METHOD FOR ACCELERATING GROWTH RATES

Abstract: Upon subjection to a low energy electrical field of varying and critical frequencies and low field strengths the growth rate of an organism disposed in a growth medium exhibits a significant increase over power-off controls.

Description: METHOD FOR ACCELERATING GROWTH RATES 1. Field of the Invention This invention relates to a method for increasing the growth rate of biosystems. More particularly, this invention provides a method for accelerating the growth rates of organisms which involves the use of low energy electrical fields produced by a unique com¬ bination of applied low field strengths and extra-low electromagnetic frequencies . 2. Prior Art The effects of electrical fields on the stimulation of growth and regeneration in various biosystems have been studied. Mcelhaney et al., J. Biomech. 1 ^ :47 (1968) disclose the use of electrical fields to stimu¬ late bone formation. The use of applied magnetic fields to accelerate growth in microorganisms is shown in U. S. Patent 3,871,961. Sewage decomposition pro¬ moting microorganisms are stated in U. S. Patent 3,336,220 to exhibit increased activity after an alter¬ nating electric current was passed through the sewage. MacKenzie in New Scientist, 28 January 1982, pp. 217-220 reports on electrical fields which develop in connection with loss of limbs and which are implicated in the resultant regeneration phenomena. The effects of electrical fields on movement in an amoeba was reported in Science, 187:357 (1975). Much of the work performed studying the effects of electrical fields on microorganisms has involved the study of mutagenesis and also sterilization, e.g., see Coate, W. B. et al. 1970, "Project Biological Effects Test Program Pilot Studies Final Report." Hazilton Laboratories, Inc., available from NTIS as AD717409, U. S. Patent 3,876,373 and Food Tehnol, 8:361 (1954). SUBSTITUTE SHEET It is an object of this invention to provide a unique and practical methodology utilizing low energy electrical fields to accelerate growth rates when applied to a living biosyste . It is a further object of this invention to increase such growth rates without producing mutants and without a measurable increase in the temperature of the biosystem. It is a still further object of this invention to provide an energy efficient, low cost and effective process for signifi- cantly reducing the growth cycle time of a biosystem. DESCRIPTION OF THE INVENTION These and the other objects of this invention are accomplished by a process for accelerating the growth rate of a live organism which comprises disposing the organism in a suitable growth or nutrient medium and subjecting the organism to an alternating electrical field at an electrical field strength and frequency combination which causes the growth rate of the orga¬ nism to increase over the growth rate of the organism when not subjected to the alternating electrical field. The desired increase in growth rate is independent of current density and is exhibited at varying frequencies and electrical field strengths. However, as far as has been tested there appear to exist certain critical fre- quencies, or regions of frequencies, at which acceler¬ ated growth occurs and outside of which there is minimal or no effect. Also at such effective frequen¬ cies there exists a threshold electrical field strength below which there is no effect. It has also been found that, within the ranges of effective frequencies and field strengths, the resultant electrical field is a low energy field which does not produce mutants of the organism; nor is there a measurable increase in the temperature of the biosystem. Furthermore, in the pre- ferred ranges of effective frequencies and strengths there is no electrolysis of the growth medium. It has found that the effective frequencies and voltages are independent of current density. However, there is a practical limitation on current density in that one would not employ currents that adversely affect the biosystem. Tests have been conducted at less than 0.1 mA (milliamp) and it is believed that absolutely no current is required to effectively increase growth rates, so long as effective field strengths and fre¬ quencies are utilized in carrying out the invention. Definition of Terms "Growth Rate (rate)" - This is obtained by taking the Klett units over time and calculating the doubling time for the culture. This is done by plotting the Klett unit number of each reading on the log scale of a semi- log graph (wherein the y axis is the semi-log scale and the x axis is time). The slope (m) of the curve is then calculated using the following formula: E xy E X Ey N m = Eχ2 .