Factors aecting the volatilization of dimethylselenide by
Enterobacter cloacae
SLD1a-1
Robert S. Dungan, William T. Frankenberger Jr.*
Department of Environmental Sciences, University of California, Riverside, CA 92521, USA
Received 7 July 1999; received in revised form 17 December 1999; accepted 21 February 2000
Abstract
The facultative anaerobic bacteriumEnterobacter cloacaeSLD1a-1 (ATCC 700258), capable of reducing selenate (SeO4 2ÿ) and
selenite (SeO32ÿ) to elemental selenium (Se0), was found to volatilize dimethylselenide (DMSe) in the presence of SeO32ÿ. The
eect of temperature, pH and electrical conductivity (EC) on the ability ofE. cloacaeSLD1a-1 to methylate selenium (Se) were investigated in a liquid medium of tryptic soy broth containing 10 mM SeO32ÿ. Optimum Se volatilization occurred at a pH,
temperature and EC of 6.5, 358C and 11 dS mÿ1
, respectively. Volatilization of Se was also found to be concentration dependent, asE. cloacae SLD1a-1 produced 11.3 times more DMSe at a SeO3
2ÿconcentration of 10 mM than at 1.0 mM. By
determining the optimum environmental conditions which stimulate Se volatilization, it may be possible to design a strategy to remediate seleniferous water.72000 Elsevier Science Ltd. All rights reserved.
Keywords:Selenium; Dimethylselenide; Biomethylation; Volatile selenium; Bioremediation
1. Introduction
Selenium (Se) is a metalloid, which is an essential trace element for animals, but can be extremely toxic if ingested in excessive amounts. Ecotoxicological eects of elevated soil Se concentrations have been recognized in many locations throughout the western U.S. (Eng-berg et al., 1998). In California's San Joaquin Valley, irrigated agriculture on seleniferous soil has resulted in the creation of Se-contaminated drainage water. Sel-enium in the agricultural drainage water has directly been linked to the death and deformity of waterfowl at California's Kesterson Reservoir (Ohlendorf et al., 1986).
Volatile methylated Se compounds are known to be formed in seleniferous soils, sediments and waters (Francis et al., 1974; Chau et al., 1976; Reamer and
Zoller, 1980; Thompson-Eagle and Frankenberger, 1990). The methylation of Se is a biological process and is thought to be a protective mechanism used by microorganisms to detoxify their surrounding environ-ment. Fungi and bacteria have been identi®ed as the predominant Se-methylating organisms in soils and sediments (Frankenberger and Karlson, 1994), while more recently, an aquatic algal species has been ident-i®ed (Fan et al., 1997). Dimethylselenide (DMSe) has been identi®ed as the major biological metabolite of Se methylation (Dungan and Frankenberger, 1999).
The biotransformation of Se to volatile Se com-pounds is considered a major process in the movement of Se from the environment (Haygarth, 1994). Although the biological signi®cance of Se methylation has yet to be elucidated, once volatile Se compounds are released to the atmosphere, Se has lost its hazar-dous potential. Therefore, microbial transformations of Se to less toxic volatile forms may ultimately prove to be an eective approach to remediate seleniferous environments.
Enterobacter cloacae SLD1a-1, a facultative
anae-0038-0717/00/$ - see front matter72000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 4 4 - 4
www.elsevier.com/locate/soilbio
* Corresponding author. Tel.: 3405; fax: +1-909-787-2954.
robe known to reduce SeO42ÿ and SeO32ÿto Se0 (Losi
and Frankenberger 1997), can volatilize DMSe in the presence of SeO32ÿ. Our study was initiated to identify
the environmental factors which aect the volatiliz-ation of Se byE. cloacaeSLD1a-1.
