METHOD FOR REMOVING AMMONIACAL NITROGEN FROM HYPERSALINE EFFLUENTS THROUGH A CONSORTIUM CONTAINING HIGH-SALINITY-RESISTANT HETEROTROPHIC NITRIFYING AND AEROBIC DENITRIFYING MICROORGANISMS

Abstract

The invention discloses a method to provide a consortium containing heterotrophic nitrifying and aerobic denitrifying microorganisms from an activated sludge originating from effluent treatment plants. The microbial consortium is capable of removing ammoniacal nitrogen from substrates with high salinity with superior efficiency. Thus, the microbial consortium from the described method can be applied to purify hypersaline effluents contaminated with nitrogen. Therefore, the use of the microorganism consortium provided to remove ammoniacal nitrogen from hypersaline effluents and a method therefor are also part of the scope of the invention.

Claims

1. A method for removing ammoniacal nitrogen from hypersaline effluents through a consortium containing high-salinity-resistant heterotrophic nitrifying and aerobic denitrifying microorganisms, of the method comprising: a) supplying an activated sludge from a biological treatment plant for an effluent; b) providing a hypersaline aqueous effluent with salinity similar to activated sludge from an effluent treatment plant; c) inoculating a reactor with the aforementioned activated sludge of item (a) and the hypersaline aqueous effluent of item (b) in a proportion ranging from 70:30 to 50:50; d) feeding/aerating the activated sludge for 4 hours, aerating for 3 hours, allowing to settle for 1 hour and discarding the supernatant for 4 hours; e) discarding 30% of the supernatant and completing the reactor volume with the effluent of item (b); f) weekly increasing the salinity of the effluent of item (b) with the addition of NaCl in the range of 5 g/L until the desired salinity is reached (between 100 and 200 g/L), provided that there is no inhibition of nitrification, that is, the residual ammoniacal nitrogen is less than 20 mg/L; g) at salinities lower than 100 g/L, adding a source of carbon to the effluent of item (b) in order to ensure a minimum ratio of COD:NH.sub.4.sup.+ from 20:1 to 40:1; or, at salinities greater than 100 g/L, maintaining the C:N ratio, measured as Total Organic Carbon (TOC)/NH.sub.4.sup.+, between 4 and 7; h) maintaining a phosphorus source at a minimum NH.sub.4.sup.+/P ratio of about 5; wherein the alkalinity of the effluent of item (b) is maintained above 900 mg/L, the pH is maintained between 6.5 and 7.5 and the temperature is maintained between 27 and 33 C.

2. The method according to claim 1, wherein the activated sludge comes from a production water treatment plant derived from oil exploration.

3. The method of claim 1, wherein the effluent of interest is production water derived from oil exploration.

4. The method of claim 1, wherein the carbon source is ethanol and the phosphate source is sodium tripolyphosphate.

5. The method of claim 1, the salinity of the effluent of interest is greater than or equal to 50 g/L.

6. The method of claim 1, wherein the temperature is maintained at about 30 C.

7. The method of claim 1, wherein the pH is maintained at about 7.

Description

[0036] FIG. 1 shows the concentrations of nitrogen compounds and the efficiency of ammoniacal nitrogen removal in bioreactors R1 (A) and R2 (B) at different salinities, through analytical monitoring of salinity, ammoniacal nitrogen (raw and treated effluent), nitrate, nitrite and removal efficiency.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The production water generated in the oil extraction process is basically formed by the combination of formation water, which is naturally present in oil reservoirs, and injection water, which is injected with the addition of adjuvants necessary to carry out the extraction. For each barrel of oil extracted, around 246 to 340 liters of effluent are generated to be treated. This production water has a complex composition, containing organic matter and several contaminants that can be toxic to the environment, particularly ammonia.

[0038] Currently, there are different technologies applied to the treatment of effluents, such as flocculation, flotation, activated sludge, membrane filtration, among others. The choice of used technology depends on the type of effluent to be treated and the contaminant to be purified. In the case of production water generated by the oil extraction process, the biological treatment using activated sludge is an option of interest, as it offers lower operating costs and allows the removal of organic matter and nutrients (Da Motta et al., 2003; Lefebvre and Moletta, 2006).

[0039] Isolated heterotrophic nitrifying and aerobic denitrifying microorganisms present in activated sludge can provide relevant advantages to the treatment of effluents with a high content of organic matter and ammonia. Such microorganisms use organic substrate and, therefore, simultaneously remove both types of contaminants from the effluent. However, the high salinity can reduce the viability of the microbiota, thus decreasing the rate of ammoniacal nitrogen purification.

