WO2010076794A1

WO2010076794A1 - Method of denitrifying brine and systems capable of same

Info

Publication number: WO2010076794A1
Application number: PCT/IL2009/001230
Authority: WO
Inventors: Michal Green; Sheldon Tarre
Original assignee: Technion Research & Development Foundation Ltd.
Priority date: 2008-12-31
Filing date: 2009-12-30
Publication date: 2010-07-08

Abstract

A method of removing nitrates from brine is disclosed. The method comprises contacting the brine with a halotolerant, anaerobic bacteria and a fluid miscible carbon source in a fluidized containment under neutral pH conditions, wherein the contacting is such that said halotolerant, anaerobic bacteria use the nitrates from the brine to generate gaseous nitrogen, and wherein the halotolerant, anaerobic 0bacteria are attached to particles as a biofilm. Systems capable of removing nitrates from brine are also disclosed.

Description

METHOD OF DENITRIFYING BRINE AND SYSTEMS CAPABLE OF SAME

FIELD AND BACKGROUND OF THE INVENTION The present invention, in some embodiments thereof, relates to a method of denitrification of brine and systems capable of same.

Found in both groundwater and surface water supplies, nitrate contamination is a result of natural geologic formations or the activities of man. Major contributors to nitrates in groundwater include fertilizers, domestic wastewater, seepage from septic systems and animal manure where there are high populations of livestock.

Recently developed membrane processes for ground-water and brackish water purification may produce brine with elevated nitrate concentrations with problematic disposal to the environment. The volume of brine produced is approximately 10 to 20 % of the water treated and the concentration of salts in the brine likewise varies between 5 to 10 times higher than the original source water.

Brines with high nitrate concentrations are also produced in armaments plants and coal-fired power plants which are equipped with a wet lime(stone) gypsum flue gas desulphurization (FGD) process.

In addition, elevated nitrate concentrations are observed with increasing frequency in closed aquaria and other seawater tanks, reflecting the accumulation of the metabolism products of animals living within the tanks.

Today, in coastline areas, the most obvious disposal method is to sea. However, the release of nitrogen compounds from the brine can cause algae bloom resulting in the negative effects on water quality of oxygen demand, color, turbidity, damage to existing the flora and fauna, etc. Other suggested methods of disposal of nitrate-rich brines include disposal to existing wastewater treatment plants, dilution, injection wells and disposal to barren areas or landfills.

Because membrane processes and the resulting brines are relatively new processes, regulations worldwide are not in place for dealing with brine disposal. However, recently the U.S. EPA has classified brines from reverse osmosis processes as industrial effluents and in the state of Florida a special permit is required for brine disposal.

Denitrifying bacteria are very common in nature and are capable of reducing unwanted nitrate into harmless nitrogen gas. Complete denitrification in nature, however, is often slow due to a limited energy source for bacterial growth. Enhanced denitrification is accomplished by stimulating indigenous denitrifying bacteria through the addition of a suitable energy source. Carbon substrates such as methanol, ethanol, acetate and sugar can significantly enhance denitrification rates by serving as electron donor and energy supply for the bacteria, while nitrate is the electron acceptor. The carbon substrate is biodegraded to carbon dioxide and water. In the metabolic denitrification process nitrate is transformed into final product nitrogen gas via a multi- step chemical reduction from NO₃ to NO₂ to NO to N₂O to N₂. Nitrite is usually the most significant intermediate while NO and N₂O are short lived.

U.S. Patent 5,482,630 teaches a process and system for the reduction of nitrate to nitrogen in a fluid medium, wherein a column of suspended beads is used as the anaerobic bacterial bed for denitrification.

Other background art includes Cyplik et al, Desalination, 207 (2007) 134-143; Van Houten et al., Biotechnology and Bioengineering, 1997, Vol. 55, No. 5, pp.807- 814; Vredenbregt et al., Water Science Technology, Vol. 36, No. 1, pp 93-100, 1997; Vrtovsek et al., Acta Chim. Slov. 2006, 53, 396-400.

