Thiourea is a typical nitrification inhibitor that shows a strong inhibitory effect against the biological nitrification process. The 50% inhibitory concentration (IC50) of thiourea on nitrification was determined to be 0.088 mg g VSS−1, and nitrifiers recovered from the thiourea inhibition after it was completely degraded. The thiourea-degrading ability of the sludge system was improved to 3.06 mg gVSS−1 h−1 through cultivation of thiourea-degrading bacteria by stepwise increasing the influent thiourea concentration. The dominant thiourea-degrading bacteria strain that used thiourea as the sole carbon and nitrogen source in the sludge system was identified as Pseudomonas sp. NCIMB. The results of this study will facilitate further research of the biodegradation characteristics of thiourea and similar pollutants.

INTRODUCTION

Sulfocarbamide (CH4N2S, CAS No. 62-56-6), which is also known as thiourea because of its similar molecular structure to urea, is as an important catalyst and chemical intermediate widely used in ore leaching and isomerization catalyzing processes. Also, thiourea has strong ability to inactivate ammonia-oxidizing bacteria (Hooper & Terry 1973), and therefore has a long history of being used as a nitrification inhibitor (Zacherl & Amberger 1990). When a large amount of industry wastewater containing thiourea is discharged to natural water bodies, it causes environmental contamination of receiving waters because of the accumulation of ammonia and thiourea itself.

Two traditional methods have been applied to prevent the inhibition of nitrification by thiourea or similar inhibitors as follows:

  1. Direct elimination of the inhibitors. Owing to the presence of C = S in its molecular structure, thiourea shows good chemical degradability and is easily decomposed into low toxic or non-toxic substances by chemical processes. Xie et al. (2012) reported that 94% of thiourea could be degraded after a 9-min reaction when the ozone flow rate, pH, and temperature were 600 mL min–1, 7.3, and 20 °C, respectively. According to McConnell (1993), 98% of thiourea could be degraded into urea and sulfate after a 3-h reaction when hydrogen peroxide was added as 4 mol/mol thiourea and the pH was greater than 12. Moreover, powdered activated carbon treatment processes have been widely applied to manage the inhibitory situation during biological treatment, and powdered activated carbon was found to perform well at eliminating the toxicity of certain types of industrial wastewater (Wong et al. 1992; Kim et al. 2008). One common disadvantage of the aforementioned treatment processes is their relatively high cost (typically USD 1–3/m3 wastewater).

  2. Enhancing nitrifier resistance to inhibitors.Vandevivere et al. (1998) found that the inhibitory effects of 0.1 mg L–1 thiourea on nitrification were greatly reduced by 59% when copper (Cu(I)/thiourea at a molar ratio of 60) was added to a bench-scale semicontinuous activated sludge reactor. But the direct addition of copper to activated sludge system would increase disposal costs of sludge containing the heavy metal. Bioaugmentation is also an approach to strengthen the activity of nitrifiers. According to Tang & Chen (2015), the nitrification rate (NR) was improved from 0.21 mg NH3-N gVSS−1 h−1 to 0.58 mg NH3-N gVSS−1 h−1 following the introduction of 6% bioaugmented nitrifiers in a municipal wastewater treatment plant suffering from poor nitrification (VSS: volatile suspended solids). Boon et al. (2003) inoculated inhibitor-degrading bacteria and successfully eliminated the inhibitory effects on nitrification by 3-chloroaniline. However, bioaugmentation in wastewater treatment also showed frequent failures and inconclusive outcomes for the inadaptability of the introduced bacteria (Boon et al. 2000; El Fantroussi & Agathos 2005).

Considering the disadvantages of aforementioned elimination methods of nitrification inhibition, a biological method is proposed to culture inhibitor-degrading microbes in an activated sludge system directly. This will avoid inadaptability and improves the degradability of the microbial community for inhibitors as well. Currently, there is little information available about eliminating thiourea inhibition by biological methods or identifying thiourea-degrading bacteria. Accordingly, the nitrification inhibition characteristics of thiourea and its biodegradability need to be evaluated.

