ELECTROCHEMICAL MODEL OF Euglena sanguinea Ehrenberg STIMULATED WITH MULTI-LEVEL ELECTROMAGNETIC FIELDS

This research aimed at studying the property of Euglena Sanguinea Ehrenberg (E. sanguinea) before and after being stimulated by multi-level electromagnetic fields (MLEM). After the stimulation, it was found that physical properties were changed, i.e., cell membrane and components deformed. Main biochemical compositions were changed in accordance with new electrochemical equation which was estimated from FTIR bands. Moreover, the estimated electrical results before and after being stimulated, i.e., impedance and complex relative permittivity of main biochemical compositions from extraction, were then measured, simulated, and compared by means of Cole-Cole plot technique which yielded the statistic correlation 0.89 at 95% confidence level. Semi-circle impedance was related to complex relative permittivity as its value decreased with the increasing frequency. These were the information for designing the new electrical equivalent circuit model of E. sanguinea with MLEM to explain many phenomena in cell. The relationship between energy from MLEM system and bond energy of main biochemical compositions of E. sanguinea as well as between electromagnetic field behaviors of MLEM system which stimulated to cell and induced voltage transmitted through each biochemical compositions of E. sanguine were also identified. Keyword: Multi-level electromagnetic fields (MLEM), Euglena sanguinea, impedance, equivalent circuit


INTRODUCTION
Most of Euglena sanguinea Ehrenberg (E.sanguinea) are found in freshwater.It forms an algae bloom which is a cause of water pollution.E. sanguinea generates the ichthyotoxin or the euglenophycin which adversely affect the economic aquatic animals 1 .For this reason, algae eradicating or make advantage from it should be focus on.The main chemical components of E. sanguinea are chlorophyll A, B and carotenoid.It does not have cell wall but cell membrane is in the form of a protein called pellicle.The stored food is a kind of carbohydrate in the form of paramylon, which is the  1-3 polymer of glucose 2 as shown in Figure 1.According to the complexity of cell components, the researchers used new electrochemical equations to explain the biochemical transforming of cells as well as developed the electrical equivalent circuit of E. sanguinea with MLEM system to explain the electro-physical change.

EXPERIMENTAL
The water sample of E. sanguinea in the form of a suspended solid 1080 mg/l was taken from a canal in Phatumtanee, Thailand.It was stimulated in 10-litre (lab scale) vessel by multi-level electromagnetic fields (MLEM).The voltage of MLEM system could be adjustable from 0 to 1000 .
Vac. Frequency could also be adjustable from 50 Hz to 20 kHz which transmitted through the MLEM coil and electrode as shown in Figure 2.This experiment consisted of 5 stages : 1) analyzed the result of physical change of E. sanguinea before and after being stimulated by MLEM using light microscope, scanning electron microscope (SEM), and transmission electron microscope (TEM), 2) analyzed major elements of E. sanguinea before being stimulated by MLEM using energy dispersive spectrometer (EDS) and analyzed the chemical components of E. sanguinea by Fourier transform infrared spectroscopy (FTIR) before and after being stimulated by MLEM as well as analyzed the chemical properties of the water sample after being stimulated by MLEM, i.e., free carbon dioxide (CO 2 ) 6 , sulfur dioxide (SO 2 ) 7 , dissolve oxygen (DO) 6 , phosphate 6 and ammonia nitrogen 6 , 3) studied the main biochemical compositions of E. sanguinea before and after being stimulated by MLEM by extraction and measured the quantity of protein 8,9 , chlorophyll 10 , carbohydrate 5 , lipid 6 and phosphorus compound 11,12 , 4) measured the electric current and changing temperature per time as shown in Figure 2, 5) measured RC impedance of extracted compositions (from 3) and E. sanguinea cell with HP 4192 ALF impedance analyzer as shown in the diagram of Figure 3, measured the viscosity 13 and specific heat capacity using differential scanning calorimetry 14 .

