Loading...

Effect of low power microwave on microbial growth and metabolism

Microwave-Microbe Interaction

by Vijay Kothari (Author) Krunal Dholiya (Author) Dhara Patel (Author)

Scientific Study 2012 78 Pages

Biology - Micro- and Molecular Biology

Excerpt

Index

List of tables

List of figures

1. Prologue
1.1 Preamble
1.2 Biological application of MW
1.3 The Importance of the Study
1.4 Statement of the Problem
1.5 Rationale of the Research Work
1.6 Aim and Objectives

2. Literature Review
2.1 Interactions of Microwave with Biological Materials
2.1.1 Thermal Mechanisms of Interaction
2.1.2 Athermal (Non-thermal) Mechanisms of Interaction
2.2 Mode of action: Molecular mechanisms
2.3 Interaction at sublethal dose
2.4 Difference between MW heat and conventional heat
2.5 Enzyme activity
2.6 Aflatoxin

3. Materials and Methods
3.1 Test organisms
3.2 Culture maintenance
3.3 Culture activation
3.4 Inoculum preparation
3.5 MW oven and its maintenance
3.6 MW treatment
3.7 Experimental outline
3.8 Growth measurement
3.9 Protease estimation
3.10 Urease estimation
3.11 Aflatoxin estimation
3.12 Statistical analysis

4. Results and discussion
4.1 Effect on growth
4.2 Effect on growth and protease activity
4.2.1 Gram positive
4.2.2 Gram Negative
4.2.3 Yeast
4.3 Effect on growth and urease activity
4.4 Effect on growth and aflatoxin production

5. Epilogue

6. Appendices
Appendix
Appendix
Appendix
Appendix
Appendix
Appendix
Appendix

7. References

List of Tables:

Table 2.1 Urease producing organisms

Table 3.1 Test organisms

Table 3.2 Experimental conditions

Table 4.1 Effect of different duration of MW on growth of E. coli (same day)

Table 4.2 Effect of different duration of MW on growth of E. coli (different day)

Table 4.3 Effect of different duration of MW on growth of different organisms

Table 4.4 Effect of MW radiation on growth of B. subtilis

Table 4.5 Effect of MW radiation on acidic and alkaline protease activity of B. subtilis

Table 4.6 Effect of MW radiation on growth of S. aureus

Table 4.7 Effect of MW radiation on acidic and alkaline protease activity of S. aureus

Table 4.8 Effect of MW radiation on growth of E. coli

Table 4.9 Effect of MW radiation on acidic and alkaline protease activity of E. coli

Table 4.10 Effect of MW radiation on growth of A. hydrophila

Table 4.11 Effect of MW radiation on acidic and alkaline protease activity of A. hydrophila

Table 4.12 Effect of MW radiation on growth of C. albicans

Table 4.13 Effect of MW radiation on acidic and alkaline protease activity of C. albicans

Table 4.14 Effect of MW radiation on growth of S. cerevisiae

Table 4.15 Effect of MW radiation on acidic and alkaline protease activity of S. cerevisiae

Table 4.16 Effect of MW on total protease activity of different organisms

Table 4.17 Effect of MW on acidic protease activity of different organisms

Table 4.18 Effect of MW on alkaline protease activity of different organisms

Table 4.19 Effect of MW radiation on growth and urease activity of M. furfur

Table 4.20 Effect of MW radiation on growth and urease activity of S. aureus

Table 4.21 Comparison of MW effect on protease and urease activity of S. aureus

Table 4.22 Effect of MW radiation on growth and aflatoxin production by A.parasiticus

Table 4.23 Effect of MW radiation on growth and aflatoxin production by of A. flavus

List of Figures:

Figure 1.1 The electromagnetic spectrum

Figure 1.2 Number of publications related to Microwaveand its application

Figure 2.1 Self-localized solitonwavefunction of trans-polyacetylene doped by a counter ion

Figure 2.2 Charges localized on the DNA base pairs

Figure 2.3 Structure of polyacetylene, torque generation due to formation of soliton

Figure 2.4 Internal spinning of two water molecule dimers, and their dissociation into water molecules and ions (H2O-H+)

Figure 2.5 Proton-driven precession of water-molecule dimers forming octal rings that compose a rowing soliton

