Identification and characterization of a cold-adapted and halotolerant nitrobenzene-degrading bacterium

Abstract

A strain named X7, which uses nitrobenzene (NB) as a carbon source at low temperature and high salinity, was domesticated and isolated from activated sludge in the aeration tank of Shenyang Northern Sewage Treatment Plant. According to its morphological, physiological and biochemical characteristics and 16S rDNA gene sequence, a preliminary identification was made. The result showed that strain X7 was Myroides odoratus. The optimal degradation conditions of strain X7 were as follows: inoculum concentration 10%, optimal temperature 15 °C, pH 7.0, rotary speed 150 rpm and salinity 1.0%–3.0%. At an initial concentration of nitrobenzene of 150 mg L⁻¹, the degradation rate was 51.50%. Moreover, the addition of glucose and peptone could increase the degradation rate to 58.92% and 60.24%, respectively. The maximum concentration of nitrobenzene that was tolerated by strain X7 was 350 mg L⁻¹.

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1 Introduction

Nitrobenzene (NB), which is an important chemical material, is widely used in the manufacture of organic products such as aniline, dyes, drugs, explosives, insecticides and synthetic rubber.¹ Nitrobenzene has been proven to be a carcinogen and a huge threat to human health.^2,3 Therefore, nitrobenzene is listed as a priority pollutant by the US Environmental Protection Agency.⁴

Much effort has been made to treat contamination by nitrobenzene.^5–8 Compared to physical and chemical methods, biological methods can mineralize nitrobenzene at a lower cost.^9–11 Bacterial strains capable of degrading nitrobenzene have been described such as Bacillus cereus, Streptomyces, Rhodotorula mucilaginosa, white rot fungi, and Methanothrix soehngenii.^12–17

Much research has described the effects of environmental factors on the degradation of nitrobenzene by microorganisms, but most of the degradation studies were conducted at room temperature.^18,19 There have only been a few reports that studied the degradation of nitrobenzene in extreme environments.²⁰ Therefore, it is essential to test the degradation of nitrobenzene by strains at low temperature and high salinity and evaluate their potential application in the treatment of nitrobenzene in wastewater.

In this study, a strain that uses nitrobenzene as a carbon source at low temperature and high salinity was domesticated and isolated from activated sludge in the aeration tank of Shenyang Northern Sewage Treatment Plant. The effects of several parameters, including inoculum, pH, temperature, salinity and auxiliary carbon/nitrogen sources, as co-substrates on the biodegradation of nitrobenzene by the strain were investigated.

2 Materials and methods

2.1 Bacterial sources

The source of the strain used in this study was obtained from activated sludge in the aeration tank of Shenyang Northern Sewage Treatment Plant.

2.2 Medium

Enriched medium contained peptone 10 g L⁻¹, beef extract 3 g L⁻¹, and NaCl 5 g L⁻¹ at pH 7.0. Mineral salt medium contained Na₂HPO₄·12H₂O 3.8 g L⁻¹, KH₂PO₄ 1 g L⁻¹, NaCl 1 g L⁻¹, MgSO₄ 0.2 g L⁻¹, and NH₄Cl 0.1 g L⁻¹. Isolation medium contained peptone 10 g L⁻¹, beef extract 3 g L⁻¹, NaCl 30 g L⁻¹, and agar gel 15%–20% at pH 7.0.

2.3 Analytical methods

Nitrobenzene concentrations were measured with a Cary 50 UV-vis spectrophotometer (Varian, US) using N-(1-naphthyl)-ethylenediamine dihydrochloride as per standard procedure. Bacterial cell concentrations were determined by a photoelectric turbidimetry method. A UV-vis spectrophotometer was used to determine the growth of bacteria by monitoring the optical density at a wavelength of 600 nm (OD₆₀₀).

