristaltic pump. Each flask's outlet was connected to a 250-mL gas washing glass bottle filled with a 150-mL alkaline peroxide trapping solution as previously described (Terry et al., 1992). Each cultivation bottle was inoculated with 10 mL of either rhizosphere suspension or cultured rhizosphere microbes (Table 1). Since microbes are well known as biomethylation agents of Se species, uninoculated bottles supplemented with 100 mg L-1 ampicillin were used as a control to estimate Se loss due to aeration or to microbial contamination. Ampicillin is a well-known antibiotic used in various biomethylation experiments because it inhibits both gram-positive and gram-negative bacteria; it interferes with bacterial cell wall synthesis and is therefore expected to have also the least effect on plant physiology. The control solutions were tested by the end of the experiment and found not contaminated.


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  		Table 1. Total number of bacteria, C content, total Se, and pH of the inoculum suspensions used in the different experiments. The microbes were isolated from the rhizosphere of common reed plants grown in the constructed wetland and used as inoculum to inoculate the culture solutions.{dagger}

 
A 2 x 2 factorial experiment was designed to test the effects of rhizosphere suspension or cultured rhizosphere microbes on the rates of Se biomethylation from selenate or selenite. The following treatment combinations were tested in four different experiments: (i) cultures supplemented with selenate and rhizosphere suspension, (ii) cultures supplemented with selenite and rhizosphere suspension, (iii) cultures supplemented with selenate and cultured rhizosphere microbes, and (iv) cultures supplemented with selenite and cultured rhizosphere microbes. All cultures were incubated in the dark at 26°C. Three replicate culture bottles were used for each treatment combination. The volatile Se coming from the headspace of each bottle was trapped continuously over a 15-d period during which the trapping solution was exchanged for a fresh one every 3 d and Se content was analyzed. Trapped Se was analyzed with HG–AAS after a two-step heating procedure of the solution in a water bath (30 min at 97°C, then cooling to room temperature and addition of concentrated HCl (1:1 v/v) followed by another heating at 97°C for 20 min). By that, all selenium species were converted into Se (IV). The HG–AAS measurements were performed in a flow injection HG–AAS mode similar to that described recently for Sb analysis (Krachler et al., 1999). In the chemifold of the flow injection system 500 µL of the sample solution was mixed with the reduction solution containing 0.4% NaBH4 in 0.04% NaOH and 10% HCl as carrier solution. The hydrogen produced in situ by the reaction of NaBH4 with the HCl reduces the Se (IV) and forms volatile gaseous SeH3, which is separated from the aqueous solution by the carrier gas flow (Ar, 60 mL min-1) and swept into the heated quartz glass cuvette (900°C). The SeH3 is thermally decomposed and the formed elemental Se is determined by measuring its atomic absorbance at 196.0 nm. Two aliquots of the same sample always were measured with three repetitions each and the analytical quality assurance for the method has already been stated above.

Potential of Rhizosphere Microbes Cultured in Hoagland Solution to Biomethylate Selenium
Rhizosphere microbes were cultured in bottles containing 300 mL of sterile half-strength Hoagland solution (Terry, 1980) adjusted to pH 7.0. A similar factorial experiment as was described with TSB with the same four treatments was performed to estimate the potential of rhizosphere suspension or cultured rhizosphere microbes to volatilize selenate or selenite. The cultures in each bottle were inoculated with 15 mL inoculum (Table 1) instead of 10 mL in the previous experiment. Uninoculated bottles supplemented with or without Se (selenate or selenite) represented control treatments. Three replicates were used for each treatment combination. Volatilized Se was sampled and trapped for a 15-d period as described earlier.

Culture Harvest
The cultures were harvested after 15 d of cultivation. Total bacteria, pH, and Se content of the remaining solutions were determined. In the half-strength Hoagland solutions the total bacteria were enumerated with tryptic soybean agar as was described earlier. Total Se was determined with inductively coupled plasma–mass spectrometry (ICP–MS) (Elan 5000; PerkinElmer). Total bacteria in TSB experiment were not determined because of the high microbial density.