SUMMARY OF THE INVENTION According to an aspect of some embodiments of the present invention there is provided a method of removing nitrates from brine, the method comprising contacting the brine with a halotolerant, anaerobic bacteria and a fluid miscible carbon source in a fluidized containment under neutral pH conditions, wherein the contacting is such that the halotolerant, anaerobic bacteria use the nitrates from the brine to generate gaseous nitrogen, and wherein the halotolerant, anaerobic bacteria are attached to particles as a biofilm.

According to an aspect of some embodiments of the present invention there is provided a system for removing nitrates from brine, the system comprising a containment comprising volcanic rock particles, the containment comprising a first port configured to allow nitrate-containing brine to flow into the containment and fluidize the volcanic rock particles and a second port being configured so as to allow an exit of nitrate-reduced brine from the containment. According to an aspect of some embodiments of the present invention there is provided a use of particles of volcanic rock for denitrification of brine.

According to an aspect of some embodiments of the present invention there is provided a method of enriching a heterogeneous bacterial population for anaerobic, halotolerant bacteria, the method comprising:

(a) culturing particles of volcanic rock with the heterogeneous bacterial population in a presence of a fluid medium under anaerobic conditions, the fluid medium comprising nitrate-containing brine and a carbon source so as to allow the anaerobic, halotolerant bacteria to form a biofilm on the volcanic rock; and (b) separating the volcanic rock from the fluid medium, thereby enriching a heterogeneous bacterial population for anaerobic, halotolerant bacteria.

According to an aspect of some embodiments of the present invention there is provided a population of anaerobic, halotolerant bacteria obtained by:

(a) culturing particles of volcanic rock with the heterogeneous bacterial population in a presence of a fluid medium under anaerobic conditions, the fluid medium comprising nitrate-containing brine and a carbon source so as to allow the anaerobic, halotolerant bacteria to form a biofilm on the volcanic rock; and

(b) separating the volcanic rock from the fluid medium, thereby enriching a heterogeneous bacterial population for anaerobic, halotolerant bacteria. According to some embodiments of the invention, the brine is generated following desalination of groundwater.

According to some embodiments of the invention, the brine is generated following desalination of waste water.

According to some embodiments of the invention, the neutral pH conditions are generated by addition of an acid to the fluidized containment.

According to some embodiments of the invention, the acid is combined with the fluid miscible carbon source prior to the contacting with the brine and the halotolerant, anaerobic bacteria to generate an acidic, fluid miscible carbon source.

According to some embodiments of the invention, the acidic, fluid miscible carbon source is selected from the group consisting of acetic acid, ethanol and hydrochloric acid, ethanol and sulfuric acid, methanol and hydrochloric acid, methanol and sulfuric acid and combinations of the above. According to some embodiments of the invention, the particles do not float in the brine.

According to some embodiments of the invention, the particles comprise porous particles. According to some embodiments of the invention, the particles comprise volcanic rocks.

According to some embodiments of the invention, the volcanic rocks comprise pumice and/or scoria.

According to some embodiments of the invention, the diameter of the particles is from about 0.5 to 5 mm.

According to some embodiments of the invention, the diameter of the particles is from about 1 to 2 mm.

According to some embodiments of the invention, the halotolerant, anaerobic bacteria comprise halotolerant bacteria H. denitrif leans. According to some embodiments of the invention, the biofilm is about 50-100 μm thick.

According to some embodiments of the invention, the first port is connected to a containment of nitrate-containing brine.

According to some embodiments of the invention, the first port is connected to a containment comprising a carbon source.

According to some embodiments of the invention, the system comprises a recirculation pump so as to further fluidize the volcanic rock particles. According to some embodiments of the invention, the system further comprises a pH meter for determining a pH of a fluid inside the containment.

According to some embodiments of the invention, the second port is connected to a filter.

According to some embodiments of the invention, the volcanic rock particles comprise a biofilm. Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced. In the drawings:

FIG. 1 is a graph illustrating nitrate nitrogen removal rate of the 9 liter fluidized bed reactor during the period of operation on brine from a reverse osmosis (RO) plant.

FIG. 2 is a graph illustrating effluent nitrate and nitrite concentrations of the 9 liter fluidized bed reactor using actual brine from a reverse osmosis (RO) plant.

FIG. 3 is a graph illustrating the acetic acid to nitrate nitrogen ratio used for effective denitrification of RO-generated brine in the 9 liter fluidized bed reactor.