2.4 Acclimation, screening and separation of strains

To obtain strains that were tolerant of nitrobenzene, 10 mL activated sludge was first inoculated into 90 mL enriched medium in a 250 mL flask containing 10 mg L⁻¹ nitrobenzene. When the culture became turbid, 10 mL of the culture was transferred to 90 mL fresh MSM in a new 250 mL flask containing 10 mg L⁻¹ nitrobenzene. The 250 mL flask was supplemented with nitrobenzene to a final concentration of 150 mg L⁻¹ and cultivated on a rotary shaker (150 rpm) at 30 °C. When the degradation rate of nitrobenzene reached a stable level, the activated sludge was domesticated at low temperature and high salinity. When the temperature decreased to 15 °C and the salinity of the medium increased to 3.0%, the culture was diluted and spread onto agar plates with isolation medium at 150 mg L⁻¹. The agar plates were incubated at 15 °C for 5–7 days. Colonies that appeared on the agar plates were subcultured. Five species of strains with nitrobenzene as a carbon source were screened after microscopic examination. After a degradation process, a strain that provided better degradation of nitrobenzene, named X7, was screened.

2.5 Strain identification

According to the strain's Gram stain, individual form, and colony, physiological and biochemical characteristics, a preliminary identification was made.

With the strain's DNA as a template, extracted DNA from the target strain X7 and a partial 16S rDNA sequence were amplified by PCR using the following primers: F27 (5′-AGACTTTGATCCTGGCTCAG-3′) as forward and R1492 (5′-ACGGTTACCTGTTACGACTT-3′) as reverse. The system contained 1 μL DNA template, 2 μL dNTP, 2.5 μL 10 × PCR buffer, 1 μL of each primer, 0.2 μL Taq polymerase, and 17.3 μL deionized water. PCR reactions were performed under the following conditions: 5 min at 94 °C; 30 cycles of 30 s at 94 °C, 30 s at 58 °C, and 90 s at 72 °C; plus an additional 10 min cycle at 72 °C. PCR fragments were ligated into the linear vector pMD18-T (TaKaRa Biotechnology, Dalian Co., Ltd, China) after purification by agarose gel electrophoresis and then transformed into competent Escherichia coli DH5α cells. The 16S rDNA gene sequence was completed by Harbin Boshi Biological Technology Co. Ltd. The partial 16S rDNA sequence has been submitted to GenBank, and BioEdit v 5.06 was used to analyze multiple sequence alignments. The sequences were analyzed by MEGA 6.0. A phylogenetic tree was obtained by a neighbor-joining method.

2.6 Growth curve of strain X7

Ten milliliters of the culture in the late exponential phase was aseptically inoculated into each 250 mL flask with 90 mL sterilized MS medium. The 250 mL flasks were supplemented with nitrobenzene (150 mg L⁻¹) and cultivated on a rotary shaker (150 rpm) at 15 °C. Samples were withdrawn from each flask at 1 day intervals and centrifuged at 12 000 rpm for 10 min. The supernatant was diluted and used to determine the nitrobenzene concentration. Three parallel samples were taken for each experiment. All experiments were performed in triplicate.

2.7 Effect of several parameters on the growth and degradation rate of X7

The effects of parameters, such as inoculum concentration (1%, 3%, 5%, 10%, 15%, and 20%), pH (4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0), rotary speed (80, 100, 120, 150, 170, and 200 rpm), temperature (5, 10, 15, 20, 25, and 30 °C), and salinity [0.5%, 1.0%, 3.0%, 5.0%, 7.0%, 9.0%, and 10.0% NaCl (w/v)] on the degradation of nitrobenzene by strain X7, were investigated. The initial concentration of nitrobenzene was 150 mg L⁻¹. Samples were withdrawn from each flask at 7 d and centrifuged at 12 000 rpm for 10 min. The supernatant was diluted and used to determine the nitrobenzene concentration. Non-inoculated cultures with the same concentrations of nitrobenzene served as controls. Three parallel samples were taken for each experiment. All experiments were performed in triplicate.

