Charge Transport in Bifidobacterium animalis subsp. lactis BB-12 under the various Atmosphere

The influence of relative humidity (RH) on quasistatic current-voltage ${(I-V)}$ characteristics of Bifidobacterium animalis subsp. lactis BB-12 thin layers have been studied for the first time. The value of electrical conductivity in 75$ \%$ RH was found to be in the order of 10$^{-7}$ (ohm cm)$^{-1}$ which was 10$^{6}$ orders of magnitude higher than that observed in dry atmosphere. Here we also demonstrated that RH played a key role in hysteresis behaviour of the measured ${(I-V)}$ characteristics. FTIR measurements showed that under water moisture environment the associated bonds for amine and carboxyl group were greatly strengthened that was the source of number of free charge carries after ionization. The type of surface charge of Bifidobacterium animalis subsp. lactis BB-12 was found to be negative by zeta potential measurements, claiming that electrons were the charge carriers.


I. INTRODUCTION
A. BB-12 and Gut Microbiota The gut microbiota contains a diverse community of commensal, symbiotic and pathogenic microorganisms [1,2]. The gut microbiota has anti-inflammatory, antioxidant, antioncogenic effects and it contributes to the immunological, hormonal and metabolic homeostasis of the host [3,4]. The genus Bifidobacterium belongs to the phylum Actinobacteria and it comprises Gram-positive, non-motile, often branched anaerobic bacteria [5]. The Bifidobacteria are one of the major species in the human colon microbiota and are frequently used as probiotic [6]. Bifidobacterium animalis subsp. lactis BB-12 is a catalase-negative, rod-shaped bacterium which was first isolated 1983 ( Figure-1). At the time of isolation, Bifidobacterium animalis subsp. was considered as belonging to the species of Bifidobacterium bifidum [7].
In 2010, the complete genome sequence of BB-12 was mapped [7]. The BB-12 genome consists of a single circular chromosome of 1,942,198 base pairs with 1642 predicted protein-encoding genes, 4 rRNA operons, and 52 tRNA genes. A physical mapping of the BB-12 chromosome revealed that the genome sequence was correctly assembled (Figure 2). BB-12 is technologically well suited, expressing fermentation activity, high aerotolerance, good stability and a high acid and bile tolerance [8] . Because of high redox potential in the colon flora ecosystem, BB-12 is highly resistant bacteria in distress condition. The BB-12 cell envelope is an electrical and physical barrier that can be * kutsalb@yildiz.edu.tr overcome by pathways that consist of redox proteins and structural proteins the main some of molecular mechanisms and electron transport systems are presented in Figure 3.

B. BB-12 and Bacterial Cellular Electron Transfer Systems
Bacterial cellular electron transfer systems (CET) are defined microbial bioelectrochemical processes in which electrons are transferred from the cytosol to the membrane of the cell [10]. Shi, L. et al showed molecular mechanisms that underlie the ability of bacterial to exchange electrons, such as c-type cytochromes and micro- bial nanoparticles [10]. Samuel, H. Light et al described food borne gut pathogen L. monocytogenes cellular electron transfer system [11]. They showed that NADH dehydrogenase enzymatic pathway is responsible mechanism for CET from aerobic respiration by channelling electrons to a discrete membrane-localized quinone pool. Although Lorena et al showed the strength of BB-12 in the event of stres, CET features of BB-12 are not completely identified. Here we first describe that the probiotic bacteria Bifidobacterium animalis subsp. lactis BB-12 strain features of CET under the various atmosphere. This work is organized as follows. Materials, Zeta-potential studies, ATR-FTIR analysis, SEM and electrical characterization of BB-12 are presented in Sec. II. Charge transfer system of Bifidobacterium animalis subsp. lactis BB-12 is discussed in Sec.III. Conclusions and outlooks are given in the last Sec. IV.

A. Material
Mean gut bifidobacterial count of 5 billions (5x10 9 ) cfu of Bifidobacterium animalis subsp. lactis BB-12 (Chr Hansen) were used in the experiment. This Bifidobacterium dissolved in 5 millilitres of distilled water and prepared sample.

