BIODEGRADATION OF ORGANIC POLLUTANTS BY A HALOPHILIC ARCHAEA

Abstract

The present invention relates to a method for reducing the content of at least one pollutant selected from the group consisting of nitrobenzene, formate, phenol, 4,4′-Methylenedianilinc (MDA) and aniline of hypersaline wastewater, said method comprising the steps of (a) providing a composition A comprising hypersaline wastewater and said at least one pollutant, and (b) contacting composition A with Haloferax mediterranei cells, 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, said at least one pollutant, and Haloferax mediterranei cells.

Claims

1.-16. (canceled)

17. A method for reducing the content of at least one pollutant selected from the group consisting of nitrobenzene, formate, phenol, methylenedianiline, in particular 4,4′-Methylenedianiline (MDA), and aniline in hypersaline wastewater, said method comprising the steps of: (a) providing a composition A comprising hypersaline wastewater and said at least one pollutant, and (b) contacting composition A with Haloferax mediterranei cells, thereby generating a composition B comprising said composition A and Haloferax mediterranei cells.

18. The method of claim 17, wherein said composition B comprises NaCl in a concentration of at least 6% (w/v), based on the total volume of composition or wastewater (e.g. of composition B).

19. The method of claim 17, wherein said composition B comprises at least 0.5 mg/l formate and/or 0.5 mg/l phenol and/or 0.5 mg/l nitrobenzene and/or 0.5 mg/l 4,4′-Methylenedianiline (MDA) and/or 0.5 mg/l aniline.

20. The method of claim 17, wherein the hypersaline wastewater is derived from the production of diaryl carbonates, the production of polycarbonates, or the production of diamines and polyamines of the diphenylmethane series.

21. The method of claim 17, wherein said composition B further comprises a substrate that allows for the growth of the Haloferax mediterranei cells, in particular wherein said substrate has been added to composition B, preferably said substrate is a carbohydrate, in particular glycerol, a sugar such as glucose or sucrose or an organic acid such as acetate, lactate, malate, succinate or citrate.

22. The method of claim 17, wherein the method further comprises step (c) of incubating composition B, thereby reducing the content of said at least one pollutant.

23. The method of claim 22, wherein the incubation in step (c) is carried out at temperature of 18° C. to 55° C. and/or wherein the incubation in step (c) is carried out at a pH value in the range of 6.0 to 8.2.

24. The method of claim 22, wherein said incubation in step (c) is carried out under aerobic conditions.

25. The method of claim 17, wherein Haloferax mediterranei cells are Haloferax mediterranei DSM.1411 cells.

26. The method of claim 17, wherein the total content of the at least on pollutant is reduced by at least 30%.

27. The method of claim 17, wherein said method further comprises the separation of the cells from composition B, thereby giving composition C, and optionally wherein the method further comprises concentrating composition C, thereby giving composition C*.

28. The method of claim 27, wherein said method further comprises the removal of inorganic components from composition C or C*, in particular of trace elements and/or salts of media components.

29. 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 27, and (ii) subjecting the composition according to (a) to a sodium chloride electrolysis process, thereby producing chlorine and sodium hydroxide and optionally hydrogen.

30. The method of claim 29, wherein the sodium chloride electrolysis is selected from membrane cell electrolysis of sodium chloride.

31. A method comprising utilizing Haloferax mediterranei cells for reducing the content of at least one pollutant selected from the group consisting of nitrobenzene, formate, phenol, methylenedianiline, in particular 4,4′-Methylenedianiline (MDA), and aniline in hypersaline wastewater.

32. A composition B comprising hypersaline wastewater, at least one pollutant selected from the group consisting of nitrobenzene, formate, phenol, methylenedianiline, and aniline, and Haloferax mediterranei cells.