2.8 Effect of co-substrate on the growth and degradation rate of X7

The effects of co-substrates, such as a carbon source (1% glucose, 1% sucrose, 1% starch, 1% lactose, 1% sodium acetate) and a nitrogen source (1% peptone, 1% ammonium sulfate, 1% ammonium chloride, 1% urea, 1% ammonium acetate) on the degradation of nitrobenzene by strain X7, were also investigated. A 250 mL flask was supplemented with nitrobenzene (150 mg L⁻¹) and cultivated on a rotary shaker (150 rpm) at 15 °C. Samples were withdrawn from each flask at 7 d and centrifuged at 12 000 rpm for 10 min. The supernatant was diluted and used to determine the nitrobenzene concentration. Three parallel samples were taken for each experiment. All experiments were performed in triplicate.

2.9 Test of the tolerance of strain X7 for nitrobenzene

One milliliter of culture in the late exponential phase was inoculated onto MSM agar plates with increasing concentrations of nitrobenzene. The plates were incubated at 15 °C for a week and the tolerance level of the culture to nitrobenzene was determined.

3 Results

3.1 Strain screening

At low temperature and high salinity, sixteen strains that use nitrobenzene as a carbon source were domesticated and isolated from activated sludge in the aeration tank of Shenyang Northern Sewage Treatment Plant. The strains were cultivated with MS medium under conditions of rotary speed 150 rpm, salinity 3.0%, and temperature 15 °C. The concentration of nitrobenzene was 150 mg L⁻¹. Nitrobenzene degradation rates of sixteen strains are shown in Fig. 1. Five (X1, X3, X7, X11 and X14) of them could grow well and their degradation rates of nitrobenzene were all over 40%. Then, those five strains were cultivated with MS medium under conditions of rotary speed 150 rpm, salinity 3.0%, and temperature 15 °C. Nitrobenzene degradation rates of five strains are shown in Fig. 2. The degradation rate of nitrobenzene of strain X7 was very high. Therefore, strain X7 was identified as a cold-adapted and halotolerant nitrobenzene-degrading bacterium.

Fig. 1 Degradation rate of different nitrobenzene-degrading bacteria.

Fig. 2 Degradation rate of five highly effective nitrobenzene-degrading bacteria.

3.2 Identification of strain X7

3.2.1 Morphological, physiological and biochemical characteristics of strain X7. Strain X7 was bar-shaped and Gram-negative with no spores and no flagella, opaque and relatively smooth. The surface of strain X7 was prominent, smooth and humid. The body of strain X7 had a regular margin. Its size was about (0.3–0.5) × (0.8–2.0) μm. Table 1 shows the results of physiological tests that were used to identify strain X7.

Table 1 Results of physiological tests used to identify the strain X7

Strain	Starch hydrolysis experiment	Glucose oxidation fermentation test	Lactose fermentation test	MR test	VP test	Indole test	Litmus milk test	Gelatin liquefaction test	Catalase	Citrate utilization test
X7	−	−	−	−	−	−	White	+	+	−

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According to the test results, strain X7 was identified as a Flavobacterium sp. by Bergey's Manual of Determinative Bacteriology and the Manual of Systematic Methods of Determinative Bacteriology.

3.2.2 16S rDNA gene sequence of strain X7. According to strain X7's morphological, physiological and biochemical characteristics and 16S rDNA gene sequence, the result showed that strain X7 was Myroides odoratus. Fig. 3 shows the phylogenetic tree of strain X7. At present, research that Myroides odoratus has been applied to degrade nitrobenzene has seldom been reported.

Fig. 3 Phylogenetic tree of strain X7.

3.3 Growth curve of strain X7

The growth curve of strain X7 and its degradation rate of nitrobenzene during 10 days are shown in Fig. 4. The strain grew slowly during the first two days, which was the adaptation period. From 3 to 6 d was the logarithmic growth phase. Both the growth of the strain and the degradation rate of nitrobenzene increased rapidly. From 6 to 8 d was the stationary phase. On the seventh day, the highest degradation rate was 51.50%. The growth of strain X7 and the degradation rate of nitrobenzene were stable after the eighth day, which may be due to inhibition by metabolic intermediate products from the degradation of nitrobenzene by strain X7.¹⁹ Finally, 7 d was determined to be the best time for degradation in this study.

