Article pubs.acs.org/IECR
Effect of Mixed Electron Donors on Autotrophic Denitrification by Pseudomonas sp. SZF15 Junfeng Su,*,†,‡ Xianxin Luo,† Tinglin Huang,† Fang Ma,‡ Shengchen Zheng,† and Sicheng Shao† †
School of Environmental and Municipal Engineering, Xi’an University of Architecture and Technology, Xi’an 710055, China State Key Laboratory of Urban Water Resource and Environment, School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China
‡
ABSTRACT: In this study, mixed electron donors of Mn(II) and Fe(II) were tested for their effects on autotrophic denitrification. The denitrification performance based on various Mn(II)/Fe(II) molar ratios was assessed via batch experiments conducted under different conditions of temperature, pH, and electron donor/electron acceptor molar ratios. Complete nitrate removal was found at high Mn(II)/Fe(II) molar ratios, 30 °C, pH 7.0, and an electron donor/electron acceptor molar ratio of 3.00. Furthermore, we assessed the expression of nirS through amplification approaches and determined N2 levels by gas chromatography. Our results demonstrated that mixed electron donors are efficient for autotrophic denitrification, and strain SZF15 is a suitable candidate for the simultaneous removal of nitrogen, Fe(II), and Mn(II) in groundwater treatment. The present work will benefit the development of autotrophic denitrification technology.
1. INTRODUCTION Excess levels of nitrate in water have become a serious concern worldwide and have exceeded the maximum allowable limits (10 mg/L) for drinking water.1 About 25% of the groundwater has exceed the maximum allowable limits concentration of nitrate in the USA.2 This is mainly due to improper treatment of wastewater and the extensive use of nitrogen fertilizers.3 Moreover, nitrate could reverted to toxic nitrite in human blood easily and result in methemoglobinemia.4 Biological denitrification can be divided into two groups: heterotrophic denitrification or autotrophic denitrification. Although heterotrophic denitrification has a high treatment capacity and high denitrification rate, some disadvantages are inevitable, such as organic remains in the effluent and nitrite accumulation when organic material is stoichiometrically insufficient.2,5 Therefore, autotrophic denitrification has been intensively studied. The advantages of autotrophic over heterotrophic denitrification include no secondary pollution, lower biomass accumulation reducing the occurrence of aquifer clogging, and being based on an inorganic carbon source and electron donors (hydrogen gas, sulfur, etc.). Alternatively, sulfur-based autotrophic denitrification has been studied in a wide variety. Reported investigation has verified that it was possible to carry out autotrophic denitrification with a high denitrification efficiency by using sulfide as an electron donor.6 © XXXX American Chemical Society
However, the main disadvantages for sulfur-based autotrophic denitrification are the following: (1) the efficiency of the process was influenced by the pH value;6 (2) the S/N ratio played a significant role in the autotrophic denitrification,19,7 and elemental sulfur accumulated when the S/N ratio was higher;7 (3) when the influent sulfide concentration was decreased to 100 mg of TDS-S/L (influent S/N: 1.4 g-S/g-N), the N2O gas emissions sharply increased to 3707.4 ppmv;8 (4) the formation of sulfate (SO42−) and H+ in the effluent was observed; limestone is often used as a low-cost alkalinity source, but it can increase the hardness of the effluent due to the release of Ca2+ during limestone dissolution.9 Meanwhile, hydrogen-based autotrophic denitrification was limited by poor H2 being poorly soluble in water, which creates waste and disconcerting security issues for an additional hydrogen source.10 As compared with sulfur- and hydrogen-based autotrophic denitrification, that based on Mn(II) and Fe(II) as inorganic electron donors is an alternative method that could simultaneously remove nitrogen, Fe(II), and Mn(II) in groundwater treatment. For example: the effluent would not Received: Revised: Accepted: Published: A
November 27, 2016 January 18, 2017 January 31, 2017 January 31, 2017 DOI: 10.1021/acs.iecr.6b04591 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
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Industrial & Engineering Chemistry Research be polluted by the byproduct; particularly, the greenhouse gas emissions in the study (5.27 ppmv) were much lower than those associated with other denitrification processes, which features an average N2O gas emission percentage of 1762.5 ppmv (heterotrophic)11 or 14.1 ppmv (sulfur-based autotrophic denitrification).8 Meanwhile, because iron is one of the most abundant metals in the earth’s crust and usually occurs along with manganese, also in the groundwater,12 autotrophic denitrification based on Mn(II) and Fe(II) as inorganic electron donors is an alternative method to treat groundwater which has high nitrogen content and low carbon content. In this study, mixed electron donors of Mn(II) and Fe(II) were tested for their effects on autotrophic denitrification. Different temperatures, pH, electron donor/electron acceptor molar ratios, and Mn(II)/Fe(II) molar ratios were employed to assess the denitrification performance via batch experiments, respectively. Furthermore, gas emissions and denitrifying enzymes genes of strain SZF15 were proposed on anaerobic autotrophic denitrification.