Figure 2.6 A molecule of water

Figure 2.7 Dipole experiencing torque in electric field

Figure 2.8 Effect of electric field on dipole molecules (Free molecules)

Figure 2.9 Rotationally excitation of polar molecules

Figure 2.10 MW heating mechanism

Figure 2.11 Dielectric polarization

Figure 4.1 Comparision of different duration of MW on growth of E. coli with three different experiment

Figure 4.2 Comparision of different duration of MW on growth of E. coli with three different experiment

Figure 4.3 Comparision of different duration of MW on growth of different organisms with three different expeiment

Figure 4.4 Comparison of effect of different duration of mw radiation on growth, total protease, acidic protease, and alkaline protease of B. subtilis

Figure 4.5 Comparison of effect of different duration of mw radiation on growth, total protease, acidic protease, and alkaline protease of S. aureus

Figure 4.6 Comparison of effect of MW radiation on growth, total protease, acidic protease, and alkaline protease of E. coli

Figure 4.7 Comparison of effect of MW radiation on growth, total protease, acidic protease, and alkaline protease of A. hydrophila

Figure 4.8 Comparison of effect of MW radiation on growth, total protease, acidic protease, and alkaline protease of C. albicans

Figure 4.9 Comparison of effect of MW radiation on growth, total protease, acidic protease, and alkaline protease S. cerevisiae

Figure 4.10 Comparison of effect of MW radiation on growth and urease activity of M. furfur

Figure 4.11 Comparison of effect of MW radiation on growth and urease activity of S. aureus

Figure 4.12 Comparison of effect of MW radiation on growth and aflatoxin production of A. parasiticus

Figure 4.13 Comparison of effect of MW radiation on growth and aflatoxin production of A. flavus

1. Prologue

1.1 Preamble

Since many years, scientists are interested in studying the interaction of electromagnetic fields (EMFs) and various bio-system and its bioprocesses. All biological systems are electrochemical in nature so EMF may influence them. Attention has been focused on different frequency range waves, of which microwave (MW) is an important part.

Microwaves (MW) are non-ionizing electromagnetic waves in 1m to 1mm wavelength range, with a wide frequency band between 300 MHz and 300 GHz [Banik et al., 2003]. They are a very important component of the electromagnetic spectrum as demonstrated by the increasing scope of applications. They have relatively short wavelengths and high frequencies compared to the extremely low frequency fields. They therefore have a greater energy which is sufficient to cause heating in conductive materials (figure. 1.1). Unlike the X-rays and gamma rays, which are ionizing, MW interaction with matter does not result in removal of orbital electrons. Rather, such interactions are known to cause effects like atomic excitations, increased atomic and molecular vibrations, rotations, andheatproduction [Davis and VanZandt, 1988]. Becauseof the nature of MW, its interaction mechanisms and the biological effects have previously not been associated with production of free radicals, oxidative modification of cell membranes, lipid peroxidation. These mechanisms are associated with ionizing radiations.

illustration not visible in this excerpt

Figure 1.1 The electromagnetic spectrum [Verschaeve and Maes, 1998]

Interest in the study of MW interaction with biological systems has been sustained for several decades. The first person to explore the bioeffects of MW fields was Antonin Gosset in 1924, when he and his co-workers used short waves to destroy tumors in plants with no damage to the plant itself [Bren, 1996]. During the 1930s,physicists, engineers, and biologists studied the effects of low frequency electromagnetic waves on biological materials. Studies of the effects of microwaves on bacteria, viruses and DNA were performed in the 1960s and included research on heating, biocidal effects, dielectric dispersion, mutagenic effects, etc. [Yaghmaee and Durance, 2005].

In recent year, the industrial MW applications have grown considerably (figure 1.2), apart from the usage of domestic MW ovens. Some of the MW applications in industries are tampering of frozen product, thawing, blanching, baking, drying/dehydration/vacuum drying/freeze-drying, pasteurization, sterilization, cooking, etc.

1.2 Biological applications of MW

Microwaves have widespread applications, among which some have already commercial acceptance while others are still about to come to the level of commercial acceptance.