Description

[0150] The Figures show:

[0151] FIG. 1. The graph illustrates formate degradation in real brine (15% w/v NaCl) after addition of 1.4 g/L Glycerol as co-substrate using cells of halophilic bacteria Haloferax mediterranei. Formate is degraded simultaneously with Glycerol (indicated with triangles). In the control experiment (indicated with Asterisks) shake flasks containing no cells neither Glycerol nor Formate was degraded.

[0152] FIG. 2. Graph represents degradation of aniline (5 and 10 mg/l) using Haloferax mediterranei cells in model media containing 15% w/v NaCl. After 240 hours of incubation and monitoring no biomass growth is observed on aniline as only substrate. In presence of a second substrate (50 mg/l phenol) increase in biomass concentration along with aniline and phenol reduction is shown. Rate of aniline degradation is dependent on the initial aniline concentration. In control experiment without bacterial cells no significant change in aniline or phenol concentration is detected.

[0153] FIG. 3. The coefficient plot shows significance of two factors initial aniline concentration and pH on aniline degradation for strain Haloferax mediterranei. NaCl concentration seem to have no significance on aniline removal, aniline degradation by HFX occurs at all NaCl concentrations.

[0154] FIG. 4. The response counter plot shows the optimal aniline degradation may occurs at lower initial aniline concentrations for the strain Haloferax mediterranei.

[0155] FIG. 5. The graph illustrates aniline degradation at various salt concentrations (0 to 20% w/v NaCl) using cells of halophilic bacteria Haloferax mediterranei. Rate of aniline degradation is higher at higher salt concentrations.

[0156] FIG. 6. The graph illustrates nitrobenzene degradation at 15% w/v salt concentrations using cells of halophilic archaea Haloferax mediterranei. 80% of total nitrobenzene content is degraded in the first 24 hours of incubation. In control experiment with 30 ppm nitrobenzene no significant reduction in nitrobenzene content is observed.

[0157] FIG. 7. Continuous processing of industrial brine containing 0.3 g/L formate using 5 g/L biomass of Haloferax mediterranei. Formate was degraded. The amount of residual formate concentration is dependent on the Dilution rate. The co-substrate glycerol is needed for biomass growth and is taken up completely in steady state condition.

[0158] 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.

[0159] 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: Degradation Experiments in Shake Flasks

[0160] Strains and Media

[0161] Haloferax mediterranei (DSM 1411) (in this study HFX) wild type strain was purchased from DSMZ—German collection of microorganisms and cell cultures. Shake-flask cultures for inoculum preparation were grown under 180 rpm and 37° C. in laboratory incubator (Infors, Switzerland) with slight modification in media no. 97 suggested by DSMZ having the following compositions (g/l): NaCl 250, MgSO.sub.4.7H.sub.2O 20.0, KCl 2.0, Na-Citrate 3.0, FeSO.sub.4.7H.sub.2O 0.05, MnSO.sub.4.H.sub.2O, yeast extract 10.0 and Glucose 5.0; pH 7.0. The 500 ml Erlenmeyer flasks and the media were always sterilized.

[0162] Analytics

[0163] Turbidity as indicator for cell growth was measured using Shimadzu UV/Vis spectrophotometer at 600 nm in various time intervals.

[0164] Residual formate, acetate and glycerol concentration in the culture supernatant was measured using HPLC. The HPLC (Thermo-Fisher) method was performed with an Aminex HPX-87H column from Bio-Rad at 30° C., an isocratic eluent of 0.1% TFA in MQ water with a flow of 0.5 ml/min followed by UV detection at 210 nm. The limit of quantification with injection volume of 20 μl was 5 mg/l for formate and acetate. The standards used for quantification were prepared in the same salty matrix as the samples.

[0165] Residual aniline, phenol, nitrobenzene and MDA 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 buffer 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 1 ppm, for phenol 0.5 mg, for nitrobenzene 1 ppm, and for 4,4′MDA 0.1 mg/l. Lower concentrations were also detectable.