Determination of Dimethylselenide Dissolved in Water Samples
The amount of DMSe dissolved in water samples was determined with helium as a purging (stripping) agent based on a method described earlier (Pecheyran et al., 1998). In brief, the glassware, tubing, and fittings used were cleaned with a common detergent, thoroughly rinsed with hot tap water, and then with distilled water before use. Water samples (200 mL) were collected from the six sampling points of the AMVF system (Fig. 1) 3 mo after Se supplementation initiation. The water sample was filled in a 250-mL gas washing glass bottle equipped with the inlet inserted inside the solution for DMSe stripping and the outlet was placed in the headspace to pass the flushed air into the trap. Helium at a rate of 250 mL h-1 was bubbled into the solution for 30 min to strip the DMSe and other volatile species. The stripped volatile species were trapped in another 250-mL gas washing glass bottle filled with 150 mL alkaline peroxide trapping solution (scrubbing solution) as previously described (Terry et al., 1992). Two water samples of 200 mL were flushed with helium from each sampling point before the Se content was determined in each trap. To eliminate the contribution of microbial biomethylation during the stripping process of DMSe from water samples, a control sample was supplemented with 100 mg L-1 ampicillin as antibiotic. The experiment was conducted at room temperature (about 22°C).

The Se concentrations in the water sample profile taken from the six sampling points were determined with ICP–MS (Elan 5000; PerkinElmer) equipped with an autosampler (AS 90; PerkinElmer) and an ultrasonic nebulizer (U-6000 AT+; Cetac Technologies, Omaha, NE). Two aliquots of each sample were measured with three repetitions and 103Re was added as internal standard to the measuring solution. Because of the problem of isobaric interferences in ICP–MS measurements with a quadrupole all Se masses were measured but only the noninfluenced signal of 82Se was used for final quantification. Therefore, the detection limit was only 10 µg L-1. Analytical quality assurance was performed by intermethod comparisons with the HG–AAS method.

Preliminary gas chromatography ICP–MS measurements were performed to determine the amounts of DMSe and DMDSe in analogy to a previously described method (Grueter et al., 2000), but without initial hydride generation. Within the frame of purging experiments we have indicated that the main volatile biomethylation product was DMSe.

Statistical Analysis
The data are presented as mean values and standard deviations. Linear regression was made to test correlation (fit) between two variants. The Duncan multiple range test was used to evaluate differences between means of the different treatments with statistically significant (p < 0.05) variation among means.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characteristics of the Microbial Rhizosphere Used as Inoculum
Table 1 summarizes the total mean number of bacteria, soluble C content, total Se, and pH of the rhizosphere inoculum suspension used to inoculate the cultures of TSB and half-strength Hoagland solutions. The different parameters of the inoculum for both experiments were relatively similar. In the TSB experiment, total bacteria used was 0.8 x 106 colony forming units (cfu) mL-1, C content 5.01 g L-1, total Se 3.4 mg L-1, and the pH was 5.7. In the half-strength Hoagland experiment, total bacteria was 1.0 x 106 cfu mL-1, C content 5.02 g L-1, total Se 3.8 mg L-1, and the pH was 5.8.

Effect of Rhizosphere Microbes Cultivated in Tryptic Soybean Broth on Selenium Volatilization
Selenium volatilization rates from cultures supplemented with selenate or selenite in both rhizosphere suspension or cultured rhizosphere microbes were relatively low when the microbes were cultivated on TSB (Fig. 2) . Selenium volatilization percentage was significantly higher (p < 0.05) in cultures supplied with selenite and inoculated with cultured microbes compared with the other three treatments. After 15 d of cultivation the mean percentage of volatilized Se from selenite in the presence of cultured microbes was 1.9%. Total mean selenate was volatilized by both microbial cultures at low rates and the percentages were only 1.2 to 1.3% (Fig. 2). No Se was detected (the detection limit of this HG–AAS procedure was 0.2 µg L-1 Se) in trap solutions connected to uninoculated cultures (controls) supplemented with selenate or selenite (data not shown). Mean pH values of all treatments were not significantly different (p < 0.05) and ranged between 8.3 and 8.6 (Table 2). The total number of cultured microbes was not measured after 15 d of growth in TSB. We noticed that after 2 to 3 d of cultivation, the culture reached stationery stage and some bacteria started to die and precipitated in the bottom of the bottles. It was clear that we had few cycles of microbial growth during the 15 d of experiment and the final number was >1010 cfu mL-1 in each cycle. Therefore, total number of bacteria in Day 15 does not represent an accurate value during the experiment and we decided not to measure it.



