Reduction of Selenium Oxyanions in Wastewater Using Two Bacterial Strains

Research Article

Reduction of Selenium Oxyanions inWastewater Using Two Bacterial Strains*

The biological reduction of selenium oxyanions is capable of reducing both sele-nate and selenite to insoluble elemental selenium. In this process, however, bac-teria inevitably require expensive chemicals such as yeast extract in almost allcases. Therefore, the reduction of selenium oxyanions with inexpensive alcoholwould be more practical. A Pseudomonas sp. strain 4C-C isolated from a sludgein a wastewater treatment facility was able to reduce selenate to selenite usingethanol as an electron donor for its anaerobic respiration, but could not reduceselenite to elemental selenium. Paracoccus denitrificans JCM-6892, on the otherhand, was observed to be able to reduce selenite to elemental selenium in the pre-sence of ethanol, but not selenate to selenite. Therefore, a mixture containing asuspension of Pseudomonas sp. strain 4C-C and P. denitrificans JCM-6892 cells al-lowed selenate to be reduced to insoluble elemental selenium via selenite in thepresence of ethanol and was also capable of reducing nitrate to nitrogen gas. Aim-ing at simplicity of the recovery process of insoluble elemental selenium, a poly-meric gel immobilized mixture of the two bacterial strains was examined usingethanol as an electron donor. The immobilized mixture could therefore reducenot only selenate to elemental selenium, but also nitrate to nitrogen gas in a sin-gle step. The gel that immobilized the microbial mixture changed its color duringthe process to bright red and no red elemental selenium was left in the waste-water. This indicates that the reduced elemental selenium was completely ab-sorbed in the gel. This simple bacterial combination would therefore be effectivein the presence of ethanol to reduce selenium oxyanions in various wastewaterscontaining selenium and the other oxyanions.

Keywords: Bacteria, Selenium, Wastewater

Received: September 15, 2006; revised: March 26, 2007; accepted: April 3, 2007

DOI: 10.1002/elsc.200620188

1 Introduction

Selenium is a useful element and widely applied in various in-dustries, such as the manufacture of glass and semiconductors.These industries eliminate therefore wastewater containing se-lenium in the form of selenate (SeO4

2–) and selenite (SeO32–).

Selenium oxyanions in industrial wastewater are strictly regu-lated, since soluble selenium oxyanions are poisonous for allliving creatures [1, 2].

At present, chemical precipitation has been used for the re-moval of selenium. This method is effective to selenite re-moval, but the efficiency in selenate removal is rather low. Themethod needs plenty of chemicals and a large amount ofsludge is produced. On the other hand, a biological techniqueis capable of reducing both selenate and selenite to insolubleelemental selenium. Fujita et al. isolated the selenate-reducingbacterium, Bacillus sp. SF-1, and investigated various factorsaffecting the reduction of selenium oxyanions [3, 4]. Thisstrain needed yeast extract for selenate and selenite reductions.Lortie et al. reported that a Pseudomonas stutzeri isolate couldreduce selenate and selenite to elemental selenium using tryp-tic soy broth [5]. A selenate-reducing bacterium, strain SES-3,was able to reduce selenate and selenite using yeast extract un-der obligately anaerobic conditions [6, 7]. In these processes,the bacteria require expensive chemicals i.e., yeast extract forthe reduction of selenium oxyanions [3–7]. Therefore, the re-duction of selenium oxyanions in the presence of inexpensive

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim http://www.els-journal.com

M. Morita1

H. Uemoto1

A. Watanabe1

1 Environmental ScienceResearch Laboratory,Central Research Instituteof Electric Power Industry(CRIEPI), Abiko-shi, Chiba,Japan.

–Correspondence: M. Morita ([email protected]), Envir-onmental Science Research Laboratory, Central Research Institute ofElectric Power Industry (CRIEPI), 1646 Abiko, Abiko-shi, Chiba 270-1194, Japan.

