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Supplementing Bacillus sp. RS1 with Dechloromonas sp. HZ for enhancing selenate reduction in agricultural drainage water
Science of the Total Environment 372 (2007) 397 – 405 www.elsevier.com/locate/scitotenv
Supplementing Bacillus sp. RS1 with Dechloromonas sp. HZ for enhancing selenate reduction in agricultural drainage water Yiqiang Zhang, William T. Frankenberger Jr. ⁎ Department of Environmental Sciences, University of California, Riverside, Riverside, CA-92521, USA Received 24 July 2006; received in revised form 11 October 2006; accepted 17 October 2006 Available online 30 November 2006
Abstract Cost and efficiency are two important factors considered in the remediation of Se-contaminated agricultural drainage water through bacterial reduction of soluble Se(VI) to insoluble Se(0). Bacillus sp. RS1 isolated from rice straw was assessed for its ability to use inexpensive molasses to reduce Se(VI) in agricultural drainage water containing NO−3 levels of 0, 50, 100, 250, and 500 mg/L. The results showed that Se(VI) (1000 μg/L) was almost entirely reduced to Se(IV) (62.7%) and Se(0) (36.4%) by Bacillus sp. RS1 in synthetic agricultural drainage (SAD) water without the presence of NO−3 . The reduction Se(VI) to Se(0) was limited in the SAD water with NO−3 levels of 100, 250, and 500 mg/L. The addition of Dechloromonas sp., a NO−3 reducer, to the SAD water not only increased NO−3 removal, but also enhanced Se(VI) reduction by Bacillus sp. RS1. During an 8-day experiment, 98–99% of the added Se(VI) was reduced to Se(0) with small amounts of Se(IV) and Se(-II) in the SAD water containing 100 and 250 mg/L NO−3 . The addition of Dechloromonas sp. HZ to the natural agricultural drainage water also significantly increased the reduction of Se(VI) (748 μg/L) by Bacillus sp. RS1, with a production of Se(0) (65%) and Se(-II) (32%). These results suggest that a combination of Bacillus sp. RS1 with Dechloromonas sp. HZ has great potential with the use of inexpensive molasses to remediate Se-contaminated agricultural drainage water containing relatively high NO−3 levels. Published by Elsevier B.V. Keywords: Bacillus sp. RS1; Dechloromonas sp. HZ; Agricultural drainage water; Nitrate; Selenate reduction; Selenium speciation
1. Introduction Selenium (Se)-rich agricultural drainage water has caused Se bioaccumulation in aquatic organisms through the food chain, which creates serious hazards to fish and waterfowl in the western San Joaquin Valley, California (Lemly et al., 1993; Ohlendorf, 1989; Presser and Ohlendorf, 1987). To protect aquatic waterfowl, the amounts of Se in agricultural drainage water needs to be ⁎ Corresponding author. Tel.: +1 951 827 5218; fax: +1 951 827 3993. E-mail address: [email protected] (W.T. Frankenberger). 0048-9697/$ - see front matter. Published by Elsevier B.V. doi:10.1016/j.scitotenv.2006.10.027
reduced or removed before it is disposed into aquatic systems. Bacterial reduction of selenate [Se(VI)] to elemental Se [Se(0)] is an important biogeochemical process in aquatic systems (Gao et al., 2000; Velinsky and Cutter, 1991; Weres et al., 1989; Zhang and Moore, 1996). In the aquatic system, Se(VI) can be used in microbial respiration as a terminal electron acceptor for growth and metabolism. Several bacteria isolated from different environments are capable of reducing Se(VI) to Se(0), i.e. Bacillus sp. SF-1, Bacillus selenitireducens, Citerobacter freundii, C. braakii, Enterobacter cloacae, E. taylorae, Sulfurodpirillum barnesiii and Thauera selenatis
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(Cantafio et al., 1996; Kashiwa et al., 2001; Losi and Frankenberger, 1997; Oremland et al., 1999; Switzer et al., 1998; Zahir et al., 2003; Zhang et al., 2003, 2004; Zhang and Frankenberger, 2006). Bacterial reduction efficiency of Se(VI) is mostly related to the electron donors and competitive electron acceptors present, as well as various environmental conditions such as salinity, pH, Eh, and temperature (Cantafio et al., 1996; Kashiwa et al., 2001; Losi and Frankenberger, 1997; Oremland et al., 1999; Switzer et al., 1998; Zahir et al., 2003; Zhang et al., 2003, 2004; Zhang and Frankenberger, 2006). Nitrate is one of the most common anions found in agricultural drainage water due to the application of fertilizers. In the San Joaquin Valley, agricultural drainage water typically contains 3 to 234 mg/L of NO3− with an average of 97 mg/L (Oswald et al., 1989); much higher than Se (140–1400 μg/L) (Cantafio et al., 1996; Sylvester, 1990) found in agricultural drainage water. The redox potential of NO3−/N2 in an aquatic system is very similar to that of Se (VI)/Se (IV) and higher than Se (IV)/Se(0) (Masscheleyn and Patrick, 1993), thus the presence of dissolved NO3− can serve as a competitive electron acceptor affecting Se(VI) reduction to Se(IV) and inhibiting Se(IV) reduction to Se(0) (Fujita et al., 1997; Masscheleyn and Patrick, 1993; Steinberg et al., 1992; Weres et al., 1990). In a study on bacterial NO3− and Se(VI) reduction, Steinberg et al. (1992) reported that NO3− reduction by an anaerobic, freshwater enrichment preceded Se(VI) reduction in an anaerobic medium with equal amounts of Se(VI) and NO3− of 20 mM. Fujita et al. (1997) reported that Se(VI) reduction by Bacillus sp. SF-1 in a basal medium with 1 mM of Se(VI) was completely inhibited when 20 mM of NO3− was added to the medium. NO3− and Se(VI) can also be reduced simultaneously in the medium with washedcell suspensions of S. barnesii and lactate as an electron donor (Oremland et al., 1999). Therefore, removing NO3− from agricultural drainage water would enhance the removal of Se(VI) by Se(VI)-reducing bacteria. Cost and efficiency are two important factors considered in the remediation of Se-contaminated water. In our previous study (Zhang and Frankenberger, 2006), we reported that C. braakii is capable of using inexpensive molasses as a carbon source to reduce Se(VI), with a reduction of 87–97% in natural river waters. However, only 20% of Se(VI) was removed from highly saline agricultural drainage water containing a higher NO3− level. Therefore, selection of salttolerant bacteria that can use inexpensive carbon sources to effectively reduce Se(VI) and NO3− would play a key role in the economics of the remediation of Secontaminated agricultural drainage water.