- Radiopharmaceuticals application [Kimura et al., 2012]
- synthesis of nanocarbon materials [Zhang and Liu 2012]
- Extraction of natural product from plants [Kothari et al.,2009; Delazar et al., 2012; Mandal et al., 2007; Gallo et al., 2010]
- Rate enhancement of biochemical reactions (low power) [Bose et al., 2002]
- Pharmaceutical application [Bedford et al., 2009]
- Utilization of Recalcitrant Biomass [Tsubaki et al 2011]
- Medical applications (low power) [Meganathan et al., 2010]
- Waste treatment [Beszédes et al., 2010]
- Enzyme immobilization [Wang et al., 2010]
- Mutagenesis in plants (low power) [Jangid et al., 2010]
- Disinfection & Sterilization [Kothari et al., 2011]
- Protein folding and unfolding(low power) [George et al., 2008]
- Moisture removal [Ozbek and Dadali, 2007]
- Diagnostic applications: (low power) [Stuchly et al., 1983]
- Tumor detection based Diagnostic applications: tumor detection based on differences in tissue electrical properties
- The rapeutic applications based on local heating: prostate hyperplasia, heart and other tissue prostate hyperplasia, heart and other tissue ablation, angioplasty
- Biological tissue processing [Emerson et al., 2006]
- Application of microwave technology to proteomics [Juan et al., 2005]
- Staining [Soans et al., 1999; Giberson et al., 2001]
- Applications of Microwaves in the Mineral Industry [Vorster, 2001]
- Protein hydrolysis and proteomics [Lill et al., 2007]
- Cancer detection [Fear and Stunchly, 2000]

illustration not visible in this excerpt

Figure 1.2 Number of publication s related to Microwaveand its application*

1.3 The Importance of the Study

Sub- lethal MW-microbes interaction:may change level of

- Growth rate [Rebrova et al., 1992; Golant et al., 1994; Shub et al., 1995]
- Metabolite activity [Dreyfuss et al., 1980]
- Plasmid amplification [Otludil et al.,2005]
- Rapid Strain identification [Spencer et al., 1985]
- Transformation efficiency [Fregel et al., 2008]
- Porosity of the cell membrane that allow uptake of drugs into MW-treated cell [Shamis et al., 2011]

* Data shown was taken from PubMed by typing keyword “Microwave”. It was done on March 12th, 2012

- Secondary metabolites such as antibiotics [Himabindu et al., 2007]
- Lose of virulence of virulent strain [Moore et al., 1979; Tsuji and Yokoigawa, 2011]
- Mutation (low power) [Li et al., 2009]

1.4 Statement of the Problem

Increasing applications of MW radiation (figure 1.2) has led to concerns globally due to the suspected bio effects associated with its exposure.

Effect of MW, thermal and/or athermal, is inconclusive,complex, and controversial investigations from literature. Thermal effect causes thermogenic effect while athermal effects are other than heat and such effects reported as somatic effect and/or genetic effect. Therefore, this study focuses on athermaleffects. This work is aimed at investigating the hypothesis that the exposure of microbial cells to MW (low power) may cause athermal effect, which affect on growth of microbes, enzyme activity, and production of aflatoxinby aflatoxin producing Aspargillus sp.

1.5 Rationale of the Research Work

There are numerous and increasing applications of MW energy and technology in the industries, in homes, in medical, research institutions etc. (figure1.2), and there is greater awareness and concern of the public over the suspected potential health hazards associated with such exposures [ICNIRP Guidelines, 1998]. There is therefore, a need for deeper understanding of the bio-effects of exposure to this radiation. Easiest and fastest way to study effect of MW on bio-system is study on microbes. Change in some properties of microorganisms through MW radiation may be useful in research and industries.

1.6 Aim and Objectives

The primary aim of this research was to verify the hypothesis that there are some athermal effects of MW on living systems (microbes).

Objectives were,

To investigate the effect of low power MW on,

I. Growth of different organisms

II. Growth and extracellular enzymes of different organisms; extracellular enzyme are,
a. Protease activity,
i. Total protease activity
ii. Acidic protease activity
iii. Alkaline protease activity
b. Urease activity