Physical property of E. sanguinea
Figure 4 shows the different forms of E. sanguinea before and after being stimulated by MLEM.Before stimulation, cell was able to move and the outer membrane orderly lined up with perfect organs.But after being stimulated, the movement stopped, the outer membrane crashed and subsided, even the organs deformed.

Chemical property of E. sanguinea
According to EDS analysis, it was found that the major elements of E. sanguinea contained carbon (C), oxygen (O), nitrogen (N), sulfur (S), phosphorus (P) and magnesium (Mg) with the atomic percentage 61.99%,24.43%, 12.22%, 0.23%, 0.9%, and 0.23%, respectively.The result of FTIR analysis is shown in Figure 5.It demonstrated the functional groups of biochemical compositions of E. sanguinea before and after stimulation which were recorded in the wave number region (650-4000 cm -1 ).The bands of IR Spectra before stimulation was approximately 3287 cm -1 which characterized OH functional group (-OH) or amino (-NH 2 ), 1641 cm -1 characterized the functional group of amide carbonyl (-CO-NH-).Both bands exhibited protein properties 15 .Two other bands at approximately 2920 and 2850 cm -1 characterized aliphatic hydrocarbon group (CH 3 CH 2 -) 16 , and 1733 cm -1 characterized the functional group of carbonyl compound (C=O) 17 .These groups exhibited the properties of carbohydrate, lipid and chlorophyll.A band at about 1451 was characteristic of functional group of alkyl, 1311 and 1154 cm -1 were characteristic of functional group of aliphatic hydrocarbon with branches.These were in agreement with reported surface function groups of -N-H, -C=O, and -CH on algae cell (Scenedesmus quadricauda) 18 .After stimulation, the bands were different.The bands of carbonyl compound (C=O) and alkyl group disappeared.The density of peak bands decreased.On the other hand, percentage of transmission increased.There were still amino group, aliphatic hydrocarbon and amide carbonyl.This result signified that MLEM destroyed some biochemical compositions of E. sanguinea which led to the decreasing intensity of chemical components or deformed to another substances.This was corresponding to the quantity of extracted biochemical compositions as shown in Table 1 which the percentages of protein, chlorophyll, carbohydrate, lipid, and phosphorus compound after being stimulated by MLEM decreased to be 83%, 47%, 45%, 30%, and 24%, respectively.When stimulated the water sample by MLEM, some chemical components occurred.There were carbon dioxide 1 ppm, sulfur dioxide < 0.001 ppm, dissolved oxygen 9 mg/l, phosphate < 0.5 mg/l and ammonia nitrogen 1.5 mg/l.sanguinea before and after being stimulated by MLEM from mean element formula 5,19 is presented in the Table 2.
According to the chemical reactions of MLEM, the complex compounds were destroyed by the energy from MLEM (P MLEM ) caused free electron releasing.Redox reaction then occurred in which the same species was both of oxidized and reduced which also called as disproportionation reaction 20 as shown by the following equation (8).

27
Where n is the number of electrons from the reaction,   is the concentration of the oxidized forms as shown in Table 2.The total value of the destroyed voltage of E. sanguinea cell cell V could be calculated with equation (10).

Complex impedance of E. sanguinea
The values of capacitance (C) and resistance (R) from stimulation measurement both before and after stimulation were very similar.Thus, in this research, the measured values without the stimulation were applied to Cole-Cole plots RC parallel circuit model technique to represent which varied in conformity with the frequency.The complex impedance of the impedance 22 of each biochemical component were calculated using equations (10)  (11), ( 12) and ( 13), respectively and shown in Figures 7 to 12. Complex impedance of cell  cell Z was calculated using equation ( 14), real part cell Z  and imaginary part cell Z   of cell 22 were calculated using equations ( 15) and ( 16), respectively as shown in Figure 13.