The objectives of the present study were: (1) to investigate the inhibitory effects of thiourea on nitrification process by determining IC50; (2) to assess the recoverability of the nitrification inhibited by thiourea and explore the biodegradability of thiourea in the activated sludge system; and (3) to isolate and identify thiourea-degrading bacteria that can use thiourea as the sole carbon and nitrogen source.

MATERIALS AND METHODS

Seed activated sludge

Activated sludge (VSS/SS = 82.7%) used in this study was collected from an aeration tank in the Changqiao municipal wastewater treatment plant (WWTP) in Shanghai, China, which uses an anoxic/oxic process to achieve organic carbon and nitrogen removal. The WWTP achieved significant nitrification, resulting in an effluent NH3-N concentration of 0.5–1.0 mg L−1 with a removal efficiency of 99.5%, with an approximately 20-day sludge retention time and a 2-day hydraulic retention time.

Determination of IC50 of thiourea on nitrification process

The inhibitory effects of thiourea on nitrification were defined as the concentration of thiourea inhibiting 50% nitrification, which was determined by a shake flask test by measuring the nitrification rates at different initial thiourea concentrations (0, 0.02, 0.05, 0.12, 0.23, 0.47 mg gVSS−1).

Before the test, a synthetic solution was prepared in the same concentration of ammonia (21.1 mg NH3 L−1) and municipal aerobic activated sludge (2.1–2.2 g MLVSS L−1). Prior to the experiment, the sludge was washed at least three times with deionized water to remove any inhibitors or nitrate present, then adjusted to pH 8.5–9.0 by adding Na2CO3 solution. Each flask was filled with 150 mL of the synthetic solution and placed in a thermostatic shaker at 20 ± 1 °C and 200 rpm for 24 h, during which time samples were taken every 30 min, centrifuged, and analyzed for NH3-N concentration. The NR, which ignored nitrogen by assimilation, was determined by the slope of the ammonia concentration profile against time.

The extent of thiourea inhibition was defined as In:
formula
where NR0 was the nitrification rate without thiourea and NRi was the nitrification rate under certain concentrations of thiourea (mg NH3-N gVSS−1 h−1). The percentage of inhibition was then plotted against the logarithm of the concentration of thiourea and the IC50 (In = 50%) was interpolated.

Determination of the recoverability of nitrification inhibited by thiourea

The recoverability of nitrification inhibition by thiourea was further determined in a 5-L sequencing batch reactor (SBR) seeded with 6.40 g SS L−1 (VSS/SS = 82.7%) activated sludge and operated at room temperature (20 ± 1 °C). The SBR was aerated for 20 h and exchanged 2.5 L of synthetic wastewater during one 24-h operation cycle. The synthetic influent wastewater was composed (mg L−1) of 0–100 thiourea, 1,400 glucose (calculated in chemical oxygen demand (COD)), 70 (NH4)2SO4 (calculated in NH3-N), and 14 KH2PO4 (calculated in P). The alkalinity consumed during nitrification was supplied by adding 1 M Na2CO3 solution to ensure that the pH of the effluent was maintained in the range of 7.0–7.4.

Cultivation of an activated sludge system with thiourea-degrading ability

To eliminate inhibition of nitrification by thiourea, activated sludge was cultivated to have thiourea-degrading ability in a 5-L SBR seeded with 4.90 g MLSS L−1 activated sludge. The SBR was operated at room temperature (20 ± 1 °C) and aerated for 20 h, during which time 2.5 L synthetic wastewater was exchanged in each 24-h operation cycle. The influent thiourea concentration increased in a stepwise manner from 5 to 50 mg L−1. Other substances added to the influent consisted (mg L−1) of 400–600 glucose (calculated in COD), 20–30 (NH4)2SO4 (calculated in NH3-N), and 4–5 KH2PO4 (calculated in P). The alkalinity consumed during cultivation was supplied by adding 1 M Na2CO3 solution to ensure that the pH of the effluent was maintained at 7.0–7.4. No sludge was discharged during cultivation, and the sludge retention time was 108 days, according to the statistical analysis.