[0040] The present invention discloses a method to obtain a microbial consortium capable of performing heterotrophic nitrification and aerobic denitrification (HN/AD) in aqueous effluents with high salinity. Further, there are encompassed by the scope of the invention, the use of said microbial consortium in a method for removing ammoniacal nitrogen from an aqueous effluent and a biological method for removing ammoniacal nitrogen from hypersaline aqueous effluents, particularly production waters from the exploration oil.

Definitions

[0041] In the context of the present invention, the term heterotrophic nitrification denotes a process of oxidation of ammonia/ammonium (NH.sub.3/NH.sub.4.sup.+) to nitrate (NO.sub.3.sup.), carried out by a microorganism, without energy production. Aerobic denitrification denotes the phenomenon of transformation of nitrates and other substances into nitrogen gas (N.sub.2), by microorganisms, in the presence of oxygen.

[0042] Microbial consortia are groups of microorganisms, mainly bacteria and fungi, acting together for the degradation of a matter. Microbial consortia in the context of the present invention are preferably consortia of bacteria from the activated sludge of a production water treatment plant derived from oil exploration.

[0043] In the context of the invention, the expression activated sludge refers to the sludge resulting from an effluent treatment process intended for the destruction of biodegradable pollutants present in wastewater and/or sewage.

[0044] Throughout the present application, the expression ammoniacal nitrogen is used to reflect the amount of ammonia in waste products, such as effluents. The expression is widely used in waste treatment and water purification systems. Ammoniacal nitrogen values in wastewater or waste liquids are measured in milligrams per liter and are used to specify water treatment systems and facilities.

[0045] According to the invention, hypersaline wastewaters, hypersaline effluents, effluents with high salinity and similar expressions denote effluents in which the NaCl concentration is greater than or equal to 5% (w/v).

[0046] In one aspect of the invention, the method for obtaining consortium of HN/AD microorganisms resistant to high salinity of the present invention is practiced on a sample of activated sludge from aqueous effluent treatment plants. Activated sludge can come from different sources, including, but not limited to, treatment plants of sewage, industrial wastewater and production water derived from oil exploration.

[0047] The activated sludge is acclimatized in appropriate reactors and in conditions of increasing salinity. The reactors can be, for example, aerated bioreactors, with a useful volume of 0.85 liters, operated in sequential batch. After sedimentation, about 18% of the supernatant (150 mL) is removed and the volume completed with crude effluent. The feeding of the bioreactors is carried out with the aid of a peristaltic pump and the disposal of the supernatant through a solenoid valve, both connected to timers. Dissolved oxygen levels in bioreactors must be maintained above 2 mg.Math.L.sup.1, through, for example, aeration by air pump and diffusers located at the bottom of the reactors. The salinity can be modulated with the addition of 2 to 10 g.Math.L.sup.1 of NaCl, until the salt concentration in the medium inhibits the ammonia removal process.

[0048] During acclimatization, the bioreactors can be operated in cycles that comprise steps of feeding, aeration, sedimentation and disposal. The feeding step with aeration can preferably last about 4 hours; the aeration step can preferably last about 3 hours; the sedimentation step can preferably last about 1 hour; and the discard step may preferably last about 4 hours. Each cycle can last, for example, 12 hours.

[0049] To favor the selection and prevalence of metabolism of HN/AD microorganisms, temperature, pH and proportion of carbon, nitrogen and phosphorus available in the reactor environment were kept stable. The temperature can be any between 27 and 33 C., preferably 30 C. The pH can be any between 6.5 and 7.5, preferably 7.0. The technician skilled on the subject knows that it is possible to adjust the pH of a solution by adding acidic components, such as, for example, H.sub.2SO.sub.4, or basic components, such as, for example, NaOH.

[0050] The ratio of carbon to nitrogen (C/N), when measured in the form of chemical oxygen demand (COD) and ammoniacal nitrogen (NH.sub.4.sup.+) can be any between 20 and 40; if measured in the form of total organic carbon (TOC)/NH.sub.4.sup.+, then the C/N ratio can be any between 4 and 7. The technician skilled on the subject will recognize that he/she can supplement the reaction with ethanol as a carbon source to achieve the C/N ratio desired.

[0051] The ratio between nitrogen and phosphorus available in the medium, with nitrogen measured in the form of ammoniacal nitrogen (NH.sub.4.sup.+) (NH.sub.4.sup.+:P), can assume different figures, but will preferably be 5:1. In addition to carbon supplementation, the medium can be supplemented with various compounds that function as sources of phosphorus. It will be apparent to the technician skilled on the subject that phosphorus supplementation can be promoted through the addition of a range of phosphorus salts including, but not limited to, for example, sodium tripolyphosphate (Na.sub.5P.sub.3O.sub.10) and monobasic potassium phosphate (KH.sub.2PO.sub.4).

[0052] In another aspect of the invention, the consortium of HN/AD microorganisms of the method described herein is capable of removing ammoniacal nitrogen from hypersaline effluents. Under certain circumstances, hypersaline effluent may be derived from sewage, industrial activity or oil exploration. In the latter case, the effluent is then production water.