Complex relative permittivity of E. sanguinea
The estimation of complex relative permittivity 23 of each biochemical composition ) ( The empty capacitor (C o ), loss tangent ) (tan and Y Bio-com which was impedance modulus of each biochemical composition are shown by the following equation (19).
Where A e is the end area and h is the thickness of E. sanguinea material from Figure 3. So, behaviors of permittivity are shown in Figure 6.The permittivity of cell was calculated using equation ( 17) but changed the complex impedance value to be that of the cell

 of each biochemical composition obtained from the calculation
According to Figure 6, the behaviors of real and imaginary permittivities varied in conformity with the frequency.Real part was the capability of electrical power storage.Imaginary part was the capability of electrical power diffusion in the condition of high frequency but the value decreased.
The capacitance (C) 24 and resistance (R) 24,25 of each biochemical composition of cell were calculated using equations ( 20) and ( 23), respectively.Consecutively, using equations (11) to (16) to calculate the impedance value then compared with the measured value.
Where A is the area of E. sanguinea cell from SEM (5.02×10 -5 cm 2 ) and  is cell thickness from SEM (70 nm).The diffusion permeability coefficient (P D(Bio-com) ) was calculated using equation ( 21) 25 depending on data collected from the experiments which the results are shown in Table 1.Vol is volume of E. sanguinea from SEM (3.51×10 -10 cm 3 ) and t is the duration that cell is destroyed by MLEM system equal to 3600 seconds.The conductivity was calculated using equation ( 22) 25 .
  is the concentration of each biochemical composition of E. sanguinea before being stimulated by MLEM (mol/l) and z is the number of valence electrons (pelli = 22, chlo = 18, pro = 22, carbo = 11, lipid = 11 and phos = 21).So, the resistance (R Bio-com ) of each part was calculated using equation (23).
The inducement of voltage coil in MLEM system was calculated using equation ( 24) 26 .
Where L coil is the inducement of MLEM voltage coil (1.58 H), and µ copper is the permeability of copper (1), N is the number of cycle of voltage coil (5.25), r coil is the radiance of voltage coil (6 mm.), l coil is the length of voltage coil (1950 mm.), f is the frequency (Hz) and the resistivity R coil is 50 .It was found that impedance from the measurement and simulation had a statistical correlation coefficient equal to 0.89 at the confidence level of 95%.Therefore, any of these values can be applied.In addition, graph appearance of impedance of each biochemical components were similar to that resembled in a semi-circle drop at high frequency.This represented the characteristic of semiconductor in the type of bimolecular and new electrical equivalent circuit can be generated as shown in Figure 14. Figure 14 shows the method to reduce the complexity of E. sanguinea with MLEM which is developed in order to facilitate MLEM system design.The electrical parameter can be concluded in Table 3. **  = mass/volume

Relation between Energy of MLEM System and Bond Energy of E. sanguinea
Energy of MLEM (P MLEM ) occurring in E. sanguinea cell of each biochemical composition could be explained from the relationship between the voltage in the Nernst equation and the electrical equivalent circuit from Figure 14 and Table 3.The output voltage of MLEM system was ) 32 .36 sin( ) as shown in Figure 15.

Fig. 15: Behavior of output voltage of MLEM system applied in stimulating to cell of E. sanguinea
Figure 15 shows the output voltage of MLEM in the form of sinusoidal as shown by the equation ( 25), applied for stimulating to electrical equivalent circuit in Figure 14 The electric current transmitted through each biochemical composition (I Bio-com ) as shown by the following equation (27).
is the amplitude of the destroyed electric current which transmits through the resistance of each component.
is the amplitude of the destroyed electric current which transmits through the capacitance of each component.In addition, I 1 , I 4 , I 7, I 10 , I 13 and I 16 are  Moreover, the energy of MLEM occurring in cell of each component P MLEM (Bio-com) was calculated using equation (29).
is the angle of resistance of each component and cos is the angle of capacitance of each component.
Bond Energy 27 of each chemical component was calculated using equation (30).

