Patent Application
20160298104
The present invention refers to treatment of fluid medium containing biological matter, preferably liquids containing plurality of biological cells distributed therein. Among the fluid medium, which can be treated in accordance with the present invention are for example various liquids containing bacteria, pathogens, viruses, algae and any other biological matter.
More particularly the present invention refers to method for electrical treatment of such liquids to cause lysis of the biological cells, their destroying and extraction of biological intracellular material from the biological cells.
Even more particularly the present invention refers to method for electrical treatment of liquids based on causing electro-kinetic flow within the liquid and near the cells, such that the flow causes destruction of membranes of the biological cells distributed in the liquid.
The method for electrical treatment and the system for its implementation in accordance with the present invention are suitable for example for treatment of liquids containing biological matter for the purpose of disinfection and/or for improving of various environmental parameters of waste waters.
Still further examples of liquid suitable for electrical treatment in accordance with the present invention are liquid waste waters originated from various industrial and agricultural installations, municipal sewage, waste waters originating from slaughter houses, from mining installations, fracturing waste waters, waste waters originating from food industry, from cosmetic industry, from pharmaceutical industry; liquids containing plant matter such as raw, dried, or powdered flowers, roots, algae, stems, peels, seeds, fruits and the like; animal sludge, municipal waste solids and water, including increase of methane production in digester, fish or poultry solid wastes. It should be taken into consideration, however, that the present invention is not limited to liquids containing solely biological cells; it is as well would be suitable for extraction of intracellular matter from liquids containing oils, fats, sugars, minerals, etc.
The method for electrical treatment according to the present invention comprises exposing electro-conductive liquid containing biological matter to discrete electrical pulses emanated from electrodes.
In accordance with some embodiments of the present invention the electrical treatment can be carried out either in batch mode or in the continuous mode.
After the treatment the biological material can undergo separation of solids from liquids by one of known separation technologies (gravitation, centrifugation, pressing, filtration, decanter, ion membrane exchange) or left as it is with improved environmental properties e.g. sprayed on fields as fertilizer, used as bedding, food, etc.
There are known in the art various methods for electrical treatment of liquids containing biological matter by means of applying electrical current thereto.
In WO 2008155315 there is described a device for cleaning and sterilizing fluids, in particular water. The device comprises elongated tubular container having an inlet and an outlet and a couple of flat electrodes installed within the container so as to be in the flow path of the fluid treated. According to the patent at least one electrode is coated with porous ceramic coating on the side facing the opposite electrode. The device comprises also an impulse generator unit electrically connected to the electrodes and capable for applying to the fluid of pulsed coronal discharges with the field strength of at least 10,000,000 V/m.
In US 2012000782 there is disclosed a uniform electrical field dielectric barrier discharge reactor for purifying of air, sterilizing of fluids or treatment of waste material. The reactor comprises an electrode unit, a dielectric catalyst container and an insulated housing. The electrode unit comprises electrode plates with discharge needles distributed on the insulated plane frame structure.
In CN 102060357 there is disclosed electrolysis reactor for treatment of high salinity waste waters. The reactor is designed as a cylindrical tube through which passes central water inlet pipe. The reactor is provided with radially installed flat electrodes.
In CN 201623198 there is described cylindrical reactor for use in microbial fuel cell. The reactor is provided with a couple of flat electrodes immersed in the electro genesis substrate within the reactor.
In JP 7299464 there is described multipurpose water treatment tank for sterilizing, cleaning and electrolyzing water. The tank is designed as a vessel of cylindrical configuration. The vessel is provided with a couple of concentric circular electrodes mounted to a cover such that they face each other.
In CN 102437360 there is disclosed multi electrode microbial fuel cell comprising a housing accommodating therein detachable circular partition plates of different diameter and detachable circular electrode plates of different diameters. Both the circular plates and the circular electrodes divide the housing into cathode chamber and anode chamber and they can be disassembled.
In CN101187038 there is described reactor for fluorination and electrolysis, which comprises electro pads, negative and positive electrode terminals, negative and positive electrode fitted rods and a generator.
In U.S. Pat. No. 6,141,905 there is described process and apparatus for utilizing animal excrement. According to this invention an aqueous mixture containing solid feed excrements from animals is subjected to treatment with an alternating electric current at a frequency of a predetermined magnitude and for a predetermined period of time, when the mixture passes through a tubular reactor.
In U.S. Pat. No. 6,344,349 there is disclosed process and system for electrical extraction of intracellular matter from biological waste materials, e.g. animal and human compost. The process comprises preparation of a mixture of biological matter with electro conductive fluid and then passing thereof through a processor unit while exposing the mixture to series of electrical pulses separated by pauses. In the process disclosed in U.S. Pat. No. 6,344,349 the electrical parameters of the applied pulses are controlled in such a manner, that the fluid medium is exposed to consequent series of electrical pulses which energy is sufficient for piercing of holes into or perforating the cell membranes of the cells. By virtue of this provision the cells membrane is destroyed and intracellular matter which releases from the cells is collected.