Figure 1. Schematic representation of the gas emission and enrichment reactors: (1) IM flow port, (2) gas equalizing port, (3) gas transfer tube, (4) water outlet port, (5) valve, (6) gas emission apparatus, (7) gas enrichment apparatus.
2. MATERIALS AND METHODS 2.1. Cultivation of Microorganisms and Batch Assays. Strain SZF15 was isolated from the sediment of Tang Yu reservoir (34.007°−34.015° N, 109.229°−109.2397640000° E), located in Lantian County, Xi’an City, Shaanxi Province, China. The strain was cultivated in 250 mL bottles at 30 °C with blue butyl stopper, where Vbacteria/Vmedium was chosen as 10%; bacteria was ready to harvest after 7 days. Then, 10 mL of bacteria with OD600 of 0.280 and 10 mL of inorganic electron donors with a concentration of 100 mg/L were added into the inorganic basal medium (IM) to carry out denitrification under anaerobic conditions. The components of IM and trace element solution used for strain SZF15 cultivation were as described in Su et al.13 The inorganic electron donors Mn(II) and Fe(II) were added by preparing an Mn(II) and Fe(II) stock solution to achieve the desired concentration with ultrapure water (=18.2 MΩ cm). The headspace of each bottle was purged with argon gas at the beginning of the experiment. The concentration of dissolved oxygen in the experiments was below 0.5 mg/L. All experiments were performed at least in triplicate. 2.2. Denitrification Gene Amplification. The primers of nirS3F/nirS5R were used to confirm the presence of the nirS gene in strain SZF15; the procedure was conducted as described in Huang et al.14 2.3. Gas Detection in Sealed Serum Bottles. A gas detection experiment was made up of a gas emission apparatus and gas enrichment apparatus. Gas emission was conducted in 1000 mL bottles with 900 mL of IM and 100 mL of bacterial suspension at 30 °C, and gas enrichment was conducted in 200 mL bottles which were filled with tap water. First, the IM was introduced to the gas emission apparatus by a pump, and then the gas was collect by a displacement method in the gas enrichment apparatus. Before adding bacteria, the IM was purged with an argon stream for 20 min. The general schematic apparatus is shown in Figure 1. After 7 days, 500 μL of gas samples were taken from the headspace of the gas enrichment apparatus by a gastight syringe to analyze H2S, CO2, N2, and H2 by GC (Agilent 6890, Japan) and N2O by GC (Clarus 600, USA), respectively. 2.4. Analytical Methods and Statistical Analysis. One milliliter solution samples were withdrawn from the medium and filtered through a 0.45 μm membrane to measure the
concentrations of nitrite, nitrate, Mn(II), and Fe(II), by using N-(1-naphthalene)-diaminoethane spectrophotometry, ultraviolet spectrophotometric methods, potassium periodate spectrophotometric method, and phenanthroline spectrophotometric method (DR5000, HACH, USA), respectively.The pH value was measured with a pH meter (MM110, HACH, American). During the growth of strain SZF15 experiment, the cultures were sampled periodically at 12 h time intervals to determine the concentration of nitrate and nitrite, optical density (OD600), the values of pH, and the levels of Mn(II) and Fe(II).
3. RESULTS AND DISCUSSION 3.1. Effect of Mn(II)/Fe(II) Molar Ratio on Autotrophic Denitrification. Batch experiments were carried out to investigate the characteristics of nitrate reduction using Mn(II) and Fe(II) as electron donors. Our initial experiments showed that the highest nitrate removal rate (0.151 mg-N·L−1·h−1) was observed at an Mn(II)/Fe(II) molar ratio of 3:7 (Figure 2). When the Mn(II):Fe(II) molar ratios were 1:9 and 9:1, nitrate removal rates were 0.088 and 0.032 mg-N·L−1·h−1, respectively. This suggested that the high nitrate reduction rate observed at the Mn(II)/Fe(II) molar ratio of 3:7 was due to the relatively high Fe(II) concentration in the IM, because Fe(II) can more easily lose an electron compared with Mn(II) and thus can be readily oxidized during the denitrification process. It is important to point out, however, that nitrate removal was complete at Mn(II)/Fe(II) (CMn:CFe) molar ratios of 9:1, 8:2, and 7:3. In contrast, the extent of nitrate reduction was only 86.59% and 70.27% when the Mn(II)/Fe(II) molar ratios were 3:7 and 1:9, respectively. Therefore, nitrate reduction at high Mn(II)/Fe(II) molar ratios was higher than that at low Mn(II)/Fe(II) molar ratios, while we observed a lag in the ratio of nitrate reduction during the initial experiment. A number of explanations can account for the fact that nitrate reduction at low Mn(II)/Fe(II) molar ratios was lower. First, the hydrolysis of Fe(II) and Mn(II) is predicted to acidify the reactions by producing hydrogen ions, according to eqs 1 and 2. This effect could inhibit denitrification, and Fe(II) could be hydrolyzed more easily than Mn(II) throughout the experiment. These events may have resulted in a lower pH that was not conducive to denitrification. Second, Hedrich et al.15 suggested that circumneutral environments increase the ability of the ferrous B
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Figure 2. Removal ratio changes of (A) the nitrate (B) and Fe(II), Mn(II) in different Mn(II)/Fe(II) molar ratios.