 
In addition, the radius of molecule com Bio r  was calculated using equation (31) 28 .
Where com Bio  is viscosity obtained from measurement of protein, chlorophyll, carbohydrate, lipid and phosphorus compound equal to 1.09, 5.6, 26, 35, and 16 (cP), respectively, c f is the critical frequency corresponding to the mid-point of relative permittivity graph and frequency.
Therefore, energy from MLEM to destroy the cell was derived from the equivalent circuit as shown in Figure 16.It was found that energy from MLEM (P MLEM ) was higher than bond energy which can be noticed from the graph in Figure 17.The bond energy depended on the critical frequency (f c ) and lined itself to the mid-point graph of relative permittivity (ε r ) of each biochemical component in equal to the bond energy of the molecule of E. sanguinea components.

ELECTROMAGNETIC WAVE AND INDUCED VOLTAGE OF MLEM SYSTEM
The electromagnetic wave varied according to the duration on a dimension if 0   z y E E which was obtained from the relation of Well-Known Maxwell.The MLEM used in stimulation depended on the dielectric or permittivity and the conductivity of material.These can be written in terms of the component notations of electric field (E) and magnetic field (H) intensities as follows: Where  is the propagating constant,  is attenuation constant and  is phase constant related to the dielectric properties of material.
 Bio-com and  Bio-com were calculated using equation ( 35) by the variation of the dielectric and loss tangent of each biochemical composition.The answers for equations (32) and (33) were: So, the energy of MLEM (P MLEM ) varied directly according to the dielectric property and the square density of the magnitude of the electric field 29 .
The occurrence of heat quantity could be derived from transmitting the energy through the material with attenuation constant in equation (38).
Then, the volumetric heat generation (Q), electric wave and the increasing of temperature which varied according to the electric current could be calculated from linear function.
was also found that equation (39) related to the electric current which transmitted through the cell in the equivalent circuit as shown in Figure 16, i 38) and (39), the result is shown in equation (41) 30 .
Replacing equation (41) with equation (40), the intensity of electric field varied according to electric current.The electric current transmitted through E. sanguinea (I cell ) was the variable of energy system of MLEM in equation (42).
Where cell E is the input value of electric field intensity transmitting through the E. sanguinea cell.
Where I Z is intrinsic impedance of material.The dielectric related to complex impedance,  Z in equation ( 17) replaced to equation (44), the intrinsic impedance of E. sanguinea cell intermediary is shown in equation ( 45). ( Thus, the electromagnetic waves which transmitted through the cell 32 are shown in the following equations ( 46) and (47).
Where E is the electric field intensity transmitting through the E. sanguinea cell, H is magnetic field intensity transmitting through the E. sanguinea cell and x is the length of media (40 µm) in xdirection at the frequency 67 Hz in the reactor tank which contained the sample water and E. sanguinea in the proportion of 1:1 as shown in Figure 2, the behavior is demonstrated in Figure 19.

