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STUDY OF THE INFLUENCE OF ELECTRIC FIELD EXPOSURE ON SOME SOIL MICROBIAL ACTIVITIES

Wessam A. Hassanein and Akram A. Ali

Departments of Botany, Faculty of Science, Zagazig University

ABSTRACT In this study, the focus on response of soil microbes to electric field (EF) different

exposure periods was detected. Exposure of different soil samples to electric field EF strength 6kV/m for 1,2 and 3 months slighty decreased the total viable counts of numbers of general bacteria, as well as Azotobacter and Azospirellum, however, the total viable counts of fungi reflected noticeable decrease comparing with bacteria. Data of unexposed soil are recorded significant increase in CTMB (total microbial biomass C), CAMB (ctive microbial biomass C), qR (metabolic quotients), BR (basal respiration), while significant decrease in qCO2 at the end of incubation period was observed . Also significant decrease in CTMB, CAMB and BR of soil under the effect of three months of exposure to electric field was indicated. Soil enzyme activities were decreased by increasing exposure period up to 3 months. Soit nitrogenase was more respond to EF followed by, acidic phosphatase, urease, dehydrogenase and alkaline phosphatase, they decreased by 30, 23.6, 20, 13 and 7.7% respectively comparing with the unexposed samples after the same exposure period. Growth rate of the selected Azotobacter isolate indicated significant decrease in the viable cell number when grown in liquid medium and exposed to the electric field for 1,2,3,4,5 and 6 days comparing with unexposed ones. Regarding to the electrophoresis analysis of extracted protein, 14 bands were obtained from exposed and unexposed Azotobacter liquid culture which incubated for 3 and 6 days. Two bands (Mw83.4 and 15.9kDa) were lost, and two others bands (Mw 86.3 and 16.2 kDa) were appeared after exposure for 3 and 6 days. The average optical density of bands decreased at exposure period 6 days compared with the unexposed ones. This study concluded that the action of EF causes an intensified slow down of either growth an activities in soil microorganisms.

Key Words: Electric field, microbial, activities, growth, soil characters.

INTRODUCTION As the result of technical and industrial development, an increasing

number of man-made electric and electromagnetic fields have appeared in human environment (Polk & Postow, 1996 and Edwards & Graham, 2002). Soil is exposed to the electric field EF near the high voltage transmission lines, primary and secondary over head utility distribution lines and electrical grounding system (Roy and Lee, 1984).

The effect of electric field EF on soil can be studied from the view of different points. An aspect of study deals with the effect of electric field on

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microorganisms in soil, where this nonionizing radiation is capable of changing everyday, the growth, division and biological function of exposed cells (Fist, 2000).

Another aspect of studying deals with the effect of EF on ionic soil composition causing ion migration and ion movement, which in turn adding another indirect effect of EF on soil microorganism. This phenomena (ion mirgation) is applied in the processes of soil decontamintion and removal of pollutants or to prevent its spreading to surrounding subsoil (Touchard et al. 1999), also in the bioremediation of soil and removal of some hazardous wastes (Ottosen et al. 2000).

The interaction of electric field to the cell result in many biological changes. The electric field exerts forces on charged particles in electrically conductive surfaces such as the surface of ground and biological cells induce electrical potentials and resultant current flows in the aqueous medium surrounding the living cells (Tenford and Kaune, 1987). Because the membranes of these cells form a dielectric barrier, only a small fraction of the induced current penetrates the cell surface producing electrochemical alterations in cell membrane. This event in turn send signals across the cell membrane barrier that produce alteration in intracellular biochemical and physiological function (Sauders et al. 1991).

Rather than the effect of EF on population and surviving fraction of microorganisms as described by the studies of Tokuda & Nakanishi (1995) and Cserhalmi et al. (2002), electrical field affects on the activity of microbial enzymes by (Grahl & Markl, 1996 and Yeom et al., 2000) soil microbial biomass and soil respiration (Robert and Tate, 1995).

In the light of the previous important facts, this research deals with studying the effect of EF strength 6kV/m on microbial soil counts, some microbial soil activities in addition to studying the effect of EF on growth rate and molecular changes in protein pattern of selected isolate of Azotobacter.