* Based on a paper presented at the ISEB/ESEB/JSEB Conference, Leipzig,Germany, July 9–13, 2006.

Eng. Life Sci. 2007, 7, No. 3, 235–240 235

alcohol is necessary for practical applications. Moreover, thereduction of selenium oxyanions was examined under suspen-sion conditions in these studies, and the bacterial strains andinsoluble elemental selenium reduced from selenate via sele-nite were mixed in the solutions [3–7]. Since these techniquesrequire an additional separation process, novel methodsshould be developed for the removal of selenium oxyanionsfrom wastewater, in which the reduction of selenium oxyan-ions and the recovery of insoluble elemental selenium (reducedfrom selenium oxyanions) could be unified in a single step.

In this study, aiming at the reduction of selenium oxyanionsin the presence of alcohol not only a new selenate-reducingbacterium was isolated but also a selenite-reducing bacteriumwas searched for, which would be capable of using ethanol asan electron donor. In addition, the artificial wastewatercontaining selenate and nitrate was treated in the presence ofethanol using the combination of two bacterial strains undersuspension conditions. Furthermore, the polymeric gel-immo-bilized mixture of two bacterial strains together was investi-gated in the presence of ethanol using an artificial wastewatercontaining selenate and nitrate in order to simplify the recov-ery process of elemental selenium.

2 Materials and Methods

2.1 Isolation of Bacterial Strain

The bacterial strain was isolated from a sludge of a wastewatertreatment facility using the following artificial wastewatercontaining [g/L]: MgSO4 · 7 H2O, 0.2; Na2HPO4 · 12 H2O, 0.9;KH2PO4, 0.15; CaCl2 · 2 H2O 0.01; NaHCO3, 0.5; EDTA-Fe0.005 (pH 7.8). The trace elements added to the wastewaterwere [mg/L]: ZnSO4 · 7 H2O, 0.1; MnCl2 · 4 H2O, 0.03; H3BO3,0.3; CoCl2 · 6 H2O, 0.2; CuCl2 · 2 H2O, 0.01; NiCl2 · 6 H2O,0.02; Na2MoO4 · 2 H2O, 0.03. All reagents were dissolved indistilled water. One gram of sludge was inoculated into 20 mLof the above artificial wastewater with 5 mM Na2SeO4 and 2 %ethanol while being gently mixed in a 50-mL vial at 30 °C un-der anaerobic conditions using nitrogen gas. After selenate wascompletely reduced, 1 mL of the enriched culture was trans-ferred into 20 mL of the new artificial wastewater and enrich-ment cultivation was continued three times. The last enrichedculture was plated on the following medium agar plates (NaCl,1.0 g/L; Na2SeO4, 1 mM; yeast extract, 1.0 g/L; agar: 2 %;pH: 7.0) under anaerobic conditions for bacterial isolation. Toensure its purity, the isolate was checked for bacterial contami-nation using the dilution agar plate method and microscopicobservations.

2.2 Bacterial Strains and Growth Conditions

The isolated strain 4C-C and P. denitrificans JCM-6892 wereused in this study. The two bacterial strains were aerobicallycultured at 30 °C in the following medium containing [g/L]:Nutrient Broth (Difco), 16.0; NaCl, 5.0 (pH 7.0). The twokinds of bacterial cells were harvested by centrifugation(20000 × g, 15 min, 4 °C) and washed twice with phosphate

buffer containing [g/L]: Na2HPO4 · 12 H2O, 9.0; KH2PO4 1.5(pH 7.5). The cells of the isolated strain 4C-C and P. denitrifi-cans were suspended in phosphate buffer for further batchexperiments.

2.3 Reductions of Selenium Oxyanions Using CellSuspensions

Batch treatments were carried out under anaerobic conditionsfor 6 days at 30 °C using an artificial wastewater containing50 mg Se/L of selenate, 50 mg N/L of nitrate and 1 % ethanol.A suspension of strain 4C-C cells (0.77 mg wet weight/mL)was injected into a 50-mL vial containing 30 mL of the above-mentioned artificial wastewater.

