In this study, the hydrogenotrophic denitrifying bacterium Ochrobactrum anthropi was added in to the process of nitrate removal by starch-stabilized nanoscale zero valent iron (nZVI) to minimize undesirable ammonium. The ammonium control performance and cooperative mechanism of this combined process were investigated, and batch experiments were conducted to discuss the effects of starch-stabilized nZVI dose, biomass, and pH on nitrate reduction and ammonium control of this system. The combined system achieved satisfactory performance because the anaerobic iron corrosion process generates H2, which is used as an electron donor for the autohydrogenotrophic bacterium Ochrobactrum anthropi to achieve the autohydrogenotrophic denitrification process converting nitrate to N2. When starch-stabilized nZVI dose was increased from 0.5 to 2.0 g/L, nitrate reduction rate gradually increased, and ammonium yield also increased from 9.40 to 60.51 mg/L. Nitrate removal rate gradually decreased and ammonium yield decreased from 14.93 to 2.61 mg/L with initial OD600 increasing from 0.015 to 0.080. The abiotic Fe0 reduction process played a key role in nitrate removal in an acidic environment and generated large amounts of ammonium. Meanwhile, the nitrate removal rate decreased and ammonium yield also reduced in an alkaline environment.
Currently, many studies are being carried out involving nitrate removal from water because excessive nitrate present in water can create many serious environmental and human health problems (Foglar et al. 2005; Grommen et al. 2006; Ghafari et al. 2008; Chaplin et al. 2009; Zhang et al. 2014). Conventional nitrate removal methods contain biological denitrification, chemical reduction, reverse osmosis, and ion exchange (Hwang et al. 2011; Martin & Nerenberg 2012; Ensie & Samad 2014).
The nanoscale zero valent iron (nZVI) reduction process as a chemical reduction has attracted a lot of attention due to the fact that nZVI exhibits high and stable reactivity (Song & Carraway 2008; Li et al. 2012) for nitrate removal. Although nZVI is effective for nitrate reduction, the major product is the undesirable ammonium (Huang & Zhang 2004; Ryu et al. 2011). For this reason, many studies have been conducted to optimize the final products of nitrate removal by nZVI (Biswas & Bose 2005; Liou et al. 2005; An et al. 2014). An et al. (2014) introduced hydrogenotrophic bacteria to an nZVI nitrate reduction system, and reported that the hydrogenotrophic bacteria decreased the Fe0 reduction reaction and thus decreased the ammonium yield. Shin and coworkers (Shin & Cha 2008) investigated the microbial reduction of the nitrate process in the presence of nZVI, and proved that the combined nZVI–cell system was efficient for the degradation of nitrate. Biswas & Bose (2005) investigated the metallic iron-assisted abiotic nitrate reduction process to minimize the ammonium yield generated by the nZVI reduction reaction, and conducted long-time batch experiments to reveal the mechanisms for nZVI-assisted biological denitrification and less ammonium production.
The hydrogenotrophic denitrification approach refers to the process whereby hydrogenotrophic denitrifying bacteria utilize clean hydrogen as an electron donor, and inorganic carbon as energy to convert nitrate into nitrogen gas (Ghafari et al. 2008; Karanasios et al. 2010). This biological method is of increasing concern because, despite its high efficiency (Karanasios et al. 2010), the hydrogen supply is a tough problem as hydrogen is highly explosive. Hydrogen generated from anoxic corrosion of metallic iron may be a satisfactory solution. Sunger & Bose (2009) studied the hydrogenotrophic denitrification process which used hydrogen generated from anoxic corrosion of metallic iron, and discussed the nitrate removal and ammonium control performance of this combined system.
In consideration of the high toxicity of nZVI to microorganisms, there is a need to study the feasibility of using combined autohydrogenotrophic bacteria and starch-stabilized nZVI to treat the nitrate-contaminated water.
As reported, Ochrobactrum anthropi was one of the main hydrogenotrophic denitrifying bacteria and exhibited effective denitrification capacity in hydrogen-dependent denitrification reactors (Szekeres et al. 2002; Karanasios et al. 2010). In the present study, starch-stabilized nZVI was used to remove nitrate-polluted water, and the autohydrogenotrophic bacterium Ochrobactrum anthropi B2 was added to control ammonium generated by the abiotic part. The ammonium control performance and cooperative mechanism of this combined process were investigated. The aim of this study was to investigate the optimization of the combined system for nitrate removal, focusing on the effects of starch-stabilized nZVI dose, biomass, and pH on nitrate reduction and ammonium control process.
MATERIALS AND METHODS
Starch-stabilized nZVI preparation
Starch-stabilized nZVI was synthesized by adding 10.0 wt% starch on nZVI, and the nZVI particles were synthesized by the reduction reaction of FeCl3 · 6H2O solution and KBH4 in polyvinylpyrrolidone (surfactant) solution. Ferric chloride hydrate (5.56 g) was dissolved into 70 mL deoxygenated de-ionized (DI) water and 30 mL ethanol mixed solution (solution A 100 mL), and 0.648 g potassium borohydride was dissolved into 100 mL deoxygenated DI water in another beaker (solution B 100 mL). After 0.5 g polyvinylpyrrolidone was added into solution A, solution B was added dropwise to solution A under continuous stirring conditions (using a mechanical stirrer). The reaction lasted 30 min under vigorously mixed conditions. After that, 10.0 wt% starch was added to stabilize the nZVI in solution. The resulting nZVI particles were then washed with DI water and anhydrous ethanol several times, and then dried under a vacuum environment, which is important for the application of the combined system.