Isolation and identification of the thiourea-degrading bacteria

To isolate and further identify the thiourea-degrading bacteria from the cultured activated sludge produced, 10 mL cultured sludge was seeded into 90 mL selective medium with a thiourea concentration of 5 mg L−1, and the seeded medium was cultured in a thermostatic shaker (150 rpm) at 30 ± 1 °C for 48 h. Next, 10 mL of the aforementioned culture medium was added into another 90 mL of selective medium with a higher thiourea concentration of 10 mg L−1 and cultured for 48 h. This process was repeated until the thiourea concentration of the medium was 25 mg L−1.

Next, 1 mL of the newly prepared culture medium was added uniformly into a solid medium that was cultured in an incubator at 35 °C. Individual strains were isolated from the solid medium by the streak plate method, after which the total community DNA was extracted and subjected to sequencing analysis (Packeiser et al. 2013).

Nucleotide sequence accession numbers

The rRNA gene sequences of the bacteria from the cultured activated sludge were deposited in the GenBank database under accession numbers KC997612–KC997615.

Analytical method of thiourea

The concentration of thiourea was determined by ultraviolet spectrophotometry. After a full wavelength scan of different concentrations of thiourea (0, 0.2, 1.0, 2.0, 4.0, 5.0, 10.0 mg L−1) using an ultraviolet spectrophotometer, 236 nm was selected as the characteristic wavelength. The thiourea concentration showed a linear correlation with its absorbance at 236 nm (linear regression equation: y = 7.105 x + 0.694, R2 = 0.9995, where x is the absorbance at 236 nm and y is the concentration of thiourea).

Other analytical methods

COD, NH3-N, NO2-N, NO3-N, mixed liquor suspended solids (MLSS), and mixed liquor volatile suspended solids (MLVSS) concentrations were determined using Standard Methods (APHA/AWWA/WEF 1998).

RESULTS AND DISCUSSION

Determination of the 50% IC of thiourea on nitrification

The 50% IC of thiourea on nitrification is shown in Figure 1. The percentage of inhibition against the logarithm of the concentration of thiourea is shown in Figure 2.
Figure 1

Nitrification processes in municipal aerobic activated sludge system under different concentrations (mg gVSS−1) of thiourea.

Figure 1

Nitrification processes in municipal aerobic activated sludge system under different concentrations (mg gVSS−1) of thiourea.

Figure 2

Simulation of the relationship between thiourea concentration and nitrification inhibition degree by Logistic function.

Figure 2

Simulation of the relationship between thiourea concentration and nitrification inhibition degree by Logistic function.

As shown in Figure 1, the NR of the activated sludge without thiourea was the largest, at 2.38 mg NH3-N gVSS−1 h−1. The NR decreased to 1.94 mg NH3-N gVSS−1 h−1 under 0.02 mg thiourea gVSS−1, while the In was 18.49%. With the thiourea loading increasing to 0.05 and 0.12 mg thiourea gVSS−1, respectively, the In increased to 40.76% and 61.34%, correspondingly. The NR continued to decrease to 0.11 mg NH3-N gVSS−1 h−1 as the concentration of thiourea increased to 0.47 mg gVSS−1, while the In increased as high as 95.38%. According to the fitting curve shown in Figure 2, the IC50 of thiourea on the nitrification process was 0.088 mg gVSS−1.

Many studies have investigated the IC of thiourea on nitrification. For example, Tomlinson et al. (1966) found that 0.076 mg L−1 thiourea would inhibit 75% of the ammonia oxidation by Nitrosomonas europaea isolated from the activated sludge in a municipal wastewater treatment plant. Vandevivere et al. (1998) found that 99% of the oxygen uptake rate of nitrifiers was inhibited after 4 h of exposure to 0.1 mg L−1 thiourea. The IC50 of thiourea was much lower than those of some recognized nitrification inhibitors or biotoxic materials, such as N-allylthiourea, 4-nitrophenol, 3,5-dichlorophenol, and cyanide (Han et al. 2014), which had IC50 values of 0.38, 43.3, 5.6, and 0.218 mg gVSS−1, respectively. Nevertheless, these findings indicate that thiourea had a strong inhibitory effect on nitrification.

