Abstract

Many studies have reported that a certain preference is obeyed by perchlorate-degrading bacteria to utilize different electron acceptors. This conclusion was stated considering only the removal rate of different electron acceptors, indicating a lack of adequate proof. This study investigated the selective utilization of different electron acceptors by a perchlorate-degrading bacterium. The results showed that the mixed population of microorganisms (containing perchlorate-degrading bacteria) obeyed a certain sequence to utilize different electron acceptors, which was oxygen > nitrate > perchlorate > sulfate. The results of high-throughput sequencing showed that the mixed population of microorganisms contained anaerobic bacteria, facultative anaerobic bacteria, and aerobic bacteria. The microbial community structure actually had been changed by adding another electron acceptor to the perchlorate-medium and the microbial genera were distinguished in terms of utilizing the specific electron acceptor (e.g., oxygen, nitrate, sulfate). The result of canonical correspondence analysis demonstrated that the abundance of microorganisms appeared as a good positive correlation with the corresponding electron acceptor. Therefore, a new viewpoint was inferred that there are two main reasons at least that make the mixed microorganisms obey a certain sequence to utilize different electron acceptors. One reason is that the perchlorate-degrading bacteria in the mixed microorganisms change their own respiratory metabolism pathway. The other reason is that the mixed microorganisms actually change their microbial community structure.

INTRODUCTION

Perchlorate is commonly used as an important material in many processes, such as the manufacture of rocket propellant, fireworks, and ammunition (Rajagopalan et al. 2006; Li et al. 2013). During manufacture and utilization, a large amount of perchlorate migrates into the water and soil environment due to leakage and waste discharge (Peng et al. 2009). Moreover, perchlorate could easily dissolve into groundwater because of its high solubility (Susarla et al. 1999; Xie et al. 2014). It has been reported that the concentration of perchlorate reached 630–3,700 mg/L in the groundwater in Las Vegas and Nevada in the USA (Bardiya & Bae 2011). Perchlorate could disturb the synthesis and secretion of the thyroid hormone, thus affecting normal metabolism and growth in humans (Smith et al. 2005). Therefore, the problem of groundwater contamination with perchlorate has attracted great attention and needs to be solved urgently. Presently, studies on perchlorate-contaminated groundwater are focused on remediation approaches, such as physical adsorption, ion exchange, chemical/electrochemical reduction, and anaerobic biological reduction. Moreover, anaerobic biological reduction has been a hot research issue (Son et al. 2006; Yu et al. 2006; Gao et al. 2012; Wang et al. 2012; Kucharzyk et al. 2013) because of its unique advantages, such as low cost, high efficiency, and no further pollution. The basic principle of anaerobic biological reduction is that functional microorganisms utilize perchlorate as the electron acceptor of the respiratory chain and perchlorate is biodegraded into chloridion and oxygen (Bender et al. 2004; Bender et al. 2005). Many studies have reported perchlorate-degrading bacteria, such as Dechlorosoma sp KJ, Pseudomonas sp., Azospira sp., and Perclace. However, most of these bacteria were facultative anaerobes and usually could utilize other electron acceptors, such as oxygen, nitrate, and sulfate. Therefore, the coexistence of oxygen, nitrate, or sulfate in perchlorate-contaminated groundwater probably affects the efficiency of remediation by anaerobic biological reduction. Unfortunately, sulfate is a conventional anion in groundwater; oxygen and nitrate are also frequently present in large quantities in groundwater from some regions (Tong et al. 2013; Pu et al. 2014; Mubarak et al. 2015; Schilling & Jacobson 2015).