__. ( Ex ) 2 N wherein m is the slope, x is time, y is the semi-log, and N is the number of individual Klett readings. "Klett units" - a unit of measurement of optical den¬ sity employing a Klett-Summerson photoelectric colori¬ meter. "LeeGs value (L)" is obtained by dividing the growth rate of the test sample by the growth rate of the control sample thus _ _ G test G cont "Correlation (coor ) " is calculated in accordance with following mathematical sequence : corr = R = Ms x s y wherein s is the standard deviation of the x and y axes of the semi-log graph referred to in the definition of growth, rate. "Dev" is an abbreviation for the standard deviation between the control groups calculated in the following manner: wherein x is the value of the growth rate of each control group and n is the number of control groups. "L-broth" - a growth medium consisting of 0.8% NaCl, 0.8% glucose and 0.7% bacto-tryptone, all percentages being weight to volume of tap water. "Biosystem" - a system comprised of the organism disposed in a medium suitable for its normal growth. "Hz" - abbreviation for hertz, a unit synonomous for cycle per second. "KHz" - 1 kilohertz or 1000 hertz. "ATCC" - American Type Culture Collection a depository for microorganisms located in Rockville, Maryland. Example 1 An overnight exponential culture of prototropic E. coli K-12 (Yale University depository number CGSC No. 4401) growing at 37°C, in L-broth was diluted with thermally equilibrated and sterilized L-broth to a den¬ sity of 1 x 10 6 cells per milliliter. Portions of this culture were then randomly placed into 10 ml. glass test tubes made of the type glass specified for the colorimeter employed. Each test tube contained two platinum electrodes, spaced 4 centimeters apart, wired in series to a variable frequency generator and in parallel to an oscilliscope, and equipped with means for oxygen input to the growth medium. The tubes con¬ taining the fractionated culture medium were maintained at constant temperature in a thermostatically controlled water bath and each subjected to an electro- motive (EmF) field. Temperature was monitored with a glass/mercury thermometer. Each tube was isolated from the field of the other tubes by brass screening. This brass screening was grounded to totally eliminate any electrical transfer from one tube to the other. The culture densities of each test tube were tested with a Klett-Summerson photoelectric colorimeter by measuring, at 20-30 minute intervals over a 3 hour period, increasing optical absorbences at 660 nanometers. Controls were also run using cells that were inac- tivated by subjecting them momentarily to 60°C and quick cooling to 37°C. Each set of controls consisted of three test tubes which were measured at 10, 100 and 1000 hertz to establish the background of optical absorbence effects due to the electroplating of cells and growth medium components onto the electrodes and also to ascertain any electrolytic effects that might be present. The EmF fields were produced by the variable frequency generator and monitored via the oscilliscope. The low energy fields were applied by passage of alternating current through the growth medium between the platinum electrodes . The results obtained with EL_ coli , following the above procedure are set forth in the following tables. Table 1 At 37°C lKHz, lOV.cm. time 0 20 40 60 80 rate coor con 37 39 42 48 52 0.37 0.99 exp 37 41 45 58 53 0.47 0.92 1.27 *************** Table 2 At 34°C, lKHz, 18V/cm