Fig. 4 Growth curve and nitrobenzene degradation rate of strain X7.

3.4 Optimal degradation conditions of X7

3.4.1 Effects of inoculum on the growth and degradation rate of X7. The effect of the initial inoculum on the biodegradation of nitrobenzene is shown in Fig. 5. With an increase in inoculum, the growth of the strain and the degradation rate of nitrobenzene increased. However, when the inoculum concentration was more than 10%, it had little influence on the growth of the strain and the degradation rate of nitrobenzene. This may be because the high density of the strain caused degradation of bacteria in the oligotrophic state.

Fig. 5 Effects of inoculum on the growth and degradation rate of X7.

3.4.2 Effects of pH on the growth and degradation rate of X7. Without exception, a microorganism has a pH that is most suitable for growth.²¹ Fig. 6 shows the growth curve of strain X7 and the degradation rate of nitrobenzene on the 7^th day. The pH has a great influence on the growth of strain X7. The strain could grow well and the degradation rate of nitrobenzene was over 40% in the pH range of 6.0–8.0. When the pH was increased to 7.0, the degradation rate of nitrobenzene approached a maximum. This result was consistent with previous studies, which have found that most degrading bacteria could degrade pollutants in the pH range of 6.0–8.0.²² Rather than alkaline conditions, acid conditions were more suitable for strain X7 to grow. It is presumed that metabolic intermediate products from the degradation of nitrobenzene by strain X7 increased the pH of the solution.

Fig. 6 Effects of pH on the growth and degradation rate of X7.

3.4.3 Effects of rotary speed on the growth and degradation rate of X7. Fig. 7 shows the growth curve of strain X7 and the degradation rate of nitrobenzene on the 7th day. When the rotary speed was less than 150 rpm, the growth of strain X7 and its ability to degrade nitrobenzene were inhibited. With an increase in rotary speed, the growth of strain X7 and its ability to degrade nitrobenzene increased. When the rotary speed was more than 150 rpm, its influence on the growth of the strain and the degradation rate of nitrobenzene was not obvious. This may be because the high rotary speed caused the bacteria that were in solution to die. Therefore, 150 rpm was determined to be the best rotary speed for degradation in this study.

Fig. 7 Effects of rotary speed on the growth and degradation rate of X7.

3.4.4 Effects of temperature on the growth and degradation rate of X7. Temperature has a great effect on the metabolism of microorganisms.²³ Fig. 8 shows the growth curve of strain X7 and the degradation rate of nitrobenzene on the 7^th day. The result demonstrates that strain X7 could grow at every temperature. When the temperature was less than 15 °C, the growth of strain X7 and its ability to degrade nitrobenzene were inhibited. This result was similar to previous studies that showed that biodegradation of nitrobenzene at low temperatures is not favorable.²⁴ With an increase in temperature, the growth of strain X7 and its ability to degrade nitrobenzene increased. As considerable cell death was observed and a small amount of degradation was achieved at high temperature, 15 °C was determined to be the best temperature for degradation.

Fig. 8 Effects of temperature on the growth and degradation rate of X7.

3.4.5 Effects of salinity on the growth and degradation rate of X7. High salinity reduces microbial metabolism.²⁵ Fig. 9 shows the growth curve of strain X7 and the degradation rate of nitrobenzene on the 7^th day. When salinity was less than 1.0%, with an increase in salinity, the growth of strain X7 and its ability to degrade nitrobenzene increased. The degradation rate of nitrobenzene was almost the same at a salinity of 1.0%–3.0%. When the salinity was more than 3.0%, the growth of the strain and the degradation rate of nitrobenzene decreased sharply. Similar phenomena have been reported in a study of the removal of nitrobenzene by a novel halophilic bacterium Bacillus licheniformis.²⁶ The growth of the strain was weak at a salinity of 10.0%, so that strain X7 could tolerate salinity as high as 10.0%.

Fig. 9 Effects of salinity on the growth and degradation rate of X7.