Many studies have reported that the coexistence of sulfate has no significant effect on the biodegradation of perchlorate, whereas oxygen and nitrate show an opposite result. The current study demonstrated that the coexistence of nitrate would reduce the perchlorate removal rate or delay perchlorate biodegradation. Particularly, nitrate and perchlorate were competitive in the anaerobic biological reduction system, and nitrate was a more preferable electron acceptor than perchlorate (Min et al. 2004; Chung et al. 2010). Current studies reported that oxygen inhibits the expression of perchlorate reductase and chlorite dismutase, the two key bio-enzymes that convert perchlorate into chloridion and oxygen (Kengen et al. 1999; Chaudhuri et al. 2002). If oxygen and perchlorate coexisted, microorganisms would choose oxygen preferentially as the electron acceptor. When the concentration of dissolved oxygen reached 2.0 mg/L, microorganisms almost lost the capacity of perchlorate degradation. However, the microorganisms (containing non-perchlorate-degrading bacteria) showed better biological activity even after the concentration of dissolved oxygen reached 4.8 mg/L (Shrout & Parkin 2006; Bardiya & Bae 2011).

The results of the above research indicated that other microbial electron acceptors, such as oxygen, nitrate, and sulfate, were competitive with perchlorate, and the problem of their coexistence with perchlorate increased as microorganisms preferred them to perchlorate. However, it is incorrect to state the above conclusion by considering only the superficial phenomenon of the removal rate of electron acceptors. It is still unclear whether the microorganisms change their own metabolic pathway or the structure of the complete microbial community changes drastically. Presently, there is no corresponding report discussing the mechanism of the effect of a multi-electron acceptor coexistence system on the biodegradation of perchlorate by the microbial community. Therefore, in this study, the community of mixed microorganisms (containing perchlorate-degrading bacteria) was investigated by high-throughput sequencing based on basic experiments of the effect of a multi-electron acceptor coexistence system on perchlorate biodegradation. This work will verify the relationship between perchlorate- biodegrading effectiveness and the microbial community affected by other electron acceptors, and provide significant biological theory for bioremediation of perchlorate-contaminated groundwater.

MATERIALS AND METHODS

Perchlorate-degrading microorganisms

Perchlorate-degrading microorganisms were successfully screened from the sediment of a river located in Changchun City using a selected liquid culture medium under anaerobic conditions. The composition of the selected culture medium is shown in Table 1.

Table 1

Composition of selected culture medium

ReagentNaClO4·2H2OKH2PO4Na2HPO4·12H2OMgSO4·7H2O(NH4)2SO4CH3COONa·3H2OCa–Fe mixed solutionMicronutrient mixed solution
Concentration 159.3 mg/L 0.95 g/L 1.5 g/L 0.1 g/L 0.3 g/L 1.2 g/L 1 mL/L 1 mL/L 
ReagentNaClO4·2H2OKH2PO4Na2HPO4·12H2OMgSO4·7H2O(NH4)2SO4CH3COONa·3H2OCa–Fe mixed solutionMicronutrient mixed solution
Concentration 159.3 mg/L 0.95 g/L 1.5 g/L 0.1 g/L 0.3 g/L 1.2 g/L 1 mL/L 1 mL/L 

The Ca–Fe mixed solution contained 1 g/L CaCl2·2H2O and 1 g/L FeSO4·7H2O. The composition of the micronutrient mixed solution is shown in Table 2.

Table 2

Composition of micronutrient mixed solution

ReagentZnSO4·7H2OMnCl2·4H2OH3BO3CuCl2·2H2ONiCl2·6H2ONa2MoO4·2H2ONa2SeO3
Concentration/(mg/L) 100 30 300 10 10 30 30 
ReagentZnSO4·7H2OMnCl2·4H2OH3BO3CuCl2·2H2ONiCl2·6H2ONa2MoO4·2H2ONa2SeO3
Concentration/(mg/L) 100 30 300 10 10 30 30 

The detailed screening process of perchlorate-degrading bacteria is as follows: 1 g river sediment was added into a glass bottle containing 100 mL selected liquid culture medium and incubated under anaerobic conditions. The dissolved oxygen in the selected liquid culture medium was expelled by nitrogen flow before culturing, and resazurin was used as the indicator for oxygen. The pH of the selected liquid culture medium was 7 and the cultivating temperature was 25°C. Unfortunately, perchlorate-degrading bacteria had a symbiotic association with other microorganisms. Therefore, a mixture of microorganisms (containing perchlorate-degrading bacteria) was used in the complete experimental process.