[0166] Formate Degradation in Shake Flask Experiments

[0167] Haloferax mediterranei (DSM 1411) is an extreme halophilic Archaeon which requires at least 10% (w/v) NaCl for growth. Optimal growth is reported for concentrations of 20-25% NaCl (w/v).

[0168] For formate studies cells were cultivated in real industrial brine containing 0.37 g/L Formate and 15% (w/v) NaCl. The media components were supplemented according to table 1 after adjusting the pH of the brine to 7.0. The medium had not been sterilized prior to fermentation.

[0169] Medium D is a synthetic medium with the composition indicated in table 2.

TABLE-US-00001 TABLE 1 Media components added to the brine at pH 7.0 for Medium A, B and C. Composition Amount g/1 Carbon Source: A) Glycerol or A) 1.4 g/L B) Acetate or B) 3.5 g/L C) Formate C) 6.0 g/L 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 MgSO4.7H.sub.2O 1.10 CaCl2.2H.sub.2O 0.55 KCl 1.66 KBr 0.50 MnCl.sub.2.4H.sub.2O 0.003 Trace elements (Tab.3) 1 ml

[0170] Shake-flasks were inoculated with preculture that does not contain any complex carbon or nitrogen source. The inoculum was free of any residual carbon source. Shake flasks were inoculated to achieve a start OD of 0.25 in a total volume of 200 ml. Cells were grown in laboratory incubator (Infors, Switzerland) with 180 rpm and 37° C. using 500 ml Shake flasks.

[0171] Experiments were done in duplicates together with a control experiment with only medium and no cells.

TABLE-US-00002 TABLE 2 Media components for synthetic medium D (pH 7.0). Composition Amount g/1 NaCl 150 Formate 6.0 g/1 NH4Cl 1.50 KH2P 04 0.15 FeCl1 0.005 MgCl2.6H2O 1.30 MgSO4.7H2O 1.10 CaCl2.2H2O 0.55 KCl 1.66 KI3r 0.50 MnCl2.4H2O 0.003 Trace elements (Tab.3) 1 ml

TABLE-US-00003 TABLE 3 Composition of trace elements stock solution. Composition Amount mg/100m1 FeSO.sub.47H.sub.2O 139 CuSO.sub.4.5H.sub.2O 100 MnCl.sub.2.4H.sub.2O  78 CoCl.sub.2.2H.sub.2O  62 ZnSO.sub.4.7H.sub.2O  86

[0172] The ability of HFX to degrade formate was investigated in shake flask experiments. Three different media were prepared with addition of A) Glycerol, B) Acetate and C) Formate. While A and B should show whether HFX can take up formate in presence of a co-substrate, trial C should show if formate supports growth of HFX. Both cell growth and formate degradation was monitored by numerous sampling.

[0173] It could be detected that formate was degraded rapidly when a second substrate (e.g. Glycerol or Acetate) was present. In those experiments formate and co-substrate were taken up simultaneously.

[0174] By supplementing additional formate to the industrial brine (medium C), it was investigated if formate could be taken up and be converted into biomass. The results showed that formate was degraded but no growth was detectable, indicating that formate is used by HFX as energy source but not for biomass production.

[0175] Further experiment showed HFX requires a second carbon source for degradation of formate. In contrast to medium C, that is an industrial waste stream that is known to contain organic pollutes like Aniline, MDA and Nitrobenzene, the medium D only contained formate as carbon source. For this medium neither growth nor degradation of formate could be detected.