Also, batch treatments were carried out anaerobically for10 days at 30 °C using an artificial wastewater containing50 mg Se/L of selenite, 50 mg N/L of nitrate and 1 % ethanol.A suspension of P. denitrificans cells (1.53 mg wet weight/mL)was injected into a 50-mL vial containing 30 mL of the above-mentioned artificial wastewater.

The wastewaters were sampled for analysis during the batchoperations.

2.4 Selenate Reduction Using a Cell SuspensionMixture

Batch treatments were carried out anaerobically for 10 days at30 °C using an artificial wastewater containing 10 mg Se/L ofselenate, 100 mg N/L of nitrate and 1 % ethanol. Cell suspen-sions of strain 4C-C (0.77 mg wet weight/mL) and P. denitrifi-cans (0.77 mg wet weight/mL) were injected into a 50-mL vialcontaining 30 mL of the above-mentioned artificial waste-water. Four experimental conditions were employed: (i) theaddition of no bacterial strain; (ii) the addition of a strain4C-C cell suspension only; (iii) the addition of P. denitrificanscell suspension only; (iv) the addition of a mixture containinga suspension of the strain 4C-C and of P. denitrificans cells.The wastewaters were sampled for analysis during the batchoperations.

2.5 Selenate Reduction Using an ImmobilizedMixture

The suspensions of strain 4C-C and P. denitrificans cells weremixed with photo-cross-linkable polymer PVA-SbQ (SPP-H-13, Toyo Gosei Kogyo Co.) in the ratio of 1 to 3. They wereapplied to double-sided non-wovens (length: 40 mm; width:20 mm) made of polyethylene terephthalate (G2260-1S, TorayCo.) at 0.7 mm thickness and solidified into a gel by irradia-tion with metal halide lamps for 20 min (1000 lmol/m/s). Thegels were kept at 0 °C in molds in order to avoid photoinacti-vation [8].

Batch treatments were carried out under anaerobic condi-tions for 10 days at 30 °C using an artificial wastewater con-taining 10 mg Se/L of selenate, 100 mg N/L of nitrate and 1 %


236 M. Morita et al. Eng. Life Sci. 2007, 7, No. 3, 235–240

ethanol. The gel-immobilized bacteria were injected into a50-mL vial containing 30 mL of the above-mentioned artificialwastewater using four experimental conditions (i) gel withoutbacterial cells; (ii) immobilized cells of strain 4C-C only(0.77 mg wet weight/mL); (iii) immobilized cells of P. denitrifi-cans only (0.77 mg wet weight/mL); (iv) immobilized cells ofstrain 4C-C (0.77 mg wet weight/mL) and P. denitrificans(0.77 mg wet weight/mL) together. The wastewaters weresampled for analysis during the batch operations.

2.6 Analytical Methods

Selenate, selenite, nitrate and nitrite concentrations were deter-mined using an ion-chromato analyzer (DX-AQ, Dionex Co.)with an IonPac AS9-HC column.

3 Results and Discussion

3.1 Isolation of Strain 4C-C

The agar plate which was inoculated with the dilute enrichedculture showed the formation of red colonies. This result indi-cated that selenate on the agar plate was reduced to elementalselenium (Se). Some colonies were picked, and the morpho-logical and physiological characteristics of the colonies wereexamined. All colonies showed identical morphological andphysiological characteristics indicating that one colony wasselected from a typical strain named 4C-C.

The morphological and physiological characteristics of thisstrain are shown in Tab. 1. The strain was a Gram-negative,motile and rod-shaped bacterium. Based on the morphologicaland physiological characteristics, this strain 4C-C was identi-fied as a Pseudomonas sp. according to Bergey’s Manual [9].

