Environ Sci Pollut Res DOI 10.1007/s11356-015-5072-8
RESEARCH ARTICLE
Perchlorate reduction from a highly concentrated aqueous solution by bacterium Rhodococcus sp. YSPW03 Sang-Hoon Lee 1 & Jae-Hoon Hwang 2 & Akhil N. Kabra 3 & Reda A. I. Abou-Shanab 4 & Mayur B. Kurade 3 & Booki Min 5 & Byong-Hun Jeon 3
Received: 21 April 2015 / Accepted: 13 July 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract A novel isolated bacterium Rhodococcus sp. YSPW03 was able to reduce high concentrations (up to 700 mg L−1) of perchlorate using acetate as electron donor. Perchlorate reduction rate increased from 2.90 to 11.23 mg L−1 h−1 with increasing initial acetate concentration from 100 to 2000 mg L−1, leading to complete removal of perchlorate (100 mg L−1) within 9 h. The bacterium also promoted complete reduction of high perchlorate concentrations (500 and 700 mg L−1) at 2000 mg L−1 of acetate within 48 and 96 h, respectively. Under semi-continuous reactor operation, efficient reduction on varied perchlorate concentrations (80– 700 mg L−1) was performed by the bacterium in presence of acetate (600–6000 mg L−1) over 140 days. The highest perchlorate reduction rate of 280 mg L−1 day−1 was observed with an initial perchlorate concentration of 570 mg L−1 at day 34. Dissolved chloride ions of 1000 mg L−1 in the semi-
Responsible editor: Bingcai Pan * Byong-Hun Jeon
[email protected] 1
Department of Environmental Engineering, Yonsei University, Gangwon-do 220-710, Republic of Korea
2
Swette Center for Environmental Biotechnology, The Biodesign Institute at Arizona State University, P.O. Box 875701, Tempe, AZ 85287-5701, USA
3
Department of Natural Resources and Environmental Engineering, Hanyang University, Seoul 133-791, Republic of Korea
4
Department of Environmental Biotechnology, City for Scientific Research and Technology Applications, New Borg El Arab, Alexandria 21934, Egypt
5
Department of Environmental Science and Engineering, Kyung Hee University, 1 Seocheon-dong, Yongin-si, Gyeonggi-do 446-701, Republic of Korea
continuous reactor (SCR) completely inhibited the biological perchlorate reduction. The findings of this study will help improve the perchlorate bioreactor design and determine the optimal conditions to maximize the perchlorate reduction efficiency. Keywords Rhodococcus sp. YSPW03 . Semi-continuous reactor . Perchlorate . Acetate . Chloride . Bioreduction
Introduction Perchlorate (ClO4−) is a substantial chemical, which is extensively used as an oxidizer in rocket propellants, fireworks, matches, and highway safety flares (US EPA 2005). The accidental release and improper disposal of materials containing perchlorate salts have led to its detection in surface water and groundwater at concentrations of 20 mg L−1 (Stroo and Ward 2009). Water sources in vicinity of the rocket propellant manufacturing industries and metal refineries have been reported to contain perchlorate at concentrations as high as 3000 mg L−1 (Hatzinger et al. 2002). Exposure to perchlorate may cause hypothyroidism by disrupting the uptake of iodide by thyroid gland, making its removal of great concern (Logan 2001). The removal of perchlorate from aqueous phase has been challenging as it is non-volatile, non-reactive due to high energy of activation, and highly soluble in water (200 g L−1 for NH4ClO4) and exhibits low adsorption on mineral surface and sediment (Urbansky 2002). Various physico-chemical techniques such as ion exchange, membrane filtration, electrochemical reduction, and adsorption have been evaluated for the removal of perchlorate (Gu et al. 2007; Kim and Logan 2001; Kumar et al. 2010; Lakshmi and Vasudevan 2013; Roquebert et al. 2000; Rusanova et al. 2006); however, such methods have
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disadvantages that include high operational and maintenance costs, secondary pollution, and complicated procedure involved in the treatment. Biological treatment of perchlorate in an engineered system can serve as a cost-effective method for complete removal of perchlorate (Attaway and Smith 1993; Hatzinger 2005; Kim and Logan 2001). Perchloratereducing bacteria (PRB) have the ability to exploit perchlorate as an electron acceptor (Coates et al. 1999; Wu et al. 2001) and reduce it to chlorite, and finally to chloride (Kim and Logan 2001; Logan 1998; Oosterkamp et al. 2011). Biological treatment of wastewater contaminated with high perchlorate concentration (120 mg L−1) has been reported (Patel et al. 2008). Extensive research on microbial reduction of high perchlorate concentration is needed to engineer the bioreactor for field application. The objective of this study was to investigate the potential of a novel bacterium isolated from sewage sludge to reduce high concentration of aqueous perchlorate. The batch and semi-continuous reactor (SCR) experiments evaluated the bacterial growth and perchlorate reduction rate at different acetate and perchlorate concentrations. The influence of oxidation-reduction potential, chloride concentration, and pH on the perchlorate reduction in SCR was also studied. Such evaluation is essential to understand the relationship between perchlorate reduction and biomass behavior based on electron donor, pH, and kinetics, for construction of a bioreactor to treat wastewaters contaminated with high perchlorate concentrations.