ferrous iron content (Mn(II)/Fe(II) molar ratio of 9:1). This result suggests that maximum Mn(II) utilization is achieved by strain SZF15 only when there is a shortage of Fe(II). 3.3. Effect of pH on Autotrophic Denitrification. In our experiments, the initial pH was set to 6.0, 7.0, 8.0, and 9.0. To compare the effect of Mn(II) and Fe(II) on denitrification, three sets of batch tests were carried out, as above. We found that the efficiency of nitrate reduction increased from pH 6.0 to 7.0, and then dropped sharply at pH 8.0 and 9.0 (Figure 4). These observations indicate that a neutral pH is necessary for autotrophic denitrification by mixed electron donors. The highest denitrification rate was approximately 0.150 mg-N·L−1· h−1 at pH 7.0 when the Mn(II)/Fe(II) molar ratio was 7:3; removal ratio of Fe(II) and Mn(II) were 93.17% and 35.64%, respectively. Of note, the denitrification rate was approximately 0.092 mg-N·L−1·h−1 at pH 6, thus pointing out a few significant differences in denitrification rates when the pH increases from 6.0 to 7.0. Increasing pH to 8.0 would decrease denitrification efficiency to 68.39%, also, denitrification efficiency would further decrease to 39.56% when the pH was set to 9.0. Zhao et al.10 suggested that the optimum pH for autotrophic denitrification is between 7.0 and 7.5; meanwhile, nitrite-N kept below 1.00 mg/L suggested that the inhibition caused by nitrite-N could be neglected. 3.4. Effect of the Electron Donor/Electron Acceptor Molar Ratio on Autotrophic Denitrification. Additional studies evaluated the effect of different electron donor/electron acceptor molar ratios on nitrate removal (Figure 5). We tested three electron donor/electron acceptor molar ratios (i.e., 0.5, 1.54, and 3.0) to study the denitrification efficiency. The nitrate removal ratio was 38.64% when the electron donor/electron acceptor molar ratio was 0.50 (Figure 5); this was likely due to the low level of electron donor provided for denitrification. In contrast, complete efficiency of nitrate removal was observed when the electron donor/electron acceptor molar ratios were 1.54 and 3.00. A higher denitrification rate was achieved when the electron donor/electron acceptor molar ratio was 3.00; the highest denitrification rate was 0.253 mg-N·L−1·h−1 at a Mn(II)/Fe(II) molar ratio of 7:3. These results were similar to those reported by Chung et al.19 who found that the nitrite removal ratio was 43.18% at an S/N ratio of 1.5 owing to the low level of electron donors. Complete nitrite removal was observed when the S/N ratios were 2.5 or 3.5. While the oxidation ratio of Fe(II) was approximately 100%, the highest
iron to act as an electron donor and yield energy because the redox potential of the ferrous/ferric couple(s) at circumneutral (and higher) pH is much lower (about +200 mV) than that observed in acidic liquors (about +770 mV). Fe2 + + 2H 2O = Fe(OH)2 + 2H+
(1)
Mn 2 + + 2H 2O = Mn(OH)2 + 2H+
(2)
3.2. Effect of Temperature on Autotrophic Denitrification. Temperature is one of the external factors that influences the growth and metabolic activity of microorganisms. In this study, we investigated the impact of various initial temperatures (20 °C, 25 °C, 30 °C, and 35 °C) on autotrophic denitrification. In addition, we determined whether varying the Mn(II)/Fe(II) molar ratio (9:1, 7:3, and 5:5) affected the efficiency of nitrate removal (Figure 3). When the Mn(II)/ Fe(II) molar ratios were 7:3 and 5:5, complete nitrate reduction was achieved at 30 °C with a denitrification rate of 0.191 mg-N·L−1·h−1. In contrast, the denitrification rate of strain SZF15 was lower (0.116 mg-N·L−1·h−1 after 6 days) when the Mn(II)/Fe(II) molar ratio was 9:1. We also observed that the denitrification rate was not significantly affected when the temperature was decreased from 30 to 25 °C. This observation is in accordance with our previous studies suggesting that the optimal denitrification temperature was 25−30 °C,16 and is in good agreement with studies