[0053] Accordingly, a conclusive aspect of the invention concerns a method for removing ammoniacal nitrogen from hypersaline effluents. In this method, the consortium of HN/AD microorganisms resistant to high salinity and a hypersaline aqueous effluent are maintained in a reactor under specific conditions of controlled pH, temperature and C:N:P ratio.

[0054] A technician skilled on the subject will recognize which hypersaline effluents require ammoniacal nitrogen purification. Among the many, a particular object of the present method is the production waters derived from oil exploration, which may have a salinity greater than 100 g.Math.L.sup.1. However, as already evident, the consortium of HN/AD microorganisms resistant to high salinity of the present invention can remove nitrogen from effluents with high efficiency in salt concentrations greater than 100 g.Math.L.sup.1, reaching up to 170 g.Math.L.sup.1. In order for the observed ammoniacal nitrogen purification efficiency to be achieved, the consortium of HN/AD microorganisms and the hypersaline aqueous effluent must be in the reactor in proportions that vary between 50:50 and 70:30.

[0055] Finally, the effluent must be supplemented with a source of phosphorus, ensuring a minimum ratio of ammoniacal nitrogen NH.sub.4.sup.+:P of 5. When the salinity of the effluent is less than 100 g.Math.L.sup.1, the reaction will need supplementation with a source ofcarbon, in order to ensure a minimum COD:NH.sub.4.sup.+ ratio of 20:1. At salinities greater than 100 g.Math.L.sup.1, the C/N ratio, measured as Total Organic Carbon (TOC)/NH.sub.4.sup.+, can be maintained between 4 and 7. The alkalinity of the medium inside the reactor must not be less than 900 mg.Math.L.sup.1 and the temperature will be 30 C. Alkalinity can be corrected with sodium bicarbonate (NaHCO.sub.3) supplementation.

[0056] A technician skilled on the subject will recognize that there are several ways to measure the demand for carbon in the mixture, either in the form of COD or in the form of TOC. For example, following the standard procedures of the Standard Methods for the Examination of Water and Wastewater, methods 5220 D and 5310 B, respectively (APHA, 2005).

[0057] There follow below are experiments illustrating the embodiments of the invention, which, as a technician skilled on the subject will understand, are only examples of how the invention can be practiced and should not limit the scope achieved by the inventive concept described herein.

EXAMPLES

Example 1: Obtaining HN/AD Microbial Consortium and Denitrification of Production Water

[0058] A) Acclimatization of the Sludge

[0059] 50% v/v of sludge from a biological treatment of production water was inoculated in two reactors (R1 and R2) with a useful volume of 0.85 L, operated in sequential batch with 12-hour cycles with the following phases: (i) feeding/aeration (for 4 hours), (ii) aeration (for 3 hours), (iii) sedimentation (for 1 hour) and (iv) disposal of the supernatant (for 4 hours). The pH of the reactors was maintained within the range of 6.5 to 7.5, with the addition of H.sub.2SO.sub.4 or NaOH. The room temperature was maintained around 30 C. and the dissolved oxygen level was maintained above 2 mg.Math.L.sup.1 by means of aeration using an air pump and diffusers located at the bottom of the reactors.

[0060] In the feeding/aeration step, a peristaltic pump was turned on (by means of a timer) and pumped raw effluent into the reactor. The pump worked with a flow rate of 0.625 mL/min. At the end of the 4 hours, the pump was turned off by the timer. Simultaneously, the air pump was turned on along with the peristaltic pump, and remained on for a period of 7 hours. At the end of 7 hours, the air pump was turned off (timer). With the air pump turned off, the system entered the sedimentation step, for a period of 1 h. After this period, a solenoid valve, positioned at the correct height of the reactor, was activated (also by a timer). When opening the supernatant effluent (treated), it was discarded. The valve remained open for a period of 4 hours. After closing the valve, a new cycle begins.

[0061] After the sedimentation step, about 18% of the supernatant (150 mL) was removed and the volume completed with the crude effluent. The effluent was enriched with a source of phosphorus (sodium tripolyphosphate, 14.5 g.Math.L.sup.1 solution) and carbon (100% ethanol) to ensure a minimum COD:NH.sub.4.sup.+:P ratio of 100:5:1 or COT:NH.sub.4.sup.+:P from 20:5:1. Alkalinity was maintained above 900 mg.Math.L.sup.1 by adding, when necessary, sodium bicarbonate (NaHCO.sub.3) to the produced water. After stabilization of the removal of ammoniacal nitrogen at a residual concentration below 20 mg.Math.L.sup.1, 5 g.Math.L.sup.1 of NaCl were added weekly to the reactor feed effluent, until 170 g.Math.L.sup.1 of NaCl was reached with removal above 79% ammoniacal nitrogen (FIG. 1). After 100 g.Math.L.sup.1 of salinity, the C/N ratio (measured as COD/NH.sub.4.sup.+) was maintained at 32. When measured as TOC/NH.sub.4.sup.+, the C/N ratio was maintained between 4 and 7. The pH was maintained between 6.5 and 7.5 by adding H.sub.2SO.sub.4 or NaOH, when necessary, and the temperature was maintained at 30 C. The nitrogen concentration in the production water ranged from 60 to 100 mg.Math.L.sup.1.