Materials and methods Enrichment and isolation of perchlorate reducing bacterium Perchlorate-reducing bacterium (PRB) was obtained by enrichment culture technique using a modified Dechlorosoma medium (ATCC #2361 Broth) supplemented with ammonium perchlorate (Hanwha Chemical, South Korea) as electron acceptor and sodium acetate (Sigma-Aldrich, USA) as electron donor. The media contained the following chemicals (g L−1): K2HPO4·3H2O 1.55, NaH2PO4·H2O 0.85, NH4H2PO4 0.50, EDTA 0.005, MgSO4·7H2O 0.03, NaCl 0.01, FeSO4·7H2O 0.001, Co(NO3)2·6H2O 0.001, CaCl2 (anhydrous) 0.001, ZnSO 4 ·7H 2 O 0.001, CuSO 4 ·5H 2 O 0.0001, AlK(SO 4 ) 2 (anhydrous) 0.0001, H 3 BO 3 0.0001, Na 2 MoO 4 ·2H 2 O 0.0001, Na 2 SeO 3 (anhydrous) 0.00001, Na 2 WO 4 ·2H 2 O 0.0001, and NiCl2·6H2O 0.0002. All the chemicals purchased were of analytical grade with highest purity available. Medium pH was adjusted to 7.0 using 0.1 M HCl or NaOH and was subjected to autoclave. The anaerobic-digested sludge obtained from municipal wastewater treatment plant (Wonju Water Supply and Drainage Center, South Korea) was used as a
source of bacterial inoculum. Perchlorate-reducing culture was enriched in a 2 L-fermentation reactor with 0.7 L of gravity-settled anaerobic sludge as a seed culture and 0.7 L of Dechlorosoma medium containing 1000 mg perchlorate L−1 and 2000 mg acetate L−1. The seed reactor was installed in a temperature-controlled chamber maintained at 35±5 °C and stirred continuously at 120 rpm under anaerobic conditions. For enrichment of PRB culture, 150 mL of medium was replaced with fresh medium every 3 days over 30 days of incubation under anaerobic conditions. PRB was isolated and enumerated using the standard agar plate dilution (APD) method. For purification of PRB, 100 μL of the diluted enrichment suspension was spread onto Dechlorosoma agar plates amended with ammonium perchlorate as electron acceptor and sodium acetate as electron donor at 30 °C for 2 weeks under anaerobic conditions to obtain single colonies. DNA extraction, PCR amplification, DNA sequencing, and phylogenetic analysis Genomic DNA of the isolated bacterium was extracted using the genomic DNA extraction kit (SolGent, Daejeon, South Korea) according to the manufacturer’s instructions. The extracted DNA was used as a template for PCR amplification of the 16S rRNA gene. The universal bacterial primers 27F (5′AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′) were used to amplify almost complete 16S rRNA gene (Marchesi et al. 1998). The PCR amplification was performed in a 25-μL total reaction volume containing 10–50 ng of the template DNA, 0.4 μM of each primer, 0.75 U of EF-Taq DNA polymerase, 0.2 mM of each dNTP, and 1×EF-Taq reaction buffer (SolGent, Daejeon, South Korea). The reaction was performed in a GeneAmp PCR system Model 9700 (Applied Biosystems, Inc., USA). The thermo-cycling protocol included the initial denaturation step at 95 °C for 15 min followed by 30 cycles at 95 °C for 20 s, 50 °C for 40 s, and 72 °C for 1.5 min with a final extension step at 72 °C for 5 min. The PCR product was separated by gel electrophoresis, visualized by a UV illuminator and purified using a SolGent PCR purification kit (SolGent, Daejeon, South Korea) according to the manufacturer’s instructions. The amplified 16S rRNA gene was sequenced using an ABI Big Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Inc., USA) and an ABI 3730XL DNA analyzer (Applied Biosystems, Foster City, Cal., USA). For identification of the isolated bacterium, the partial 16S rRNA gene sequence was compared with full sequences available in the GenBank database of National Center for Biotechnology Information (NCBI) using Basic Local Alignment Search Tool (BLAST) (Maidak et al. 2001). Phylogenetic analysis of the sequence data was also performed using the software package MEGA5 (version 5.05; http:// www.megasoftware.net/) after multiple alignments of the
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data using CLUSTAL X (version 1.83). A distance matrix method (with distance options according to the Jukes & Cantor distance model) was employed using the clustering obtained with the neighbor joining method (Jukes and Cantor 1969; Saitou and Nei 1987). Bootstrap values were calculated on the basis of 1000 replications (Felsenstein 1985). Perchlorate reduction in batch experiment Batch experiment was performed in 150-mL serum bottles using 100 mL of sterile Dechlorosoma broth medium supplemented with perchlorate and acetate. Acetate was used as an electron donor and a carbon source for microbial perchlorate reduction in all the experiments. An acetate and perchlorate theoretical ratio of 1.217 mg acetate mg perchlroate−1was calculated using the thermodynamic approach by Rittmann and McCarty (2001). To study the effect of initial acetate concentrations (100, 500, and 2000 mg L−1), experiment was performed with aqueous solution