E. sanguinea varying according to x axis
The electric field intensity transmitted through water was bigger than the electric field intensity of E. sanguinea (in the right) due to the difference of dielectric property of water and E. sanguinea.The loss tangent of the cell was as high as 10 representing the penetration level of electric field conducting the heat well causing the cell to be easily destroyed.The induced voltage transmitted through cell of E. sanguinea (∆V cell ) and each biochemical composition (∆V Bio-com ) on a spherical cell 33 which related to the intensity of the electric field as described in equation ( 48) and (49), respectively.
Where r cell is the radius of E. sanguine cell from SEM (2×10 -5 cm 2 ), E Bio-com is the intensity of the electric field transmitting each cell component that can be calculated with equation (42) by changing the parameter applied for the calculation of cell to be Bio-com.The Induced voltage varied according to the angle (  ).Amplitudes were different depending on the conductivity which was the electrical property of each component of E. sanguinea as shown in Figure 20.
According to Figure 20, the induced voltage transmitted through cell of E. sanguinea on a spherical cell would be similar to the Nernst equation which was proved by the electrochemical method.But the induced voltage was proved by the electro-physical method and can be applied to other fields of calculation.By means of stimulating the cell with MLEM, the voltage of cell changed while it was induced on cell membrane, it caused the other molecules unable to move through the cell.As a result, when the electric field was stimulated to the cell with some intensity, cell would be unstable which resulting in cell death 33  The E. sanguinea cell stimulated by MLEM would be deformed.The FTIR result presents a chemical proof that the composition of the cell changed after MLEM stimulation which caused cell death.
According to this experiment, the new electrochemical equation was correlated and total destroyed voltage of cell was equal to 1.655 V. Z  , in addition, Z   behaviors from the extraction of protein, chlorophyll, carbohydrate, lipid, and phosphorus compound of E. sanguinea were measured, simulated, and compared resulting in a statistics relation of 0.89 at the confidence level 95 %.These were directly related to the complex relative permittivity.Minimum energy from MLEM system causing E. sanguinea cell death would be at least higher than bond energy of each biochemical composition at the critical frequency (f c ).The electrical equivalent circuit of E. sanguinea with MLEM was applied to the calculation result of destroyed electric current of E. sanguinea which was equal to 492 µA.Electric field intensity varied according to the dielectric value and electric current.Magnetic field intensity varied indirectly to the intrinsic impedance.In addition, induced voltage which transmitted through E. sanguinea cell on a spherical cell was similar to the destroyed voltage which derived from Nernst equation.Consecutively, MLEM system was a new principle in applying electro-physical method to destroy the algae cell without using any chemical substance.This method would be beneficial and more accurate for MLEM waste water treatment design.Eventually, this research can be applicable for the bio-electronic engineering as well as other related fields.

Fig. 1 : 3 , c as carbohydrate 4 , d as chlorophyll 4 , e as lipid 3 ,
Fig. 1: A; SEM of E. sanguinea B; Model of biochemical compositions of cell with a as pellicle, b as protein 3 , c as carbohydrate 4 , d as chlorophyll 4 , e as lipid 3 , f as phosphorus compound 5 and C;

Fig. 7 :
Fig. 7: A; Cole-Cole plots of pellicle obtained from simulation and measure B;

Fig. 12 :Fig. 13 :
Fig. 12: A; Cole-Cole plots of phosphorus compound obtained from simulation and measure B; phos Z  and phos Z   obtained from the simulation and C;

Fig. 14 :
Fig. 14: Electrical equivalent circuit model of MLEM.Left; the RL impedance of coil, Right; the RC Impedance of E. sanguinea with water

Fig. 18 :
Fig. 18: Relation between the electric current and the temperature

Fig. 19 :
Fig. 19: Behavior of electric field ) (E transmitting through reactor consisting of water and

Fig. 20 :
Fig. 20: The induced voltage transmitted through cell of E. sanguinea and each biochemical composition stimulated with MLEM and varying according to  of spherical cell

Table 1 :
Quantity of E. sanguinea before and after being stimulated by MLEM

Table 2 :
Concentration of biochemical compositions of E. sanguinea before and after being stimulated by MLEM

03 0.E+00 6.E-04 1.E-03 2.E-03 2.E-03 3.E-03 Z'' pro (MΩ) Z' pro (MΩ)
chlo z' chlo B C Fig. 8: A; Cole-Cole plots of chlorophyll obtained from simulation and measure B; chlo Z  and pro B C Fig. 9: A; Cole-Cole plots of protein obtained from simulation and measure B; pro Z  and pro Z   obtained from the simulation and C; pro Z  and pro Z   obtained from the measurement

Table 3 :
The parameter applied for the calculation of electrical equivalent circuit and electric field * R and C are the values obtained from the calculation.
The electric current transmitted through each E. sanguinea cell component was calculated by assuming RL of MLEM coil as a very good conductor.As a result, the straddling voltage of each component had an angle equal to the voltage from MLEM with the same amplitude as the voltage in the Nernst equation.