Mixed microorganisms in multi-electron acceptor coexistence system

Four experimental groups were maintained simultaneously. As the blank, the first group contained 100 mL original selected liquid culture medium (containing 100 mg/L of perchlorate) inoculated with 2 mL of mixed microorganisms without any other electron acceptor. The second group was supplemented with dissolved oxygen into the selected liquid culture medium (containing 100 mg/L of perchlorate) through pure-oxygen aeration and the concentrations of dissolved oxygen were adjusted to 2.65 mg/L, 4.00 mg/L, 5.80 mg/L, 8.14 mg/L, and 10.18 mg/L, respectively, followed by inoculation with the mixed microorganisms. The third group was supplemented with nitrate instead of oxygen and the concentrations of nitrate were adjusted as 14.54 mg/L, 21.79 mg/L, 57.34 mg/L, 101.20 mg/L, 221.11 mg/L, 314.78 mg/L, and 581.08 mg/L, respectively. The fourth group was supplemented with sulfate instead of oxygen (or nitrate) and the concentrations of sulfate were adjusted as 56.54 mg/L, 130.70 mg/L, 224.21 mg/L, 329.33 mg/L, and 551.45 mg/L, respectively. Then, the concentrations of perchlorate and other competitive electron acceptors were determined at various time-points during culturing.

Test of high-throughput sequencing

DNA extraction

The culture of microbial cells belonging to four groups, such as the original selected liquid culture medium, 10.18 mg/L of dissolved oxygen, 500 mg/L of nitrate, 500 mg/L of sulfate, respectively, were collected to extract total DNA using a bacterial DNA extraction kit produced by Shanghai Sangon, and samples were named as Perchlorate, Oxygen, Nitrate, and Sulfate.

Barcoded PCR and amplicon sequencing

The V3–V4 regions of 16S rDNA were amplified from the above total DNAs using the unique primers 341F and 805R. Then, the polymerase chain reaction (PCR) products were sequenced on the Illumina MiSeq 2*300 bp Sequencing Platform. The whole process of PCR and sequencing, and the subsequent data analysis were done by Shanghai Sangon Biological Engineering Technology & Service Co., Ltd. The significance level of sequencing was set as p < 0.01.

Canonical correspondence analysis

The sequences whose similarity was more than 97% were considered as one operational taxonomic unit (OTU). Then, canonical correspondence analysis (CCA) was applied to demonstrate the relationships between microbial OTU composition and the electron acceptors using the software program CANOCO (version 4.5). The data involving the abundance of microbial OTU and the concentrations of electron acceptors were log-transformed before CCA to eliminate the influence of extreme values on ordination scores.

Analytical methods

The concentration of dissolved oxygen was determined by an electrochemical probe method. Perchlorate, nitrate, and sulfate were monitored using ion chromatography and the chromatographic condition was set as follows: 10 μL sample volume, AS 20 model of separation column (4 mm × 250 mm), 30°C column temperature, 35 mmol/L KOH in mobile phase, 1.0 mL/min flow velocity of mobile phase, 87 mA detecting electric current, and 20 min run time.

RESULTS AND DISCUSSION

Effect on perchlorate biodegradation by multi-electron acceptor coexistence system

Oxygen

When perchlorate was used as the sole microbial electron acceptor, biodegradation followed the first-order kinetic model (R2 = 0.900) and the degradation rate constant was about 0.098 h−1. In the incipient 12 h, about 89.41% of perchlorate was biodegraded effectively. Moreover, 98.93% of perchlorate was exhausted at the end of incubation at 60 h (Figure 1).