The recoverability of nitrification after adding thiourea

The ability of the nitrifiers to recover from thiourea inhibition was also investigated in this study. As shown in Table 1, the reactor was operated for 40 days divided into six phases. No thiourea was added to the influent in phases ii, iv, and vi, respectively.

Table 1

Variation of operational parameters in the experiment of the inhibition of thiourea on nitrification and its recoverability

ParameterPhases
iiiiiiIvvvi
Time (day) 1–5 6–10 11–17 18–24 25–32 33–40 
The influent thiourea concentration (mg L−120 50 100 
ParameterPhases
iiiiiiIvvvi
Time (day) 1–5 6–10 11–17 18–24 25–32 33–40 
The influent thiourea concentration (mg L−120 50 100 

Figure 3 shows, in phase i, thiourea effluent increased from 3.25 to 7.85 mg L−1 on day 1 to day 3, then decreased slowly to 2.88 mg L−1 on day 5, when the average thiourea degradation rate was 0.056 mg gVSS−1 h−1. During the same period, the ammonia effluent concentration gradually increased from 6.7 to 20.8 mg L−1, and the NR was 0.19 mg NH3-N gVSS−1 h−1 at day 5. In phase ii, the thiourea effluent concentration from the reactor decreased gradually to undetectable limits, which agrees with a matching decline in ammonia effluent concentration, indicating the sludge nitrification ability to be recovered.
Figure 3

Variation of operational parameters in the experiment on the inhibition of thiourea on nitrification and its recoverability (n = 5).

Figure 3

Variation of operational parameters in the experiment on the inhibition of thiourea on nitrification and its recoverability (n = 5).

In phase iii, when thiourea concentrations decreased from 7.5 to 4.2 mg L−1, the ammonia concentration firstly increased to maximum, 11.7 mg L−1, on day 12, then decreased to 7.3 mg L−1 on day 17. In phase iv, the nitrification was recovered rendering undetectable ammonia effluent concentrations. A similar variation trend was observed in phase v and vi: with thiourea levels decreasing from 33.4 mg L−1 to undetectable, while ammonia increased to 15.3 mg L−1, then decreased to be undetectable. With no thiourea added (phases ii, iv and vi), the effluent ammonia concentration remained undetectable.

Based on the results described above, with residual concentrations of thiourea higher than its IC50 (0.088 mg gVSS−1), a strong inhibitory effect against nitrification was observed. As a result, the effluent ammonia concentration was as high as 20.8 mg L−1 in phase i. However, after the added thiourea was completely degraded, the nitrification ability of the activated sludge recovered slowly, indicating that thiourea only inhibited the activities of nitrifiers and was not lethal.

The inhibitory effect in phase iii and v was weaker than that in phase i, and the nitrification of the system recovered more quickly in phases iv and vi than in phase ii. This phenomenon can be attributed to the following reasons: (1) microbes that could degrade thiourea originally existed in the seeded activated sludge, and the thiourea-degrading ability of the activated sludge system was enhanced by continuous dosing of thiourea for a period of time, resulting in weakening of the inhibitory effects; (2) the nitrifiers' resistance to thiourea was enhanced after exposure to thiourea for a period of time.

Cultivation of thiourea-degrading bacteria

Based on the conjecture above, the influent thiourea concentration was increased stepwise from 0.5 mg L−1 to 150 mg L−1 to cultivate thiourea-degrading bacteria and eliminate the thiourea inhibitory effect on nitrification. The influent concentration of carbon and nitrogen sources was then reduced stepwise to screen the thiourea-degrading bacteria that used thiourea as their only carbon and nitrogen source. For these experiments, the reactor was operated for 100 days (Figure 4).
Figure 4

Cultivation of thiourea-degrading bacteria.

Figure 4

Cultivation of thiourea-degrading bacteria.