2. Materials and methods
2.1. Chemicals
Tryptic Soy Broth (TSB) was obtained from Difco (Detroit, MI); sodium selenite (Na2SeO3) and sodium
selenate (Na2SeO4), dimethyl diselenide (DMDSe) and
dimethyl disul®de (DMDS) from Aldrich Chemical (Milwaukee, WI); Na275SeO3 from Amersham Life
Sciences (Arlington Heights, IL); and DMSe from Strem Chemical (Newburyport, MA). Dimethyl selene-nyl sul®de (DMSeS) was prepared according to the method of Chasteen (1993).
2.2. Organism
Enterobacter cloacaeSLD1a-1 was isolated from the San Luis Drain, San Joaquin Valley, California, as described by Losi and Frankenberger (1997). The bac-terium was maintained in TSB that contained 0.5 mM Na2SeO3. It was transferred to fresh media every 2 d
to maintain the culture. A standard inoculum for each of the experiments was prepared by transferring 100ml of the maintenance culture to fresh TSB containing no Se. The inoculum was incubated at 208C on an orbital shaker (150 rev minÿ1
) for 24 h before being used.
2.3. Factors aecting selenium volatilization
Tryptic soy broth was added in 100-ml quantities to 250-ml Erlenmeyer ¯asks and autoclaved (1218C, 20 min) before the addition of SeO32ÿ. The pH and EC of
the TSB was 7.3 and 11 dS mÿ1
, respectively (except when pH and EC were the variables). All reagents were prepared in deionized water and ®lter-sterilized (0.2mm membrane ®lter) prior to their addition to the growth cultures. The non-radiolabeled SeO32ÿ
concen-tration used in all experiments was 10 mM (except when the SeO32ÿ concentration was the variable) and
3.7103Bq of 75Se as Na275SeO3 was added to each
¯ask. In addition, all ¯asks were inoculated with 100 ml of the standard inoculum and incubated at 208C (except when temperature was the variable) under sta-tic conditions. The data in each ®gure represents the average of three replicates; the last datum point rep-resents the standard error of the data.
To assess the in¯uence of various SeO32ÿ
concen-trations on the volatilization of Se by E. cloacae
SLD1a-1, non-radiolabeled SeO3
2ÿat concentrations of
0.01, 0.1 and 1.0 mM were tested. The cultures were sampled for volatile Se during a 10-d incubation.
To test the eect of temperature, cultures ofE. cloa-cae SLD1a-1 were incubated at 12, 20, 30, 35 or 408C and sampled for volatile Se during a 5-d incubation. The temperature coecient (Q10) values were calcu-lated by using the following equation:
Q10
The in¯uence of pH on the volatilization of DMSe by
E. cloacae SLD1a-1 was evaluated by adjusting the pH of the TSB to 6.0, 6.5, 7.0, 7.5, 8.0 or 9.0. The pH was adjusted with NaOH or HCl and pH measurements were made using an Accument 910 pH meter. The cul-tures were sampled for volatile Se during a 9-d incu-bation.
The eect of salinity was tested by adjusting the EC of the TSB to 11, 20, 25, 30, 35 or 40 dS mÿ1 with
NaCl. Electrical conductivity measurements were per-formed at room temperature using a YSI model 34 conductance/resistance meter. The cultures were sampled for volatile Se during a 9-d incubation.
2.4. Air sampling of volatile selenium
Moistened air was ¯ushed through two-hole stop-pered 250-ml Erlenmeyer ¯asks at an approximate ¯ow rate of 100 ml minÿ1. The outlet air from each
¯ask was passed through two stacked activated carbon cartridges (14 mm o.d., 55 mm long, containing ap-proximately 1.8 g of activated carbon) to trap the vol-atile Se. Activated C as an adsorbent to trap volvol-atile
75
Se, speci®cally DMSe, has successfully been employed by Evans et al. (1968) and Karlson and Frankenberger (1988). Activated C traps were collected approximately every 24 h and samples which could not be analyzed on the same day were frozen at ÿ208C until analyzed. The second cartridge was used to detect breakthrough and was analyzed separately. Each of the activated C traps were analyzed directly for 75Se without any further preparation.