MATERIALS AND METHODS The electric field

Electric field of 50 Hz frequency and 6 kV/m strength was used. The electric field was generated between two parallel aluminum electrode of dimensions 60 x 50 x 2cm, fixed horizontally above and below the samples. The electric field was derived directly from 50 Hz high voltage step-up transformer, manufactured by Center of Scientific and Electronic Equipment Maintenance, Faculty of Science, Cairo University.

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Exposure of soil to the electric field Cultivated soil samples were collected, divided in plastic bags (200 gms

in each), placed in the electric field and exposed to 6 kV/m strength for 1, 2 and 3 months. The control samples for each period were placed away from the field in the same atmospheric conditions. Unexposed and exposed soil samples were irrigated continuously to maintenance 75% humidity. After each period the soil samples were subjected to the different studies. Soil microbial counts

The total viable counts of bacteria and fungi (CFU/g dry soil) were obtained using pour plates method. Nutrient agar and Dox agar media were used for bacterial and fungal counts respectively. By using the most probable number method the number of Azotobacter and Azospirillum were determined. Modified Ashby5s medium for Azotobacter and Azospirillum medium of Dobereiner et al. (1976) were used.

Azotobacter medium (g/L), Glucose 2.0; K2HPO4 0.5; MgSO4 .7H2O 0.2; NaCl 0.5; CaSO4.2H2O 0.1; CaCO3 0.5; MnSO4. 4H2O 0.002; FeCl3. 6H2O 0.002; Na2M0O4. 2H2O 0.002; distilled H2O 1L and pH 7.0.

Azospirillum medium (g/L), Malic acid 5; KOH 4; K2HPO4 0.5; FeSO4. 4H2O 0.05; Mn SO4. 4H2O 0.01; Mg SO4. 7H2O 0.1; NaCl 0.02, CaCl2 0.01; Na2 MoO4. 2H2O 0.002; bromothymol blue (0.5% in C2H5OH) 2ml, agar 1.75, tap water 1L and pH 7.0. Soil microbial activities:

Total microbial biomass (CTMB): The CTMB (M CO2-C m-3) was measured by the carbon field index (CFI) method (Jenkinson and Powlson, 1976).

Active microbial biomass (CAMB): The CAMB (M CO2- C m-3) of soil was measured by the stimulated basal respiration method (Van de Werf and Verstrate, 1987).

Metabolic quotients (qR): A number of metabolic quotients (qR), such as CTMB Corg

-1, CAMB Corg-1 and CAMB CTMB were calculated (Insam and

Domsch, 1988). Basal respiration (BR): The BR (M CO2-C m-3 day-1) was measured as

the average CO2-evolution of 2 mm sieved non-amended homogenized soil (unfumigated) after an incubation period of 10 days (Islam, 1996).

Specific maintenance respiration rate (qCO2): The qCO2 was calculated as mean daily BR CTMB (M CO2-C d-1 CTMB

-1) by the method of Anderson and Gray, (1991).

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Microbial Soil enzyme activities

- Dehydrogenase activity : According to the assay method of Casida et al. (1964) dehydrogenase activity was calculated as ug TPF/g dry soil/day (TPF is triphenyl farmazon).

- Urease activity: According to the assay method of Tabatabai and Bremner (1972) urease activity was calculated as ug NH+

4-N/g dry soil/hr. - Nitorgenase activity: Nitrogenase activity was determined by GLC

and the method of acetylene reduction activity according to Hardy et al. (1973) as ul C2H4/g dry soil/hr.

- Phosphatase activity: The enzyme is classified as acid phosphatase and alkaline phosphatase because they show their optimum activities in acid and alkaline ranges respectively. According to the assay method of Eivazi and Tabatabai (1977) phosphatase activity was determined as ug P/g dry soil/hr. Isolation of Azotobacter

From the unexposed positive tubes of the most probable number, selected isolate of Azotobacter was subcultured, purified on soild Ashby’s medium. The pure colonies were identified according to Holt, (1984) and subjected to further experiments. Exposure of Azotobacter to electric field

The growth rate was carried out by inoculation 1 ml of 72 hrs old bacterial liquid culture to 5ml liquid Ashby’s medium in test tubes. Two groups of inoculated tubes were prepared and then incubated for 1,2,3,4,5 and 6 at 28- 30oC under exposure and unexposure conditions. After each period the growth was determined at 600 nm and the total viable counts (CFU/g dry soil) were calculated. Protein banding pattern using SDS-PAGE:

Total cellular protein of both exposed and unexposed Azotobacter liquid culture (incubated for 3 and 6 days) were separated on the basis of molecular weight by sodium Dodecyl sulfate polycrylamide Gel Electrophoresis (SDS-PAGE) (Laemmli, 1970).