In this study, we have investigated the effect of NO3− on the bacterial reduction of Se(VI) in agricultural drainage water, and have evaluated the enhancement of Se(VI) reduction by the addition of a NO3− reducer to remove NO3− from agricultural drainage water. The removal of Se(VI) and NO3− in agricultural drainage water was characterized in a series of batch experiments. 2. Materials and methods 2.1. Materials Natural agricultural drainage water was collected from the western San Joaquin Valley, California. The water, with a pH of 8.2 and salinity [electrical conductivity (EC)] of 11.56 dS/m, contained 782 μg/L Se(VI) and 191 mg/L NO3− . Synthetic agricultural drainage (SAD) water was prepared with the following constituents (in g/L): Na2SO4, 5.92; NaCl, 0.989; NaHCO3, 0.138; CaCl2·2H2O, 0.917; MgSO4, 1.238; Na2B4O7·10H2O, 0.529; NaNO3, 0.069; K2HPO4, 0.091; FeCl2, 0.0002; molasses 1; and trace element solution (Focht, 1994), 1 mL/L. The SAD water was autoclaved (18 psi at 121 °C) for 20 min after a pH adjustment to 7.5. The Se(VI) standard stock solution (10,000 mg/L) was passed through a sterile 0.2 μm membrane filter prior to its addition to the SAD water. 2.2. Isolation of the Se(VI)-reducing bacterium Rice straw as an effective organic carbon source and a carrier of Se(VI)-reducing bacteria has been tested to remove Se(VI) from agricultural drainage water (Zhang and Frankenberger, 2003a,b). Air-dried rice straw used to isolate Se(VI)-reducing bacteria was obtained from the San Joaquin Valley, California. Two grams of rice straw and 200 mL of sterile DI water were added into a 250-mL sterile Erlenmeyer flask, spiked with Se(VI) to a concentration of 10 mg/L. The flask was capped with a sterile rubber stopper and incubated at 20 °C for 5 days when a clear red color of Se(0) appeared in the flask. An aliquot of the water was serially diluted in sterile deionized water and spread onto tryptic soy agar (TSA; Difco, Detroit, Mich.) plates containing 50 mg/L of Se(VI). The plates were incubated at 30 °C for 48 h when several colonies with red Se(0) precipitates were observed on the TSA plates. The colonies were restreaked on TSA plates with and without Se(VI) to ensure that the red color of the colony was not due to a bacterial pigment. One isolate was identified as Bacillus sp. based on 16S rRNA gene sequence (MIDI Labs, Newark, DE), and designated strain RS1. Dechloromonas sp. strain HZ, an
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organism widely used to reduce perchlorate to chloride in groundwater (Xu et al., 2003) was used in this study to reduce NO3− in the SAD water and natural agricultural drainage water. Dechloromonas sp. HZ was obtained from the Department of Environmental Engineering, University of California at Riverside. Bacillus sp. RS1 and Dechloromonas sp. HZ were were initially grown on TSA plates without Se(VI) and incubated (30 °C) for 2–3 days. The colonies of each bacterium were separately resuspended into the sterile SAD water and incubated overnight to give an OD600 range of 0.55– 0.57 for a series of experiments in this study. 2.3. Reduction of Se(VI) in the SAD water The first experiment was conducted in the laboratory to determine the effect of the addition of Dechloromonas sp. HZ on reduction of Se(VI) to Se(0) by Se(VI) reducers in the SAD water. Se(VI)-reducing bacteria used in this study included C. braakii isolated from agricultural drainage water (Zhang and Frankenberger, 2006) and Bacillus sp. RS1 isolated from rice straw. In the experiment, 38 mL of the sterile SAD water containing 1000 mg/L of molasses was added to each 40-mL glass vial. The vials were spiked with Se(VI) to give a final concentration of 1000 μg/L and inoculated with 0.2 mL of each bacterial cell suspension (C. braakii and Dechloromonas sp. HZ or Bacillus sp. RS1 and Dechloromonas sp. HZ). The vials were capped with sterile caps and incubated under a static condition at room temperature (20 °C). Another batch experiment without the addition of Dechloromonas sp. HZ was conducted for comparison. The experiments were run in triplicate for 8 days. The water samples were collected at an interval of 1–2 days for analysis of Se species. 2.4. Reduction of Se(VI) in the SAD water with varying amounts of NO3− After obtaining the results from the first experiment described above, Bacillus sp. RS1 proved better than C. braakii for using molasses to effectively reduce Se (VI) to Se(0). Therefore, the second experiment was conducted in the laboratory to test the effect of varying amounts of NO3− on the reduction of Se(VI) to Se(0) in the SAD water. In this experiment, 230 mL of the SAD water containing a NO3− level of 100, 250, and 500 mg/L was added to each 250-mL Erlenmeyer flask after an amendment with 1000 μg/L of Se(VI) and 1000 mg/L of molasses. The terms “100 mg/L NO3− SAD water”, “250 mg/L NO3− SAD water”, and “500 mg/L NO3− SAD