2.5. Radioactivity measurements
All activated C samples were measured for 75Se using a gamma counter (Norland 5000 MultiChannel Analyzer System, Fort Atkinson, WI) with a NaI crys-tal (Bicron, Newbury, OH). The window setting was 93±440 KeV, which produced a typical background count rate of 220 counts minÿ1
2.6. Headspace analysis
The detection of DMSe in the headspace above cul-tures of E. cloacae SLD1a-1 was determined as fol-lows: 50 ml of TSB, at a SeO32ÿ concentration of 10
mM (non-radiolabeled Se only) was placed in 125-ml screw-cap Erlenmeyer ¯asks. The ¯asks were inocu-lated with 50 ml of the standard inoculum and incu-bated under aerobic conditions at 288C for 48 h on a rotary platform shaker (120 rev minÿ1). After 48 h the
¯asks were sealed with 70% ethanol disinfected Mini-nert valve-septa (Dynatech, Baton Rouge, LA) and incubated for another 48 h. At the end of the second incubation a 1-ml sample of the headspace was with-drawn using a gas-tight syringe (Pressure-Lok, Baton Rouge, LA) and injected into a gas chromatograph (GC) equipped with a ¯ame-ionization detector (FID). The syringes were cleaned between injections by heat-ing them to 558C. The GC used was a Hewlett-Pack-ard (Avondale, PA) model 5890 GC, connected to a Hewlett-Packard 3396A Series II integrator. The oper-ational conditions were as follows: stainless steel col-umn (10 m long and 2.2 mm I.D.); liquid phase, 10% Carbowax 1000; solid support, Chrom W-AW; particle size, 0.18±0.24 mm (mesh 60/80); column temperature, 658C; injector and detector temperature, 1058C; carrier gas, He, 30 ml minÿ1; H
2, 33 ml minÿ1; air, 320 ml
minÿ1
.
Volatile organo-Se compounds in the headspace of cultures of E. cloacae SLD1a-1 were detected with the use of pure standards. A standard retention time was established by injecting a 1-ml volume of the head-space above each of the pure standards. The only gas
found in the headspace of E. cloacae SLD1a-1 cultures was DMSe. To further verify the presence of DMSe in the headspace, a 100-ml sample was also analyzed by GC-mass spectrometry (GC-MS). The GC (Hewlett-Packard model 5890), equipped with a 30 m, 0.25 mm i.d. HP-5 capillary column, was connected to a Hew-lett-Packard model 5971 Mass Selective Detector. The operating conditions were as follows: carrier gas, He, 1 ml minÿ1; injector temperature, 2508C; column
tem-perature, 358C rising to 808C at 108C minÿ1 after 3
min; electron energy, 70 eV.
3. Results and discussion
Cultures of E. cloacae SLD1a-1 volatilized Se in an inverse relationship to the Se concentration (Fig. 1). The volatile Se product was positively identi®ed as DMSe. The highest amount of volatile Se was pro-duced at a SeO32ÿ concentration of 10 mM. At SeO32ÿ
concentrations of 0.1 and 1.0 mM, E. cloacaeSLD1a-1 volatilized 1.8 and 11.3 times less Se, respectively, than at 10 mM. In contrast, resting cell suspensions of a
Corynebacterium sp. volatilized approximately 10 times more DMSe at a SeO32ÿ concentration of 0.63 mM
than at 0.06 mM (Doran and Alexander, 1977). Fan et al. (1997) isolated a euryhaline alga (Chlorellasp.) that volatilized 6±10 times more Se (as DMSe, DMDSe and DMSeS) at a SeO32ÿ concentration of 1.3 mM
than at 0.01 mM. Unlike the Corynebacterium and
Chlorella species, it appears that high SeO32ÿ
concen-trations are toxic to E. cloacae SLD1a-1, which may explain why Se volatilization rates decreased at higher Se concentrations. Smith (1959) found that a SeO32ÿ
concentration of 70 mM was toxic to E. coliand other enteric species of bacteria. Selenite is a powerful oxi-dizing agent, which readily denatures sulfhydryl enzymes and oxidizes sulfhydryl groups to disul®des and selenotrisul®des (Doran, 1982).