time 0 30 60 rate coor conl 66 68 72 0.12 0.98 con2 50 52 55 0.14 0.99 ex l 49 53 58 0.24 1.00 exp2 93 98 105 0.17 1.00 cont avg . -*•= 0.13, dev = 0.01 LI - 1.85, L2 - 1.31 *************** Table 3 At 37°C, 10KHZ, 5V/cm time ) 30 60 rate coor conl 65 76 88 0.44 1.00 con2 65 78 89 0.46 1.00 expl 71 84 99 0.48 1.00 exp2 66 81 98 0.57 1.00 cont avg. = 0.45, dev _= 0.01 LI - 1.07, L2 - 1.27 *************** . Table 4 At 37 < 'C , power off time 60 90 rate coor conl 88 107 0.56 1.00 con2 89 108 0.56 1.00 ex l 99 112 0.36 1.00 exp2 98 112 0.39 1.00 cont avg. = 0.56, dev = 0.00 Ll - 0.64, L2 - 0.70 *************** Table 5 At 37°C, IKHz, 5V/cm time 120 150 rate coor conl 141 154 0.25 1.00 con2 130 148 0.37 1.00 expl 137 151 0.28 1.00 exp2 135 151 0.32 1.00 cont avg. = 0.31, dev = 0.08 Ll - 0.90, L2 - 1.03 *************** Table 6 At 37°C, IKHz, 5V/cm time 0 10 95 110 rate coor conl 62 64 91 94 0.34 1.00 con2 58 61 89 95 0.42 1.00 expl 77 82 123 117 0.79 0.98 exp2 64 68 94 95 0.65 0.99 cont avg. = 0.38, dev = 0.06 Ll - 2.08, L2 - 1.71 *************** Table 7 At 36°C, 100Hz, 5V/cm conl 133 138 153 0.20 0.96 con2 140 144 152 0.12 0.98 expl 135 139 152 0.17 0.96 exp2 144 149 159 0.14 0.98 cont avg. = 0.16, dev = 0.06 Ll - 1.06, L2 - 0.88 *************** Table 8 At 37°C, IKHz, lOV/cm time 30 60 90 120 rate coor conl 120 135 153 174 200 0.36 1.00 con2 132 141 158 179 200 0.30 1.00 expl 128 141 164 185 200 0.34 1.00 exp2 125 137 160 180 200 0.35 1.00 cont avg. = 0.33, dev = 0.04 Ll - 1.03, L2 - 1.06 *************** Table 9 At 34°C, 500Hz, 5V/cm time ' 0 30 60 rate coor conl 132 144 158 0.26 1.00 con2 129 145 155 0.27 0.99 expl 121 136 146 0.27 0.99 exp2 112 120 133 0.25 0.99 cont avg. = 0.27, dev = 0.01 Ll = 1.00, L2 = 0.93 *************** SUBSTI Table 10 At 40°C, 5KHz, 5V/cm time 0 30 90 rate coor conl 108 119 142 0.26 1.00 con2 107 113 139 0.25 0.99 expl 107 121 140 0.26 0.99 exp2 113 126 142 0.22 0.99 cont avg. = 0.26, dev = 0.01 Ll = 1.00, L2 = 0.85 *************** Table 11 At 37°C, IKHz, 8V/cm time 0 30 60 90 120 rate coor conl 98 103 106 115 122 0.16 0.99 con2 103 105 109 116 125 0.14 0.97 expl 95 100 109 120 122 0.20 0.98 exp2 95 102 115 118 120 0.18 0.95 cont avg. = 0.15, dev = 0.01 Ll = 1.33, L2 = 1.20 *************** Table 12 At 37°C, 100Hz, 5V/cm time 70 90 rate coor conl 107 127 129 0.19 0.99 con2 93 111 115 0.21 1.00 expt 95 116 113 0.19 0.94 cont avg. = 0.20, dev = 0.01, L = 0.95 *************** Table 13 At 37°C, IKHz, 3V/cm time 0 70 90 rate coor conl 107 127 129 0.19 0 . 99 con2 93 111 115 0.21 1. 00 expt 84 100 107 0.23 1. 00 cont avg. = 0.20, dev _= 0.01, L = 1.15 *************** Table 14 At 37°C, lOKHz, 5V/cm time 0 30 60 rate coor conl 33 34 37 0.16 0.96 con2 33 34 37 0.16 0.96 expt 33 34 39 0.23 0.94 cont avg. = = 0.16, dev = 0.00, L = 1.44 *************** Table 15 At 37°C, IKHz, 4V/cm time 0 30 60 rate coor conl 33 34 37 0.16 0.96 con2 33 34 37 0.16 0.96 expt 38 42 48 0.33 1.00 cont avg . = = 0.16, dev = 0.00, L = 2.06 ******* ***** *** Table 16 At 35°C, IKHZ, 4V/cm time 30 60 90 120 rate coor conl 36 41 44 48 54 0.28 1.00 con2 39 44 50 62 79 0.48 0.99 ex t 39 45 59 78 103 0.67 0.99 cont avg. = 0.38, dev = 0.14, L = 1.76 *************** Table 17 At 34°C, IKHz, 2V/cm time 0 30 60 90 120 rate coor conl 36 41 44 48 54 0.28 1.00 con2 39 44 50 62 79 0.48 0.99 expt 44 47 53 60 65 0.29 1.00 cont avg. = 0.38, dev = 0.14, L = 0.76 *************** Table 18 At 37°C, .0Hz, 4V/cm time 0 30 60 85 rate coor conl 42 43 44 42 0.01 0.18 con2 37 38 39 34 -0.07 -0.46 ex t 35 48 55 70 0.70 0.99 *************** Table 19 IKHz, IV, 0.4% NaCl time 46 61 75 rate coor cont 23 31 34 36 0, ,49 0, .98 expt 23 31 36 42 0. .66 0, .99 (Power was inadvertently on on the control at lKHz . , 0.5V until the time of the 46 minute reading, therefore the valid reading would start at that time. Starting from 46 minutes the results turn out that the growth rate of the control turns out to be 0.45 gen/hr with a correlation of 0.99 and the rate of the experimental is 0.90 gen/hr with 1.00 correlation. This gives L=2.00 which corresponds with previous data.) *************** Table 