FIG. 4 is a graph illustrating the Effluent TSS (Total Suspended Solids) and VSS (Volatile Suspended Solids) of the 9 liter fluidized bed reactor during the period of operation on RO-generated brine.

FIG. 5 is a graph illustrating Daily biomass (VSS) content in effluent and biomass removed during FB reactor cleaning during the period of operation on RO- generaled brine.

FIG. 6 is a graph illustrating effluent soluble total organic carbon (TOC) of the 9 liter fluidized bed reactor during the period of operation on RO-generated brine. FIG. 7 is a graph illustrating the effluent of total BOD (Biological Oxygen demand) (suspended + soluble) of the 9 liter fluidized bed reactor during the period of operation on RO-generated brine.

FIG. 8 is a diagram of an exemplar system according to embodiments of the present invention.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to a method of denitrification of brine and systems capable of same.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

The process currently most used for nitrate removal from ground water is the ion exchange process; specifically - anion exchange. Since this process does not destroy the nitrate, it eventuates in a more concentrated form in the waste streams. Since these waste streams inherently comprise considerable amounts of salt, disposal of nitrate- contaminated brine has become a relevant environmental issue.

Biological denitrification of brine necessitates use of halotolerant bacteria which are capable of maintaining osmotic balance with a surrounding salinated environment. Such salt-tolerant bacteria do not readily adhere to particle surfaces and accordingly it is difficult to achieve the desired biomass concentration for effective denitrification using a fluidized bed biological reactor (FBBR).

Whilst searching for particles on which halotolerant bacteria can form biofilms for use in the FBBR, the present inventors unexpectedly found that macrovesicular volcanic rock (e.g. scoria) was a suitable candidate. The present inventors found that biofilms formed on such carriers could withstand the high fluidization conditions prevailing in the reactor.

Thus, according to one aspect of the present invention there is provided a method of removing nitrates from brine, the method comprising contacting the brine with a halotolerant, anaerobic, bacteria and a fluid miscible carbon source in a fluidized containment under neutral pH conditions, wherein the contacting is such that the halotolerant, anaerobic bacteria use the nitrates from the brine to generate gaseous nitrogen, and wherein the halotolerant, anaerobic bacteria are attached to particles as a biofilm.

As used herein the term "brine" refers to water comprising salt, having a salinity between about 5 to 40 ppt (parts per thousand) which is about 0.5 to 4 % salt. Exemplary sources of nitrate-containing brines include brines from membrane desalination of brackish water that contain nitrogen compounds, brines from well reclamation plant treating well water rich in nitrogen compounds, and brines from membrane desalination of treated municipal wastewater. Brines with high nitrate concentrations are also produced in armaments plants and coal-fired power plants which are equipped with a wet lime (stone) gypsum flue gas desulphurization (FGD) process.

The term "halotolerant bacteria" refers to microbes that grow (i.e. propagate) in brine. According to one embodiment, the microbes grow optimally in the relevant salinity range and poorly or not at all in lower concentrations of electrolyte.

The present invention contemplates use of a heterogeneous population of halotolerant (i.e. salt-tolerant), anaerobic bacteria or a homogeneous population of a single strain of halotolerant, anaerobic bacteria. Examples of such bacteria include, but are not limited to H. denitrificans (ATCC 35960) and H. chitinovorans. Other examples of halotolerant, anaerobic bacteria are taught in Ollivier et al., Microbiol MoI Biol Rev. 1994 March; 58(1): 27-38, incorporated herein by reference. According to one embodiment a heterogeneous population of bacteria being enriched for anaerobic and halotolerant bacteria is generated by culturing particles of the volcanic rock with the heterogeneous bacterial population in a presence of a fluid medium under anaerobic conditions, the fluid medium comprising nitrate-containing brine and a carbon source so as to allow the anaerobic, halotolerant bacteria to form a biofilm on the volcanic rock.

As used herein, the phrase "volcanic rock" refers to a rock formed from magma which crystallize at the earth's surface.

Preferably, the volcanic rock comprises a large surface area: volume ratio. According to one embodiment, the volcanic rock is a vesicular rock - i.e. comprises many vesicles (cavities) and whose surface is rough with crevices and channels so as to expedite biofilm growth and attachment. Rock types that display a vesicular texture include pumice and scoria. According to one embodiment, the volcanic rock particles used are between about 0.5 to 5 mm and more preferably between about 1 to 2 mm.