In addition to DMSe production, cultures contain-ing SeO3
2ÿat concentrations of 0.1 and 1.0 mM turned
the distinctive brick-red color that is associated with elemental Se (Se0). The formation of Se0in association with the production of DMSe, may support Doran's proposed pathway, which suggests that DMSe is formed via the intermediate Se0 (Doran, 1982). Trans-mission electron micrographs of washed-cell suspen-sions of E. cloacae SLD1a-1 exposed to 0.63 mM SeO42ÿ, revealed the presence of Se0associated with the
outer cell membrane (Losi and Frankenberger, 1997), possibly the periplasmic space. Selenite is known to be transported into the cell of some microbes by distinct permeases, dierent from those for SeO42ÿ and SO42ÿ
or SO32ÿ(Brown and Shrift, 1980; Hudman and Glenn,
1984; Bryant and Laishley, 1988). Garbisu et al. (1995) found thatBacillus subtiliscultures grown in a medium Fig. 1. In¯uence of various SeO32ÿconcentrations on Se volatilization
containing 1 mM SeO32ÿ, deposited Se0 between the
cell wall and the plasma membrane. SeO32ÿ may be
taken into the periplasmic space ofE. cloacae SLD1a-1 via outer membrane permeases and reduced to Se0 by membrane-associated reductases. Eventually the Se0 may be reduced to the Se2ÿ form, which can then be
incorporated or transformed into either DMSe and or selenoamino acids. Since free Se0can be found in cul-tures of E. cloacae SLD1a-1 containing SeO32ÿ, it
appears that excess Se0 which cannot be reduced to Se2ÿ is released extracellularly. It has been speculated
that the bacterial reduction of SeO32ÿto Se0is a
detox-i®cation mechanism, which operates independent of dissimilatory reduction (Lortie et al., 1992; Garbisu et al., 1995). This may be true, since Se0 is insoluble and therefore biologically unavailable. The biological con-version of Se oxyanions to Se0is currently being inves-tigated as a remediation technique to treat Se-contaminated water. In addition, microbial Se volatil-ization may also be a detoxi®cation mechanism, because DMSe was found to be less toxic to mammals than SeO4
2ÿ and SeO 3
2ÿ (McConnell and Portman,
1952).
The optimum temperature for Se volatilization was 358C, with the rate of Se volatilization increasing as the temperature increased from 12 to 358C (Fig. 2). At 408C, the rate of Se volatilization was slightly less than 358C, but greater than at 308C. Optimal reduction of SeO32ÿ to Se0, by E. cloacae SLD1a-1, occurred at
408C (Dungan and Frankenberger, 1998). At 358C, the Se volatilization rate was 78-fold higher than at 128C and the average Q10 was 5.6. The Q10 value predicts that with every 108C rise in temperature a 5.6-fold in
Se volatilization will occur. The highest Q10 value of 10.7 occurred between 20 and 308C. The Q10 values progressively decreased from 10.7 to 0.9 as the tem-perature increased above 308C. Since biomethylation of Se is a temperature-dependent process, volatilization of Se in the environment is highest in the warmer spring and summer months. In seleniferous agricultural evaporation pond water and sediments, maximum Se volatilization occurred at a temperature of 358C (Fran-kenberger and Karlson, 1989; Thompson-Eagle and Frankenberger, 1990; Calderone et al., 1990).