20 Testing of 0.4 & 0.2% NaCl to show that the effect is not due to current. IKHz. , IV time 0 30 60 70 80 90 rate coor 0.4% 20.0 22 .0 25.0 27.5 29 .5 32 .0 0.43 0.98 ' 0.2% 9.0 10 .0 10.0 11.5 12 .5 14 .0 0.36 0.91 power off time 0 30 60 rate coor 0.4% 32.0 35.5 39.5 0.29 0.90 0.2% 14.0 15.0 17.0 0.28 0.99 IKHz. , IV time 0 10 20 30 rate coor 0.4% 39.5 42 1.5 47.5 49, .0 0.66 0.98 0.2% 17.0 21 ..0 22.5 24. .5 1.07 0.96 0.4% rate avg. = 0.54, div = 0.16, L = 1.86 0.2% rate avg. = 0.72, div, div = 0.50, L = 2.57 That mutagenesis was not implicated in the accel¬ eration of growth rates for EL_ coli was demonstrated by presence of carbon dioxide evolution when a culture medium from Example 1 was added to a test tube con¬ taining lactose and mineral salts at the temperature of 43°c (Eijkmann-Durham Test for contaminants). Example 2 The procedure of Example 1 was followed except that the microorganism was Bacillus subtilus (ATCC 6051). The following results were obtained: power off (control) temp. = 22c time 0 30 60 rate coor A 0.126 0.132 0.145 0.200 0.980 B 0.028 0.033 0.036 0.370 0.980 power IKHz, iV/cm time 30 60 90 rate coor A 0. .145 0. .162 0, .182 0. .206 0, .336 1. .000 B 0. .036 0, .041 0, .053 0, .064 0, .553 0, .993 A L = 1.68; B L = 1.50 L = 1.59 Example 3 The procedure of Example 1 was employed except that 2% Bacto yeast extract (Fleichman's fresh active yeast) was employed in place of E^ coli and was grown at 25°C in an aqueous solution of 0.3% Bacto-Tryptone and 4.0% glucose (percentages are weight to volume water). The following results were obtained: power off ( control ) time 60 120 rate coor 1 67 69 71.5 0 .05 1.00 2 99 100 105 0. .04 0.93 IKHz. , IV/cm time 0 60 120 180 rate coor 1 71.5 75 81 83 0.08 0.98 2 105 110 113 118 0.05 1.00 14 power off (control) time 0 60 rate coor ch-1 83 85 0.03 1.00 ch-2 118 120 0.02 0.99 power 100 Hz., IV/cm. time 60 100 rate coor 1 85 87.5 89 0.04 1.00 2 120 123 124 0.03 0.99 cont ch-1 avg = 0.04, dev = 0.01 cont ch-2 avg = 0.03, dev = 0.01 rate ch-1 IKHz. = 0.08, L = 2.00 rate ch-2 lKHz. = 0.05, L = 1.67 L IKHz. avg = 1.84 rate ch-1 100 Hz. = 0.04, L = 1.00 rate ch-2 100 Hz. = 0.03, L = 1.00 Example 4 This example demonstrates the efficacy of the pro¬ cess of the invention in accelerating plant growth rates . Glass or plastic containers containing soil and bean seeds (Bountiful Green Bush variety of Pinto) were configured for generation of low energy electrical fields in the following four modes: 1. parallel brass screens above and below the soil between which the beans are positioned without touching the screens 2. one brass screen parallel to the side of the container with the seeds spaced apart therefrom with intervening soil in between the seeds and the screen 3. parallel wires in the horizontal plane of the bean seeds and spaced apart therefrom 4. one brass screen at the bottom of the container below the bean seeds. Each container had from 4-8 bean seeds planted therein. All wires and screens were connected in series to an alternating current generator capable of producing multiple functions (e.g., sine or square waves). For each planted container connected to the generator there was a power-off control container of the same con¬ figuration. All other conditions were the same for controls and test groups. To each test container were applied 22 volts (approximately 2.5 volts/cm. ) at 1 KHz over a period of one week. At the end of the test period the test groups averaged about 8.5 inches in height, whereas the controls had just commenced to ger- minate. The test group had significantly higher ger¬ mination success than the controls. At comparable stages of development the test group was more vigorous in appearance than the controls and hence appeared to exhibit an increase in developmental success. Example 5 Into a glass container was added a brass screen upon which was placed fruit fly medium containing freshly laid eggs. On top of the medium was placed another screen which along with the screen on the bot- torn of the container, was connected in series with a variable