Biodegradation of aniline from hypersaline environments using halophilic microorganisms

Abstract

The present invention relates to a method for reducing the aniline content of hypersaline wastewater, said method comprising the steps of (a) providing a composition A comprising hypersaline wastewater and aniline, and (b) contacting composition A with cells of at least one halophilic microbial strain, thereby generating a composition B comprising said composition A and cells of said at least one halophilic microbial strain. The present invention further concerns a method for the production of chlorine and sodium hydroxide. Further encompassed by the present invention is a composition comprising hypersaline wastewater, aniline, and cells of at least one halophilic microbial strain.

Claims

1. A method for reducing the aniline content of a composition comprising hypersaline wastewater, said method comprising the steps of: (a) providing a composition A comprising hypersaline wastewater and aniline, and (b) contacting composition A with cells of at least one halophilic microbial strain, selected from Halomonas alimentaria cells and/or Haloferax sp. D1227 cells, thereby generating a composition B comprising said composition A and cells of said at least one halophilic microbial strain, wherein said composition B comprises NaCl in a concentration of more than 6% (w/v), and (c) incubating composition B, thereby reducing the aniline content of the composition.

2. The method of claim 1, wherein said composition B comprises NaCl in a concentration of at least 8% (w/v).

3. The method of claim 1, wherein said composition B comprises NaCl in a concentration of a least 10% (w/v).

4. The method of claim 1, wherein the aniline content of the composition B comprises at least 0.5 mg/l aniline.

5. The method of claim 1, wherein the step (a) comprises isolating the hypersaline wastewater from methylene diamine production.

6. The method of claim 1, wherein said composition B further comprises a substrate that allows for the growth of the cells of at the least one halophilic microbial strain.

7. The method of claim 1, wherein the incubation in step (c) is carried out at temperature of 18° C. to 45° C. and/or wherein the incubation in step (c) is carried out at a pH value in the range of 5.8 to 8.5.

8. The method of claim 1, wherein said incubation in step (c) is carried out under aerobic conditions.

9. The method of claim 1, wherein composition B further comprises formate.

10. The method of claim 1, wherein said method further comprises the separation of the cells of the at least one halophilic microbial strain from composition B, thereby giving composition C, and optionally wherein the method further comprises concentrating composition C, thereby giving composition C.

11. A method for the production of chlorine and sodium hydroxide, comprising the steps of (i) providing a composition C or C* according to the method of claim 10, and (ii) subjecting the composition according to (a) to a sodium chloride electrolysis process, thereby producing chlorine and sodium hydroxide.

12. The method of claim 11, wherein the sodium chloride electrolysis is selected from membrane cell electrolysis of sodium chloride.

13. A composition B comprising hypersaline wastewater, aniline, and cells of at least one halophilic microbial strain, selected from Halomonas alimentaria cells and/or Haloferax sp. D1227 cells, wherein said composition B comprises NaCl in a concentration of more than 6% (w/v).

14. The method of claim 1, wherein said composition B comprises NaCl in a concentration of a least 12% (w/v).

15. The method of claim 1 wherein the aniline content of the composition B comprises at least 5 mg/l aniline.

16. The method of claim 1, wherein said composition B further comprises a substrate that allows for the growth of the cells of at the least one halophilic microbial strain, wherein the substrate is a carbohydrate.

17. The method of claim 1, wherein said composition B further comprises a substrate that allows for the growth of the cells of at the least one halophilic microbial strain, wherein the substrate is selected from the group consisting of glycerol, acetate, glucose, sucrose, lactate, malate, succinate, and citrate.

18. The method of claim 11, wherein the sodium chloride electrolysis is membrane electrolysis using oxygen consuming electrodes, diaphragm cell electrolysis of sodium chloride and mercury cell electrolysis of sodium chloride.

Description

(1) The Figures show:

(2) FIG. 1 Graph shows aniline reduction by strain Halomonas alimentaria at NaCl concentration of 15% w/v in synthetic media containing 10 and 20 mg/l aniline. Biomass was reduced and aniline was degraded. In presence of a second substrate 50 mg/l phenol still no increase in biomass concentration occurred.