In order to ensure anaerobic culturing conditions, the heterogeneous bacterial population is typically cultured in a non-aerated medium in the presence of the volcanic rock. Accordingly, the volcanic rock or size thereof, is selected according to one that docs not float i.e. has a specific gravity greater than 1.

Culturing is effected in the presence of a nitrate-containing brine (so as to ensure selection of halotolerant denitrifiers) and a carbon source (for use as an electron donor).

Examples of carbon sources contemplated for this embodiment of the present invention include, but are not limited to ethanol, acetic acid and methanol.

Following sufficient time to allow for initial biofilm formation (e.g. 1 day) in the bioreactor, the culturing medium is typically flushed out and all suspended bacteria are removed. This process may be repeated for a length of time sufficient for adequate biofilm formation on the volcanic rock. According to one embodiment, under initial operation or "start-up mode", the bioreactor is operated for about one day followed by continuous flow operation (e.g. 10 days) at low loading (e.g. 0.1-0.5 g N/L reactor / day).

According to one embodiment, the thickness of the biofilm on the volcanic rock is between about 20-200 μm, and more preferably between about 50-100 μm. As mentioned, the denitrification method of the present invention is effected by contacting the nitrate-containing brine with a biofilm of halotolerant, anaerobic, bacteria and a fluid miscible carbon source in a fluidized containment under neutral pH conditions.

Exemplary fluid carbon sources include, but are not limited to methanol, ethanol or acetic acid. The amount of fluid carbon source is selected such that the halotolerant, anaerobic bacteria remain viable and propagate and are capable of converting at least 80

%, at least 90 %, at least 95 %, at least 99 % of the nitrates and its intermediate reduction byproducts into nitrogen gas under appropriate conditions. Further, the amount of fluid carbon source is selected such that it is not present in a contaminating amount in the effluent e.g. no greater than 10mg/L.

Any method of directing the nitrate containing brine and the fluid miscible carbon source is envisaged by the present invention so long as the system remains at neutral pH conditions and the bacterial culture is capable of converting the nitrates in the brine into nitrogen gas.

According to one embodiment, the nitrate-containing brine (either in the presence or absence of the carbon source) is streamed (directly) into the bacterial culture. The brine may be directed at the bottom of the culture thereby creating turbulence, mixing the culture so that all the particles are fluidized. Preferably, the nitrate-containing brine is streamed into the bacterial culture without allowing air to enter the system.

According to one embodiment the minimal loading rate is about 0.5 g N per liter reactor per day, more preferably about 2 g N per liter reactor per day, more preferably about 4 g N per liter reactor per day and more preferably about 6.5 g N per liter reactor per day. It will be appreciated that higher minimal loading rates are also contemplated for the present system (e.g. up to 10 g N per liter reactor per day).

As used herein, the phrase "neutral pH" refers to a pH of about 6.5-7.5 (e.g. pH 7). The exact pH is determined such that not more than about 10 mg/L of CaCO₃ is present in the bacterial culture, more preferably not more that about 5 mg/L of CaCO₃ is present in the bacterial culture and even more preferably less than about 5 mg/L of

CaCO₃ is present in the bacterial culture.

Since the risk of scaling is more pronounced when applying high nitrate loads, the present inventors found maintenance of the system at a neutral pH prevents mineral (e.g. calcium carbonate) precipitation. In order to maintain neutral pH conditions, an acid is typically added to the fluidized containment. The acid may be added separately from the fluid miscible carbon source or alternatively together with the fluid miscible carbon source as an acidic, fluid miscible, carbon source. Contemplated acids which may be added to maintain a neutral pH include hydrochloric acid, acetic acid or sulfuric acid. Exemplary acidic, fluid miscible carbon sources include but are not limited to acetic acid alone, ethanol and hydrochloric acid, ethanol and sulfuric acid, methanol and hydrochloric acid, methanol and sulfuric acid and combinations of the above. The fluidized containment of the present invention is typically a containment

(e.g. a column) with a first port allowing nitrate-comprising brine to enter and a second port which allows the exit of nitrate-reduced brine. According to another embodiment the nitrate-reduced brine comprises less than

20 % of the nitrates present in the incoming nitrate-comprising brine.