Optimum Se volatilization by E. cloacae SLD1a-1 occurred at a pH of 6.5, with a pH of 6 and > 6.5 pro-moting signi®cantly less Se volatilization (Fig. 3). The volatilization of Se was 8.1 times higher at pH 6.5 than at pH 9. Maximum Se volatilization occurred at the same pH (i.e., 6.5) that E. cloacae SLD1a-1 maxi-mally reduced SeO32ÿto Se0(Dungan and
Frankenber-ger, 1998). These results may indicate the reduction of SeO3
2ÿto Se0and production of DMSe occurs through
a similar pathway and possibly the same enzyme sys-tem. In aqueous environments, neutral to alkaline con-ditions increase the solubility and availability of Se for microbial transformations. Rael and Frankenberger (1996) found that Aeromonas veronii volatilized Se (principally DMSe) between pH values of 7.9±8.5, with decreases toward the higher pH values. In contrast,
Alternaria alternata, a Se methylating fungus isolated from evaporation pond water, was found to maximally volatilize Se at a pH of 6.5 (Thompson-Eagle et al., 1989). Baker et al. (1983) found that the optimal pH range for arsenic and mercury methylation by sediment microorganisms was 3.5±7.5 and 5.5±6.5, respectively.
Fig. 3. In¯uence of pH on Se volatilization byE. cloacaeSLD1a-1. Fig. 2. In¯uence of temperature on Se volatilization by E. cloacae
Apparently, most indigenous micro¯ora have the high-est biomethylation activity when they are exposed to the pH they presumably are adapted to in their natural environment (Frankenberger and Karlson, 1989).
The EC of the San Luis Drain water from whichE. cloacaeSLD1a-1 was isolated was 9.3 dS mÿ1.
Electri-cal conductivity values may range from 10 to 15 dS mÿ1 in agricultural drainage water, to around 40 and
as high as 200 dS mÿ1 in evaporation ponds.
Maxi-mum amounts of Se were volatilized by E. cloacae
SLD1a-1 at an EC of 11 dS mÿ1(Fig. 4). Signi®cantly,
lower amounts of Se were volatilized over the EC range of 20±40 dS mÿ1
and the volatilization of Se was 3.1 times higher at an EC of 11 dS mÿ1
than at 40 dS mÿ1
. The volatilization of DMSe by A. veronii
occurred over the EC range of 3.9±40.2 dS mÿ1
, but was signi®cantly reduced at the highest EC value of 40.2 dS mÿ1
(Rael and Frankenberger, 1996). Appar-ently, the decrease in Se volatilization was attributed to the increase in osmotic potential. Karlson and Fran-kenberger (1990) found that as the salinity of soils increased over a range of 5±20 dS mÿ1, the rate of
mi-crobial Se volatilization decreased. They reported that soil microorganisms have less tolerance towards Na+ and Clÿ ions as opposed to SO
4
2ÿ and Ca2+ ions.
Apparently, the Na+ and or Clÿ ions have similar
eects on E. cloacae SLD1a-1, which may explain the signi®cantly lower Se volatilization rates seen over the EC range of 20±40 dS mÿ1
. Since the minimum EC of the TSB used in this study was 11 dS mÿ1
, lower EC values were not tested. However, based on our results, we would expect more Se to be volatilized by E. cloa-cae SLD1a-1 at EC values below 11 dS mÿ1
. In
ad-dition, our results indicate that the organism is probably best suited to treat agricultural drainage water prior to discharge in evaporation ponds.
4. Conclusion
Our work indicates that E. cloacae SLD1a-1 is capable of converting SeO32ÿ into DMSe, in addition
to Se0under aerobic conditions. Other volatile Se com-pounds commonly produced by Se methylating micro-organisms such as DMDSe, DMSeS and methaneselenol, were not produced by E. cloacae
SLD1a-1. The volatilization of DMSe by E. cloacae
SLD1a-1 is dependent upon temperature, pH, EC and Se concentration. By adjusting these variables it is possible to optimize the amount of Se volatilized by E. cloacae SLD1a-1. As a result, it may be possible to de-sign a low-cost strategy to remediate Se-contaminated water through simultaneous Se reduction and volatiliz-ation reactions.
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