(3) FIG. 2 Shows aniline degradation by strain Haloferax sp. D1227 in synthetic media containing 15% w/v NaCl and 20 mg/l aniline. 50% of Aniline is degraded after 48 hours of incubation. Reduction in optical density at 600 nm is shown. In presence of a second substrate 50 mg/l phenol increase in biomass concentration as well as reduction in phenol and aniline is detected.

(4) FIG. 3 Aniline degradation at various salt concentrations (0 to 20% w/v NaCl) using cells of halotolerant bacteria Halomonas alimentaria. Rate of aniline degradation is higher at higher salt concentrations. Best aniline degradation occurs at NaCl concentration of 18% w/v.

(5) FIG. 4 The graph illustrates aniline degradation at various salt concentrations (0 to 20% w/v NaCl) using cells of halophilic bacteria Haloferax sp. D1227. Rate of aniline degradation is higher at higher salt concentrations. Best aniline degradation occurs at NaCl concentration of >14% w/v.

(6) FIG. 5 The coefficient plot shows significance of two factors initial aniline concentration and NaCl concentration on aniline degradation for strain Halomonas alimentaria. At lower salt and aniline concentration better removal can be observed. pH seem to have no significance on aniline removal. Aniline degradation by H. alimentaria occurs at all NaCl concentrations.

(7) FIG. 6 The response counter plot shows the optimal aniline degradation may occur at lower initial aniline concentrations and lower NaCl concentrations for the strain Halomonas alimentaria.

(8) FIG. 7 Shows formate uptake by strain Halomonas alimentaria on an industrial residual media containing 15% w/v NaCl. Formate is reduced and biomass increases over time.

(9) All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

(10) The invention will be merely illustrated by the following Examples. The said Examples shall, whatsoever, not be construed in a manner limiting the scope of the invention.

EXAMPLES

Example 1: Aniline Degradation in Shake Flask Experiments

(11) Strains and Media

(12) Halomonas alimentaria (DSM 15356) (in this study H. alimentaria) wild type was purchased from dsmz. Shake-flask cultures for inoculum preparation were grown under 120 rpm and 30° C. in laboratory incubator (Infors, Switzerland) on media no. 514 suggested by dsmz with some modifications and following compositions (g/l): Yeast extract 5.0, Fe (III) citrate 0.1, NaCl 19.45, MgCl.sub.2 5.9, Na.sub.2SO.sub.4 3.24, CaCl.sub.2 1.80, KCl 0.55, NaHCO.sub.3 0.16, KBr 0.08 and trace elements in mg/l SrCl.sub.2 34.0, H.sub.3BO.sub.3 22.0, Na-Silicate 4.0, NaF 2.40, (NH.sub.4)NO.sub.3 1.60, Na.sub.2HPO.sub.4 8.0; pH 7.6.

(13) Haloferax sp. D1227 (ATCC 51408) (in this study D1227) was purchased from American type culture collection. Shake-flask cultures for inoculum preparation were grown under 170 rpm and 35° C. with following media compositions (g/l): (NH.sub.4).sub.2SO.sub.4 0.33, KCl 6.0, MgCl.sub.2.6H.sub.2O 12.1, MgSO.sub.4. 7H.sub.2O 14.8, KH.sub.2PO.sub.4 0.34, CaCl.sub.2.2H.sub.2O 0.36, NaCl 100.0, Yeast extract 3.0, Tryptone 3.0; pH 6.8.

(14) Analytics

(15) Turbidity as indicator for cell growth was measured using Shimadzu UV/Vis spectrophotometer at 600 nm in 12 hour intervals. Residual aniline concentration in the culture supernatant was measured using HPLC. The HPLC (Thermo-Fisher) method was performed with an Acclaim PA C-16-3 μm column (Thermo-Fisher). Acetonitrile, 25 mM KH.sub.2PO.sub.4 pH 3.5 and MQ were used as the mobile phase and detection was done with UV at 190 nm. With an injection volume of 5 μl the limit of quantification for aniline is 1 ppm. Lower concentrations of aniline were also detectable.