Example 2: Aniline and Phenol Uptake Studies in Shake Flask

[0176] For the aniline uptake studies synthetically defined media was prepared. The media composition is listed below:

TABLE-US-00004 TABLE 4 Synthetic defined media and trace elements composition to study phenol and aniline uptake by Haloferax mediterranei Composition Amount g/1 NaCl 150 NH4Cl 1.50 KH2PO4 0.15 FeCl3 0.005 MgCl2.6H2O 1.30 MgSO4.7H2O 1.10 CaCl2.2H2O 0.55 KCI 1.66 NaHCO3 0.20 KI3r 0.50 MnCl2.4H2O 0.003 Trace elements l ml Aniline 99% 5-100 mg Phenol 50 to 100 mg Trace Elements Composition Amount mg/100m1 FeSO4.7H2O 136 CuSO4.5H2O 100 MnCl2.4H2O 50 CoCl2.2H70 44 ZnSO4.71-120 86

[0177] The strain was studied for 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 shake-flasks containing 100 ml of the respective synthetic defined media and 15% w/v NaCl with aniline as only carbon source and were incubated at temperature of 37° C. and agitation. At zero hour OD600 was measured and one ml sample was stored for HPLC analysis as reference. Growth on aniline and the residual aniline concentration was monitored. The strains Haloferax mediterranei fail to use aniline as a source for growth however the residual aniline concentration over time show 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 increase in biomass concentration and better aniline degradation was detected. Growth of phenol was also detected in absence of aniline. The degradation of aniline and phenol 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 3: Optimum Culture Conditions for Aniline Degradation

[0178] Aniline Studies Using Multivariate Design of Experiments

[0179] In order to find the optimum conditions for aniline degradation by HFX multivariate design of experiments was used. A fractional factorial design of experiment was carried out to evaluate the influence of three factors (pH, aniline concentration and NaCl concentration) on three parameters (delta biomass, residual aniline concentration and pH). The factors studied in this experiment along with respective ranges are given in (Table 5).

TABLE-US-00005 TABLE 5 The factors and responses studied for aniline degradation by HFX Factor name Ranges pH 6.2 to 8.2 aniline concentration 15 to 50 mg/l NaCl 12 to 18% w/v

[0180] Eleven experiments were suggested by the statistical tool, Modde for this study. The experiments were performed in shake flasks on synthetic defined media at 37° C. and 170 rpm strokes. 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.

[0181] A valid model was obtained for delta aniline. Aniline was degraded in all experiments. The coefficient plot showed significance of initial aniline concentration and pH on aniline degradation. The best aniline degradation by HFX cells occurred at pH 6.2, 12% w/v NaCl and 15 mg/l aniline where after 144 hours 94% aniline was degraded.

[0182] Aniline Degradation at Various Salt Concentrations

[0183] Degradation of aniline was studied 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 HFX better aniline degradation occurred at a higher NaCl concentration of above 14% w/v where the best degradation was at 20% w/v NaCl.

Example 4: Nitrobenzene Degradation by Haloferax mediterranei

[0184] Primary Shake Flask Experiments on Nitrobenzene

[0185] For nitrobenzene uptake studies synthetically defined media was prepared. The media composition used is given in (Table 1) where instead of aniline, 30 and 50 ppm nitrobenzene was used as only substrate. Cells previously grown on the complex media were harvested by centrifugation at 3000 rpm, for 5 minutes. Cells were washed and dissolved in shake-flasks containing 100 ml of the respective synthetic defined media and 15% w/v NaCl with nitrobenzene as only carbon source and were incubated at temperature of 37° C. and agitation of 170 rpm. At zero hour OD600 was measured and one ml sample was stored for HPLC analysis as reference. Growth on nitrobenzene and the residual nitrobenzene concentration was monitored. The strain Haloferax mediterranei did not use nitrobenzene as a source for growth however the residual nitrobenzene concentration over time show it was completely removed from the culture media both on synthetic media and on actual brine. Highest degradation rate was observed during the first 24 hours. The degradation of nitrobenzene 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.

[0186] Nitrobenzene Studies Using Multivariate Design of Experiments

[0187] In order to find the optimum conditions for nitrobenzene degradation by HFX it was studied using multivariate design of experiments. A fractional factorial design of experiment was carried out to evaluate the influence of three factors (pH, nitrobenzene concentration and NaCl concentration) on three parameters (delta biomass, residual nitrobenzene concentration and delta pH). The factors studied in this experiment along with respective ranges are given in (Table 6).