amended with 100 mg L−1 of perchlorate, giving an acetate and perchlorate ratio of 1, 5, and 20, respectively. In addition, the PRB was also subjected to high concentration of 500 and 700 mg L−1 perchlorate at 2000 mg L−1 of acetate. The serum bottles were inoculated with 50 mL of PRB (YSPW03) at with an optical density 0.16 (OD600nm) and were capped with thick butyl rubber stoppers using an aluminum crimp seal. Bottles were successively pressurized with pure nitrogen gas for 15 min to create anaerobic conditions and were incubated in a shaking incubator (Seyoung Sci., South Korea) at 200 rpm and 25±1 °C for 5 days. All experiments were performed in triplicates, and the average values were reported with standard error of mean (±SEM). The perchlorate reduction rates corresponded well to the theoretical values calculated by the Monod Eq. (1), which can be expressed as (Rittmann and McCarty 2001): Substrate mass balance −
ds q ⋅S ¼ max X dt Ks þ S
ð1Þ
where S is the substrate concentration (mg L−1), qmax is the maximum specific substrate reduction rate (mgsubstrate mg-dry cell weight(DCW)−1 h−1), Ks is the half saturation constant (mg-substrate L−1), and X is the microbial concentration (mg-DCW L−1). Perchlorate reduction in semi-continuous reactor (SCR) Perchlorate reduction experiment was carried out using a SCR (2 L) containing 1.5 L of bacterial culture (0.028 OD600nm) in Dechlorosoma broth medium supplemented with 80 mg L−1 perchlorate and 670 mg L−1 acetate. The culture medium in
SCR was stirred with a magnetic stirrer at 50 rpm, and the perchlorate reduction was examined at room temperature (25 ±2 °C). When perchlorate concentration in SCR was decreased to 0 mV), indicating a strong influence of ORP on perchlorate reduction (Fig. 3). Shrout and Parkin (2006) reported that high concentration of electron donor (acetate) was required under high ORP (>0 mV) conditions for perchlorate reduction. The acetate concentration in reactor was thus increased up to 600 mg L−1 by replacing 10 % volume of the reactor medium with fresh medium at day 13, which resulted in three complete reduction cycles of perchlorate (80 mg L−1) in phase II (13th–19th day). The medium pH during phase II ranged between 6.9 and 7.1. Previous studies have reported a pH range of 6.0–9.0 to be appropriate for perchlorate reduction as it supports the PRB growth (Bruce et al. 1999; Nor et al. 2011; Wang et al. 2008). The ORP was dropped to −42 mV at day 15 after first perchlorate reduction cycle, but it again increased to 37 mV due to intermediate injection of perchlorate. The bacterium performed six reduction cycles of perchlorate ranging from 200 to 600 mg L−1 at an initial acetate concentration of 6000 mg L−1 in phase III (20th–52nd day). The highest perchlorate reduction rate of 280 mg L−1 day−1 was observed with chloride concentration of 350 mg L−1 at day 34. The bacterial growth (OD 600 nm) increased from 0.27 to 0.45 with the increase of medium pH from 7.1 to 7.4 throughout phase III. The OD and pH values were comparable with values reported in previous studies for perchlorate reduction (Nor et al. 2011; Shrout and Parkin 2006; Wang et al. 2008). During phase IV (53rd–103rd day), perchlorate concentration was leveled to 700 mg L−1 at day 54 and acetate concentration was adjusted to 6200 mg L−1 at day 63 (upon the depletion of acetate) by replacing 10 % volume of the reactor medium. Initially, in phase IV, low perchlorate reduction was observed which coincides with a significantly decreased perchlorate reduction rate. A lower perchlorate reduction rate was observed even after addition of fresh acetate on day 63. High chloride concentration (800–1000 mg L−1) was observed in phase IV compared to phase III (200–800 mg L−1). An excess
amount of chloride inhibits the microbial growth, leading to a decrease in perchlorate reduction (Okeke et al. 2002). The effect of chloride concentration on perchlorate reduction rate was further investigated in phases V (103rd–125th days) and VI (126th–143rd days). At initiation of phase V, 30 % working volume of SCR was replaced with fresh medium, which decreased the chloride concentration from 1000 to 700 mg L−1. The OD of the reactor medium decreased by 0.7-fold due to replacement of the SCR medium. Perchlorate reduction rate was still lower than 10 mg L−1 day−1 in phase V, compared to reduction rate at high chloride concentration (>800 mg L −1 ) in phase IV. This might be due to prolonged exposure of the bacterium to higher chloride toxicity prior to phase V. Niu et al. (2013) reported that the bacterial activity which is inhibited after prolonged exposure to toxic substance can be regained by decreasing the concentration of the toxic substance to a level where the bacterium can demonstrate optimum activity. Thus, 50 % working volume of SCR was replaced with fresh medium at day 126 (initiation of phase VI) to decrease the chloride concentration