According to another embodiment the nitrate-reduced brine comprises less than 10 % of the nitrates present in the incoming nitrate-comprising brine. According to another embodiment the nitrate-reduced brine comprises less than 5

% of the nitrates present in the incoming nitrate-comprising brine.

According to another embodiment the nitrate-reduced brine comprises less than 2.5 % of the nitrates present in the incoming nitrate-comprising brine.

According to another embodiment the nitrate-reduced brine comprises less than 1 % of the nitrates present in the incoming nitrate-comprising brine.

According to another embodiment the nitrate-reduced brine comprises less than 0.5 % of the nitrates present in the incoming nitrate-comprising brine.

According to another embodiment the nitrate-reduced brine is totally devoid of nitrate.

It will be appreciated that the nitrate-reduced brine of the present invention may be further treated in order to remove additional impurities. Thus, for example, the present invention contemplates filtration of the nitrate-reduced brine following the denitrification process of the present invention to remove any suspended solid contents such as excess biomass. Such a filter would preferably reduce the amount of suspended solid contents in the nitrate-reduced brine to less than 20 mg/litre. An exemplary poie size of such a filter would be about 20 microns. The filtrate from such a filter could be used as an additional carbon source or alternatively disposed of in an appropriate fashion. Other methods of cleaning the bacteria-comprising containment include bubbling nitrogen gas through the reactor followed by passing clean water through the column to purge the stripped biomass. Typically such cleaning processes are performed on a daily basis to prevent accumulation of suspended solid contents.

Thus, according to another aspect of the present invention there is provided a system for removing nitrates from brine. The system comprises a containment filled with the volcanic rock particles. The containment has a first port configured to allow nitrate-containing brine to enter and fluidize the volcanic rock particles and a second port configured to allow an exit of nitrate-reduced brine from the containment.

Reference is now made to Figure 8, which is a schematic illustration of a system configured to implement an exemplary system of the present invention. System 10 comprises a containment 12 for growing bacteria as a biofilm on particles of volcanic rock 14. The containment 12 comprises a first port 26 configured to allow nitrate containing brine 15 to stream therein. The nitrate containing brine 15 may be stored in containment 32 and may be generated following a desalination procedure. Containment 32 and containment 12 are fluidically interconnected. In addition, containment 12 comprises a second port 30 configured to allow an outflow of nitrate-reduced brine 17 from the containment 12. The second port 30 may be connected to a filter 18 to filter the nitrate-reduced brine 17 of suspended solid contents. The filtered nitrate-reduced brine may be collected in containment 20. Accordingly containment 12 and containment 20 are fluidically interconnected. In order to facilitate fluidization of the particles of volcanic rock 14, recirculation line 28 may be outfitted with a pump 29 for reactor water recirculation (fluidization of the carrier particles). The recirculation line 28 may originate from the top part of containment 12 and connect to the bottom part of containment 12 via the pump 29. Pump 29 may also be used to effect cleaning of containment 12.

Port 26 may be further outfitted with a feed pump 34 which, together with pump 29 may generate a total upflow velocity - e.g. up to 65-75 m/hour. Pump 34 may be equipped with two pump heads, one for the brine and a second for the addition of a carbon source 36 from containment 38. The ratio of brine feed water to the carbon source may be fixed at a particular ratio using pump 34, depending on the carbon source.

The combination of flows of influent brine and recirculation fluid is sufficient to fluidize the volcanic rock particles.

It will be appreciated that the carbon source may be fed through port 26 or alternatively through an additional port. The carbon source may be acidified in containment 36 or alternatively acid may be added through an additional port. Yet alternatively, the carbon source may be an acid (e.g. acetic acid). The dimensions of containment 12 is selected to be sufficient, for a given desired fluidizalion and to provide sufficient contact time to provide a desired level of nitrate consumption. According to one embodiment the minimal retention time is 12.5 minutes. It will be appreciated that higher retention times are also contemplated for the system of the present invention - for example up to 90 minutes.

According to one embodiment, containment 12 is cylindrical in shape (i.e. a column). Typically, the ratio of length to width may be greater than 20:1, and may exceed 100:1. It will be appreciated that containment 12 may also be other shapes such as square or circular. Typically, the particles of volcanic rock 14 fill at least 50 % of the containment

12, preferably at least 60 % and even more preferably at least 70 % of the containment.