B. Zeta Potential
The Zeta potential (ZP) was measured for BB-12 in water using a Zetasizer Nano ZS (Malvern Instruments Ltd) at room temperature. The voltage applied to the driving electrodes of the capillary electrophoresis cell was 149V .

C. FTIR Spectral Method
The structural change of BB-12 was recorded by an Agilent Technologies Cary 630 FTIR apparatus at room temperature. Spectra of all samples have been recorded for 400 and 4000 cm −1 at a resolution of 4 cm −1 with 100 scans.

D. Electrical Characterization of Bifidobacterium animalis subsp. lactis BB-12
Photolitographically patterned interdigital arrays of tin (Sn) electrodes on plexiglass substrate were used for the electrical characterization of Bifidobacterium animalis subsp. lactis BB-12. The interdigital electrodes used in the electrical characterization experiments consisted of 20 finger pairs of electrodes with a width of 100 µm and a space of 100 µm between adjacent electrodes.
FIG. 3. Main molecular mechanisms involved in the response of BB-12 to different stresses. A Bile is detoxified from the cytoplasm by the activity of bile efflux pumps and/or multidrug transporters. Conjugated bile acids are deconjugated by the bile salt hydrolase, although the relationship of this enzyme with the resistance to bile is unclear. B Both bile and heat shock induce protein aggregation and misfolding, which is counteracted by the action of chaperones and proteases. C The F1F0-ATPase is used by bifidobacteria for counteracting the cytoplasm acidification that occurs in acidic environments. In addition, production of branched-chain amino acids is coupled with glutamine deamination, rendering ammonia that acts as a cytoplasmic buffer. From Lorena et al [9] with permissions from Springer-Verlag , Copyright 2011.
Thin film of BB-12 was obtained by spin coating double distilled water solution of the BB-12 over the electrode arrays to obtain devices suitable for electrical characterization in various ambient conditions. During the deposition of the BB-12 film, the temperature of the interdigitated substrate temperature was kept constant at 300 K during deposition of the materials over the electrodes. A home made stainless steel test chamber of 1 × 10 −5 liters capacity was used during these experiments. Dry nitrogen was used as carrier gas and the desired concentration of relative humidity inside the test chamber was obtained by bubbling of dry nitrogen gas through double distilled water. Well defined levels of relative humidity was obtained by mixing dry nitrogen gas and water vapor using computer driven mass flow controllers (Alicat Scientific, Inc.). D.c conductivities (σ) of the BB-12 film was obtained from the measured current-voltage (I − V ) characteristics by using following equation; where I is the measured current, V is the bias voltage, d is the electrode spacing, m is the number of electrode finger pairs, l is the overlap length of the electrode fingers and h is the thickness of the electrodes.
In resumed conditions, absorption measurements were also carried out (not shown), leading to non-variation of absorption spectra and hence the ambient did not any change in structural properties of the BB-12 in terms of absorption analysis.

A. Zeta-potential studies
The zeta potential of BB-12 was measured using a Zeta master (Malvern Instruments, Malver, UK) at room temperature. The zeta potential for BB-12 was found to be negative (−7.85mV ), which means that in the cell surface is predominantly anionic compounds, such as strong acids, the phosphate based (lipo-) teichoic acids, weak acids, the carboxylate containing acidic polysaccharides and proteins [12]. These negative value is in accordance with the previous studies performed considering that (i) CIDCA 5310, 537 and NCC 189 grown in bile-containing medium [13] and (ii) free L. rhamnosus GG cells as a function of pH [14]. Moreover, similar range of zeta potentials have also been reported for microencapsulation in Alginate and Chitosan Microgels to Enhance Viability of Bifidobacterium [15]. Then, the zeta potential of BB-12 is increased from (−7.85mV ) to (−10.4mV ) as a function of time, which can be expressed as an indication that the acidic character of the surface is reduced [12].

