Mechanism of Electric Field Assisted Screening for Candida Tropicalis

: 8 Electric field assisted technology has been widely used in many fields. Candida tropicalis 9 degrading thiophene has been successfully screened by electric field assisted screening (EFAS). 10 However, the mechanism of EFAS is yet unclear. In this study, the cell concentration of C. 11 tropicalis at different locations in the electric field was studied. In the electric field, C. tropicalis 12 cells migrate towards the cathode plate. When the loading electric field intensity was 0.6 V /cm, 13 the concentration of cells near the cathode plate reached the maximum and it was nearly stable at 14 15 minutes of loading time. Analysis of the cell morphology at different locations showed that 15 only the budding cells could migrate in the electric field. The charged characteristic of the budding 16 cells in the electric field explained the screening mechanism of thiophene-degrading C. tropicalis 17 by EFAS. The results can provide a theoretical foundation for EFAS in screening other 18 biodegradable microorganisms. 19


Introduction 23
Electric and magnetic fields can convert to each other, and they are closely related to the growth 24 and metabolism of living things [1]. Almost all living creature has some changes in properties and 25 physiological activities under a certain electric field(EF) [2]. Therefore, in recent years, 26 electric-field assisted (EFA) technology has been gradually applied in various researches [3,4]. 27 Through application of EFA technology, the efficiency of soil remediation was improved [5,6], the 28 growth and trace metal content of aquatic plants are affected [2], and the harmful contaminants, 29 which strictly limited the land application of the sludge, were reduced from sludge [7]. The EFA 30 technology was also being used in drug delivery research [8]. In order to improve the activity and 31 degradation efficiency of microorganisms, the EFA technology has been applied more and more in 32 biodegradation[9,3,6]. 33 In our previous studies[10], the EFA technology was used to screen functional microorganisms, 34 and a Candida tropicalis biodegrading thiophene was obtained. The EFA screening (EFAS) 35 method is simple, timesaving, and can also improve the activity of C. tropicalis at the same time. 36 The screened strains showed excellent biodegradation efficiency in study of thiophene 37 biodegradation. However, the mechanism that EFAS can screen for the microbes remains unclear. 38 Single molecule electrophoresis under the EF has been proved to be a feasible method for DNA 39 sequencing [11]. At low voltages (1.5-3.0V), the EF could provide electron donors and receptors 40 for microbial degradation of PCBs [12]. These researches explained the microbial aggregation and 41 migration in the EF. 42 Therefore, the objective of this study was to investigate the migratory and aggregate behavior of mechanism of EFAS was explored by the migratory behavior of the cells under different EF. 45

Strain and medium 47
needle could be used to sample through the sampling hole. The anode plate and the cathode plate 67 were two platinum plates located at the ends of the culture tank and connected to a direct current 68 power supply with adjustable voltage and current.

Experiment of aggregation or/and migration in the EF 74
The experimental device was placed in the clean workbench, the inside and electrode of the 75 device were cleaned with alcohol, and then ultraviolet sterilization was conducted for 30min. 76 Afterwards, 1000mL of yeast suspension of a certain concentration was added to the EFA culture 77 tank. Then at 0.3 V/cm of EF strength, power was applied for 5min, and 1mL C. tropicalis 78 suspensions at different sampling holes(0, 3, 6, 9, 12, 15, 18, and 21cm distance from the anode 79 plate) were quickly collected into the sampling tube with a pipette. The OD 600 value of 200 µL C. 80 tropicalis suspension was measured by a microplate reader in a 96-well plate. The OD 600 value 81 was repeated three times and the mean value was obtained. The above operations were repeated 82 when the loading EF strength was changed to 0.4, 0.5, 0.6 and 0.7 V/cm, respectively. The 83 suspension must be shaken well before the loading voltage was changed each time. According to the measured OD 600 value, the cell concentrations at different location of the culture tank under 85 different loading voltages were calculated. The loading EF that were most conducive to C. 86 tropicalis migration or/and aggregation were obtained. 87 After the loading EF was determined, the loading time was explored under the condition of the 88 optimum EF. The selected loading time was 5, 10, 15, 20, 25 and 30min, respectively. The cell 89 concentration at different location of the culture tank under different loading times was obtained 90 according to the above mentioned method. The loading time to facilitate C. tropicalis migration 91 or/and aggregation was determined. 92

Verification of counting method 94
In this study, the number of samples was large, and the count of C. tropicalis cell needed to be 95 fast and accurate, so the hemocytometer method could not meet the requirements. The microplate 96 reader that could simultaneously determine 96 samples at a high speed was selected for counting

3.2The behavior under different EF 104
The loading voltage at both ends of the culture tank was changed, and the concentration of C. 105 tropicalis at different locations was sampled and analyzed. The results were shown in Fig. 3a. 106 Under a certain loading voltages(in the test range), the cell concentration of C. tropicalis 107 fluctuated slightly with the distance (distance from anode plate) in the culture tank. However, at 108 the loading EF of 0.3-0.6 V/cm, the concentration of C. tropicalis at 21cm distance(near the 109 cathode plate) was significantly higher than that at other distance. And the concentration of C. 110 tropicalis at 21 cm distance increased with the increase of the loading EF. When the loading EF 111 reached 0.6 V/cm, the cell concentration at 21 cm distance reached its maximum value. When the 112 loading EF reached 0.7 V/cm, the cell concentration at 21 cm distance decreased again. The 113 overloading EF, whose strenght was 0.7 V/cm, might cause damage to the C. tropicalis cells.  locations, except the cell concentration near the cathode plate(21cm of distance) was 145 increased (Fig. 4a). The cell concentration near the anode plate(0cm of distance) was the lowest 146 after 5min of EF loading. As the cells migrated, the concentration of cells near the anode plate did 147 not change. And then at a distance of 12cm, the concentration of the cells didn't change when the 148 positions did not change. Except near the cathode plate(21cm of distance), the cell concentration 150 at all positions was almost the same and lower than the initial value. This was the concentration of 151 non-budding cells in the solution. At this time, almost all the budding cells gathered near the 152 cathode plate. And the cell concentration at a distance of 21cm was significantly higher than that 153 at other distance. With the extension of the loading time, the cell concentration near the cathode 154 plate became higher and higher, however the increasing trend slowed down (Fig.4 b). It might be 155 due to a decrease in the number of budding cells in the solution other than near the cathode plate.  Compliance with ethical standards 182

Conflict of interest 183
All authors declare that there are no conflict of interests. 184

Ethical guidelines 185
All applicable international, national, and/or institutional guidelines for the care and use of 186 animals were followed. 187