Figure 1

Effect on biodegradation when perchlorate was used as the sole electron acceptor

Figure 1

Effect on biodegradation when perchlorate was used as the sole electron acceptor

When oxygen was introduced into the reductive biodegradation system of perchlorate, its effects on perchlorate biodegradation were significant (Figure 2). When the initial concentration of dissolved oxygen was 2.65 mg/L, perchlorate biodegradation was evidently inhibited in the incipient 24 h. In case of 4.00 mg/L, 5.80 mg/L, and 8.14 mg/L initial concentrations of dissolved oxygen, the perchlorate biodegradation was inhibited for around 36 h. In the case of 10.18 mg/L dissolved oxygen, perchlorate biodegradation was almost inhibited. Except for the sample with 10.18 mg/L of dissolved oxygen, all the other perchlorate biodegradation reactions occurring at the incubation time of 36–70 h followed first-order kinetic models. Although oxygen inhibited perchlorate biodegradation, the rate constant of perchlorate biodegradation with dissolved oxygen was significantly higher than that of perchlorate when used as the sole electron acceptor. It could be inferred from the above results that the microbial community preferentially utilized oxygen as the electron acceptor, followed by perchlorate when oxygen was exhausted.

Figure 2

Effect of dissolved oxygen on perchlorate biodegradation.

Figure 2

Effect of dissolved oxygen on perchlorate biodegradation.

Nitrate

According to the theory of redox potential, nitrate is more readily available for microorganisms as the electron acceptor than perchlorate. When the initial concentration of nitrate was lower than 21.79 mg/L, the coexistence of nitrate did not affect perchlorate biodegradation (Figure 3(a)). However, the rate constant of perchlorate biodegradation decreased significantly along with the increase of initial concentration of nitrate, and the removal rate of perchlorate decreased even at the incubation time of 60 h indicating that nitrate is a competitive electron acceptor and slowed down the rate of perchlorate biodegradation.

Figure 3

Effect of different levels of nitrate on perchlorate biodegradation. (a) Perchlorate biodegradation based on using perchlorate and nitrate as competitive electron acceptors. (b) Nitrate biodegradation based on using perchlorate and nitrate as competitive electron acceptors.

Figure 3

Effect of different levels of nitrate on perchlorate biodegradation. (a) Perchlorate biodegradation based on using perchlorate and nitrate as competitive electron acceptors. (b) Nitrate biodegradation based on using perchlorate and nitrate as competitive electron acceptors.

In addition, the concentration of nitrate decreased gradually and the biodegradation curves followed the first-order kinetic models (Figure 3(b)). Moreover, the removal rate of nitrate was invariably higher than that of perchlorate during the whole incubation period, which adequately verified that nitrate was more preferred by microorganisms as an electron acceptor than perchlorate.

Sulfate

The rate constant of perchlorate biodegradation in the sulfate-coexisting systems had no significant change compared with that when perchlorate was used as the sole electron acceptor (Figures 1 and 4(a)), indicating that sulfate had no obvious effect on perchlorate biodegradation. When the initial concentration of sulfate increased up to 581.08 mg/L, the rate constant of perchlorate biodegradation decreased significantly and the removal rate of perchlorate dropped distinctly. These results could demonstrate that low concentrations (less than 314.78 mg/L) of sulfate did not affect perchlorate biodegradation, whereas high concentrations (more than 581.08 mg/L) of sulfate affected perchlorate biodegradation to some extent. This can be explained by two reasons: that a high concentration of sulfate improved the redox potential and that it increased the probability of contact between sulfate and microorganisms.

In addition, sulfate was biodegraded to some extent and this process followed a zero-order kinetic model even when different initial concentrations of sulfate were used (Figure 4(b)). However, the rate of sulfate reduction was slower than that of perchlorate biodegradation and decreased gradually with the decrease in initial concentration of sulfate. Moreover, the removal rate of sulfate was about 20.65–73.08%, and some amount of sulfate remained at the end of incubation (60 h), indicating that perchlorate had a priority over sulfate to be biodegraded.

Figure 4

Effect of sulfate on perchlorate biodegradation. (a) Perchlorate biodegradation based on using perchlorate and sulfate as competitive electron acceptors. (b) Sulfate reduction based on using perchlorate and sulfate as competitive electron acceptors.

Figure 4

Effect of sulfate on perchlorate biodegradation. (a) Perchlorate biodegradation based on using perchlorate and sulfate as competitive electron acceptors. (b) Sulfate reduction based on using perchlorate and sulfate as competitive electron acceptors.