3.4.6 Effects of carbon sources on the growth and degradation rate of X7. To improve the degradation of nitrobenzene, this experiment studied the growth of the strain and the degradation rate of nitrobenzene by adding other auxiliary carbon sources. Fig. 10 shows the growth curve of strain X7 and the degradation rate of nitrobenzene on the 7th day. The growth of strain X7 was improved by adding glucose, therefore improving the degradation of nitrobenzene. The degradation rate increased from 51.50% to 58.92%. This result was similar to that of Wang et al., who detected that a small amount of a second carbon source as co-substrate could slightly enhance the biodegradation of nitrobenzene.²⁷ However, there were few influences on the growth of the strain and the degradation of nitrobenzene by adding other carbon sources. The reason may be that glucose stimulated strain X7 to generate a corresponding inducible enzyme, which led to improved degradation of nitrobenzene.

Fig. 10 Effects of carbon sources on the growth and degradation rate of X7.

3.4.7 Effects of nitrogen sources on the growth and degradation rate of X7. Nitrogen sources play an important role in the process of microbial protein synthesis. Fig. 11 shows the growth curve of strain X7 and the degradation rate of nitrobenzene on the 7^th day. The growth of strain X7 was improved by adding peptone, therefore improving the degradation of nitrobenzene. The degradation rate increased from 51.50% to 60.24%. However, the growth of strain X7 and its ability to degrade nitrobenzene were inhibited by adding ammonium acetate. That was because the pH of the solution was increased by adding ammonium acetate, which led to inhibition of the degradation of nitrobenzene.

Fig. 11 Effects of nitrogen sources on the growth and degradation rate of X7.

3.4.8 Test of the tolerance of strain X7 for nitrobenzene. Nitrobenzene is a harmful and toxic substance, which is difficult to degrade by microorganisms. Therefore, to investigate the tolerance for nitrobenzene of strain X7 has great significance for its practical application. Table 2 shows the growth of strain X7 at different concentrations of nitrobenzene on the 7^th day. The result showed that a high concentration of nitrobenzene had a large inhibitory effect on the growth of strain X7. The maximum tolerated concentration of nitrobenzene for strain X7 was 350 mg L⁻¹. Strain X7 had a high ability to tolerate nitrobenzene.

Table 2 Growth of strain X7 at different concentrations of nitrobenzene on the 7^th day

Strain	Concentration of nitrobenzene (mg L^−1)
	100	150	200	250	300	350	400
X7	+	+	+	+	±	±	−

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4 Conclusions

(1) Five (X1, X3, X7, X11 and X14) strains that use nitrobenzene as a carbon source were domesticated and isolated from activated sludge in the aeration tank of Shenyang Northern Sewage Treatment Plant. At an initial concentration of nitrobenzene of 150 mg L⁻¹, the degradation rate of nitrobenzene was over 40%. The nitrobenzene degradation rate of strain X7 was uniformly high. Therefore, strain X7 was identified as a cold-adapted and halotolerant nitrobenzene-degrading bacterium.

(2) According to strain X7's morphological, physiological and biochemical characteristics and 16S rDNA gene sequence, the result showed that strain X7 was Myroides odoratus.

(3) From tests on the growth and degradation processes of strain X7, the optimal degradation conditions of strain X7 were as follows: inoculum concentration 10%, optimal temperature 15 °C, pH 7.0, rotary speed 150 rpm and salinity 1.0%–3.0%. At an initial concentration of nitrobenzene of 150 mg L⁻¹, the degradation rate was 51.50%. However, the addition of glucose and peptone could increase the degradation rate to 58.92% and 60.24%, respectively. The maximum tolerated concentration of nitrobenzene of strain X7 was 350 mg L⁻¹.

Acknowledgements

This study was supported by National “12·5” water special sub-topics (2012ZX07202003-05); Funded projects of the 211 key discipline of Environmental Institute, Liaoning University; Research of teaching reform project of undergraduate, Liaoning University (No. JG2013ZD0010).

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