TABLE-US-00006 TABLE 6 The factors and responses studied for nitrobenzene degradation by HFX Factor name Ranges pH 6 to 8 nitrobenzene concentration 5 to 25 mg/1 NaCl 15 to 20% w/v

[0188] Eleven experiments were suggested by the statistical tool, Modde for this study. The experiments were performed in shake flasks on synthetic defined media at 37° C. and 170 rpm strokes. Biomass concentration, pH changes and the residual nitrobenzene concentration were determined at 24 hour intervals. The measurements obtained after 72 hours were analyzed by Modde, shown in table 7.

TABLE-US-00007 TABLE 7 Shows the results obtained for nitrobenzene degradation by HFX. NaCl % Exp pH Nitrobenzene w/v Δ OD600 Degradation % N1 6 5 15 −0.099 60.1 N2 8 5 15 −0.098 80 N3 6 25 15 −0.093 81 N4 8 25 15 −0.08 64.5 N5 6 5 20 −0.117 80.4 N6 8 5 20 −0.136 80.4 N7 6 25 20 −0.124 89.3 N8 8 25 20 −0.128 87.7 N9 7 15 17.5 −0.106 100 N10 7 15 17.5 −0.094 100 N11 7 15 17.5 −0.108 100 Control 7 15 17.5 0.01 13

[0189] Table 7 shows the experimental matrix and the results obtained. Nitrobenzene was degraded in almost all experiments. Reduction in biomass concentration was observed in all experiments. In control experiment without cells 13% nitrobenzene oxidation occurs in compare to 100% removal in experiments N9 to 11 which show 87% higher degradation in presence of HFX cells. The best nitrobenzene degradation by HFX cells occur at CenterPoint experiments with pH 7.0, 17.5% w/v NaCl and 15 mg/l nitrobenzene where after 72 hours 100% nitrobenzene was degraded.

Example 5: Degradation of Aniline, Phenol, Nitrobenzene and 4, 4′MDA in Actual Brine in Batch Mode

[0190] Cultivation was established in bioreactor in order to check applicability of degrading strain in actual processes on an industrial residual water. For this experiment HFX cells were used for the process using an actual brine containing 15% w/v NaCl. In this case process parameters and culture conditions were controlled and the experiments were carried out in special corrosion resistant bioreactor equipment suitable for cultivation at hypersaline environments.

[0191] The special non-corrosive Labfors PEEK (Infors, AG, Switzerland) reactor was utilized with the following specifications: [0192] Borosilicate glass culture vessel: 1 L volume [0193] Borosilicate glass exhaust gas cooling [0194] Special corrosion resistant Polymer (PEEK) bioreactor top lid [0195] Special corrosion resistant Polymer (PEEK) thermometer holder [0196] Borosilicate glass sampling tube and gas inlet tube [0197] Special corrosion resistant agitator [0198] Borosilicate glass jacket on the [0199] reactor vessel

[0200] Online Analytics of: [0201] Exhaust gas CO2 [0202] Exhaust gas O2 [0203] Glass pH probe [0204] Hastelloy Clark pO2 and [0205] Thermal Mass flow controller for air

[0206] The following media components were added to the brine: KCl 0.66 g/l, NH4Cl 1.5 g/l, KH.sub.2PO4 0.15 g/l, MgCl2.6H2O 1.3 g/l, MgSO4.7H2O 1.1 g/l, FeCl3 0.005 g/l, CaCl.sub.2).2H2O 0.55 g/l, KBr 0.5 g/l, Mn stock 3 ml and trace elements 1 ml. [0207] Temperature: 37° C. [0208] pH: 7.2 (either 0.5 M HCl and 0.5 M NaOH were used for pH control)

[0209] As the aromatic compounds in this study are mostly degraded and not used as substrate to facilitate biomass growth in order to increase the biomass concentration in bioreactor a batch cultivation was done on brine with the addition of media components and glycerol as substrate for growth (Table 1). Once enough biomass was obtained (about 3 g/l) a master mix containing aniline 5 mg/l, phenol 5 mg/l and 4,4′MDA 3 mg/l was added to the reactor as a pulse. During the batch cultivation complete degradation of the aromatic compounds took up to 96 hours.