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  		Fig. 2. Effect of rhizosphere suspension (Rhiz) or cultured rhizosphere microbes (Cul) on total Se volatilized in 15 d. The 200-mL TSB cultivation solutions were supplemented with either 20 µM sodium selenate or sodium selenite. Each data point represents the mean value of three replicates. Mean values of total volatilized Se ± standard deviation are presented only in Day 15. The mean percentage of Se volatilized during 15 d of incubation is shown in the bar graph along with ± standard deviation. Different letters accompanying mean values of total Se volatilized (left side) and in the bar graph represent means with significant differences (Duncan multiple range test, p < 0.05).

 

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  		Table 2. Effects of Se species and aeration of the cultivation medium on total number of bacteria and on pH determined after 15 d of growth. Rhizosphere suspension (Rhiz) and cultured rhizosphere microbes (Cul) were cultivated for 15 d either in liquid tryptic soybean broth (TSB) medium or in half-strength Hoagland solution. The colony forming units (cfu) in TSB medium were not determined because of high microbial density.{dagger}

 
Effect of Rhizosphere Microbes Cultivated in Hoagland Solution on Selenium Volatilization
Total Se volatilized during 15 d of cultivation and the percentage of biomethylated Se from selenate or selenite in the presence of cultured rhizosphere microbes was significantly higher (p < 0.05) compared with rhizosphere suspension cultures (Fig. 3) . Total Se volatilized in the presence of selenite and cultured rhizosphere microbes during 15 d of incubation was the highest (p < 0.05) compared with the other treatments. The total mean percentage of volatilized Se from culture solutions supplemented with selenate or selenite in the presence of cultured rhizosphere microbes was 7.9 and 49.1%, respectively. The total mean percentage of volatilized Se from selenate or selenite in the presence of rhizosphere suspension was only 1.7 and 3.0%, respectively (Fig. 3). The results show that after 9 d of incubation, the volatilization rate of Se in cultures inoculated with cultured rhizosphere microbes increased substantially specially in the presence of selenite, and after 15 d of growth the mean rate of volatilized Se was significantly higher (p < 0.05) compared with cultures inoculated with rhizosphere suspension (27.5 and 0.9 µg Se d-1, respectively). No Se was detected in trap solutions connected to uninoculated cultures (controls) supplemented with selenate or selenite (data not shown). These cultures were supplemented with antibiotics to avoid microbial contamination and by the end of the experiments were tested to be sterile.



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  		Fig. 3. Effect of rhizosphere suspension (Rhiz) or cultured rhizosphere microbes (Cul) on total Se volatilized in 15 d. The 300-mL half-strength Hoagland cultivation solutions were supplemented with either 20 µM sodium selenate or sodium selenite. Each data point represents the mean value of three replicates. Mean values of total volatilized Se ± standard deviation are presented only in Day 15. The mean percentage of Se volatilized during 15 d of incubation is shown in the bar graph along with ± standard deviation. Different letters accompanying mean values of total Se volatilized (left side) and in the bar graph represent means with significant differences (Duncan multiple range test, p < 0.05).

 
The total number of cultured microbes after 15 d of cultivation was 84- to 340-fold greater in cultures inoculated with cultured rhizosphere microbes compared with rhizosphere suspension (Table 2). All treatments inoculated with rhizosphere cultures contained a significantly smaller microbial population (p < 0.05) compared with those supplemented with cultured microbes (Table 2). A linear regression was applied, which revealed that there was a relative best fit (r = 0.87) between total number of rhizosphere and cultured microbes and the percentage of volatilized Se when these microorganisms were cultured in Hoagland solution.