Six markers of known molecular weights were used as standard proteins, bovine albumin (218, 131, 86, 43.8, 33 and 19.3 kDa). Data were identified

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and analyzed by using gel proanalyzer version 3 media cybernetic imaging exerts software, which compare the molecular weight (kDa), the relative forwent (RF) and average optical density (OD) of each band for all samples relative to the standard marker. Statistical analysis :

Statistical analyses were carried out using the SPSS BASE 10.0 (SPSS inc., Chicago, IL) packages. Data were tested by ANOVA. F-protected LSD separated means at p< 0.05 levels. Mean, standard deviations and variance separation between different exposure periods was calculated by using the means of individual measurements.

RESULTS AND DISCUSSION

Electric fields are non-ionizing radiation (NIR) emitted by electric power stations, transmission lines, electric blankets and other sources (Edwards and Graham 2002). There is a wide range of data documenting the ability of NIR to affecting living cells, including changing in the biochemical and molecular mechanisms of cells both in vitro and in vivo (Barnes, 1996), cell metabolism and cell poliferation inducing potentially damaging in all cell components from the cytoplasmic membrane, where the distribution of proteins is modified (Bersani et al. 1997) to cytoplasm and nucleus, where the activities involving intracellular enzymes and molecules regulating cell growth (Hill 1998). Results of the present research reflect some of these changes.

In the present investigation, the effect of exposure periods (1, 2 and 3 months) of electric field strength 6 kV/m on microbial soil counts (CFU/g dry soil) was detected. Table (1) indicated that, the total viable counts of bacteria as well as Azotobacter and Azospirillum were slightly decreased by increasing the exposure period to three months comparing with the unexposed sample, however the total viable counts of fungi reflected noticeable decrease compared with bacteria. In this connection previous researches found that the surviving fraction of E.coli suspended in buffer solution decreased to 1% of the initial value after 1h 0.33A DC, also this field was effective for B.subtilis, Ps.areuginosa and S.aureus cells (Tokuda and Nakanishi, 1995). Saccharomyces cerevisiae was inactivated 4 log reduction in apple juice using 10.4 pulse at 20 kV/cm (Cserhalmi et al. 2002). The biological influence of external fields depends on their transfer into electroconductive surfaces such as the surface of the ground and biological bodies (Tenford and Kaune, 1987) resulting in current flow in the medium surrounding the cells causing considerable hyper or

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hypopolarization of the membrane resulting in membrane electric breakdown and membrane electroporation, when the membrane loses its property as diffusional barrier, its electrical resistance breaks down leading to full destruction of the cell by consequent lysis (Chang and Myers, 1996).

Biotic properties of selected soil exposed to different exposure periods of 6 kV/m EF are listed in table (2). It is clear from the results that, unexposed soil recorded significant increase in CTMB (total microbial biomass), CAMB (active microbial biomass), qR (metabolic quotients, and BR (basal CAMB/CTMB respiration) while significant decrease in qCO2 at the end of incubation periods was observed. Also significant decrease in CTMB, CAMB , BR and qCO2 of soil under the effect of three months of exposure to electric field was indicated. In this relations reported that, significant in activation of microorganisms in response to EF because of three types of factors 1) the process (EF intensity, pulse width, treatment time, temperature, pulse wave shapes); 2)microbial entity (type concentration, growth stage of microorganisms) and treatment media (pH, anti-microbial, ionic compounds, conductivity and medium ionic strength.