(16) Aniline Uptake Studies in Shake Flask

(17) For the aniline uptake studies synthetically defined media was prepared for every strain. The media composition are listed below:

(18) TABLE-US-00001 TABLE 1 Synthetic defined media (top table) and trace elements (bottom table) composition for strain Haloferax sp. D1227 Composition Amount g/l NaCl 140 NH.sub.4Cl 1.50 KH.sub.2PO.sub.4 0.15 FeCl.sub.3 0.005 MgCl.sub.2•6H.sub.2O 1.30 MgSO.sub.4•7H.sub.2O 1.10 CaCl.sub.2•2H.sub.2O 0.55 KCl 1.66 NaHCO.sub.3 0.20 KBr 0.50 MnCl.sub.2•4H.sub.2O 0.003 Trace elements   1 ml Aniline 99% 5-100 mg Composition Amount mg/100 ml FeSO.sub.4•7H.sub.2O 136 CuSO.sub.4•5H.sub.2O 100 MnCl.sub.2•4H.sub.2O 50 CoCl.sub.2•2H.sub.2O 44 ZnSO.sub.4•7H.sub.2O 86

(19) TABLE-US-00002 TABLE 2 Synthetic defined media composition for the strain Halomonas alimentaria Composition Amount g/l NaCl 140 MgCl.sub.2 5.90 Na.sub.2SO.sub.4 3.24 CaCl.sub.2 1.80 KCl 0.55 KBr 0.08 SrCl.sub.2  34 mg H.sub.3BO.sub.3  22 mg Na-Silicate 4.0 mg NaF 2.4 mg (NH.sub.4)NO.sub.3 1.6 mg Na.sub.2HPO.sub.4 8.0 mg Aniline 99% 5-100 mg 

(20) The strains were individually studied to check aniline uptake. Cells previously grown on the complex media were harvested by centrifugation at 3000 rpm, for 5 minutes. Cells were washed and dissolved in 500 ml shake-flasks containing 100 ml of their respective synthetic defined media and 15% w/v NaCl with aniline as only carbon source and were incubated at the respective temperature and agitation mentioned previously. The zero hour OD.sub.600 was measured and one ml sample was stored for HPLC analysis as zero hour reference. Growth on aniline and the residual aniline concentration was monitored. The strains D1227 and H. alimentaria used in this study fail to use aniline as a source for growth, however, the residual aniline concentration over time showed that aniline was completely removed from the culture media both on synthetic media and on actual brine depending on initial aniline concentration. In presence of a second substrate in this case phenol 50 to 100 mg/l for D.1227 increase in biomass concentration and better aniline degradation was detected. The degradation of aniline was investigated in more details in more experiments in shake flasks as well as bioreactor in order to be able to control other process parameters.

Example 2: Optimum Culture Conditions for Aniline Degradation

(21) Aniline Studies Using Multivariate Design of Experiments

(22) In order to find the optimum conditions for aniline degradation by halophilic strain Halomonas alimentaria multivariate approach for design of experiments was used.

(23) For the strain H. alimentaria a fractional factorial design of experiment was carried out to evaluate the influence of three factors (pH, initial aniline concentration and NaCl concentration) on three parameters (delta biomass, residual aniline concentration and delta pH). The factors and their ranges studied are given in the following table 3:

(24) TABLE-US-00003 TABLE 3 The factors and responses studied for aniline degradation by H. alimentaria Factor name Ranges pH 6.5 to 8.5 aniline concentration 10 to 50 mg/l NaCl 5 to 15% w/v

(25) The experiments were performed in the shake flasks at 30° C. and 120 rpm strokes. Increase in biomass concentration, pH changes and the residual aniline concentration were determined at 24 hour intervals. The measurements obtained after 144 hours were analyzed by Modde (statistical tool).

(26) In case of H. alimentaria better aniline degradation was observed at lower NaCl concentrations of 5 and 10% w/v and lower pH and aniline concentration of 10 mg/l respectively.

Example 3: Aniline Degradation at Various Salt Concentrations

(27) Degradation of aniline was studied for three different strains at various salt concentrations of 0 to 20% w/v in synthetic media containing 30 mg/l aniline as carbon source in shake flask experiments. The residual aniline concentration was determined by HPLC at 24 hour intervals. For the strain D1227 better aniline degradation occurred at NaCl concentrations of above 10% where the best degradation was at 16% w/v NaCl. For the strain H. alimentaria best aniline degradation was 70% of total aniline content at 18% w/v NaCl after 192 hours of incubation.

