Denitrification of saline industrial waste water

Abstract

The present invention concerns a biochemical process for the denitrification of a hypersaline wastewater composition.

Claims

1. A biochemical process for denitrification of a hypersaline wastewater composition comprising a nitrate concentration of at least 0.1% (w/v) and a chloride concentration of at least 5% (w/v), comprising a step of using a community of halophilic and/or halotolerant bacteria comprising at least the following genera: Pseudomonas, Bacillus and Halomonas, for the denitrification of said hypersaline wastewater composition.

2. The process according to claim 1, wherein said community is selected from a sludge mix consisting of about 85 to 95 weight % of activated sludge from the denitrification step of a municipal waste water treatment plant and of about 5 to 15 weight % of saline sludge from a crystallizer pond of a solar saltern.

3. The process according to claim 1 for treatment of hypersaline waste water originating from an ion exchange process.

4. The process according to claim 1, wherein the nitrate concentration is at least 0.20% (w/v).

5. The process according to claim 1 for treatment of hypersaline waste water originating from a production process utilising potassium chloride and calcium nitrate to produce a calcium chloride and nitrate containing waste water stream.

6. The process according to claim 1 for treatment of hypersaline waste water originating from a production process utilising sodium chloride and calcium nitrate to produce a calcium chloride and nitrate containing waste water stream.

7. The process according to claim 1, wherein said community is selected from a sludge mix consisting of about 90 weight % of activated sludge from a denitrification step of a municipal waste water treatment plan and of about 10 weight % of saline sludge from a crystallizer pond of a solar saltern.

8. The process according to claim 1, wherein the halophilic bacteria are selected from the genera of Pseudomonas (abundance: 19 weight %), Bacillus (abundance: 4 weight %) and Halomonas (abundance: 3 weight %), and optionally with minor (1 weight % or less) abundance Rhodobacter, Arthrobacter, Flexibacter, Propionibacterium, Enterobacteriaceae, Flavobacterium, Bradyrhizobium, Hyphomicrobium, Lysobacter, Sinorhizobium, Azospirillum, Thiobacillus, Sphingobacter, Paracoccus, Aeromanas, Ochrobacterium, Nitrosomonas, Herbaspirillum, Janthinobacterium, Lactobacillus, Nitrobacter, Cellulomonas, Streptomycetes, Cytophaga, Thiomicrospira, Beggiatoa, Cellvibrio, Moraxella, Alteromonas, Kingella, Aquaspirillium, Norcadia and Azoarcus.

9. The process according to claim 1, wherein said process operates at 35? C. to 40? C.

10. The process according to claim 1, wherein potassium acetate is used as a carbon source.

11. The process according to claim 1 wherein an initial nitrate concentration of 1.5 to 3.0 g/l is reduced to a concentration of about 0.001 g/l within 24 to 48 hours.

12. The process according to claim 1, wherein the process is performed in a bioreactor of a suspended sludge reactor type.

13. The process according to claim 1, wherein the process is performed in a bioreactor of a floating bed reactor type.

14. The process according to claim 4, wherein the nitrate concentration is 0.25% (w/v).

15. The process according to claim 9, wherein said process operates at about 37? C.

Description

(1) The following figures are referred to in this description.

(2) FIG. 1: Denitrification under standard conditions (no addition of carbon source, room temperature) in the 2-L microcosm setup as described (Experiment A).

(3) FIG. 2: Denitrification in the 2-L microcosm setup with addition of MeOH (10 ml/l) as carbon source at room temperature (Experiment B).

(4) FIG. 3: Denitrification in the 2-L microcosm setup at a nitrate concentration of 260 mg/l (Experiment C).

(5) FIG. 4: Denitrification in the 2-L microcosm setup at a nitrate concentration of 2,200 mg/l (Experiment D).

(6) FIG. 5: Assessing the effect of temperature on denitrification rate in an experimental 2-L microcosm setup (Experiment E).

(7) FIG. 6: Assessing the effect of KAc as a carbon source (Experiment F).

(8) FIG. 7: Denitrification under high salt conditions (Experiment G) without significant halophilic and/or halotolerant bacteria (not according to one or more embodiments of the invention).

(9) FIG. 8: Total bacterial abundance (x) and relative abundance of denitrifying bacteria (?) in Experiment G, as measured photometrically (left) and using DAPI stains (right).

(10) FIG. 9: Denitrification rates for different sediment substitution pro-portions according to one or more embodiments of the invention (Experiment H).

(11) FIG. 10: Total bacterial abundance (x) and abundance of denitrifying bacteria (?) in Experiment H, as measured photometrically (left) and using DAPI stains (right).