Unfortunately this mechanism requires electrical pulses with relatively large energies. This requirement, in its turn, is associated with excessive heating of the fluid medium, which might be not desirable or in some cases even not allowed at all. To prevent or at least to reduce the heating of the liquid the supplied electrical pulses are controlled in such a manner that intervals between consecutive series of pulses are kept long enough to let the fluid medium to cool down. It can be readily appreciated that in some cases this might render the process of treatment too long.
Furthermore, in order to generate electrical pulses with energy sufficient for perforating the cell membrane a relatively large power supply unit would be required, which might render this method and the system for its implementation too expensive, cumbersome and inefficient.
Thus it can be appreciated that despite of many attempts to solve the problem of electrical treatment of liquids containing organic matter there is still needed a new and improved method and system which would be suitable for fast, efficient and inexpensive treatment of various liquid wastes and in particular would be suitable for the extraction of intracellular matter from fluid biological waste materials.
The main object of the present invention is to provide for a new and improved method and system suitable for inexpensive, efficient and fast extracting intracellular matter from fluids containing organic matter by exposing thereof to electrical pulses.
The further object of the present invention is to provide for a new and improved method and system for extracting intracellular matter from fluids containing organic matter by exposing thereof to electrical pulses by inducing electro-kinetic flow in the fluids resulting in lysis of wall of the cells.
Still further object of the present invention is to provide a new and improved method and system for extracting intracellular matter from fluids containing biological cells by exposing thereof to electrical pulses capable to induce electro-kinetic flow in the fluid resulting in acceleration of the cells and establishing shear stress in vicinity of the cell walls, while the shear stress stretches the cell walls.
Yet another object of the present invention is to provide a new and improved method and system for extracting intracellular matter from fluids containing biological cells by exposing thereof to bipolar, discrete electrical pulses separated by pauses, while duration of a single pulse being equal or shorter than duration of a pause between two consecutive pulses, and duration of the single pulse being equal or longer than the time of acceleration of the cells.
Still further object of the present invention is to provide a new and improved method and system for extracting intracellular matter from electrically conductive fluids containing organic matter by exposing thereof to discrete electrical pulses, resulting in establishing electrical field having electrical strength of 30 V/m to 30,000 V/m.
Yet another object of the invention is to provide a new and improved method and system for extracting intracellular matter from electrically conductive fluids containing organic matter while said fluids being defined by electrical resistance of 0.05 Ohm·m-5 Ohm·m and said fluids have weight concentration of organic matter of 0.1%-10%.
The electrical pulses employed in the method of the present invention are defined by a very short time period and by a low electrical strength. This specific combination allows inducing the electro-kinetic flow resulting in establishing shear stresses in vicinity of the cell's wall. Those cells which initially were in rest start moving. At the beginning of the motion, when the cells accelerate the flow velocity is relatively high A shear stress establishes which is applied to the cell's wall. As soon as the shear stress exceeds ultimate tensile stress of the cells walls it ruptures the cell's wall and eventually causes lysis of the cells. This process requires relatively low energy. Furthermore, the electro-kinetic flow influences on the cell membrane and this could create liposomes by virtue of vesicle biogenesis. The liposomes can be used for destroying viruses and for drug delivery due to blood circulation to various parts of the body, thus enhancing vesicle uptake by target cells.
The intracellular material could be separated from solids by any known separation means, e.g. pressing, gravitational separation, centrifugation, filtration or other means.
Before explaining the present invention in more details some definitions will be given for various terms which will be used in the future through disclosure of the present invention.
Under the term “lysis” here will be understood destruction, destroying or any other termination of integrity of biological cell which is caused by tearing off of the cell's outside wall and results in liberation of intracellular matter and its exit from the cell.
Under the term “cell wall” here will be understood an outside wall that surrounds and separates biological cells.
Under the term “intracellular matter” here is understood content of an organic cell delimited by its outer wall.
Under the term “electro-kinetic” flow here will be understood flow of a liquid, in which the cells are distributed, while this flow is induced upon exposure the liquid to an electrical field.
Under the term rms here is meant “mean root square”
With reference to
In
With reference to
Proteins embedded within the phospholipid bi-layer carry out the specific functions of the plasma membrane, including selective transport of molecules and cell-cell recognition. The phospholipids are arranged in a bi-layer, with their polar, hydrophilic phosphate heads facing outwards, and their non-polar, hydrophobic fatty acid tails facing each other in the middle of the bi-layer. This hydrophobic layer acts as a barrier to all but the smallest molecules, effectively isolating the two sides of the membrane. Different kinds of membranes can contain phospholipids with different fatty acids, influencing on the strength and flexibility of the membrane, and animal cell membranes also contain cholesterol linking the fatty acids together and so stabilizing and strengthening the membrane.