According to an embodiment of this aspect of the present invention containment 12 comprises a cover 22. Cover 22 may extend over the entire surface of containment 12, or cover a part of containment 12. It will be appreciated that if containment 12 comprises a cover 22, the cover 22 must comprise a port 24 for release of the nitrogen gas 16 into the atmosphere.

Containment 12 may be maintained at certain temperatures or temperature ranges suitable or optimal for productivity using a temperature controlling device 29. These specific, desirable temperature ranges for operation will, of course, depend upon the characteristics of the bacterial species used within system 10, the type of containment, etc. Typically, it is desirable to maintain the temperature of the liquid medium between about 5 ⁰C and about 45 ⁰C, more typically between about 15 ⁰C and about 37 ⁰C, and most typically between about 15 ⁰C and about 25 ⁰C. In one embodiment, the temperature of the containment is maintained at about 20 ⁰C. Containment 12 may further comprise a pH monitoring device 34 so as to determine the amount of acid to be added to the system to ensure that the system does not allow mineral precipitation.

Conditions of the culture in the containment 12 may be monitored using any suitable type monitoring devices e.g. a computer-implemented system 31. Variables that may be tracked include without limitation, pH, temperature, conductivity, turbidity, dissolved nitrate concentration, dissolved oxygen, as well as the concentrations of ammonia and chloride. These variables may be recorded through out system 10. Monitoring device 31 may also be used to monitor and control the operation of the various components of the systems disclosed herein, including valves, sensors, weirs, blowers, fans, dampers, pumps, etc.

The term "port" as used herein, refers to a path for distributing liquid or gas, either on or above ground surface or underground, which may include but is not limited to ducts, pipes, channels, tubes, troughs or other means for distribution.

The term "fluidically interconnected", when used in the context of two containments refers to the existence of a continuous coherent flow path from one of the containment to the other. In this context, two containments can be "fluidically interconnected" if there is, or can be established, liquid and/or gas flow through and between the ports even if there exists a valve between the two conduits that can be closed, when desired, to impede fluid flow there between.

Ports 26 and 30 may be made from any material capable of transporting and withstanding the pressure and temperature of the incoming nitrate containing brine or outgoing nitrate-reduced brine such as aluminium, steel, PVC, glass or rubber. Contemplated materials suitable for fabricating containment 12 include, but are not limited to PVC, steel, etc.

It is expected that during the life of a patent maturing from this application many relevant desalination techniques will be developed and the scope of the term desalinating is intended to include all such new technologies a priori. As used herein the term "about" refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of means "including and limited to". The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof. Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements. Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion. Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", VoIs. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley- Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. L, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

EXAMPLE 1 Removal of nitrates from reverse osmosis generated brine

MATERIALS AND METHODS

Brine Characteristics: Table 1, herein below presents the average values for the relevant chemical characteristics of brines (including antiscalant) received from a reverse osmosis plant on two separate occasions. Comparison of these brines revealed that they are very similar in chemical composition particularly in regard to nitrate (about 50 mg/L) and electrical conductivity (about 10.6 mS/cm). No phosphate (PO₄ ^'), ammonium or nitrite (NO₂ ^") was present in the brine.

Table 1- brine characteristics

Fluidized Bed Reactor Configuration and Operational Characteristics: Table 2 herein below gives the dimensions and the operational characteristics of the 9 liter fluidized bed reactor. The fluidized bed reactor was constructed out of transparent PVC piping and was outfitted with 2 peristaltic pumps, one for reactor water recirculation (fluidization of carrier) and another for influent feeding. The influent feeding pump was equipped with two pump heads, one for the brine and the second for the addition of carbon source (acetic acid) and phosphate. The ratio of brine feed water to the nutrient feed water was about 10 to 1. Due to similar cost, the choice of carbon source for denitiϊfication was changed from methanol plus hydrochloric acid (to prevent mineral precipitation) to acetic acid.

The biofilm carrier use in the duration of the experiments was tuff (pumice) from the Ramat Hagolan, Israel, with a particle size of between 1 and 2 mm. The tuff carrier is a porous, irregularly shaped carrier allowing for biomass retention and was shown to give more consistent results than sand particles particularly at lower nitrogen loading rates.