reported by Park et al.17 Since the temperature was rarely higher than 30 °C in groundwater environments, subsequent experiments were conducted at 25 °C. Our studies also showed that at 35 °C the maximum nitrate-N removal efficiency was approximately 80%. At a much lower temperature (20 °C), the denitrification efficiency was only 43.47%. This may be because lower temperatures can significantly decrease membrane fluidity and limit the ability of the substrates to enter the cell.18 These results suggest that moderate temperatures are advantageous for denitrification by strain SZF15. Although nitrite might impose inhibition, nitrite-N was accumulated to about 1.00 mg/ L in the test and bacteria were able to reduce the nitrite completely. In addition to the above observations, we found that Fe(II) concentrations decreased dramatically during the test period and were eventually completely depleted. Strain SZF15 utilized Mn(II) less readily than Fe(II). Interestingly, the highest Mn(II) removal efficiency (41.59%) was achieved under low C
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Figure 3. Profiles of nitrate concentration and Fe(II), Mn(II) removal ratios in different temperature periods. (A) Mn(II)/Fe(II) = 9:1, (B) Mn(II)/ Fe(II) = 7:3, (C) Mn(II)/Fe(II) = 5:5.
3.5. Strain SZF15 Utilizes Nitrate for Growth under Anaerobic Conditions. Growth studies under anaerobic conditions at a molar ratio of Mn(II)/Fe(II) of 7:3 showed that strain SZF15 grew by using nitrate as the terminal electron acceptor and sodium bicarbonate as the substrate (Figure 6). We observed a lag of 16 h during which the denitrification rate was approximately 0.116 mg-N·L−1·h−1. This rate was lower than that observed during the later phase (0.304 mg-N·L−1· h−1). In addition, during the initial 32 h, the oxidation rates of
Mn(II) oxidation ratio of 55.3% was achieved when Fe(II) was almost completely removed at the electron donor/electron acceptor molar ratio of 0.50 (Mn(II)/Fe(II) = 1:9); this occurred because a low level of electron donor was provided. This observation indicates that the bacteria began to utilize Mn(II) once Fe(II) was completely consumed, a result similar to that reported in section 3.5. In addition, nitrite-N production (below 1.00 mg/L) did not impose any inhibitory effect for denitrification. D
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Figure 4. Profiles of nitrate concentration and Fe(II), Mn(II) removal ratios in different pH periods. (A) Mn(II)/Fe(II) = 9:1, (B) Mn(II)/Fe(II) = 7:3, (C) Mn(II)/Fe(II) = 5:5.
Fe(II) and Mn(II) were 0.943 mg-Fe(II)·L−1·h−1 and 0.094 mg-Mn(II)·L−1·h−1, respectively. This indicated that strain SZF15 first utilized Fe(II) as electron donor because Fe(II) was more readily oxidized than Mn(II). Subsequently, between 32 and 72 h, the oxidation rates of Fe(II) and Mn(II) were 0.092 mg-Fe(II)·L−1·h−1 and 0.498 mg-Mn(II)·L−1·h−1, respectively. This indicated that Mn(II) was oxidized by the bacteria when there was a shortage of Fe(II). Nitrite accumulation (0.902 mg· L−1) was observed as soon as nitrate was completely utilized (72 h), in agreement with Sun et al.,20 and did not impose any
inhibitory effect. As shown in Figure 6b, the bacteria managed to decrease nitrite dramatically (from 0.902 mg·L−1 to 0.042 mg·L−1) suggesting that nitrite might be further converted to nitrogen gas. In this regard, we verified expression of nirS, which encodes nitrite reductase (section 3.6). We also found that strain SZF15 reached stationary growth after 64 h with OD600 of 0.224 and that the pH increased gradually from 6.81 to 7.45, likely owing to bacterial adaptation to the new environment and alkali production during denitrification. These responses were similar to those of our previous experiments.21 E
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Figure 5. Effect of electron donor/electron acceptor ratio on nitrate removal by SZF15. (A) Mn(II)/Fe(II) = 9:1, (B) Mn(II)/Fe(II) = 7:3, (C) Mn(II)/Fe(II) = 5:5.