Effect of electric field on the microbial soil enzyme activities is indicated in table (3). The results revealed that, all enzyme activities were decreased by increasing exposure period up to 3 months comparing with the unexposed samples. Nitrogenase was more respond to EF followed by acidic phosphatase, urease, dehydrogenase and alkaline phosphatase, they decreased by 30, 23.6, 20, 13 and 7.7% respectively comparing with the unexposed samples after 3 months of exposure to the electric field. Most previous researches were carried out on the effect of EF on microbial enzymes in foods samples and suspensions but not in soil. Vega Mercado et al. (1995), achieved the inactivation of microbial alkaline phosphatase higher than 90% after HI PEF treatment of milk. Grahl and Markl, (1996) observed a slight depletion of microbial alkaline phosphatase in milk, similar to results that obtained by Ho et al. (1997) in a buffer solution. Yeom et al. (2000) reported that, an electric field strength 35 kV/cm for 59 µs results in significant inactivation of microorganism and pectin methyl esterase in orange juice. Bendicho et al. (2002) proved that the activity of bacterial thermoresistant lipases of Ps. fluorescences can be reduced by HI PEF. The influence of electric field on soil enzyme may be due to its effect on the proteinaceous nature of enzyme causing change in the secondary and tertiary protein structure of enzyme (which optimizes interaction of enzymes with substrates for the reaction) causing denaturation of enzyme protein (Robert and Tate, 1995), or may be due to the effect of EF on the enzyme activities and biochemical conversions process of organic matter.

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The change in enzyme activities may be correlated with the probability of electroporation (Reina et al. 1998) and irreversible cell membrane break down of microorganisms (Dunne, 2000).

The change in microbial enzyme activity in soil means, change in soil fertility where there is a relation between basic enzyme activity and soil fertility, soil microbial biomass and soil respiration (Robert and Tate, 1995), where alkaline phosphatase, amidase, alpha-glucoisdase and dehydrogenase activities correlated with microbial respiration and alkaline phosphatase, amidase and catalase activities correlated with microbial biomass (Frankenberger and Dick, 1983).

An Azotobacter isolate which isolated from unexposed soil sample was subjected to further experiments to study the influence of EF on the bacterial cell. Results in Fig. (1) showed that, the growth of Azotobacter was increased by increasing the incubation period up to 6 days under exposure and unexposure conditions. Further more, it was observed that, the viable counts of exposed culture was decreased comparing with the unexposed ones at all exposure period. In this connection, study of (Reina et al. 1998) noted that Listeria was inactivated in skim milk with pulsed EF by 1 to 3 log cycle, the vegetative bacterial population in dairy products and juices was decreased by 5 to 6 logs by using PEF and hydrostatic pressure pasteurization (Dunne, 2000). Also the population of B. cerens and Saccharomyces cerevisiae suspensions in saline solution were reduced more than 1 log cycle using 10.4 pulse at 20kV/cm (Cserhalmi et al. 2002), moreover, Ps. aeroginosa and S.aureus growth were decreased and increased, respectively, when the suspensions were subjected to electric field for 1h at frequencies in range 0.7 to 20Hz (El-Hag, 2003). It could be mentioned that, the inactivation of microorganisms due to high voltage pulse of (PEF) exposure may be due to the production of electroporation of the cell membranes resulting in the cell inactivation (Jeyamkondan et al., 1998). The criteria leading to the lethal breakdown of microorganisms suspended in continuos medium depend on two parameters, applied electric field and joule energy, the 1st initiates reversible breakdown and the second one, the completion of irreversible electrical breakdown leading to death of the cell (Kekez et al. 1996).

Protein banding pattern and gel electrophoresis analysis of extracted protein of unexposed and exposed Azotobacter is shown in Fig. (2) and Table (4). The results indicated that, 14 bands were obtained from protein analysis of both exposed and unexposed cultures. Two bands (of molecular weight 83.4 and 15.9 kDa) were lost after 3 and 6 days exposure period and two others bands (of molecular weight 86.3 and 16.2 kDa) were appeared

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as a result of exposure to electric field for 3 and 6 days. The average optical density which reflects the intensity of bands were decreased at exposure period 6 days compared with the unexposed ones. This results reflect molecular change in protein as a response of the electric field exposure. In this relation El-Hag, (2003) studies the effects of electric field at frequencies (0.3 and 0.5 Hz) for Pseudomonas on molecular level, he found that, the electric field can produce molecular changes in water soluble protein and plasmid DNA subjected to agarose gel electrophoresis analysis. Moreover, decrease in band intensity means decrease in amount of prtoein of the band, distruction of certain protein molecules and changing in secondary and tertiary protein structure leading to protein denaturation (Robert and Tate, 1995).