The mean pH of cultures inoculated with rhizosphere suspension ranged between 6.1 and 6.3; however, in treatments inoculated with cultured rhizosphere microbes it ranged between 8.6 and 8.7 (Table 2). In the presence of cultured rhizosphere microbes the pH was at least 2.0 units higher compared with rhizosphere suspension cultures.

Concentration of Dimethylselenide in Water Samples
The DMSe was indirectly detected in water samples taken from two sampling points (Fig. 1, Points 1 and 4) purged with helium where the mean Se concentrations were 2.4 and 1.7 µg Se L-1, respectively (Table 3). Water samples from Point 1, which contained the highest concentration of Se in the water sample profile had the highest level of stripped DMSe (Table 3); however, in the other two sampling points (2 and 3) that contained high to medium levels of Se in water, the DMSe was not detected (the detection limit of this HG–AAS procedure was 0.2 µg L-1 Se). In Point 4, which contained significantly low (p < 0.05) Se level, the amount of stripped DMSe was detected at a concentration of 1.7 µg Se L-1. In the outlet no DMSe was detected. Preliminary gas chromatography ICP–MS measurements from the purging experiment have shown that the main volatile biomethylation product was DMSe; DMDSe was detected in smaller peaks (data not shown). Addition of antibiotics to the water samples before purging did not affect the amount of stripped DMSe (data not shown). Therefore, the bacterial population had no effect on the amount of stripped DMSe.


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  		Table 3. The measured Se content of the sample profile and the concentration of dimethylselenide (DMSe) detected in water samples collected from aquatic microcosm vegetation facility (AMVF) solution supplemented with selenate after purging with helium. The measurements were conducted in water samples taken 12 wk after the initiation of Se supplementation to the common reed plants from six sampling points (see Fig. 1) along the constructed wetland.{dagger}

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study showed that rhizosphere microbes isolated from roots of common reed plants grown in a subsurface-flow constructed wetland are involved in Se volatilization. Selenite was easily volatilized by the rhizosphere microbes compared with the selenate in all treatments (Fig. 2 and 3). Rhizosphere microbes isolated from bulrush [Schoenoplectus acutus (Muhl. ex Bigelow) A. Love & D. Love] grown in a surface-flow constructed wetland showed a similar trend in Se biomethylation (Azaizeh et al., 1997). In our current experiments, around 50% of the supplied Se as selenite was volatilized during 15 d of incubation when the Hoagland culture solutions were inoculated with cultured rhizosphere microbes (Fig. 3), which indicates that these microorganisms have a high potential of Se volatilization. The applied linear regression showed relative best fit (r = 0.87) between total number of rhizosphere or cultured microbes and the percentages of volatilized Se when these microorganisms were cultured in Hoagland solution, which indicates the importance of the size of the microbial population on Se biomethylation when cultivated under low C content. When microbes were cultured in the presence of rich medium containing C source (TSB) the volatilization yields of selenate and selenite were relatively low (Fig. 2). The volatilization rates of selenate are usually very low by microbes compared with selenite (Azaizeh et al., 1997); however, in this current work, Se volatilization from selenate was relatively high when culture solutions were inoculated with cultured rhizosphere microbes under low C content (Fig. 3). The means of the volatilization rates in the last 3 d of incubation, Days 12 through 15 (Fig. 3), were calculated for the cultures supplemented with selenite and inoculated with cultured microbes, supplemented with selenate and inoculated with cultured microbes, supplemented with selenite and inoculated with rhizosphere suspension, and supplemented with selenate and inoculated with rhizosphere suspension. The calculated means were 27.5, 1.8, 0.9, and 0.8 µg Se d-1, respectively. This suggests that these microbes are better adapted to volatilize Se from selenite when the culture solutions were inoculated with cultured microbes isolated from rhizosphere plants supplied for more than 2 mo with 100 µg Se L-1 as selenate compared with the other three treatments (Fig. 3). The pH of the TSB cultivation solution (with high C content) after 15 d of cultivation was more than 8.0, which is considered optimal for Se volatilization, but the volatilization yield in this cultivation medium was low (Fig. 2). In Hoagland cultures (with low C content), the mean pH of the cultivation solution inoculated with rhizosphere suspension ranged between 6.1 and 6.3, compared with cultures inoculated with cultured rhizosphere microbes, which ranged between 8.6 and 8.7 (Table 2). The optimum pH for Se biomethylation from seleniferous Kesterson sediments and rhizosphere bacteria isolated from wetland plants was 8.0, but it ranges between 6 and 8.5 (Karlson and Frankenberger, 1989; Azaizeh et al., 1997). The final microbial population in cultures inoculated with cultured rhizosphere microbes exceeded in many folds the population in cultures inoculated with rhizosphere suspension (Table 2), which resulted in higher Se volatilization yields when the cultures were supplemented with either selenate or selenite (Fig. 3). Our rhizosphere microbes were isolated from plant roots of common reed grown in gravel without soil, and the only C source in this type of wetland comes from the decomposed organic matter in each cultivation bed and from plant exudates, which mainly include sugars, amino acids, and organic acids (Azaizeh et al., 1995). However, in the previous experiments (Azaizeh et al., 1997), the rhizosphere microbes were isolated from plants grown in soil; therefore, the total number of rhizosphere microbes used as inoculum in these experiments were 73-fold more compared with our current experiments (Table 1). The total amount of Se that was lost from inoculated cultures over 15 d could be accounted for volatile Se and more than 80% was detected in the alkaline peroxide trapping solution (data not shown). Very low percentages (1.2–1.7%) of the supplied selenate in the culture solutions were biomethylated by microbes into DMSe (Fig. 2 and 3) except when cultures were inoculated with cultured rhizosphere microbes where 7.9% of the supplied Se as selenate was biomethylated (Fig. 3). This suggests that these adapted microbes could be multiplied in the laboratory and then reintroduced into the contaminated sites to bioremediate wastewater containing high levels of selenate or selenite.