Effect of multi-electron acceptor coexistence system on microbial community

Microbial diversity

According to the statistical data, the sequences with more than 97% similarity were considered as one OTU, which also represented a microbial genus. The ecological indexes were calculated based on the OTU and are shown in Table 3. OTU number, ACE index, and Chao1 index showed that the number of species in a mixed cultured system was the largest with sulfate, followed by nitrate or perchlorate, and the smallest with oxygen as electron acceptor, which initially demonstrated that the genus number of anaerobic bacteria was significantly more than that of aerobic bacteria, and the species of bacteria utilizing sulfate was quite abundant in the mixed culture system. The α-diversity indexes, including the Shannon index and Simpson index, showed that the diversity of microbial community cultured with sulfate or oxygen was relatively high, whereas that of the microbial community cultured with perchlorate or nitrate was relatively low. An unusual phenomenon was that microorganisms utilizing oxygen had lowest OTU number, yet they had the highest diversity. The reason for this phenomenon was that the distribution of microbial genus using oxygen as an electron acceptor was relatively homogeneous. Overall, all the Shannon indexes were greater than 3, indicating that the mixed microbial colonies biodegrading perchlorate could keep better species diversity with different electron acceptors.

Table 3

Effect of multi-electron acceptor coexistence system on microbial diversity

Sample IDSequence numberOTU numberShannon indexACE indexChao1 indexCoverageSimpson index
Perchlorate 19,161 1,900 3.80 4,773 3,793 0.9474 0.2026 
Oxygen 13,996 1,361 3.87 2,904 2,416 0.9534 0.1519 
Nitrate 20,736 1,967 3.65 4,597 3,752 0.9505 0.2306 
Sulfate 49,981 3,251 3.96 8,632 6,447 0.9670 0.1882 
Sample IDSequence numberOTU numberShannon indexACE indexChao1 indexCoverageSimpson index
Perchlorate 19,161 1,900 3.80 4,773 3,793 0.9474 0.2026 
Oxygen 13,996 1,361 3.87 2,904 2,416 0.9534 0.1519 
Nitrate 20,736 1,967 3.65 4,597 3,752 0.9505 0.2306 
Sulfate 49,981 3,251 3.96 8,632 6,447 0.9670 0.1882 

Genus composition of microbial community

Generally, the genus composition of microbial communities cultured with nitrate, perchlorate, and sulfate were similar, and were distinguished from the microbial communities cultured with oxygen significantly (Figure 5). The dominant genera of microorganisms degrading perchlorate under anaerobic conditions with nitrate, or perchlorate, or sulfate as electron acceptors were Sulfurospirillum, Enterobacter, Klebsiella, Dysgonomonas, etc. Moreover, the proportion of Sulfurospirillum, Enterobacter, Klebsiella, and Dysgonomonas could reach 64.44–69.70%, 23.74–27.81%, 4.25–4.95%, and 0.93–2.51%, respectively.

Figure 5

Effect of different electron acceptors on the genus composition of microbial community.

Figure 5

Effect of different electron acceptors on the genus composition of microbial community.

When oxygen was introduced as the electron acceptor, the microbial communities was mainly composed of Enterobacter, Klebsiella, and Citrobacter. The proportion of Enterobacter, Klebsiella, and Citrobacter was 83.24%, 13.61%, and 1.14%, respectively. The above results adequately demonstrated that the community of perchlorate-degrading bacteria changed significantly when different electron acceptors were introduced.

A heat map could show a better specific variation in microbial communities which were cultured with different competitive electron acceptors, such as oxygen, nitrate, and sulfate, respectively (Figure 6). In the multi-electron acceptor coexistence systems, five microbial genera, including Sulfurospirillum, Enterobacter, Klebsiella, Dysgonomonas, and Citrobacter, were predominant. Under anaerobic conditions with perchlorate, or nitrate, or sulfate as the microbial electron acceptor, Sulfurospirillum and Dysgonomonas were relatively dominant, whereas Enterobacter, Klebsiella, and Citrobacter were more dominant under aerobic conditions with oxygen as the microbial electron acceptor.

Figure 6

Heat map based on the distribution and abundance of microbial genera cultured with different electron acceptors.

Figure 6

Heat map based on the distribution and abundance of microbial genera cultured with different electron acceptors.