Example 6: Degradation of Formate, MDA, Nitrobenzene, Aniline and Phenol in Actual Residual Water in Continuous Bio-Processing Using Cell Retention System

[0210] Bioreactor Setup with Cell Retention

[0211] Continuous degradation of pollutants in actual brine was performed using a cell retention system. The industrial brine was supplemented with media components given in table 1 and glycerol as co-substrate. The amount of glycerol in the medium was adjusted in such a way to achieve a specific growth rate of 0.026 h-1. Cultivation was established in the bioreactor as described for shake flask experiments. Fermentation in the bioreactor was performed at 450 rpm agitation and 37° C. The cell retention system was set in the bioreactor using a polysulfone (PSU) hollow fiber microfiltration membrane cartridge having an area of 420 cm.sup.2 and a pore size of 0.2 μm. Feed flows of 130 to 610 g/h led to a dilution rate of 0.1 to 0.6 h-1. By adjusting the feed flow in relation to the cell containing bleed flow and the cell free harvest, a constant biomass in the fermenter could be achieved.

[0212] Turbidity as indicator for cell density and HPLC analytics of the residual formate, acetate and glycerol were measured during the entire process.

[0213] Residual MDA, Nitrobenzene, Aniline and Phenol were also measured by HPLC.

[0214] Fermentations

[0215] HFX was cultivated in the 1 L bioreactor to degrade pollutants in brine with a continuous flow of ca. 0.3 g/L formate. For the experiments the two parameters Biomass concentration (g/L) and Dilution rate (h-1) were varied in the range of 2-5 g/L and 0.1-0.6 h-1 respectively.

[0216] Significant formate degradation could be observed for all continuous cultivations. Experiments with higher Biomass concentration resulted in lower residual formate concentration. In experiments with higher flow rate a higher residual formate concentration could be measured.

[0217] Analytics of the culture supernatant sampled from the reactor showed that the amount of MDA and aniline could also be decreased during bioprocessing. Phenol and nitrobenzene were degraded to a concentration below detection limit. HFX can degrade formate, MDA, aniline, phenol and nitrobenzene in batch as well as continuous mode. Rate of degradation of the mentioned pollutants depends on the biomass concentration, the dilution rate and the concentration of the pollutants in the brine.

[0218] Summary—Conclusions:

[0219] Hypersaline wastewaters frequently comprise organic pollutants such as formate aniline, phenol, nitrobenzene and 4,4′Methylenedianilin. Several physical and chemical methods for example sorption, ozonation and electrochemical treatment are used to treat residual water containing organic compounds. However, 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.

[0220] In this invention, it was discovered that Haloferax mediterranei DSM.1411 can actively degrade toxic organic pollutants from their hypersaline environment of up to 200 g/l salinity. Further, it was discovered that Haloferax mediterranei DSM.1411 can degrade formate aniline, phenol, nitrobenzene and 4,4′Methylenedianiline from hypersaline environments.

[0221] This invention can be directed to optimal and efficient treatment of any hypersaline water containing any or the combination of the following components formate, phenol, nitrobenzene, 4,4′-Methylenedianiline (MDA) and aniline intending to reduce the total organic carbon content.

[0222] 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, makes the pretreatments of these hypersaline residual streams, absolutely crucial. Our invention helps achieving this cheap, quick and efficient pretreatment to meet the requirement for membrane electrolysis to produce chlorine gas and/or sodium hydroxide from treated brine.