The amounts of DMSe stripped from the water samples collected from Points 1 and 4 were 2.4 and 1.7 µg L-1, respectively (Table 3), which are relatively high and indicate that the rhizosphere microbes of common reed are active in selenate biomethylation. Our preliminary results showed that the main detected volatile compound was DMSe, and the amounts of DMDSe were relatively very low. Produced DMSe is dissolved in water before it is released as gas into the atmosphere, and it might be that part of that remains in the solution because of the subsurface flow of water in this type of AMVF system. Common reed represents a helophyte with highly developed gas exchange (aerenchyma) facilitating gas transport to the atmosphere via the intercellular lacunar system (Armstrong and Armstrong, 1990; Brix et al., 1996; Salhani and Stengel, 2001), which may indicate that part of the biomethylated DMSe is released into the atmosphere through plant culms. The solubility of DMSe in water was determined to be 24.4 g L-1 at 35°C (Chasteen, 1998), which may indicate that part of the produced DMSe is converted into other Se species, depending on the various environmental conditions in the matrix. In another study conducted to determine the amount of volatile Se species in the Gironde estuarine environment in France with on-line purging cryofocusing technique (Pecheyran et al., 1998), DMSe was also found to be the main volatile Se species in concentrations ranging between 0.8 and 6.3 pmol L-1.

The current results show that rhizosphere microbes isolated from common reed plants grown in the AMVF system have high potential to biomethylate Se in cultures supplied with selenate or selenite. These microbes are culturable organisms, and could be cultivated in growth media and reintroduced into the water inlet of the wetland for bioremediation purposes. The subsurface-flow constructed wetland is a suitable system for Se biomethylation and volatilization where certain plants are grown in gravel without soil and the decomposed organic matter and root exudates represent the organic C source required for microbial growth and proliferation.


   ACKNOWLEDGMENTS
 
We thank Mrs. Carola Mohl (Institute of Phytospheric Research, Research Center, Juelich, Germany) for her assistance in determining Se content with HG–AAS and ICP–MS, and Dr. Eberhard Stengel (Research Center Juelich) for discussions about the AMVF facility.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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