It is known in the art to expose the liquid containing biological cells to electrical pulses for the purpose of destroying the biological cells. This method is known as electroporation.
The parameters of electrical field strength, E, used for bacteria electroporation are in the range of 100,000-2,500,000 V/m while the pulse duration is less than 0.001 s.
In the present invention is also employed exposure biological cells to electric pulses for the purpose of destroying the cells, nevertheless the electrical field strength of the pulses is several orders of magnitude lower in comparison with electroporation and it lies in the range of 30 V/m-30,000V/m. The range of the pulse duration is 0.00005-0.005 s.
It has been empirically revealed that it is possible efficiently destroy the cells by electrical pulses defined by much lower electrical field strength. This is possible by virtue of electro-kinetic flow induced in the liquid. The electro-kinetic flow causes establishing of shear stress in vicinity of the cell wall, which stretches it and eventually causes lysis of the cell as soon as the shear stress exceeds the ultimate tensile strength of the cell wall.
The bacteria cell membrane carries a net negative charge under most physiological conditions. This charge interacts with ions in the surrounding liquid, while generating different layers. Among them is the Stern layer which is a layer with ions charged oppositely to the surface charge of the cell wall. This layer is immobilized due to strong interaction with the cell wall. There is also the Gouy-Chapmen layer that consists of diffuse ions of net same sign as the Stern layer, but it is mobile.
As shown in
With reference to
Referring now to
Referring now to
In
When electrical field is applied, first the electro-kinetic flow in a EDL is generated. At the beginning of the pulse, the cell due to its inertia is at rest. The electro-kinetic flow induces shear stress in vicinity of the outer cell wall due to viscosity of the liquid in which the cell is distributed. The shear stress causes acceleration of the cell during a characteristic time τac.
During this time period the shear stress applied to the cell is maximal.
However as soon as the cell starts' moving with a steady state velocity, the shear stress reduces. The shear stress established during the acceleration period τac stretches the outer wall and tears it off and brings to lysis of the cell as soon as the shear stress exceeds ultimate tensile strength of the outer wall. In accordance with the present invention in order to enable efficient lysis of the cell the pulse (signal) characteristic time, τs, should be equal or be less than the acceleration time, i.e.
τs≦τac
This would be the first criterion, which should be satisfied in order to cause lysis of the cell by electro-kinetic flow in accordance with the present invention.
Now with reference to
In
Both forces remain equal until tensile force reaches ultimate tensile strength of the outer wall and further increase of the shear force results in rupture of the outer wall. Thus, the condition for rupture of the outer wall would be:
F
w
=F
σ
From this expression it is possible to derive the radius, RL, of the line of rupture of the cell as follows:
where
S is area of the outer wall of the cell to which shear stress is applied,
σw is ultimate tensile strength of the area S
σ is shear stress applied to the area S
We can further assume that
S≈4πRc2
And thus
Thus the lowest value of RL is equal to about diameter of lipid molecules, Dlipid, but it should be not more than radius of the cell, and therefore the second criterion for establishing the cell lysis is:
This criterion depends on different physical parameters of the biological system, like zeta-potential of a cell, temperature, ion's concentration, ultimate tensile strength of the cell wall and it also depends on the applied electrical field strength. The applied electrical strength and the ion concentration define the electrical current density. Therefore, the second criterion can be rewritten as a relationship between electrical field strength E and a density of electrical current J, or as electrical power E·J, which should be supplied to the cells during the treatment:
In the above expression the numbers are obtained empirically from the tests and m is meter, V—volt, A—amper.
In practice electrical pulses defined by electrical field strength of 30 V/m-30,000 V/m would be suitable for inducing electro-kinetic flow to cause rupture of walls of various biological cells and their lysis.
Furthermore it has been empirically revealed that after exposure the cells to a pulse the outer wall of the cells is stretched and a pause is required for relaxation of the cell wall before it is exposed to the next pulse. This relaxation or pause time, τp, should be equal or not shorter than the signal time and therefore the third criterion for achieving lysis of the cell in accordance with the present invention is:
τp≧τs
Now with reference to
A system shown in
It is not shown specifically but should be appreciated that processor unit is provided with appropriate inlet and outlet port for entrance of liquid containing biological cells to be treated and for exit of the liquid with distributed therein intracellular matter liberated from the cells during the treatment. The power modulated by the convertor unit is supplied to the electrodes, which in response emanate electrical pulses into liquid to be treated. It is also shown schematically a feedback line 6, which enables adjustment of electrical parameters of the pulses.