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water” refer to the experimental set up containing 100, 250, and 500 mg/L NO3− in SAD water at the beginning of the experiments, respectively. The flasks were inoculated with 1 mL of the Bacillus sp. RS1 cell suspension. The flasks were capped with sterile rubber stoppers and incubated under static conditions at room temperature (20 °C). In order to examine the effect of the addition of Dechloromonas sp. HZ on reduction of Se(VI), another batch of the same experiment described above was added with 1 mL Dechloromonas sp. HZ to remove NO3− in the SAD water. The experiment without the addition of NO3− was run as control. The experiment was performed in triplicate for 8 days. The water samples were collected at an interval of 1–2 days for analysis of Se species and NO3−. 2.5. Reduction of Se(VI) in natural agricultural drainage water In order to observe whether Bacillus sp. RS1 and Dechloromonas sp. HZ could survive in natural agricultural drainage water and reduce Se(VI) to Se(0), 38 mL of the non-sterile agricultural drainage water was added to a 40-mL glass vial. The vials were supplemented with 1000 mg/L of molasses, and inoculated with 0.2 mL of a cell suspension of Bacillus sp. RS1, or 0.2 mL each of Bacillus sp. RS1 and Dechloromonas sp. HZ cell suspension. The water devoid of the inoculum served as a control. All vials were capped with sterile caps and incubated at room temperature (20 °C). The experiment was run in triplicate for 6 days. The water samples were collected at an interval of 1–2 days for analysis of Se species. 2.6. Analysis Selenium species in the water samples were determined using a method developed by Zhang and Frankenberger (2003a) and Zhang et al. (1999) after removal of Se(0) from the solution by centrifugation at 12,000 for 14 min. Directly measured Se species included total soluble Se, Se(IV), and Se(IV) plus Se (-II) [organic Se(-II) plus inorganic Se(-II)]. Se(VI), Se(0) and Se(-II) were determined by the difference method (Zhang et al., 1999; Zhang and Frankenberger, 2003a). Se concentrations in all the prepared solutions were analyzed by hydride generation atomic absorption spectrometry (HGAAS) (Zhang et al., 1999). Nitrate was determined by a NO3− ion electrode (ColeParmer InC). Nitrate standards were made using sodium nitrate (NaNO3) and the sterile SAD water without the addition of NO3− . The Se detection limit of
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the HGAAS and the experimental water was 0.4 and b10 μg/L, respectively. 3. Results 3.1. Bacterial Se(VI) reduction The reduction of Se(VI) in the SAD water containing 1000 μg/L of Se(VI) and 50 mg/L of NO3− is illustrated in Fig. 1. During an 8-day experiment, Se (VI) was not reduced in the SAD water without the addition of C. braakii or Bacillus sp. RS1 (Fig. 1A and B). The extent of Se(VI) reduction differed in the SAD water added with different bacteria. In the SAD water inoculated with C. braakii, Se(VI) was not reduced (Fig. 1C). Se(VI) reduction by C. braakii occurred when Dechloromonas sp. HZ was added to the SAD
water, with 60% Se(VI) reduction to Se(IV) and Se(0) (Fig. 1E). In contrast, Bacillus sp. RS1 is capable of using molasses to reduce Se(VI) in the SAD water, with a 57% Se(VI) reduction at day 8 (Fig. 1D). Se(VI) reduction by Bacillus sp. RS1 was significantly enhanced when Dechloromonas sp. HZ was added to the SAD water (Fig. 1F). Se(VI) was completely removed at day 3. On the final day of the experiment, Se(0) was the major Se form in the SAD water, with a small amount of Se(-II). 3.2. Effect of NO3− concentrations on Se(VI) reduction The effect of NO3− concentrations on Se(VI) reduction by Bacillus sp. RS1 with or without the presence of Dechloromonas sp. HZ is shown in Figs. 2–6. In the SAD water without the presence of NO3− (Fig. 2A),
Fig. 1. Reduction of Se(VI) (1000 μg/L) in the SAD water by C. braakii (left figures; A, C, and E) and Bacillus sp. RS1 (right figures; B, D, and F). Top figures (A and B), control experiments without the addition of bacteria; middle figures (C and D), experiments with Se(VI)-reducing bacteria; bottom figures (E and F), experiments with Se(VI)-reducing bacteria and Dechloromonas sp. HZ. Error bars indicating one standard deviation (n = 3).
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Fig. 2. Reduction of Se(VI) (1000 μg/L) in the no-NO−3 SAD water by Bacillus sp. RS1 with Dechloromonas sp. HZ (right figure; B) or without Dechloromonas sp. HZ (left figure; A). Error bars indicating one standard deviation (n = 3).
Se(VI) was almost entirely reduced at day 4. At day 8, Se(0) and Se(IV) accounted for 36 and 63% of the added Se in the SAD water. Addition of Dechloromonas sp. HZ to the SAD water enhanced the reduction of Se(VI) (Fig. 2B). On the last day of the experiment, Se(0) was the major Se form (83%) in the SAD water, with a small amount of Se(-II). With the increase of NO3− to levels of 100, 250, and 500 mg/L in the SAD water, reduction of Se(VI) by Bacillus sp. RS1 slowed down (Figs. 3A, 4A, and 5A). On the final day of the experiment, Se(VI), Se(IV), and Se(0) in the 100 and 250 mg/L NO3− SAD water
accounted for 56.5–68.6, 24.7–37.7, and 5.92–7.18%, respectively. The addition of Dechloromonas sp. HZ significantly increased removal of Se(VI) in the 100 and 250 mg/L NO3− SAD water (Figs. 3C and 4C). At day 8, 98–99% added Se(VI) was reduced to Se(0), with small amounts of Se(IV) and Se(-II). In the 500 mg/L NO3− SAD water with or without the presence of Dechloromonas sp. HZ (Fig. 5C), only 10% of the added Se(VI) was reduced to Se(IV) and Se(0) during an 8-day experiment. Both Bacillus sp. RS1 and Dechloromonas sp. HZ were capable of reducing NO3− in the SAD water. During
Fig. 3. Reduction of Se(VI) (1000 μg/L) and NO−3 in the 100 mg/L NO−3 SAD water by Bacillus sp. RS1 with Dechloromonas sp. HZ (bottom figures; C and D) or without Dechloromonas sp. HZ (top figures; A and B). Error bars indicating one standard deviation (n = 3).
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Fig. 4. Reduction of Se(VI) (1000 μg/L) and NO−3 in the 250 mg/L NO−3 SAD water by Bacillus sp. RS1 with Dechloromonas sp. HZ (bottom figures; C and D) or without Dechloromonas sp. HZ (top figures; A and B). Error bars indicating one standard deviation (n = 3).
an 8-day experiment, 92.5, 51.7, and 29.7% of the added NO3− was removed from the 100, 250, and 500 mg/L NO3− SAD water, respectively (Figs. 3B, 4B, and 5B).