III. Growth rate and production of aflatoxinby aflatoxin producing Aspargillus sp.

2. Literature Review

2.1 Interactions of MW with Biological Materials

The interactions can be considered at various levels of organization of a living organism: atomic, molecular, subcellular, cellular, entire organism. These interactions with bio-system arise because of three processes: (i) penetration by electromagnetic waves and their propagation into the living system, (ii) primary interaction of the waves with bio-system; and (iii) possible secondary effects arising from the primary interaction. One of the first fundamental steps in evaluating the effects of a certain exposure to radiation in a living organism is determination of the induced internal electromagnetic field and its spatial distribution. Further, various possible biophysical mechanisms of interaction can be applied. Any such interactions, which may be considered primary, elicit one or more secondary reactions in the living system. For instance, when MW energy absorption results in a temperature increase within cell (a primary interaction), the activation of the thermoregulatory, compensatory mechanism is a possible secondary interaction [Czerski, 1975; Czerski, 1975; Baranski and Czerski, 1976]. While the primary interactions are becoming better understood, there is still insufficient attention devoted to the interaction mechanisms involving molecular level.

Studies on the biological effects of MWs reveal several areas of established effects and mechanisms on the one hand and speculative effects on the other. There are known thermal and athermal interaction mechanisms of MWs with biological systems.

2.1.1 Thermal Mechanisms of Interaction

Thermal Effect is the heating effect that produces alterations in the dielectric properties of the dipole molecules. The electric field of the MWs exerts a force on the charged molecules of the material [Jacob et al., 1995]. Thermal effect is involved in MW dielectric heating. The quantity of free water in a sample strongly influences its biological properties. With abundant free water, thermal effect would be dominating any electromagnetic response [Wayland et al., 1977].

When ions interact strongly with MWs, reacting system containing ionized products and reagents undergo an unforeseen increase of temperature [Fini and Breccia, 1999]. This is especially applicable for solvents, which undergo MW treatment, reaching a temperature exceeding its normal boiling point. This phenomena is called superheating or superboiling [Barghurst et al., 1992; Chemat et al,. 2001]. Superheating along with non-thermal effects may lead to lack of reproducibility associated with MW operations.

2.1.2 Athermal (Non-thermal) Mechanisms of Interaction

MW radiation seems to affect system in a manner, which cannot be explained by thermal effects alone [Spencer et al., 1985]. MW have ability to destroy bacterial cells at specific parameters withoutcausing heating of the substrate [Barnabas et al., 2010]. MW plays role in dielectric saturation [Hyland, 1988], formation of oxidative stress [Sokolovic et al., 2008], protein unfolding[George et al., 2008], changing the structures by differentially partitioning the ions [Asadi et al., 2011], others chemical transformation of small molecules such as chemical bond cleavage [Oslen, 1966], vibrational resonance in DNA molecules [Edwards et al., 1985]. The oscillating EMF of MW couples energy into large biomolecules with several oscillations. When a large number of dipoles are present in one molecule (DNA, protein, RNA etc.) and kept under MW, enough energy can be transferred to the molecules, which would be able to break the bond.

Formation of soliton, interactions of phonons and excitons along linear molecules may produce nonlinear molecular vibrations in the form of soliton waves (figure 2.1), as a means of energy transport over molecular distance (Davydovsoliton theory). Solitons exist in a minimal energy state and are extremely long-lived in comparison to linear oscillations. Solitons may convey energy released by chemical reactions from one site to another in enzymes of other long-chain proteins [Lawrence and Adey, 1982] .The direct charge transfer along the protein molecule may result from the capture of an extra electron by the moving acoustic soliton (electrosoliton) [Kadantsev and Savin, 1997] These nonlinear waves may also couple reaction-diffusion processes in the intracellular and extracellular domains.

illustration not visible in this excerpt

Figure 2.1 Self-localized solitonwavefunction of trans-polyacetylene doped by a counter ion

(Black ball)

illustration not visible in this excerpt

Figure 2.2 Charges localized on the DNA base pairs, which seem to form small polarons, which would be transported via the pi stacking of these base pairs.

A polaron is a quasiparticle composed of a charge and its accompanying polarization field. A phonon is a collective excitation in a periodic, elastic arrangement of atoms or molecules in condensed matter.

illustration not visible in this excerpt

Figure 2.3 Structure of polyacetylene, torque generation due to formation of soliton

The basic science is that the conventional one-dimensional soliton is in fact strongly coupled to three-dimensional conformations of macromolecules (figure 2.3), through non-linear electron-electron and electron-phonon interactions [Lin et al., 2005].