The 100-day experimental period was divided into two phases. In phase 1 (day 1 to day 58), the influent thiourea concentration was increased from 0.5 mg L−1 to 150 mg L−1 in a stepwise manner and the concentrations of COD and NH3-N were 400 mg L−1 and 20 mg L−1, respectively, resulting in effluent concentrations of thiourea, COD, and NH3-N below 5 mg L−1, 30 mg L−1, and 0.2 mg L−1, respectively. Moreover, every time the influent thiourea concentration increased there was a slight increase in the effluent NH3-N concentration, but it still remained below 5 mg L−1. From days 46 to 58, the influent concentrations of thiourea, COD, and NH3-N were 150 mg L−1, 600 mg L−1, and 30 mg L−1, while the effluent concentrations were below 0.5 mg L−1, 30 mg L−1, and 0.2 mg L−1, respectively.

Phase 2 was designed to cultivate thiourea-degrading bacteria that used thiourea as the sole carbon and nitrogen source. During this phase, the influent thiourea concentration remained 150 mg L−1 while the concentrations of COD and NH3-N were decreased stepwise from 400 mg L−1 and 20 mg L−1 to 0 mg L−1, respectively. These culture conditions were maintained for another 25 days, during which time the effluent concentrations of thiourea, COD, and NH3-N were below 0.5 mg L−1, 30 mg L−1, and 0.2 mg L−1, correspondingly.

Average thiourea-degrading rate increased to 26.6 mg gVSS−1 d−1 gradually as the influent thiourea concentration was increased stepwise to 150 mg L−1 in phase 1, and it was enhanced to 32.6 mg gVSS−1 d−1 after cultivation and enrichment of thiourea-degrading bacteria in phase 2 (results not shown). However, no inhibitory effect by thiourea was observed. The sludge concentration was increased from 3.66 to 5.87 g VSS L−1 during phase 1. In this phase, there was no obvious sludge wash-out and the sludge system performed steadily, which was likely because of the extra glucose added to the reactor. However, the dose of glucose had the potential to influence cultivation of thiourea-degrading bacteria; therefore, in phase 2, the influent concentration of glucose and ammonium sulfate was decreased stepwise to remove organisms that used glucose as their only carbon source or ammonium sulfate as their only nitrogen source. As a result, the sludge concentration of the system decreased from 5.87 g VSS L−1 to 3.63 g VSS L−1 and plateaued at the end of the cultivation period, after which thiourea-degrading bacteria that used only thiourea as their carbon and nitrogen source were believed to become the dominate microbes.

The thiourea-degrading ability improved greatly throughout the 100-day cultivation period. To calculate the maximum degradation rate of thiourea and investigate the nitrification process in this reactor, samples were collected on day 55 and day 95, after which the plots were analyzed for thiourea, COD, and NH3-N (Figure 5).
Figure 5

Variations of CS(NH2)2 (initial concentration of 75 mg L−1) and NH3-N (a) in the presence of glucose and ammonium sulfate with the influent COD and NH3-N concentrations of 600 mg L−1 and 30 mg L−1, and (b) in the absence of glucose and ammonium sulfate.

Figure 5

Variations of CS(NH2)2 (initial concentration of 75 mg L−1) and NH3-N (a) in the presence of glucose and ammonium sulfate with the influent COD and NH3-N concentrations of 600 mg L−1 and 30 mg L−1, and (b) in the absence of glucose and ammonium sulfate.

Figure 5(a) and 5(b) show the theoretically calculated initial concentrations for 0 h after the feeding period of the SBR. As shown in Figure 5(a), the COD decreased to 56 mg L−1 on day 55 in the presence of glucose and ammonium sulfate in the first half hour, then fell to equal or less than 30 mg L−1 in the next 5.5 h. The thiourea concentration decreased rapidly from 75 mg L−1 to 2 mg L−1 within 5 h, then decreased to 0.5 mg L−1 after 7 h of degradation. The NH3-N concentration decreased to 12.1 mg L−1 in the first 2 h, then increased to 19.6 mg L−1 in the next 2 h because of the joint effects of sludge absorption and rapid thiourea degradation, after which it linearly decreased to below 0.20 mg L−1, corresponding to the nearly complete degradation of thiourea. As shown in Figure 5(b), the thiourea concentration decreased below 0.5 mg L−1 within 7 h of the absence of glucose and ammonium sulfate. On day 95, the NH3-N increased to 5.34 mg L−1 gradually in the first 6 h, then declined to below the detection by 9 h.