The addition of Dechloromonas sp. HZ to the water enhanced the removal of NO3− (Figs. 3D, 4D, and 5D). Nitrate was almost entirely removed at day 3 in the
Fig. 5. Reduction of Se(VI) (1000 μg/L) and NO−3 in the 500 mg/L NO−3 SAD water by Bacillus sp. RS1 with Dechloromonas sp. HZ (bottom figures; C and D) or without Dechloromonas sp. HZ (top figures; A and B). Error bars indicating one standard deviation (n = 3).
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Fig. 6. Removal of Se(VI) from agricultural drainage water by Bacillus sp. RS1. Top figures, control experiment without the addition of bacteria and molasses (left figure; A) and only without bacteria (right figure; B). Bottom figures, experiment with Bacillus sp. RS1 plus Dechloromonas sp. HZ (right figure; D) or without Dechloromonas sp. HZ (left figure; C). Error bars indicating one standard deviation (n = 3).
100 mg/L NO3− SAD water. In the SAD water with 250 and 500 mg/L NO3−, 96.7 and 83.1% of the added NO3− was reduced within 8 days, respectively. 3.3. Reduction of Se(VI) in natural agricultural drainage water Reduction of Se(VI) in agricultural drainage water with and without inoculation of Bacillus sp. RS1 and Dechloromonas sp. HZ is shown in Fig. 6. In the absence of the bacteria, there was little change in Se(VI) (748 μg/L) concentration in the agricultural drainage water with and without the addition of molasses (Fig. 6A and B). 81% of Se(VI) in the agricultural water was reduced to Se(IV) (12.3%), Se(0) (50.6%), and Se(-II) (17.8%) in the presence of Bacillus sp. RS1 (Fig. 6C). Se(VI) was almost entirely removed when Bacillus sp. RS1 and Dechloromonas sp. HZ were added to the water (Fig. 6D). At day 6, Se(IV), Se(0), and Se(-II) accounted for 2.8 (21 μg/L), 65 (486 μg/L), and 32.2% (241 μg/L), respectively. 4. Discussion Reduction of Se(VI) to Se(0) by Se(VI)-reducing bacteria that are capable of using inexpensive organic carbon sources is a useful remedial technique
for removing Se from agricultural drainage water. C. braakii is capable of using inexpensive molasses as a carbon source to reduce Se(VI) in natural river waters (Zhang and Frankenberger, 2006), but, its ability to reduce Se(VI) in the SAD water is limited. In conrast, Bacillus sp. RS1 has performed better using molasses to reduce Se(VI). The pathway of Se(VI) reduction by Bacillus sp. RS1 can be characterized as Se(VI) → Se(IV) → Se(0) → Se(-II). Several researchers have reported that different Bacillus species can reduce Se(VI) or Se(IV) in different media. Kashiwa et al. (2000) reported that 1 mM Se(VI) was completely reduced by Bacillus sp. SF-1 in a basal salt medium containing 0.1% yeast extract and 20 mM sodium lactate. Garbisu et al. (1995) used Bacillus subtilis to reduce Se(IV) in a minimal chemically defined liquid medium containing 70 mM glucose. However, added Se(VI) in the same medium had little change during a 4-day experiment. Oremland et al. (1999) found that B. selenitireducens was only able to reduce Se(IV) to Se(0) and did not respire Se(VI) in a salt medium containing lactate. Bacillus sp. RS1 was also able to reduce NO3− in the SAD water with a reduction of 92, 52, 30% of the added NO3− within 8 days, respectively in the 100, 250, and 500 mg/L NO3− SAD water. Addition of Dechloromonas sp. HZ to the SAD water increased the removal of NO3−
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to 100% at day 3 in the 100 mg/L NO3− SAD water, and 97 and 83% at day 8, respectively in the 250 and 500 mg/L NO3− SAD water. Addition of Dechloromonas sp. HZ also enhanced the Se(VI) reduction from 31– 58% to 98–100% in the 50, 100, and 250 mg/L NO3− SAD water. Nitrate is a competitive electron acceptor affecting Se(VI) reduction in aquatic systems due to their similar redox potentials (Masscheleyn and Patrick, 1993). Therefore, addition of Dechloromonas sp. HZ to the NO3− containing SAD water to remove NO3− would enhance reduction of Se(VI) by Se(VI)-reducing bacteria although Dechloromonas sp. HZ itself did not reduce Se(VI). It might be likely that the higher amounts of organic carbon sources are needed to treat Se in agricultural drainage water having a high level of NO3−. In this study, reduction of Se(VI) by Bacillus sp. RS1 decreased with an increase of NO3− concentrations in the SAD water with or without the addition of Dechloromonas sp. HZ. In the 500 mg/L NO3− SAD water, Se(VI) reduction was almost totally inhibited. The reason might be attributed to amount of organic carbon added to the SAD water. In this study, we added 1000 mg/L molasses (liquid phase). We do not know exact amounts of the available organic carbon in it, which can be used by bacteria to reduce NO3− and Se(VI). Competition of NO3− with Se(VI) for electron donors in the SAD water with a high ratio of NO3− (500 mg/L) to Se(VI) (1 mg/L) would most likely consume most of the available organic carbon of molasses for NO3− reduction. Thus, there would be little organic carbon left for Se(VI) reduction. Kashiwa et al. (2000) also reported that Se(VI) was not reduced in a basal salt medium when NO3− concentration increased from 1, 5 to 10 mM. Their explanation is that most of the added lactate in the medium was consumed as the electron donor for NO3− reduction, and consequently, little lactate remained for utilization in Se (VI) reduction. This study revealed that Bacillus sp. RS1 not only reduce Se(VI) in the SAD water, but also effectively reduce Se(VI) in natural agricultural drainage water, with 80% reduction of Se(VI). Upon the addition of Dechloromonas sp. HZ to natural agricultural drainage water, Se(VI) was almost entirely reduced by Bacillus sp. RS1 during a 6-day experiment. Agricultural productivity in the San Joaquin Valley, California generates agricultural drainage water containing a high level of Se(VI) and NO3− (Oswald et al., 1989; Sylvester, 1990). These elevated Se levels need to be reduced before discharge to nearby wetlands and lakes. The results of this study indicate that a combination of Bacillus sp. RS1 with Dechloromonas sp. HZ are capable of using