It could be mentioned that, the electric field and EMF may have different targets affecting the biological systems. Plasma membrane is a possible way to evidentiate these biological effects. An important role is played by the intermembrane protein (IMP) located in the lipid bilayer of the cell membrane which function as ion channels, enzymes or receptors (Marinelli et al. 1996).

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طة ميكروبات التربةدراسة المجال الكهربى على بعض أنش

و أآرم محمد عبدالمنعم وسام عبدالغنى على حسانين جامعة الزقازيق – آلية العلوم –قسم النبات

الملخص العربى

يعتبر المجال الكهربى من العوامل الطبيعية التى اظهرت تـأثيرات خطيـرة وواضـحة علـى

البحث دراسة تأثير المجال الكهربـى علـى بعـض العديد من األنظمة الحيوية ، ولذلك استهدف هذا فأظهرت نتائج تعريض عينات مختلفة من التربة لمجال كهربـى . العمليات الحيوية لميكروبات التربة

أشهر الى تناقص قليل فى اعداد البكتريا وكذلك األزوتوباكتر ٣ ، ٢ ، ١متر لمدة / كيلو فولت ٦شدته لفطريات بنسبة أعلى ومن ناحية أخرى تزايدت قيم الكتلة الميكروبية واالزوسبيرلم بينما تناقصت أعداد

ومعدل ايض الميكروبات ) BR(والتنفس القاعدى ) CAMB(والكتلة الميكروبية النشطة ) CTMB(الكلية )qR ( مع تناقص معدل صيانة التنفس)qCO2 ( فى عينات التربة الغير معرضة للمجال بعـد ثالثـة

٢ ، ١ا تناقصت هذه القيم بشكل ملحوظ بعد التعريض للمجال الكهربى لفترات أشهر من التحضين بينم . أشهر٣،

وكان للمجال الكهربى تـأثير مثـبط علـى نشـاط بعـض انزيمـات ميكروبـات التربـة ، ويعتبر النيتروجينيز هو أكثر االنزيمات تأثرا بالمجال الكهربى ثم يليه انزيم الفوسفاتيز الحمضـى ثـم

وكان انخفاض نشاط هذه االنزيمات نتيجة تعرضها . اليورياز ثم الديهيدروجينيز ثم الفوسفاتيز القاعدى على التـوالى عنـد % ٧,٧ ، ١٣ ، ٢٠ ، ٢٣,٦ ، ٣٠ أشهر هى ٣ ، ٢ ، ١للمجال الكهربى لفترات

عزلة ازوتوبكتر وقد امتدت الدراسة الى تأثير المجال الكهربى على . مقارنتها بالعينات الغير معرضة معزولة من تربة غير معرضه للمجال فأظهر منحنى النمو لالزوتوبكتر أن العدد الكلى للخاليا الحيـة

يوم بالمقارنة بالعينـات غيـر ٦ ، ٥ ، ٤ ، ٣ ، ٢ ، ١تناقص فى المزارع المعرضه للمجال لفترات ئى لمزارع االزوتوبكتر المعرضـة أظهرت دراسة أنماط البروتين باستخدام التفريد الكهربا . المعرضه

شريط من الشرائط البروتينية ١٤ يوم أن كل منها يحتوى على ٦ ، ٣وغير المعرضة للمجال لفترات ذات (فقد اختفت بينما الشـرائط ) كيلو دالتون ١٥,٦ ، ٨٣,٤ذات وزن جزئى (وأن اثنان من الشرائط

يوم ، باإلضافة الى ٦ ، ٣يض للمجال لفترة قد ظهرت بعد التعر) كيلو دالتون١٦,٢ ، ٦٨,٣ون جزئى ذلك انخفضت معدالت الكثافة الضوئية للشرائط البروتينية لمزارع االوزتوبكتر المعرض للمجال عنـد

يوم، وعلى ذلك يتضح أن للمجال الكهربى تـأثير واضـح ٦مقارنتها بالمزارع غير المعرضة لفترة .على نمو ونشاط الميكروبات بالتربة