Ballistic protons and microwave-induced water solitons can transform energy in biological reaction [Tirosh, 2006]. Consider the cleavage of a high-energy complex into two products of relatively small and large masses (m). By energy and momentum conservation, the two products gain an equal and opposite momentum (p), while the low-mass product carries most of the kinetic energy (EK) released, namely:

illustration not visible in this excerpt

The same law can be imposed to energy transfer from a water into ballistic proton, product in water is a ballistic H+ released from H2O-H+ . The de Broglie quantum-mechanical relation entails a classical view of a protonic molecular orbit enclosing the two lone-pair electrons of the oxygen atom in H2O-H+ (figure 2.4).

illustration not visible in this excerpt

Figure 2.4 Internal spinning of two water molecule dimers, and their dissociation into water molecules and ions (H2O-H+ ).

illustration not visible in this excerpt

Figure 2.5 Proton-driven precessions of water-molecule dimers forming octal rings that compose a rowing soliton.

A ballistic H+ may be released from H2O-H+ at a velocity of 10km/sec, carrying a kinetic energy of 0.5 proton* volt. By coherent exchange of microwave photons during 10-10 sec, the ballistic proton can induce cooperative precession of about 13300 electrically-polarized water molecule dimers, extending along 0.5 µm. The dynamic dimers rearrange along the proton path into a pile of non-radiating rings that compose a persistent rowing-like water soliton (figure 2.5). During a life-time of 20 ms, this soliton can generate and overcome a maximal pressure head of 1 kgW/cm2 at a streaming velocity of 25 µm/sec and intrinsic power density of 5 W/cm3 . This energy may change metabolic activity of cell.

2.2 Mode of action: Molecular mechanisms

MW is part of electromagnetic spectrum and ranging in frequency from 300 MHz to 300 GHz, which correspond to a wavelength range from 1 m to 1 mm [Banik et al., 2003]. Energy of photon is function of frequency.

illustration not visible in this excerpt

illustration not visible in this excerpt

Where, [illustration not visible in this excerpt] is energy of photon, [illustration not visible in this excerpt] is frequency and [illustration not visible in this excerpt] is plank constant (4.1356 × 10-15 eV). From (eq. 2), energy of MW is ranging from 1.24 µeV – 1.24 meV. This is considerably lower than energies required producing any intramolecular alterations or intermolecular bond breaking [Yaghmaee and Durance, 2005]. As typical covalentbond energy is 5 eV and even hydrogen bond energy is 0.01 eV, and is even one order of magnitude lower than thermal motion energy 0.025 eV (k T , k-Boltzman's constant (8.617×10-5 eVK-1 ), T-temperature in K) at 37° [Kramers, 1940]. Therefore, direct MW effects at a molecular level can be ruled out unless some cooperative mechanisms can be invoked.

illustration not visible in this excerpt

Figure 2.6 A molecule of water, whichis polar because of the unequal sharing of its electrons in a "bent" structure.

One of the basic mechanisms underlying interactions of MW with biological systems at molecular level is the field-induced rotation of polar molecules such as water (figure 2.6), proteins, and DNA [Takashima, 1984]. Such molecules experience a torque when placed in an external electric field and attempt to align with the field to minimize the potential energy of the dipoles (Principle of minimum energy) [Hovgaard, 1923] (figure 2.7 and 2.8).

Figure 2.7 Dipole experiencing torque in electric field

illustration not visible in this excerpt

The thermal (Brownian) motion of the molecules hinders orientation. These frictional forces, that are dependent upon the rate of orientation (the frequency of the applied field) and the relaxation time, determine the degree of orientation. The relaxation time depends on the viscosity of the solvent, the size, and shape of the dipoles, temperature, and the nature of the solute-solvent bonds [Zhang et al., 1996].