frequency generator. To the test sample was applied a field of IKHz, 22V (about 2.5 V/cm. ) ; the control was a power off sample. The flies (Drosophila melanogaster) were then observed to eclosure (reaching of adulthood) , and observed to show an earlier eclosure in the test sample than the control. The test sample also exhibited a greater hatching and eclosure success and also was more vigorous (i.e., observably signifi¬ cantly more active than the control.) The foregoing examples demonstrate that 1000 Hz and 10,000 Hz are highly preferred effective frequencies for growth rate acceleration with widely differing spe¬ cies of organisms at field strengths, which can be expressed in terms of volts per centimeter, ranging upwards of from about 1 volt per centimeter to about 22 volts per centimeter at the expressed frequencies. However, positive growth rate acceleration can be achieved at selected frequencies ranging upwards from 10 Hz to about one megahertz (MH Z ), the only practical limitation being that point at which mutagenesis occurs. A wide range of field strengths can be employed at each selected effective frequency with a concomitant threshold field strength below which no effect occurs. Again practicality dictates that the upper limit for such field strengths is that point at which a measurable temperature increase occurs in the biosystem. Selection of these critical frequencies and field strengths can routinely be accomplished by per¬ forming a simple series of tests as follows: Set up electrodes so that electrical field is applied across the biosystem, to which electrodes are applied selected frequencies at a set minimal field strength of IV/cm. Upon discovery of an effective frequency for growth rate increase, various field strengths are applied at the set effective frequency to determine the effective field strength range. With microorganisms it is recom¬ mended that the system be designed essen¬ tially as described in Example 1. With plant or animal species any one of the systems described in Example 4 can be utilized. If no effective frequency is found at 1 volt/cm. , repeat procedure at 5 volts/cm. and at 5 volt increments thereafter until an effective growth rate increasing frequency is found or until a measureable temperature increase occurs in the biosystem using a standard glass mercury thermometer. Increase of growth rates while improving develop- mental success and vitality of some organisms in accor¬ dance with this invention has important commercial implications for industrial fermentation, as well as, agricultural and pharmaceutical production industries. Improved yields and increased production capacities are direct advantages. Shorter organism growth cycle could open new vistas in diagnostic medicine as well as other areas of medicine such as bone marrow regeneration, organ regeneration, tissue revitilization and the like.

Claims: What is claimed is: 1. A method of increasing the growth rate of a living organism which comprises disposing the organism in a growth medium and subjecting the organism to a low energy electrical field produced by a combined field strength and frequency effective for increasing the growth rate of said organism. 2. The method of claim 1 wherein said field strength is above about 1 volt per centimeter and said frequency is selected from about 10 to about 10 * 5 Hz. 3. The method of claim 1 wherein said organism is a microorganism or plant or animal. 4. The method of claim 3 wherein said voltage is above about 1 volt per centimeter and said frequency is selected from about 1000 Hz and about 10,000 Hz and the resulting electrical field does not increase the tem¬ perature of the medium. 5. The method of claim 3 wherein said voltage is selected from above about 1 volt per centimeter to about 22 volts per centimeter and said frequency is about 1000 Hz or about 10,000 Hz.

Inventors: SWEENEY GEORGE WILLIAM JR, SWEENEY, GEORGE WILLIAM, JR.
Applicants: SWEENEY GEORGE WILLIAM JR, SWEENEY, GEORGE WILLIAM, JR.
Application year: 1986
Main classification: C12N13
IPC codes: A01G7, A61K41, C12N13