3.6. Detection of Gaseous Nitrogen Compounds and nirS Gene Amplification. Nitrite is an intermediate in the denitrification process. Ghafari et al.22 reported that reduction of nitrate into nitrogen gas proceeds according to the following sequence of events: NO3− → NO2− → NO → N2O → N2. We found evidence suggesting that strain SZF15 expresses cd1-NIR as amplification reactions showed the presence of a 512 bp nirS amplification product (Figure 7 (a)).23 Throbäck et al.24 reported that the reduction of nitrite to nitric oxide distinguishes denitrifiers from other nitrate-respiring bacteria. To further investigate the mechanism whereby strain SZF15 removes nitrate, we analyzed gas production during autotrophic
denitrification. To ensure an adequate supply of nitrogen sources for strain SZF15, the initial NO3−-N concentration was set to 20.00 mg·L−1. The production of N2 was monitored using a gas chromatography (GC) instrument equipped with a thermal conductivity detector and argon as the carrier gas. The results (Figure 7b) show that 98.99% of N2 was produced in the enclosed environment. Meanwhile, slight N2O (5.27 ppmv) was detected by PE600; this was in accordance with our previous study that N2 was the end product with slight N2O being detected, and, as Yao et al.25 reported, N2 increased to the predominant product over time. As suggested by Yang et al.,8 higher S/N mass ratios favor greater biomass-specific N2O F
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Figure 6. Variation of (A) nitrate, Fe(II), Mn(II), OD600 and (B) nitrite, pH by strain SZF15 under anaerobic conditions.
Figure 7. Amplification of nirS gene. Mark: DL 2000 DNA Marker (TaKaRa, Japan) (a) and the gas production (b) by strain SZF15.
investigated. The mechanism whereby strain SZF15 removes nitrate was investigated; the results suggested that nitrite is an intermediate in the denitrification process, and could be further reduced to N2 by nitrite reductase (nirS). Complete nitrate removal was found at high Mn(II)/Fe(II) molar ratios, 30 °C, pH 7.0, and an electron donor/electron acceptor molar ratio of 3.00. Eventually, a pathway of nitrate reduction was elaborated, nitrite was an intermediate product, and N2 was the end product. Therefore, a promising candidate in the extensive application of groundwater was found under optimal conditions.
reduction rates. For example, the dissolved N2O concentration quickly decreased from 12.0 mg-N/L to 0.5 mg-N/L with an initial S/N mass ratio of 5.0 g-S/g-N. This is 4 times greater than that at the S/N mass ratio of 0.8 g-S/g-N. In the study, slight N2O (5.27 ppmv) off-gas was detected in the end, and 98.99% of N2 was produced in the enclosed environment. Therefore, the greenhouse gas emissions in the study were much lower (5.27 ppmv) than those associated with other denitrification processes, which features an average N2O gas emission percentage of 1762.5 ppmv11 or 14.1 ppmv.8 Overall, a mechanism process of denitrification followed by reduction of nitrate to nitrite (a intermediate product of denitrification) and subsequently to N2O and N2 was elaborated completely. This study provides a novel method for the simultaneous removal of nitrogen, Fe(II), and Mn(II) in groundwater treatment process, this would benefit the development of autotrophic denitrification technology. For example, a bioreactor which immobilized cells could be used in an extensive application of nitratecontaminated groundwater treatment, as suggested by Bai et al.,26 Pseudomonas was the dominant bacteria in a bioaugmented reactor. The amplification product of nitrite reductase (nirS) further confirmed that N2 was the end product; meanwhile, the greenhouse gas emissions in the study were much lower than those associated with other denitrification processes. This would not produce the effect of global warming. Therefore, strain SZF15 is a promising candidate for extensive application to groundwater treatment.
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AUTHOR INFORMATION
Corresponding Author
*Tel.: +86 29 82202520. Fax: +86 29 82202520. E-mail:
[email protected]. ORCID
Xianxin Luo: 0000-0002-4913-7257 Fang Ma: 0000-0002-8849-0803 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research work was partly supported by the National Key Technology Research and Development Program of the Ministry of Science and Technology of the People’s Republic China (NO.2012BAC04B02), the National Natural Science Foundation of China (NSFC) (No. 51678471), and supported by Open Project of State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology
4. CONCLUSIONS In this study, the effect of mixed electron donors on autotrophic denitrification by Pseudomonas sp. SZF15 was G
DOI: 10.1021/acs.iecr.6b04591 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
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(NO.QA201518) and the Key Laboratory of the Education Department of Shan Xi Province (NO.12JS051).
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