[0062] B) Characterization of the Microbial Diversity

[0063] Samples of sludge from the bioreactors, R1 and R2, were collected, performed the extraction of total DNA and subjected to sequencing of the RNA16S region and analysis of microbial diversity. The extraction of total DNA from the microbial community was performed according to the protocol described by Silva et al. (Silva, C. C., Jeus, E. C., Tones, A. P. R., Sousa, M. P., Santiago, V. M. J., Oliveira, V. M. (2010) Investigation of bacterial diversity in membrane bioreactor and conventional activated sludge processes from petroleum refineries using phylogenetic and statistical approaches. J. Microbiol. Biotechnol., 20(3), 447-459.) with modifications.

[0064] A 4 mL aliquot of the sludge samples was washed with 10 mL of SET buffer and centrifuged for 10 minutes at 3,000 g. The washing procedure was performed five times. After washing, the sludge sample was suspended in 600 L of SET buffer and homogenized in the tube shaker. Subsequently, lysozyme (GE Health Care) was added (50 uL of a 100 mg/mL solution), and the solution was incubated in a water bath at 37 C., with agitation every 10 minutes. Then, proteinase K (Sigma) was added (50 L of a 10 mg/mL solution) and 200 L of 10% SDS (Cf=2%), the solution was incubated at 60 C. for 30 minutes, with agitation every 10 minutes. The sample was then subjected to three freeze-thaw cycles, 2 minutes in liquid nitrogen and 2 minutes at 65 C. Afterwards, an equal volume of saturated phenol pH 8.0 was added to the solution, homogenized for 2 minutes and centrifuged at 10,000 g for 5 minutes. The supernatant was collected, an equal volume of chloroform-isoamyl alcohol (24:1) was added and centrifuged at 10,000 g for 5 minutes. Again, the supernatant was collected, and a 5M NaCl solution (10% of the total volume) was added to the same, followed by the addition of 2 volumes of cold absolute ethanol. The microtube was centrifuged for 20 minutes at 10,000 g. The pellet was washed with 70% ethanol and after drying, eluted in 25 L of H2O-Milli-Q.

[0065] The genomic DNA of the two samples was sent to NGS (Next Generation Sequencing) of the variable region V1-V2 and V3 of the 16S rRNA gene, using the 27f/338r primer, at Molecular Research DNA (www.mrdnalab.com, Shallowater, TX, USA) by the MiSeq platform (Illumina). Sequence data were processed using the MR DNA analysis pipeline (MR DNA, Shallowater, TX, USA). Barcodes were removed, sequences smaller than 150 bp and chimeras were removed. The contigs were assembled and an OTU (Operational Taxonomic Unit) file was generated. OTUs were defined by grouping at 3% divergence. A representative sequence from each OTU was taxonomically classified using BLASTn against a curated database derived from GreenGenes, RDPII, and NCBI (www.ncbi.nlm.nih.gov, DeSantis et al. 2006, http://rdp.cme.msu.edu). The diversity indices were obtained using the PAST (Paleontological Statistics Software Package for education and data analysis) software (Hammer, ., Harper, D. A. T., Ryan, P. D. (2001) Past: Paleontological statistics software package for education and data analysis. Palaeontol. Electron., 4(1), 9).

[0066] C) Evaluation of Ammonia Removal

[0067] To evaluate the performance of ammoniacal nitrogen removal in the reactors, the following parameters were monitored and evaluated: COD, TOC, CI, ammoniacal nitrogen, nitrite, nitrate, total nitrogen, phosphorus, total suspended solids, volatile suspended solids, pH, alkalinity and conductivity.

[0068] The COD, ammoniacal nitrogen, nitrite, nitrate, total nitrogen and phosphorus procedures were performed using the TNT Plus Hack Kit, the others were performed following the standard procedures of the Standard Methods for the Examination of Water and Wastewater (APHA, 2005).

[0069] Although preferred embodiments of the present invention have been demonstrated throughout this specification, it will be obvious to a technician skilled on the subject that these embodiments are provided by way of example only. Several variations, alterations and substitutions will occur to those skilled in the art without departing from the invention. Accordingly, it is expected that the following claims define the scope of the invention, encompassing its possible equivalents.