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Supplementing Bacillus sp. RS1 with Dechloromonas sp. HZ for enhancing selenate reduction in agricultural drainage water Yiqiang Zhang, William T. Frankenberger Jr. ⁎ Department of Environmental Sciences, University of California, Riverside, Riverside, CA-92521, USA Received 24 July 2006; received in revised form 11 October 2006; accepted 17 October 2006 Available online 30 November 2006
Abstract Cost and efficiency are two important factors considered in the remediation of Se-contaminated agricultural drainage water through bacterial reduction of soluble Se(VI) to insoluble Se(0). Bacillus sp. RS1 isolated from rice straw was assessed for its ability to use inexpensive molasses to reduce Se(VI) in agricultural drainage water containing NO−3 levels of 0, 50, 100, 250, and 500 mg/L. The results showed that Se(VI) (1000 μg/L) was almost entirely reduced to Se(IV) (62.7%) and Se(0) (36.4%) by Bacillus sp. RS1 in synthetic agricultural drainage (SAD) water without the presence of NO−3 . The reduction Se(VI) to Se(0) was limited in the SAD water with NO−3 levels of 100, 250, and 500 mg/L. The addition of Dechloromonas sp., a NO−3 reducer, to the SAD water not only increased NO−3 removal, but also enhanced Se(VI) reduction by Bacillus sp. RS1. During an 8-day experiment, 98–99% of the added Se(VI) was reduced to Se(0) with small amounts of Se(IV) and Se(-II) in the SAD water containing 100 and 250 mg/L NO−3 . The addition of Dechloromonas sp. HZ to the natural agricultural drainage water also significantly increased the reduction of Se(VI) (748 μg/L) by Bacillus sp. RS1, with a production of Se(0) (65%) and Se(-II) (32%). These results suggest that a combination of Bacillus sp. RS1 with Dechloromonas sp. HZ has great potential with the use of inexpensive molasses to remediate Se-contaminated agricultural drainage water containing relatively high NO−3 levels. Published by Elsevier B.V. Keywords: Bacillus sp. RS1; Dechloromonas sp. HZ; Agricultural drainage water; Nitrate; Selenate reduction; Selenium speciation
1. Introduction Selenium (Se)-rich agricultural drainage water has caused Se bioaccumulation in aquatic organisms through the food chain, which creates serious hazards to fish and waterfowl in the western San Joaquin Valley, California (Lemly et al., 1993; Ohlendorf, 1989; Presser and Ohlendorf, 1987). To protect aquatic waterfowl, the amounts of Se in agricultural drainage water needs to be ⁎ Corresponding author. Tel.: +1 951 827 5218; fax: +1 951 827 3993. E-mail address: [email protected] (W.T. Frankenberger). 0048-9697/$ - see front matter. Published by Elsevier B.V. doi:10.1016/j.scitotenv.2006.10.027
reduced or removed before it is disposed into aquatic systems. Bacterial reduction of selenate [Se(VI)] to elemental Se [Se(0)] is an important biogeochemical process in aquatic systems (Gao et al., 2000; Velinsky and Cutter, 1991; Weres et al., 1989; Zhang and Moore, 1996). In the aquatic system, Se(VI) can be used in microbial respiration as a terminal electron acceptor for growth and metabolism. Several bacteria isolated from different environments are capable of reducing Se(VI) to Se(0), i.e. Bacillus sp. SF-1, Bacillus selenitireducens, Citerobacter freundii, C. braakii, Enterobacter cloacae, E. taylorae, Sulfurodpirillum barnesiii and Thauera selenatis
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(Cantafio et al., 1996; Kashiwa et al., 2001; Losi and Frankenberger, 1997; Oremland et al., 1999; Switzer et al., 1998; Zahir et al., 2003; Zhang et al., 2003, 2004; Zhang and Frankenberger, 2006). Bacterial reduction efficiency of Se(VI) is mostly related to the electron donors and competitive electron acceptors present, as well as various environmental conditions such as salinity, pH, Eh, and temperature (Cantafio et al., 1996; Kashiwa et al., 2001; Losi and Frankenberger, 1997; Oremland et al., 1999; Switzer et al., 1998; Zahir et al., 2003; Zhang et al., 2003, 2004; Zhang and Frankenberger, 2006). Nitrate is one of the most common anions found in agricultural drainage water due to the application of fertilizers. In the San Joaquin Valley, agricultural drainage water typically contains 3 to 234 mg/L of NO3− with an average of 97 mg/L (Oswald et al., 1989); much higher than Se (140–1400 μg/L) (Cantafio et al., 1996; Sylvester, 1990) found in agricultural drainage water. The redox potential of NO3−/N2 in an aquatic system is very similar to that of Se (VI)/Se (IV) and higher than Se (IV)/Se(0) (Masscheleyn and Patrick, 1993), thus the presence of dissolved NO3− can serve as a competitive electron acceptor affecting Se(VI) reduction to Se(IV) and inhibiting Se(IV) reduction to Se(0) (Fujita et al., 1997; Masscheleyn and Patrick, 1993; Steinberg et al., 1992; Weres et al., 1990). In a study on bacterial NO3− and Se(VI) reduction, Steinberg et al. (1992) reported that NO3− reduction by an anaerobic, freshwater enrichment preceded Se(VI) reduction in an anaerobic medium with equal amounts of Se(VI) and NO3− of 20 mM. Fujita et al. (1997) reported that Se(VI) reduction by Bacillus sp. SF-1 in a basal medium with 1 mM of Se(VI) was completely inhibited when 20 mM of NO3− was added to the medium. NO3− and Se(VI) can also be reduced simultaneously in the medium with washedcell suspensions of S. barnesii and lactate as an electron donor (Oremland et al., 1999). Therefore, removing NO3− from agricultural drainage water would enhance the removal of Se(VI) by Se(VI)-reducing bacteria. Cost and efficiency are two important factors considered in the remediation of Se-contaminated water. In our previous study (Zhang and Frankenberger, 2006), we reported that C. braakii is capable of using inexpensive molasses as a carbon source to reduce Se(VI), with a reduction of 87–97% in natural river waters. However, only 20% of Se(VI) was removed from highly saline agricultural drainage water containing a higher NO3− level. Therefore, selection of salttolerant bacteria that can use inexpensive carbon sources to effectively reduce Se(VI) and NO3− would play a key role in the economics of the remediation of Secontaminated agricultural drainage water.