EXPERIMENTAL

(12) General

(13) A production process of potassium nitrate is performed by ion exchange from potassium chloride and calcium nitrate, the process delivering a wastewater stream of high salinity due to calcium chloride and high contamination of nitrate. A biological treatment of this wastewater stream with a maximum conversion of the nitrate to gaseous nitrogen was to determine.

(14) Goal of this experiment is the assessment and establishment of a microbial environment capable of complete denitrification at high salt concentrations. Wastewater specifications include 2.5 g nitrate/l wastewater (0.25% w/v), 91 g CaCl.sub.2/l (5.7% w/v of Cl-ions) wastewater and temperatures exceeding 50? C. and lack of carbon source in the incoming industrial wastewater. Potassium acetate as an additional carbon source was added as 30 ml/l.

(15) The proposed strategy to conduct the experiment was the establishment of a closed controlled laboratory system (2 liter microcosm, in the following 2-L-microcosm) mimicking activated sludge bioreactor conditions. The experimental setup of the 2-L microcosm included the possibilities to control oxygen concentrations (maintain anoxia), pH values (a range between 6.5 and 8.5 is mandatory for efficient denitrification) and temperature. Therefore, the setup was designed with permanent online pH-, oxygen- and temperature sensors, a stirring unit, access for sub-sampling without oxygen contamination, and access for argon flushing in case of accidental oxygenation events.

(16) Activated sludge from the Wastewater Treatment Plant (WWTP) of Kaiserslautern (Germany) was used as a basis for a microbial environment (2.5 L volume in each experiment). For the sediment containing the natural halophilic denitrifying bacteria, sediment from the crystallizer pond of a solar saltern from Ses Salines, Spain, was used.

(17) During experiments, samples were taken at different time intervals for nitrate and nitrite measurements (ion chromatography and photometry), total bacterial abundances (using DAPI stains), relative abundance of nitrite reductase (nir) genes, which indicate the abundance of denitrifying bacteria, and molecular microbial community profiling (Illumina ribosomal RNA sequencing and statistical community structure analyses). During the experiments, the pH had to be adjusted with KOH due to slight acidification of the microbial environment.

(18) Nitrate concentrations were measured photometrically under low salt loadings, and using ion chromatography under high salt loadings.

(19) Experiment A: Standard Denitrification without Sample Manipulation in a Controlled 2-L Microcosm Setup.

(20) This experiment served as a control for the functioning of the 2-L microcosm system under standard conditions at ambient (room) temperature. Denitrification was performed under standard conditions (no addition of carbon source, room temperature) in the 2-L microcosm setup. 20 mg nitrate/l were added, corresponding to nitrate concentrations usually prevailing in the incoming wastewater in the WWTP.

(21) The results are shown in FIG. 1. In the established experimental setup, 20 mg nitrate/l wastewater was denitrified within 85 minutes. Denitrification rate was 14.1 mg/l/h.

(22) Conclusion:

(23) The experimental setup is ideally suited to conduct the experiments because denitrification in the 2-L microcosm is as efficient as under usual conditions in a well-functioning WWTP.

(24) Experiment B: Addition of Methanol (MeOH, 10 ml/l) as Additional Carbon Source to Assess the Effect of this Carbon Source on Denitrification Rate.

(25) The experimental conditions are as in Experiment A with slightly elevated nitrate concentrations (33 mg/l). The results are shown in FIG. 2. Complete denitrification of 33 mg nitrate/l is completed after 50 min. Slope of trendline is steeper compared to experiment under standard conditions (without addition of extra carbon source). The denitrification rate is 39.8 mg/l/h.

(26) Conclusion:

(27) Additional carbon source (methanol) enhances the denitrification distinctly. Variation of carbon source concentration did not positively affect the denitrification rate in further experiments.

(28) Experiment C: Increase of Nitrate Concentration to 260 mg/l (Exceeding Natural Conditions Ca. 10-Fold)

(29) The results are shown in FIG. 3. Complete denitrification is still possible at a highly elevated nitrate concentration. However, the time for the complete denitrification of 260 mg nitrate/l runs up to 34 hours. The denitrification rate is 7.4 mg/l/h.

(30) Conclusion:

(31) Microbial processes at such elevated nitrate concentrations are distinctly lower compared to Experiment A with nitrate concentrations similar to a microbial environment. It seems likely that the abundance of naturally-occurring bacteria in the experimental setup is too low for an efficient denitrification.

(32) Experiment D: Increase of Nitrate Concentration to 2,200 mg/l

(33) The results are shown in FIG. 4. Complete denitrification is still possible at a highly elevated nitrate concentration. However, the time for the complete denitrification of 2,200 mg nitrate/l runs up to 200 hours. The denitrification rate is 9.3 mg/l/h.