3.2 Selenate Reduction Using a Cell Suspensionof the Strain 4C-C

When the artificial wastewater containing 50 mg Se/L of sele-nate, 50 mg N/L of nitrate and 1 % ethanol was treated using acell suspension of the Pseudomonas sp. strain 4C-C, the sele-nate concentration in the wastewater decreased gradually,whereas the concentration of selenite, which was reduced fromselenate, in the wastewater increased gradually (see Fig. 1A).As a result, the total selenium (the sum of selenate and sele-nite) concentration did not change (see Fig. 1A). On the otherhand, the nitrate in the wastewater was completely reducedand some nitrite was detected after 26 hours. The total nitro-gen (the sum of nitrate and nitrite) concentration decreasedrapidly.

The artificial wastewater treatment using 1.0 g/L yeast ex-tract instead of ethanol showed a similar result (data notshown). The isolated strain 4C-C was able to reduce selenatecompletely, but selenite accumulated in both cases of yeast ex-tract and ethanol. As a result, only small amounts of selenatewere reduced to elemental selenium via selenite.

3.3 Selenite Reduction Using a Cell Suspensionof P. denitrificans

In order to completely reduce selenate to elemental seleniumvia selenite in the presence of ethanol, selenite accumulated bythe cells of Pseudomonas sp. strain 4C-C has to be reduced toelemental selenium by means of another method. One method


Table 1. Morphological and physiological characteristics of strain4C-C.

Gram stain negative

Shape rod

Cell diameter 0.5–0.6 lm

Cell length 1.5–2.0 lm

Spore –

Motile +

Growth temperature at 37 °C +

Growth temperature at 45 °C +

Catalase reaction +

Oxidase reaction +

Acid from glucose –

Denitrification +

Sulfate reduction –

Indole production –

Arginine dihydrolase –

Urease –

Gelatin hydrolysis –

Glucose utilization +

L-Arabinose utilization –

D-Mannose utilization –

D-Mannitol utilization +

D-Maltose utilization +

Figure 1. Time-dependent changes in (A) selenium and (B)nitrogen oxyanion concentrations when artificial wastewatercontaining selenate and nitrate was treated using Pseudomonassp. strain 4C-C.Symbols: selenate (�), selenite (D), total selenium (�) inFig. 1A; nitrate (�), nitrite (�), and total nitrogen (�) in Fig. 1B.The vertical lines indicate standard deviations (n = 3).

Eng. Life Sci. 2007, 7, No. 3, 235–240 Reduction of Selenium Oxyanions 237

is the use of a bacterial strain capable of reducing selenite toelemental selenium in the presence of ethanol. It was observedthat P. denitrificans JCM-6892 cells could reduce selenite toelemental selenium in the presence of ethanol although thisstrain could not reduce selenate to selenite or elemental sele-nium at all in preliminary experiments. Therefore, P. denitrifi-cans JCM-6892 cells seemed to be an appropriate candidate toreduce the selenite in the wastewater.

When an artificial wastewater containing 50 mg Se/L of sele-nite, 50 mg N/L of nitrate and 1 % ethanol was treated using P.denitrificans JCM-6892 cell suspensions, the selenite concen-tration, which was equivalent to the total selenium concentra-tion in this case, in the wastewater decreased gradually (seeFig. 2A). On the other hand, the nitrate in the wastewater wascompletely reduced and no nitrite was detected. The total ni-trogen (the sum of nitrate and nitrite) concentration decreasedrapidly.

These results suggested that a mixture of Pseudomonas sp.strain 4C-C and P. denitrificans JCM-6892 cells would be ableto reduce selenate to insoluble elemental selenium in thepresence of ethanol. The mixture would also reduce nitrate tonitrogen gas. However, as the selenite reduction rate by P. deni-trificans JCM-6892 cells was smaller than the selenate reduc-tion rate by Pseudomonas sp. strain 4C-C cell, selenite mightaccumulate when the mixture would be used.