In this study, we have investigated the effect of NO3− on the bacterial reduction of Se(VI) in agricultural drainage water, and have evaluated the enhancement of Se(VI) reduction by the addition of a NO3− reducer to remove NO3− from agricultural drainage water. The removal of Se(VI) and NO3− in agricultural drainage water was characterized in a series of batch experiments. 2. Materials and methods 2.1. Materials Natural agricultural drainage water was collected from the western San Joaquin Valley, California. The water, with a pH of 8.2 and salinity [electrical conductivity (EC)] of 11.56 dS/m, contained 782 μg/L Se(VI) and 191 mg/L NO3− . Synthetic agricultural drainage (SAD) water was prepared with the following constituents (in g/L): Na2SO4, 5.92; NaCl, 0.989; NaHCO3, 0.138; CaCl2·2H2O, 0.917; MgSO4, 1.238; Na2B4O7·10H2O, 0.529; NaNO3, 0.069; K2HPO4, 0.091; FeCl2, 0.0002; molasses 1; and trace element solution (Focht, 1994), 1 mL/L. The SAD water was autoclaved (18 psi at 121 °C) for 20 min after a pH adjustment to 7.5. The Se(VI) standard stock solution (10,000 mg/L) was passed through a sterile 0.2 μm membrane filter prior to its addition to the SAD water. 2.2. Isolation of the Se(VI)-reducing bacterium Rice straw as an effective organic carbon source and a carrier of Se(VI)-reducing bacteria has been tested to remove Se(VI) from agricultural drainage water (Zhang and Frankenberger, 2003a,b). Air-dried rice straw used to isolate Se(VI)-reducing bacteria was obtained from the San Joaquin Valley, California. Two grams of rice straw and 200 mL of sterile DI water were added into a 250-mL sterile Erlenmeyer flask, spiked with Se(VI) to a concentration of 10 mg/L. The flask was capped with a sterile rubber stopper and incubated at 20 °C for 5 days when a clear red color of Se(0) appeared in the flask. An aliquot of the water was serially diluted in sterile deionized water and spread onto tryptic soy agar (TSA; Difco, Detroit, Mich.) plates containing 50 mg/L of Se(VI). The plates were incubated at 30 °C for 48 h when several colonies with red Se(0) precipitates were observed on the TSA plates. The colonies were restreaked on TSA plates with and without Se(VI) to ensure that the red color of the colony was not due to a bacterial pigment. One isolate was identified as Bacillus sp. based on 16S rRNA gene sequence (MIDI Labs, Newark, DE), and designated strain RS1. Dechloromonas sp. strain HZ, an
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organism widely used to reduce perchlorate to chloride in groundwater (Xu et al., 2003) was used in this study to reduce NO3− in the SAD water and natural agricultural drainage water. Dechloromonas sp. HZ was obtained from the Department of Environmental Engineering, University of California at Riverside. Bacillus sp. RS1 and Dechloromonas sp. HZ were were initially grown on TSA plates without Se(VI) and incubated (30 °C) for 2–3 days. The colonies of each bacterium were separately resuspended into the sterile SAD water and incubated overnight to give an OD600 range of 0.55– 0.57 for a series of experiments in this study. 2.3. Reduction of Se(VI) in the SAD water The first experiment was conducted in the laboratory to determine the effect of the addition of Dechloromonas sp. HZ on reduction of Se(VI) to Se(0) by Se(VI) reducers in the SAD water. Se(VI)-reducing bacteria used in this study included C. braakii isolated from agricultural drainage water (Zhang and Frankenberger, 2006) and Bacillus sp. RS1 isolated from rice straw. In the experiment, 38 mL of the sterile SAD water containing 1000 mg/L of molasses was added to each 40-mL glass vial. The vials were spiked with Se(VI) to give a final concentration of 1000 μg/L and inoculated with 0.2 mL of each bacterial cell suspension (C. braakii and Dechloromonas sp. HZ or Bacillus sp. RS1 and Dechloromonas sp. HZ). The vials were capped with sterile caps and incubated under a static condition at room temperature (20 °C). Another batch experiment without the addition of Dechloromonas sp. HZ was conducted for comparison. The experiments were run in triplicate for 8 days. The water samples were collected at an interval of 1–2 days for analysis of Se species. 2.4. Reduction of Se(VI) in the SAD water with varying amounts of NO3− After obtaining the results from the first experiment described above, Bacillus sp. RS1 proved better than C. braakii for using molasses to effectively reduce Se (VI) to Se(0). Therefore, the second experiment was conducted in the laboratory to test the effect of varying amounts of NO3− on the reduction of Se(VI) to Se(0) in the SAD water. In this experiment, 230 mL of the SAD water containing a NO3− level of 100, 250, and 500 mg/L was added to each 250-mL Erlenmeyer flask after an amendment with 1000 μg/L of Se(VI) and 1000 mg/L of molasses. The terms “100 mg/L NO3− SAD water”, “250 mg/L NO3− SAD water”, and “500 mg/L NO3− SAD