(34) Conclusion:

(35) The denitrification rate is in the same order of magnitude as in Experiment C with an about 10-fold lower nitrate concentration. This is too low for a standard application in a WWTP process for removal of such high nitrate loads. Also, from this experiment it seems likely that the abundance of naturally occurring bacteria in the experimental setup is too low for an efficient denitrification. Therefore, further experiments are conducted in order to increase bacterial abundance and activity.

(36) Experiment E: Assessing the Effect of Temperature on Denitrification Rate in an Experimental 2-L Microcosm Setup.

(37) Several experiments were conducted with an initial nitrate concentration of about 1000 mg/l to assess the effect of temperature on denitrification rate in an experimental 2-L microcosm setup. The results are shown in FIG. 5. The highest denitrification rates were achieved at 37? C. Denitrification rate: 20.4 mg/l/h. Denitrification is inhibited at higher temperatures.

(38) Conclusion:

(39) A temperature of 37? C. results in increased bacterial densities and activities in an experimental setup. As a consequence, denitrification rates are in the same order of magnitude as under standard conditions (see Experiments A and B) even at extremely elevated nitrate concentrations. However, this rate is still too low for an efficient nitrate removal of such high concentrations in industrial processes. Further experiments were conducted.

(40) Experiment F: Assessing the Effect of the Carbon Source.

(41) MeOH is not appropriate as carbon source in industrial application (cost and safety issues). Therefore, Experiment F is conducted in order to evaluate potassium acetate (KAc) as an alternative carbon source. In all experiments KAc was added at a defined ratio: C:N=1.5:1. The results are shown in FIG. 6. The denitrification rate is 102 mg/l/h, with KAc as carbon source, temperature at 37? C. and extreme nitrate concentration.

(42) Conclusion:

(43) This is the highest denitrification rate obtained this far and significantly exceeds standard values for denitrification in WWTP reported in the scientific literature. Therefore, we conclude that KAc as carbon source and an optimal temperature of 37? C. is very efficient to enhance microbial denitrification.

(44) Experiment G: Denitrification Under High Salt Conditions without Significant Halophilic and/or Halotolerant Bacteria (not According to One or More Embodiments of the Invention).

(45) Denitrification under high salt conditions was done with a CaCI.sub.2 concentration of 91 g/l (5.7% w/v of CI-ions), a nitrate concentration of 2.5 g/l (0.25% w/v), KAc as a carbon source and at a temperature of 37? C. The results are shown in FIG. 7. The denitrification rate is 43.7 mg/l/h. A significant increase in bacterial abundance and denitrifier abundance in the wastewater during the course of the experiment was observed. This was determined using quantitative real time PCR of the nir gene (nitrite reductase, see Saggar et al. Sci. Total Environ), encoding a part of the denitrification process, and total bacterial load, as determined by DAPI-abundance, calculated from 5 replicates per sample. The results are shown in FIG. 8. The figure displays (relative) abundance data in the experimental setup for Experiment G.

(46) Conclusion:

(47) The biomass of specifically denitrifying bacteria increases 2.5 fold over the course of the experiment.

(48) Experiment H: Substitution of Activated Sludge with Solar Saltern Sediment Including Natural Halophilic Denitrifiers.

(49) Activated sludge from the Waste Water Treatment Plant (WWTP) of Kaiserslautern (Germany) was substituted with sediment from the crystallizer pond of a solar saltern from Ses Salines, Spain, including natural halophilic denitrifiers. The results are shown in FIG. 9. The substitution with 10 volume % of sediment containing natural halophilic denitrifiers increased the denitrification rate compared to the Experiment G. The denitrification rate was 51.8 mg/l/h. Surprisingly, increasing the relative proportion of sediment containing natural halophilic denitrifiers (experiments were done for 20 and 30 volume %, see FIG. 9) did not increase the denitrification rate, but significantly inhibited the denitrification instead.

(50) Furthermore, the biomass of specifically denitrifying bacteria increased 3.5 fold over the course of the experiment (see FIG. 10). It can be seen that with the process according to one or more embodiments of the invention, using 10% saline sediment, an initial nitrate concentration of 2.5 g/l is reduced to a concentration of about 1 g/l after 24 hours, and to a concentration of about 0.001 g/l after 48 hours.

(51) Although the disclosure has been described with respect to only a limited number of embodiments, those skilled in the art, having benefit of this disclosure, will appreciate that various other embodiments may be devised without departing from the scope of the present invention. Accordingly, the scope of the invention should be limited only by the attached claims.