Figure 2.8 Effect of electric field on dipole molecules

(Free molecules)

illustration not visible in this excerpt

The degree of orientation of the dipoles in the directing field decreases with the frequency of the field above a certain frequency [Friedrich and Herschbach, 1999]. At these frequencies the combined effects of the viscosity, molecular size and shape, and solute-solvent bonding prevent the molecular motion from being in phase with the applied field. As the frequency is further increased, a stage is reached where none of the dipoles is able to keep up with the field and the system then behaves like a non-polar material. The orientation of the molecules and their rotation in the electric field are only partial in directing field of order of a few V/cm (figure 2.9).The rotational energy is converted into heat (figure 2.10) [Leadbeater, 2010]. This type of interaction is not suggestive of irreversible effects in biological media other than those resulting from the increase in temperature.

illustration not visible in this excerpt

Figure 2.9 Rotationally excitation of polar molecules, Polar molecules rotate as electric fields change direction in MW (only electric field is shown, magnetic field is not shown). This spinning happens 2.45 billion times per second for 2.45 GHz of MW.

illustration not visible in this excerpt

Figure 2.10 MW heating mechanism. Panels 1-3 show a molecule “a” that has been rotationally excited by MW radiation being approach by a second molecule “b”. In panel 3, rotational energy of molecule “a” is converted to translational movement of molecule “b”. In panel 4, the increase in translational vector magnitude, the consequence of which increase in molecular collision (kinetic energy), this is resulted as heat [Leadbeater, 2010].

If the charged particles that experience a force through the electric field of MWs are able to move freely, a current is induced. However, if the charged particles are bound in the compound, restricted in their movements, they merely reorient themselves in phase with the electric field. This is termed dielectric polarization (figure 2.11) [Jacob et al., 1995].

illustration not visible in this excerpt

Figure 2.11 Dielectric polarization

Water, which constitutes about 70% of the total weight of any biosystem exhibits rotational relaxation at MW frequencies. Water appears in two forms as free water and bound water. The relaxation of free water takes place at a frequency of approximately 25 GHz at 37°C, and the relaxation frequency is a function of temperature. The rotational motion of free water in cell, which may be converted into translational and vibrational excitations by collision leads to an increase in the cell temperature. However, no specific biological effects are expected from this mechanism since the molecular structure remains unchanged [Penn state university article, 2001].

Bound water as water molecules held in non-random orientations at or near the surface of a macromolecule through H bonding [Dailey, 1952]. The bound water constitutes about 30% of the total weight of protein molecules. The relaxation frequency of bound water depending on the molecule the water is attached to and the solvent viscosity [Takashima, 1984].

The rotational motions of biological molecules such as amino acids, peptides, and proteins have been reported, including pioneering work by Oncley [Oncley, 1942]. Protein molecules are highly charged with the number of positive and negative charges nearly equal and the charge distribution on the surface uniform. Due to large dimensions of the molecules, a relatively small asymmetry in the charge distribution results in a large dipole moment. Typical values of dipole moments are 200-1000 Debye units (1 D = 3.33 x 10-30 C/m).The relaxation frequencies are between 100 kHz and 50 MHz [Takashima et al., 1984].

2.3 Interaction at sublethal dose

The studies reviewed demonstrate effects of low-intensity MW (10 mW/cm2 and less) on cell growth and proliferation, activity of enzymes, state of cell genetic apparatus, function of excitable membranes, peripheral receptors, and other biological systems .

MW radiation increases the enzyme activity of enterobacteria [Spencer et al., 1985].The effects of MW radiation were examined on the metabolism of members of enterobacteriaceae. In presence of particular substrate, MW irradiation of bacteria caused increase in enzyme activity and it show some biochemical tests were positive with the majority of strains by the MW method but negative by conventional tests: Serratia with raffinose, rhamnose, dulcitol; and Enterobacter with adonitol. Therefore, MW treatment uses as rapid method for identification of particular strain [Spencer et al., 1985].

MW exposure (6mW/g) alters process in enzyme-loaded unilamellar liposome. The enzyme carbonic anhydrate acetate entrapped into cationic unilamellarvesicle, which passes p-nitrophenyl acetate (PNPA) across intact liposome bilayer with low diffusion rate. A twofold increase in the diffusion rate of PNPA through the lipid bilayer after 120 min of MW radiationwas observed compared to temperature control sample. The enzyme activity, as function of increased diffusion of PNPA, increasesover 120 min from 22.3% to 80% [Orlando et al., 1994].

[...]

Details

Pages
78
Year
2012
ISBN (eBook)
9783656270669
ISBN (Book)
9783656271178
File size
1.9 MB
Language
English
Catalog Number
v201075
Institution / College
Nirma University
Grade
A
Tags
effect microwave-microbe interaction

Authors

Share

Previous

Title: Effect of low power microwave on microbial growth and metabolism