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water” refer to the experimental set up containing 100, 250, and 500 mg/L NO3− in SAD water at the beginning of the experiments, respectively. The flasks were inoculated with 1 mL of the Bacillus sp. RS1 cell suspension. The flasks were capped with sterile rubber stoppers and incubated under static conditions at room temperature (20 °C). In order to examine the effect of the addition of Dechloromonas sp. HZ on reduction of Se(VI), another batch of the same experiment described above was added with 1 mL Dechloromonas sp. HZ to remove NO3− in the SAD water. The experiment without the addition of NO3− was run as control. The experiment was performed in triplicate for 8 days. The water samples were collected at an interval of 1–2 days for analysis of Se species and NO3−. 2.5. Reduction of Se(VI) in natural agricultural drainage water In order to observe whether Bacillus sp. RS1 and Dechloromonas sp. HZ could survive in natural agricultural drainage water and reduce Se(VI) to Se(0), 38 mL of the non-sterile agricultural drainage water was added to a 40-mL glass vial. The vials were supplemented with 1000 mg/L of molasses, and inoculated with 0.2 mL of a cell suspension of Bacillus sp. RS1, or 0.2 mL each of Bacillus sp. RS1 and Dechloromonas sp. HZ cell suspension. The water devoid of the inoculum served as a control. All vials were capped with sterile caps and incubated at room temperature (20 °C). The experiment was run in triplicate for 6 days. The water samples were collected at an interval of 1–2 days for analysis of Se species. 2.6. Analysis Selenium species in the water samples were determined using a method developed by Zhang and Frankenberger (2003a) and Zhang et al. (1999) after removal of Se(0) from the solution by centrifugation at 12,000 for 14 min. Directly measured Se species included total soluble Se, Se(IV), and Se(IV) plus Se (-II) [organic Se(-II) plus inorganic Se(-II)]. Se(VI), Se(0) and Se(-II) were determined by the difference method (Zhang et al., 1999; Zhang and Frankenberger, 2003a). Se concentrations in all the prepared solutions were analyzed by hydride generation atomic absorption spectrometry (HGAAS) (Zhang et al., 1999). Nitrate was determined by a NO3− ion electrode (ColeParmer InC). Nitrate standards were made using sodium nitrate (NaNO3) and the sterile SAD water without the addition of NO3− . The Se detection limit of
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the HGAAS and the experimental water was 0.4 and b10 μg/L, respectively. 3. Results 3.1. Bacterial Se(VI) reduction The reduction of Se(VI) in the SAD water containing 1000 μg/L of Se(VI) and 50 mg/L of NO3− is illustrated in Fig. 1. During an 8-day experiment, Se (VI) was not reduced in the SAD water without the addition of C. braakii or Bacillus sp. RS1 (Fig. 1A and B). The extent of Se(VI) reduction differed in the SAD water added with different bacteria. In the SAD water inoculated with C. braakii, Se(VI) was not reduced (Fig. 1C). Se(VI) reduction by C. braakii occurred when Dechloromonas sp. HZ was added to the SAD
water, with 60% Se(VI) reduction to Se(IV) and Se(0) (Fig. 1E). In contrast, Bacillus sp. RS1 is capable of using molasses to reduce Se(VI) in the SAD water, with a 57% Se(VI) reduction at day 8 (Fig. 1D). Se(VI) reduction by Bacillus sp. RS1 was significantly enhanced when Dechloromonas sp. HZ was added to the SAD water (Fig. 1F). Se(VI) was completely removed at day 3. On the final day of the experiment, Se(0) was the major Se form in the SAD water, with a small amount of Se(-II). 3.2. Effect of NO3− concentrations on Se(VI) reduction The effect of NO3− concentrations on Se(VI) reduction by Bacillus sp. RS1 with or without the presence of Dechloromonas sp. HZ is shown in Figs. 2–6. In the SAD water without the presence of NO3− (Fig. 2A),
Fig. 1. Reduction of Se(VI) (1000 μg/L) in the SAD water by C. braakii (left figures; A, C, and E) and Bacillus sp. RS1 (right figures; B, D, and F). Top figures (A and B), control experiments without the addition of bacteria; middle figures (C and D), experiments with Se(VI)-reducing bacteria; bottom figures (E and F), experiments with Se(VI)-reducing bacteria and Dechloromonas sp. HZ. Error bars indicating one standard deviation (n = 3).
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Fig. 2. Reduction of Se(VI) (1000 μg/L) in the no-NO−3 SAD water by Bacillus sp. RS1 with Dechloromonas sp. HZ (right figure; B) or without Dechloromonas sp. HZ (left figure; A). Error bars indicating one standard deviation (n = 3).
Se(VI) was almost entirely reduced at day 4. At day 8, Se(0) and Se(IV) accounted for 36 and 63% of the added Se in the SAD water. Addition of Dechloromonas sp. HZ to the SAD water enhanced the reduction of Se(VI) (Fig. 2B). On the last day of the experiment, Se(0) was the major Se form (83%) in the SAD water, with a small amount of Se(-II). With the increase of NO3− to levels of 100, 250, and 500 mg/L in the SAD water, reduction of Se(VI) by Bacillus sp. RS1 slowed down (Figs. 3A, 4A, and 5A). On the final day of the experiment, Se(VI), Se(IV), and Se(0) in the 100 and 250 mg/L NO3− SAD water
accounted for 56.5–68.6, 24.7–37.7, and 5.92–7.18%, respectively. The addition of Dechloromonas sp. HZ significantly increased removal of Se(VI) in the 100 and 250 mg/L NO3− SAD water (Figs. 3C and 4C). At day 8, 98–99% added Se(VI) was reduced to Se(0), with small amounts of Se(IV) and Se(-II). In the 500 mg/L NO3− SAD water with or without the presence of Dechloromonas sp. HZ (Fig. 5C), only 10% of the added Se(VI) was reduced to Se(IV) and Se(0) during an 8-day experiment. Both Bacillus sp. RS1 and Dechloromonas sp. HZ were capable of reducing NO3− in the SAD water. During
Fig. 3. Reduction of Se(VI) (1000 μg/L) and NO−3 in the 100 mg/L NO−3 SAD water by Bacillus sp. RS1 with Dechloromonas sp. HZ (bottom figures; C and D) or without Dechloromonas sp. HZ (top figures; A and B). Error bars indicating one standard deviation (n = 3).
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Fig. 4. Reduction of Se(VI) (1000 μg/L) and NO−3 in the 250 mg/L NO−3 SAD water by Bacillus sp. RS1 with Dechloromonas sp. HZ (bottom figures; C and D) or without Dechloromonas sp. HZ (top figures; A and B). Error bars indicating one standard deviation (n = 3).
an 8-day experiment, 92.5, 51.7, and 29.7% of the added NO3− was removed from the 100, 250, and 500 mg/L NO3− SAD water, respectively (Figs. 3B, 4B, and 5B).