3.4 Selenate Reduction Using a Mixture of BothBacterial Strains

In order to verify the above-mentioned prediction, a mixtureof strain 4C-C and P. denitrificans cells was examined using anartificial wastewater containing 10 mg Se/L of selenate and100 mg N/L of nitrate. The selenate concentration in the was-tewater decreased gradually under the addition of cells ofstrain 4C-C only or the addition of a mixture of strain 4C-Cand P. denitrificans cells, whereas the selenate concentration

was invariable under the addition of no bacterial strains or theaddition of P. denitrificans cells suspension only (see Fig. 3A).The concentration of selenite, which was reduced by strain4C-C, increased to 2.8 mg Se/L and decreased gradually whenthe mixture of cells from strain 4C-C and P. denitrificans wereadded, whereas the selenite concentration increased sharplywithin 39 hours and decreased slightly under the addition ofcells of the strain 4C-C only (see Fig. 3B). As a result, selenateand selenite were completely reduced by the addition of themixture of cells of strain 4C-C and P. denitrificans, and no totalselenium (the sum of selenate and selenite) concentration(<0.05 mg Se/L) was detected after 231 hours (see Fig. 3C).

On the other hand, the nitrate in the wastewater was com-pletely reduced except for the case of no bacterial strains pres-ent (see Fig. 3D), and the total nitrogen (the sum of nitrateand nitrite) concentration also decreased rapidly (see Fig. 3F).


Figure 2. Time-dependent changes in (A) selenium and (B) ni-trogen oxyanion concentrations when artificial wastewater con-taining selenite and nitrate was treated using P. denitrificansJCM-6892.Symbols: selenate (�), selenite (D), total selenium (�) inFig. 2A; nitrate (�), nitrite (�), and total nitrogen (�) in Fig. 2B.The vertical lines indicate standard deviations (n = 3).

Figure 3. Time-dependent changes in (A) selenate concentra-tion, (B) selenite concentration, (C) total selenium concentra-tion, (D) nitrate concentration, (E) nitrite concentration, and (F)total nitrogen concentration when suspensions of Pseudomonassp. strain 4C-C and P. denitrificans JCM-6892 cells were used.Symbols: the addition of no bacterial strain (�), the addition ofa 4C-C cell suspension only (�), the addition of a P. denitrificanscell suspension only (�), and the addition of a mixture contain-ing a suspension of strain 4C-C and P. denitrificans cells (�). Thevertical lines indicate standard deviations (n = 3).


Some nitrite accumulated when cells from strain 4C-C wereonly added after 15 hours (see Fig. 3E).

As predicted, the mixture of Pseudomonas sp. strain 4C-Cand P. denitrificans JCM-6892 cells could reduce selenate to in-soluble elemental selenium via selenite in the presence of etha-nol and the color of the artificial wastewater turned red. Themixture was also capable of reducing nitrate to nitrogen gas.

3.5 Selenate Reduction Using an ImmobilizedMixture

As the bacterial strains and the elemental selenium reducedfrom selenate via selenite were mixed in the wastewater, a sepa-ration process was necessary to recover the elemental selenium.Aiming at the simplicity of a recovery process, the immobi-lized mixture of cells of strain 4C-C and P. denitrificans wasused.

The selenate concentration in the wastewater decreased withcells of the immobilized strain 4C-C only or with cells of theimmobilized mixture of strain 4C-C and P. denitrificans,whereas the selenate concentration was invariable when nobacterial strain or immobilized P. denitrificans cells were em-ployed only (see Fig. 4A). The concentration of selenite, whichwas reduced by strain 4C-C, increased to 8.9 mg Se/L and de-creased gradually with the immobilized mixture of strain 4C-Cand P. denitrificans cells, whereas the selenite concentration in-creased within 63 hours and decreased slightly with cells of theimmobilized strain 4C-C only (see Fig. 4B). As a result, sele-nate and selenite were reduced completely by the immobilizedmixture of strain 4C-C and P. denitrificans cells, and no totalselenium (the sum of selenate and selenite) concentration(< 0.05 mg Se/L) was detected after 231 hours (see Fig. 4C).