9 Liter Reactor Startup and Operation: The startup period was conducted using simulated laboratory prepared brine at the same nitrate concentration. The startup included operation up to 2 g N/1 reactor/day. In reactor startup, no problems of nitrogen removal were observed at low loading rates and nitrogen concentrations in the effluent were always below 1 mg/1. Particular attention was given to controlling the carbon (acetic acid) to nitrate ratio in the startup period to effectively remove all nitrate while minimizing excess effluent total organic carbon (TOC). The stoichiometric ration of 3.64 mg acetate per milligram nitrate was found to give good results. Excess biomass from the reactor was removed on a daily basis by first bubbling nitrogen gas through the reactor followed by passing clean water through the column to purge the stripped biomass. High upflow velocities were used in order to fluidize the slightly larger tuff particles used and to ensure mixed conditions.

Table 2. Fluidized Bed Characteristics

9 liter Fluidized Bed Operation Using RO generated Brine

FB Nitrogen Removal Rate: As mentioned above, operation of the 9 liter fluidized bed reactor using actual brine from the reverse-osmosis plant began with the reactor already operating at 2 g N/1 reactor/day. Using actual brine, the reactor continued to operate at 2 g N/1 reactor/day for two weeks in order to establish base line data before increasing the nitrogen loading rate (Figure 1). Following the two week period, the loading rate of the reactor was steadily increased over the next 25 days to just over 5 g N/1 reactor/day. No decrease in nitrogen removal rate was observed as the loading rale was increased. The average nitrogen removal rate for the 40 day period was 98.7±1.2 %.

FB Effluent Nitrate and Nitrite Concentrations: Effluent nitrate and nitrite concentrations from the 9 liter fluidized bed reactor are shown in Figure 2. The figure shows that the concentration of nitrate and nitrite throughout the experimental period were very low, nearly always below 1 mg/1. The average nitrate concentration was 0.44±0.4 mg/1, while the average nitrite concentration was 0.26±0.6 mg/1. Concentrations of nitrite or nitrate in excess of 1 mg/1 in the effluent could always be attributed to slight fluctuations between the flow rates of the brine and nutrient feed. Carbon Source Requirement for Denitrification: As mentioned above in the startup section, careful attention was given to determining the optimal dose of acetic acid (carbon source) for efficient denitrification. Too much dosing of carbon can result in an effluent with total removal of nitrates, but residual TOC can be present requiring post treatment. Too little dosing of carbon will result in excessive residual nitrate and possibly nitrite in the effluent water. The acetic acid to nitrate nitrogen ratio was measured on a daily basis and shown in Fig. 3. Slight increases and decreases in the C/N ratio were made according to the nitrite and nitrate concentrations found in the effluent (Figure 3). The average acetic acid to nitrate nitrogen ratio for 99 % removal of nitrate without overdosing was 3.64±0.15 mg/L which is in good agreement to literature values. Bacterial Biomass Production and Removal in the 9 liter FB Reactor:

Bacterial biomass is produced as a byproduct of biological denitrification. The biomass produced builds up as excess biofilm in the reactor and is also washed out with the effluent. Overall, in the 9 liter FB reactor treating RO-generated brine, it was found that for every mole of NO₃-N reduced, 0.849 mole acetic acid were consumed and 0.086 mole biomass were produced (biomass yield of about 0.3 g COD cells per g COD utilized). This translates into 39.5 mg biomass production measured per liter of Granot brine fed to the reactor.

Effluent TSS and VSS concentrations from the 9 liter fluidized bed reactor are shown in Figure 4. The average TSS value was 35.9±5.5 mg/1, while the average VSS value was 32.5±4.8 mg/1. Therefore, the majority or 82.3% of the biomass produced in the FB reactor was washed out with the effluent stream. A further smaller fraction of 17.8 % was removed during daily reactor cleaning (see reactor startup) as shown in Figure 5. The relatively high amount of TSS in the effluent was a result of the higher upflow velocities used to fluidized the tuff particles and ensure completely mixed conditions in the reactor for low effluent nitrate.