The addition of Dechloromonas sp. HZ to the water enhanced the removal of NO3− (Figs. 3D, 4D, and 5D). Nitrate was almost entirely removed at day 3 in the
Fig. 5. Reduction of Se(VI) (1000 μg/L) and NO−3 in the 500 mg/L NO−3 SAD water by Bacillus sp. RS1 with Dechloromonas sp. HZ (bottom figures; C and D) or without Dechloromonas sp. HZ (top figures; A and B). Error bars indicating one standard deviation (n = 3).
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Fig. 6. Removal of Se(VI) from agricultural drainage water by Bacillus sp. RS1. Top figures, control experiment without the addition of bacteria and molasses (left figure; A) and only without bacteria (right figure; B). Bottom figures, experiment with Bacillus sp. RS1 plus Dechloromonas sp. HZ (right figure; D) or without Dechloromonas sp. HZ (left figure; C). Error bars indicating one standard deviation (n = 3).
100 mg/L NO3− SAD water. In the SAD water with 250 and 500 mg/L NO3−, 96.7 and 83.1% of the added NO3− was reduced within 8 days, respectively. 3.3. Reduction of Se(VI) in natural agricultural drainage water Reduction of Se(VI) in agricultural drainage water with and without inoculation of Bacillus sp. RS1 and Dechloromonas sp. HZ is shown in Fig. 6. In the absence of the bacteria, there was little change in Se(VI) (748 μg/L) concentration in the agricultural drainage water with and without the addition of molasses (Fig. 6A and B). 81% of Se(VI) in the agricultural water was reduced to Se(IV) (12.3%), Se(0) (50.6%), and Se(-II) (17.8%) in the presence of Bacillus sp. RS1 (Fig. 6C). Se(VI) was almost entirely removed when Bacillus sp. RS1 and Dechloromonas sp. HZ were added to the water (Fig. 6D). At day 6, Se(IV), Se(0), and Se(-II) accounted for 2.8 (21 μg/L), 65 (486 μg/L), and 32.2% (241 μg/L), respectively. 4. Discussion Reduction of Se(VI) to Se(0) by Se(VI)-reducing bacteria that are capable of using inexpensive organic carbon sources is a useful remedial technique
for removing Se from agricultural drainage water. C. braakii is capable of using inexpensive molasses as a carbon source to reduce Se(VI) in natural river waters (Zhang and Frankenberger, 2006), but, its ability to reduce Se(VI) in the SAD water is limited. In conrast, Bacillus sp. RS1 has performed better using molasses to reduce Se(VI). The pathway of Se(VI) reduction by Bacillus sp. RS1 can be characterized as Se(VI) → Se(IV) → Se(0) → Se(-II). Several researchers have reported that different Bacillus species can reduce Se(VI) or Se(IV) in different media. Kashiwa et al. (2000) reported that 1 mM Se(VI) was completely reduced by Bacillus sp. SF-1 in a basal salt medium containing 0.1% yeast extract and 20 mM sodium lactate. Garbisu et al. (1995) used Bacillus subtilis to reduce Se(IV) in a minimal chemically defined liquid medium containing 70 mM glucose. However, added Se(VI) in the same medium had little change during a 4-day experiment. Oremland et al. (1999) found that B. selenitireducens was only able to reduce Se(IV) to Se(0) and did not respire Se(VI) in a salt medium containing lactate. Bacillus sp. RS1 was also able to reduce NO3− in the SAD water with a reduction of 92, 52, 30% of the added NO3− within 8 days, respectively in the 100, 250, and 500 mg/L NO3− SAD water. Addition of Dechloromonas sp. HZ to the SAD water increased the removal of NO3−
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to 100% at day 3 in the 100 mg/L NO3− SAD water, and 97 and 83% at day 8, respectively in the 250 and 500 mg/L NO3− SAD water. Addition of Dechloromonas sp. HZ also enhanced the Se(VI) reduction from 31– 58% to 98–100% in the 50, 100, and 250 mg/L NO3− SAD water. Nitrate is a competitive electron acceptor affecting Se(VI) reduction in aquatic systems due to their similar redox potentials (Masscheleyn and Patrick, 1993). Therefore, addition of Dechloromonas sp. HZ to the NO3− containing SAD water to remove NO3− would enhance reduction of Se(VI) by Se(VI)-reducing bacteria although Dechloromonas sp. HZ itself did not reduce Se(VI). It might be likely that the higher amounts of organic carbon sources are needed to treat Se in agricultural drainage water having a high level of NO3−. In this study, reduction of Se(VI) by Bacillus sp. RS1 decreased with an increase of NO3− concentrations in the SAD water with or without the addition of Dechloromonas sp. HZ. In the 500 mg/L NO3− SAD water, Se(VI) reduction was almost totally inhibited. The reason might be attributed to amount of organic carbon added to the SAD water. In this study, we added 1000 mg/L molasses (liquid phase). We do not know exact amounts of the available organic carbon in it, which can be used by bacteria to reduce NO3− and Se(VI). Competition of NO3− with Se(VI) for electron donors in the SAD water with a high ratio of NO3− (500 mg/L) to Se(VI) (1 mg/L) would most likely consume most of the available organic carbon of molasses for NO3− reduction. Thus, there would be little organic carbon left for Se(VI) reduction. Kashiwa et al. (2000) also reported that Se(VI) was not reduced in a basal salt medium when NO3− concentration increased from 1, 5 to 10 mM. Their explanation is that most of the added lactate in the medium was consumed as the electron donor for NO3− reduction, and consequently, little lactate remained for utilization in Se (VI) reduction. This study revealed that Bacillus sp. RS1 not only reduce Se(VI) in the SAD water, but also effectively reduce Se(VI) in natural agricultural drainage water, with 80% reduction of Se(VI). Upon the addition of Dechloromonas sp. HZ to natural agricultural drainage water, Se(VI) was almost entirely reduced by Bacillus sp. RS1 during a 6-day experiment. Agricultural productivity in the San Joaquin Valley, California generates agricultural drainage water containing a high level of Se(VI) and NO3− (Oswald et al., 1989; Sylvester, 1990). These elevated Se levels need to be reduced before discharge to nearby wetlands and lakes. The results of this study indicate that a combination of Bacillus sp. RS1 with Dechloromonas sp. HZ are capable of using
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