On the other hand, the nitrate in the wastewater was re-duced completely except for the case of no bacterial strains(see Fig. 4D), and the total nitrogen (the sum of nitrate andnitrite) concentration also decreased (see Fig. 4F). Nitrite wasaccumulated through 63 hours and decreased gradually underthe immobilization of strain 4C-C cell only (see Fig. 4E).

These results were similar to the results using a cell suspen-sion mixture of strain 4C-C and P. denitrificans cells thoughthe reduction rates of selenium and nitrogen oxyanions in thepresence of an immobilized mixture of strain 4C-C and P. de-nitrificans cells were smaller than those using a cell suspensionmixture of strain 4C-C and P. denitrificans cells. This differencewould be caused by slow diffusion of oxyanions into the poly-meric gel, where the bacterial strains were immobilized.

The immobilized mixture of Pseudomonas sp. strain 4C-Cand P. denitrificans JCM-6892 cells could reduce selenate toinsoluble elemental selenium via selenite in the presence ofethanol. The immobilized mixture was also capable of reduc-ing nitrate to nitrogen gas. The photographs of the vials withthe gel-immobilized cell mixture of strain 4C-C and P. denitri-ficans are presented in Fig. 5. The non-woven immobilizedmixture of strain 4C-C and P. denitrificans cells was pigmen-tized to bright red and there was no red elemental selenium inthe wastewater after 231 hours. This means that the reducedelemental selenium was completely captured in the non-wovengel. On the contrary, the color of the non-woven gel was ivory

yellow at the start time. The non-woven immobilized strain4C-C cells only or P. denitrificans cells only also remained ivoryyellow even at the end of the experiment (data not shown).

4 Conclusions

The combination of both Pseudomonas sp. strain 4C-C andP. denitrificans JCM-6892 cells enabled the reduction of selenateto elemental selenium via selenite in a single step. This simplebacterial combination would be effective in the presence ofinexpensive ethanol to reduce selenium oxyanions in variouswastewaters containing selenium and the other oxyanions suchas nitrate or sulfate. In this study, the artificial wastewater wasadjusted to anaerobic conditions using nitrogen gas ahead of


Figure 4. Time-dependent changes in (A) selenate concentra-tion, (B) selenite concentration, (C) total selenium concentra-tion, (D) nitrate concentration, (E) nitrite concentration, and (F)total nitrogen concentration when the gel-immobilized Pseudo-monas sp. strain 4C-C and P. denitrificans JCM-6892 cells wereused.Symbols: Gel without bacterial cells (�), immobilized cells ofstrain 4C-C cell only (�), immobilized cells of P. denitrificans only(�), and immobilized cells of strain 4C-C and P. denitrificans to-gether (�). The vertical lines indicate standard deviations (n = 3).

Eng. Life Sci. 2007, 7, No. 3, 235–240 Reduction of Selenium Oxyanions 239

treatment. However, the use of nitrogen gas was not practical.The polymeric gel could possibly maintain anaerobic condi-tions in the inner space of the gel without nitrogen gas becausethe molecular oxygen in the wastewater probably would beconsumed in the outer surface of the gel. In fact, when poly-meric beads [10, 11] or polymeric tubular gels [12, 13], inwhich nitrifier and denitrifier were co-immobilized, were ap-plied to remove nitrogen, nitrification, namely consumption ofmolecular oxygen, occurred in the outer layer of the beads ortubular gels and denitrification took place in the core of thebeads or tubular gels. The artificial wastewater containingselenate and nitrate will be treated under aerobic conditionsusing the immobilized mixture of cells of Pseudomonas sp.strain 4C-C and P. denitrificans in a further experiment.

Acknowledgements

The authors would like to thank Ms. Naho Kitazawa andMs. Yumi Kotake for their help with analysis.

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Figure 5. Photographs of vials with the gel-immobilized Pseudo-monas sp. strain 4C-C and P. denitrificans JCM-6892 cells togeth-er at (A) the start and (B) the end of the experiment.


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Reduction of Selenium Oxyanions in Wastewater Using Two Bacterial Strains