ReactorEffluent TOC and BOD of the 9 Liter Reactor: Reactor soluble TOC (Total Organic Carbon) shown in Figure 6 and total (soluble and suspended) BOD (Figure 7) was monitored closely during the operation of the 9 liter reactor with RO- generated brine. TOC was measured on the filtrate (0.45 micron filter) of the effluent and is a good indication of carbon dosing for the denitrification process. An average soluble TOC low value of 4.3±2.1 mg/L was obtained meaning there was little or no excess acetic acid added to the process with the residual TOC probably originating from biomass. BOD was measured on the total effluent and an average value of 24.3±3.7 mg/L was obtained. This is in line with the VSS values observed in the effluent.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

WHAT IS CLAIMED IS:

1. A method of removing nitrates from brine, the method comprising contacting the brine with a halotolerant, anaerobic bacteria and a fluid miscible carbon source in a fluidized containment under neutral pH conditions, wherein said contacting is such that said halotolerant, anaerobic bacteria use the nitrates from the brine to generate gaseous nitrogen, and wherein said halotolerant, anaerobic bacteria are attached to particles as a biofilm.

2. The method of claim 1, wherein said brine is generated following desalination of groundwater.

3. The method of claim 1, wherein said brine is generated following desalination of waste water.

4. The method of claim 1, wherein said neutral pH conditions are generated by addition of an acid to said fluidized containment.

5. The method of claim 1, wherein said acid is combined with said fluid miscible carbon source prior to said contacting with said brine and said halotolerant, anaerobic bacteria to generate an acidic, fluid miscible carbon source.

6. The method of claim 5, wherein said acidic, fluid miscible carbon source is selected from the group consisting of acetic acid, ethanol and hydrochloric acid, ethanol and sulfuric acid, methanol and hydrochloric acid, methanol and sulfuric acid and combinations of the above.

7. The method of claim 1, wherein said particles do not float in the brine.

8. The method of claim 1, wherein said particles comprise porous particles.

9. The method of claim 1, wherein said particles comprise volcanic rocks.

10. The method of claim 9, wherein said volcanic rocks comprise pumice and/or scoria.

11. The method of claim 1, wherein a diameter of said particles is from about 0.5 to 5 mm.

12. The method of claim 1, wherein a diameter of said particles is from about 1 to 2 mm.

13. The method of claim 1, wherein said halotolerant, anaerobic bacteria comprise halotolerant bacteria H. denitrificans.

14. The method of claim 1, wherein said biofilm is about 50-100 μm thick.

15. A system for removing nitrates from brine, the system comprising a containment comprising volcanic rock particles, said containment comprising a first port configured to allow nitrate-containing brine to flow into said containment and fluidize said volcanic rock particles and a second port being configured so as to allow an exit of nitrate-reduced brine from said containment.

16. The system of claim 15, wherein said first port is connected to a containment of nitrate-containing brine.

17. The system of claim 15, wherein said first port is connected to a containment comprising a carbon source.

18. The system of claim 16, wherein said first port is connected to a containment comprising a carbon source.

19. The system of claim 15, comprising a recirculation pump so as to further fluidize said volcanic rock particles.

20. The system of claim 15, further comprising a pH meter for determining a pH of a fluid inside said containment.

21. The system of claim 15, wherein said second port is connected to a filter.

22. The system of claim 15, wherein said volcanic rock particles comprise a biofilm.

23. The system of claim 15, wherein said volcanic rock particles comprise pumice and/or scoria.

24. Use of particles of volcanic rock for denitrification of brine.

25. A method of enriching a heterogeneous bacterial population for anaerobic, halotolerant bacteria, the method comprising:

(a) culturing particles of volcanic rock with the heterogeneous bacterial population in a presence of a fluid medium under anaerobic conditions, said fluid medium comprising nitrate-containing brine and a carbon source so as to allow said anaerobic, halotolerant bacteria to form a biofilm on said volcanic rock; and

(b) separating the volcanic rock from said fluid medium, thereby enriching a heterogeneous bacterial population for anaerobic, halotolerant bacteria.

26. The method of claim 25, wherein said volcanic rock comprises pumice and/or scoria.

27. The method of claim 25, wherein said halo tolerant, anaerobic bacteria comprise halotolerant bacteria H. denitrificans.

28. A population of anaerobic, halotolerant bacteria obtained according to the method of claim 25.