Example 4: Formate Removal Experiment by Halomonas alimentaria in Shake Flask

(28) Formate studies were done on an actual brine containing 15% w/v NaCl and 270 mg/l formate. For this experiment cells were previously grown on complex media (composition given in Example 1 strain and media section) at 30° C. with 120 rpm strokes. Cells from their late exponential growth phase were harvested by centrifugation for 5 minutes at 3000 rpm and 25 OC. Cells were washed and re-suspended in the brine with formate as only carbon source and media components given in Table 2; pH 7.2. Biomass concentration at zero hour was determined spectrophotometrically and the initial formate concentration was determined by HPLC. Residual formate concentration and biomass concentration were determined at 24 hour intervals. The results obtained showed slight increase in biomass concentration along with formate reduction over time. The results obtained suggest formate was removed and slight biomass increase was detected on formate as only substrate.

Example 5: Formate Degradation by Haloferax sp. D1227

(29) A multivariate design of experiment approach was chosen to check formate uptake by Haloferax sp. D1227. Influence of two factors formate concentration and NaCl concentration (see table 4) was studied on formate uptake and biomass concentration. Experiments were performed in shake flasks on synthetic media with formate as only substrate and media components given in Example 1 at pH 6.8, temperature 35° C. and 150 rpm strokes. Residual formate concentration and biomass were determined in 24 hour intervals. HPLC results obtained up to 168 hours of incubation suggest formate stayed intact during the experiment and biomass showed decrease in all experiments. It could be concluded that formate is neither degraded by Haloferax sp. D1227 nor taken up by these cells as energy source. In table 5 list of the halophilic and halotolerant strains which we have studied for formate degradation are given.

(30) TABLE-US-00004 TABLE 4 Factor name Studied range NaCl concentration   10 to 20% w/v Formate concentration 0.25 to 1 g/l

(31) TABLE-US-00005 TABLE 5 Formate NaCl concentration Strain name degradation % w/v Halomonas alimentaria DSM 514 Yes 2 to 20 Haloferax sp. D1227 No Non ATCC Natronobacterium gregoryi No Non DSM 3393 Halobacterium salinarum No Non DSM 669 Haloferax Volcanii No Non DSM

Example 6: Aniline Degradation in Additional Shake Flask Experiments

(32) Additional halophilic and halotolerant strains were studied for Aniline degradation at various salt concentrations. The results are shown in the following table 6.

(33) TABLE-US-00006 TABLE 6 Halophilic and halotolerant strains studied for Aniline degradation NaCl conc. Ranges Strain name Aniline degradation % w/v Halomonas alimentaria Yes about 2 to 20% DSM 514 Haloferax D.1227 Yes about 2 to 25% ATCC 51408 Halobacterium salinarum No — DSM 669 Natronobacterium gregoryi No — DSM 3393

SUMMARY—CONCLUSIONS

(34) Hypersaline wastewaters frequently comprise aniline and formate.

(35) Aniline is the major industrial chemical intermediate which is used in the manufacturing of herbicides, developers, perfumes, medicine, rubber and dyes. Several physical and chemical methods, for example adsorption, ozonation and electrochemical treatment, are used to treat aniline-containing salty residual streams. Most of the mentioned treatments are not able to reduce the total organic carbon content in the salty residual streams down to the required maximum level.

(36) In this invention, it was discovered that Halomonas alimentaria cells can degrade aniline from hypersaline environments. Further, it was discovered that Halomonas alimentaria cells and Haloferax sp. D1227 can degrade aniline and formate from hypersaline environments. This invention can be directed to optimal and efficient treatment of any hypersaline water containing aniline intending to reduce the total organic carbon content.

(37) The other aspect of this current invention comprises the concept of residual to value. On one hand the environmental issues caused by highly saline residual streams enriched with considerable amount of unwanted organic contaminants and on the other hand need for high quality saline water as precursor for other industrial processes, such as membrane electrolysis, make the pretreatments of these hypersaline residual streams, absolutely crucial. The invention helps achieving this cheap, quick and efficient pretreatment to meet the requirement for membrane electrolysis to produce chlorine and/or sodium hydroxide.