Ochrobactrum anthropi cultivation
The autohydrogenotrophic denitrifying bacterium Ochrobactrum anthropi B2 (GenBank: KJ957921) was isolated from our laboratory. Ochrobactrum anthropi B2 was isolated from a biofilm reactor, which was used to remove nitrate, and the bacteria were obtained through multiple separation and purification processes. The bacteria possess the ability of autohydrogenotrophic denitrification. The compositions of the bacteria culture media were (mg L−1): ZnCl2 0.68, CoCl2 · 6H2O 0.19, MnSO4 · 7H2O 0.12, NiCl2 · 6H2O 0.27, CuCl2 · 2H2O 0.32, Na2MoO4 · 2H2O 0.36, MgCl2 · 6H2O 0.28, H3BO3 0.35, NaHCO3 (inorganic carbon source) 550, KH2PO4 350, NaNO3 (nitrogen source) 400, pH 7.0–7.5. The bacteria were cultured in a biochemical incubator in order to maintain reactivity.
In order to investigate the ammonium control performance and cooperative mechanism of this combined system, batch experiments were carried out in Erlenmeyer flasks in an incubator shaker. Twenty millilitres of bacteria solution (OD600 = 0.015), 1.0 g/L starch-stabilized nZVI, 20 mL nitrate solution (initial nitrate concentration = 100 mg/L) and 160 mL distilled water were added into the Erlenmeyer flask. The pH of the mixed liquor was adjusted to 7.0. Then the Erlenmeyer flask was put into the incubator shaker at 25 °C and 200 rpm.
To investigate the effects of starch-stabilized nZVI dose on the combined system, the starch-stabilized nZVI doses were adjusted to 0.5, 1.0, 1.5, and 2.0 g/L at OD600 0.015 and pH 7.0 condition for 100 mg/L nitrate removal.
The biomass was adjusted to OD600 0.015, 0.030, 0.050 and 0.080 at 1.0 g/L starch-stabilized nZVI and 7.0 pH condition to evaluate the effects of biomass on the combined system.
In order to evaluate the influences of pH on the combined system, the pH of the mixed solutions was adjusted to 5.0, 6.0, 7.0, 8.0, and 9.0 at OD600 0.015 and starch-stabilized nZVI 1.0 g/L condition for 100 mg/L nitrate removal.
Total nitrogen, ammonia nitrogen , nitrate , and nitrite were determined according to Standard Methods for the Examination of Water and Wastewater (APHA 2005). The pH was determined using a pH meter (PHS-3C, Kexiao Instrument, China). Biomass was determined by the turbidity (OD600) by means of a spectrophotometer (UV-2802H, Shanghai Optical Company, China). The batch experiments were conducted in duplicate in order to control quality.
RESULTS AND DISCUSSION
Ammonium control performance and cooperative mechanism on nitrate removal
OD600 increased from 0.015 to 0.190 in the combined system, meaning that autohydrogenotrophic bacteria grew well for nitrate degradation, which also proved that Ochrobactrum anthropi could harmonically cooperate with starch-stabilized nZVI corrosion for nitrate degradation.
Effects of starch-stabilized nZVI dose on nitrate removal
After reaction, OD600 0.015 changed to 0.213, 0.190, 0.117, and 0.046 under 0.5, 1.0, 1.5, and 2.0 g/L starch-stabilized nZVI conditions, respectively, which also indicated that the biotic reactivity was gradually weakening with increasing biomass. On the other hand, the biotic reaction was slower than the abiotic reaction, so that the nitrate could be rapidly reduced and thus large amounts of ammonium would be generated. Therefore, increasing starch-stabilized nZVI dose could increase nitrate removal efficiency and increase ammonium yield.
Effects of biomass on nitrate removal
Effects of pH on nitrate removal
The autohydrogenotrophic bacterium Ochrobactrum anthropi exhibited excellent performance on the control of the generation of ammonium, which was the by-product of nitrate reduction by starch-stabilized nZVI. XRD demonstrated the corrosion products of nZVI may be non-crystalline. Increasing starch-stabilized nZVI dose could increase nitrate removal efficiency and increase ammonium yield. Higher biomass lowers the reduction of nitrate by nZVI. When pH increased from 5.0 to 9.0, nitrate removal rate gradually decreased and ammonium yield decreased from 49.32 to 7.24 mg/L. The autohydrogenotrophic Ochrobactrum anthropi was proved to exhibit effective capacity for ammonium control under appropriate conditions in the process of nitrate removal by starch-stabilized nZVI. These outcomes demonstrate the feasibility of using combined autohydrogenotrophic bacteria and starch-stabilized nZVI to treat the water contaminated by nitrate, and offer a guide for the application of the combined system for nitrate removal. Further investigations are needed to promote the efficiency of nitrate removal.
This work was financially supported by the National Natural Science Foundation of China (NSFC) (No. 51378400), the National Science and Technology Pillar Program during the Twelfth Five-year Plan Period (2014BAL04B04, 2015BAL01B02).