As shown in Figure 5(a) and 5(b), in the presence of thiourea, the degradation of thiourea was prior to nitrification at the start of day 55 and day 95, which corresponded to the rise of NH3-N concentration. The NH3-N declined rapidly after the concentration of thiourea decreased to below 0.5 mg L−1, which again confirmed that the inhibition of thiourea was recoverable and the activities of nitrifiers were recovered when thiourea was completely degraded. The thiourea-degrading rate was 2.56 mg gVSS−1 h−1 on day 55 and 3.06 mg gVSS−1 h−1 on day 95, which was 55 times higher than that of the uncultured sludge system. The thiourea-degrading ability was greatly improved through cultivation in which the influent thiourea concentration was increased in a stepwise manner. These findings indicate that thiourea-degrading bacteria multiplied preferentially and occupied a larger proportion in the sludge system during this period. Hence, thiourea was degraded more rapidly and nitrification recovered more quickly.

The dominant thiourea-degrading bacteria strain was identified as Pseudomonas sp. NCIMB

To isolate and further identify the thiourea-degrading bacteria in the cultured activated sludge, samples were streaked for isolation and four colonies with the following characteristics were recovered from the HM plate: (a) white and oily; (b) white and dry; (c) faint yellow and oily; (d) yellow and oily. These colonies were denoted TDB1–TDB4, respectively. The dominant bacteria in these colonies was identified as Pseudomonas sp. (Figure 6).
Figure 6

Phylogeny of bacterial sequences obtained from the cultured activated sludge based on the neighbor-joining method. Bootstrap values (n = 1,000 replicates) of ≥50% are reported.

Figure 6

Phylogeny of bacterial sequences obtained from the cultured activated sludge based on the neighbor-joining method. Bootstrap values (n = 1,000 replicates) of ≥50% are reported.

None of the identified bacteria were reported to degrade thiourea, but all four could degrade compounds with structures similar to thiourea. Herzog et al. (2013) found that Pseudomonas sp. SMX345 could use sulfamethoxazole and that it relied on sulfonyl as its sole nutrient source. According to a study by Aguilar et al. (2008), sulfide could be oxidized by Pseudomonas stutzeri. Peressutti et al. (2008) found that a bacterial consortium containing Pseudomonas alcaliphila strain D11 was capable of completing the degradation of linear alkylbenzene sulfonate. Kumar et al. (2008) found that endosulfan could be oxidized by Pseudomonas sp. BI205, and inorganic sulfide could be oxidized by the Gram-negative bacterium W1G-2, which was very similar to the results reported for Pseudomonas sp. BI205 in a study by Duncan et al. (2001). However, this is the first report of the thiourea degradation potential of bacterial isolates.

The results of this study indicated that four identified thiourea-degrading bacteria isolated from a cultured activated sludge system contributed to rapid thiourea degradation, achieving complete elimination of thiourea inhibition of nitrification.

CONCLUSIONS

Thiourea showed a strong inhibitory effect on nitrifiers, and the IC50 of thiourea on nitrification was determined to be 0.088 mg gVSS−1. The inhibitory effect of thiourea on nitrification was recoverable, and the nitrification process recovered after thiourea was completely degraded. The dominant thiourea-degrading bacteria strain identified was Pseudomonas sp. NCIMB. The results presented herein will facilitate future studies investigating the biological treatment of thiourea wastewater and the biodegradation characteristics of thiourea and similar pollutants.

ACKNOWLEDGEMENTS

This study was financially supported by National Environmental Protection Public Welfare Science and Technology Research Program of China (No. 201309047) and National Natural Science Foundation China (No. 41201302). In addition, the authors would like to thank all group members for their assistance with experiments.

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