As mentioned above the power source unit supplies sinusoidal electrical wave with specific voltage V and current I, depending on resistivity of the liquid media to be treated. In practice liquids having resistivity varied between 0.05 Ohm·m and 5 Ohm·m can be treated in accordance with the present invention. Therefore, the power should be modulated by the signal convertor unit 2 so as to supply a pulse with characteristic times, maximum amplitude voltage and amplitude current and with minimum rms current as might be required for treatment of particular biological material. By virtue of a feedback line 12 the minimum rms current is set by variation of the pulse characteristic time depending on the media electrical resistivity.
An example of electronic schematics, which is responsible for modulation of the power and supplying to electrodes pulses with required parameters, is depicted in
Now with reference to non-limited examples 1, 2 and 3 and to
Full scale operation unit was tested in close-loop system for treatment anaerobic digested sludge with concentration of solids about 1-2%. The sludge was exposed to electrical pulses cut from a standard sinusoidal wave having frequency of 60 kHz. The pulses were cut from the voltage 120 V supplied by the power source unit 1.
The maximum electrical strength of a single pulse was around E=250-400 V/m and the characteristic time, τs, was about 0.0006-0.001 s. The pause time, τp,
was about 0.0073-0.0077 s. The electrical resistivity of the media varied between 0.5 and 0.7 Ohm·m. Reduction of pathogens due to the electrical treatment is presented in
The tests were carried out using an experimental lab unit that could supply pulses cut from standard sinusoidal wave of 120V having frequency 60 kHz as supplied by the power source unit. The pulse pattern was obtained by cutting thereof from the sinusoidal wave at close to zero voltage point. The maximum electrical strength of a single pulse was around E=250-400 V/m and the characteristic time were about 0.0006-0.001 s. In the second case the pulse pattern was obtained by cutting from the sinusoidal wave near the maximum voltage and the maximum electrical strength of a single pulse was around E=1,100 V/m and the pulse characteristic time, τs, was about 0.0002-0.0005 s. The pause time,
was about 0.0078-0.0081 s. The used pulse patterns are seen in
The treated material was the waste activation sludge (WAS) with solids concentration 1-3%. The material was treated during short periods, up to one minute and sent for analysis. In contrast to the first example it was batch treatment and not a close loop treatment.
In the first case, when the pulse pattern was as shown in
The tests with algae distributed in water have been carried out with a purpose to destroy the cell membrane and to extract oils from the algae cells. The tests were performed using the both patterns of electrical pulses cut from standard sinusoidal wave of 220V having frequency 50 kHz as supplied by the power source unit, as in the example 2.
We have performed several tests that we can divide into three categories as follows:
When we treated the algae solely with benzene we found no reaction. (Note: benzene was chosen as a product known for its extraction capabilities) In other words there was no oil liberation from the algae that naked eye could observe. The mixture looked the same before and after treatment. The picture labeled Sample “A” as seen in
The next treatments involved first heating the algae mixture to at least 90° C., then adding benzene. At no point during this treatment did we observe any change in the appearance of the algae mixture. The results of this process are shown in
Finally, we conducted experiments in which we first exposed the algae mixture to electrical pulses, and then added benzene. The pulses of the pattern shown in
To enhance our study we used a microscope to better investigate appearance of the algae mixture as an indication of the impact of the treatment on the algae. In
We then treated the algae by processing it in a centrifuge. What happened is that virtually all solid matter was separated and collected to one side and the water part was clear, meaning being free from all solids associated with the algae.
Next, we treated the mixture of algae with water by exposing it to electrical pulses in accordance with the present invention and again ran the mixture through the centrifuge. Remarkably, we could notice a significant difference in the “water” portion of the mixture that now obviously contained parts of the algae. Pictures of the liquid portion are shown below in
The main conclusion from this experiment with the centrifuge and microscope is that the electrical treatment causes parts of the algae cells to break down and be left aside. No other extraction technique was used; neither did we estimate what percentage of algae was found in the liquid portion.
From the above examples it is evident that the method and system for electrical lysis of biological cells distributed in a liquid medium according to the present invention has advantages in terms of efficiency of the treatment and of convenience in exploitation.
It should be appreciated also that the present invention is not limited by the above described embodiments and that one ordinarily skilled in the art can make changes and modifications without deviation from the scope of the invention as will be defined below in the appended claims.
It should also be appreciated that features disclosed in the foregoing description, and/or in the foregoing drawings, and/or examples, and/or tables, and/or following claims both separately and in any combination thereof, be material for realizing the present invention in diverse forms thereof.
When used in the following claims the terms “comprise”, “contain”, “have” and their conjugates mean “including but not limited to”.