According to available literature and Bergey's manual of determinative bacteriology, it is known that: (1) Sulfurospirillum is strictly anaerobic and can biodegrade perchlorate, nitrate, and sulfate simultaneously; (2) Enterobacter is aerobic or facultatively anaerobic and can biodegrade nitrate; (3) Klebsiella can biodegrade nitrate and sulfate; (4) Dysgonomonas is anaerobic and cannot biodegrade nitrate and sulfate, but can biodegrade perchlorate; (5) Citrobacter is facultatively anaerobic and can biodegrade perchlorate and nitrate.

The above information preliminarily suggested that the coexistence of multi-electron acceptors in a perchlorate-biodegrading system seemingly made the corresponding electron acceptor-degrading bacteria the dominant species. More specifically, under aerobic conditions with oxygen as the electron acceptor, strictly anaerobic bacteria, Sulfurospirillum and Dysgonomonas, were not detected, aerobic or facultatively anaerobic bacteria, Enterobacter, easily consumed oxygen as the electron acceptor and were the most dominant species, and facultatively anaerobic bacteria, Citrobacter, seemingly preferred oxygen as the electron acceptor and were more dominant under aerobic conditions than under anaerobic conditions. Under anaerobic conditions in the presence of perchlorate, nitrate, and sulfate as microbial electron acceptors, strictly anaerobic bacteria, Sulfurospirillum, biodegrading perchlorate, nitrate, and sulfate, were the most dominant species, the proportions of aerobic or facultatively anaerobic bacteria, Enterobacter and Klebsiella, were significantly decreased compared with that under aerobic conditions, anaerobic bacteria, Dysgonomonas, which do not biodegrade nitrate and sulfate, but degrade perchlorate, were relatively more dominant with perchlorate as the sole electron acceptor, and the dominance of facultatively anaerobic bacteria, Citrobacter, was decreased as a matter of course.

The relationship between microbial genera and electron acceptor

CCA was used to investigate the relationship between microbial genera and electron acceptor and the result is shown in Figure 7. The dominant genera, including Enterobacter, Klebsiella, and Citrobacter, had a better positive correlation with oxygen, whereas Sulfurospirillum and Dysgonomonas had a negative correlation with oxygen but had a positive correlation with nitrate to some extent. These results were consistent with the results of the biodegrading effects of various electron acceptors and the results of analysis of genus composition of the microbial community. Further, it was demonstrated that the outcomes of biodegradation of different electron acceptors obeyed a certain sequence and were mainly caused by the variation in the microbial community, and not by the sequence of utilizing different electron acceptors with the same kind of bacterium.

Figure 7

Correlation between microbial species and electron acceptor based on CCA.

Figure 7

Correlation between microbial species and electron acceptor based on CCA.

CONCLUSIONS

Perchlorate-degrading microorganisms usually were a mixed population composed of anaerobic, facultative anaerobic, and aerobic bacteria. When multi-electron acceptors coexisted in the culture, the mixed microorganisms (containing perchlorate-degrading bacteria) showed a definite sequence to utilize different electron acceptors, and a more preferred sequence of mixed microorganisms was oxygen > nitrate > perchlorate > sulfate. According to the analysis on the genus composition of microbial community and the relationship between microbial genera and electron acceptors, it can be concluded that one main reason why the mixed microorganisms (containing perchlorate-degrading bacteria) obeyed a certain sequence to utilize different electron acceptors was that the coexistence of multi-electron acceptors caused the microbial community actually to change significantly. For example, the abundance of perchlorate-degrading bacteria Sulfurospirillum was extremely low in the aerobic condition, and the abundance of perchlorate-degrading bacteria Dysgonomonas was relative high when perchlorate was the sole microbial electron acceptor. Of course, the changes of microbial respiratory metabolism pathway actually is the other main reason.

ACKNOWLEDGEMENTS

This study was supported by the Natural Science Foundations of China (No. 41072170 and No. 41572217) and the ‘Twelfth Five-Year’ Science & Technology Research Project of the Education Department in Jilin Province (2014B027).

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