B. ATR-FTIR Analysis
The analysis of surface of the cells of BB-12 were examined by ATR-FTIR spectroscopy. The changes in the peaks of all the samples was recorded as a function of time and spectrum is shown in Fig. 4. New bonds and increase in the bond intensity were determined in the BB-12 spectra after treatment distilled water. Then, peak losses were detected depending on time. Powder BB-12, a small broad band at 3301.81 cm −1 was seen, this band intensity increased after treatment distilled water, which is corresponds to the hydroxyl stretching vibration of the polysaccharide [16,17]. The vibrational small peak at 2910.32 cm −1 are related to the alkyl-hydrocarbons groups, the so called fatty acid region [18,19]. After contact with the pure, an increase in the intensity of this peak was observed, indicating that the bacterium began to live with water. As is shown in Fig.4, the very-very small peak at 1636.62 cm −1 were related to the carbonyl stretching of secondary amides (Amide I) [18,20]. But, it was observed that after distilled water demonstrated the increase for the amide I absorption band.
The very small peaks at 1407 and 1354 cm −1 are assigned to the bending of −CH 3 and −CH 2 groups of bacterium [20]. The between 900-1200 cm −1 was observed wide and intensive polysaccharides and carbohydrates in the spectrum of in the BB-12 after distilled water, with maximum at 995 cm −1 caused by valence C − O − C group vibrations in the cyclic structures [17][18][19]. But, after 432 h distilled water treatment the contribution of carbohydrate compounds on the surface decreased. The band are seen the at 852 cm −1 , which is attributed to indicate to configurations exist in the polysaccharides [16]. The change of spectral peaks as a fraction of time is in agreement with the zeta-potential results of a BB-12 that has passed into the endogenous phase.

C. SEM analysis
The surface morphologies of powder BB12 before/after treatment distilled water were characterized using a scan-ning electron microscope (SEM) and the results are presented in Figure 5. It is seen in Fig. 5 that the bacterial integrity is maintained and non-spherical structure before purified water.

D. Charge transport in Bifidobacterium animalis
subsp. lactis BB-12 Charge transport in BB-12 film under dc condition were investigated by means of current-voltage (I − V ) measurements. The measurements were carried out at room temperature under various RH conditions. Fig. 6 X1 shows the I −V characteristics of the sample under investigation at indicated RH levels. In this measurements the voltage has been incremented in steps of 50mV from −1 to +1 V and back again. It should be mentioned here that the measured I − V curves exhibit considerable hysteresis between increasing and decreasing voltage sweeps. It is important to point out that the area within the loop decreased with an increase in RH level. The interpretation of appearance of the hysteresis behavior in dry nitrogen atmosphere and low RH level requires to take into account many elementary physical processes such as, density of mobile charges, and delay time between two successive measurements separated by a potential step. A considerable increase in film conductivity with the increase in RH level, compared to dry nitrogen atmosphere, is also clear. It should be mentioned here that the effect of the film conductivity is reversible for all RH levels investigated, it means that when the film surface is purged with carrier gas (dry nitrogen in our case) the conductivity of the film returns to the initial value. Increase in dc conductivity of the film reveals that the interaction between the water molecules and the BB-12 film is based on charge transfer. When the water molecules interact with the surface of the BB-12 film, and one should keep in mind that BB-12 exhibit n-type character, the zeta potential becomes more negative because of the formation of functional groups such as COO and N H 2 .

IV. CONCLUSIONS
Charge transport behavior and the effect of the RH level on it in the BB-12 film have been investigated by means of I − V measurements. Within water moisture environment, electrical conductivity of the BB-12 increased more than six decades while under N 2 environment conductivity returns to the initial current value. This behaviour in conductivity modulation was reversible at least in the three cycles. This experimental findings showed us that there was no structural transformation under relative humidity. On the other side, increase in the conductivity was interpreted by the increase in the population of charge carries, supplied by the interaction BB-12 with the water moisture, monitored by amine and carboxyl group through FTIR and Zeta potential measurements. Overall, obtained result in this study indi-cated that Bifidobacterium animalis subsp. lactis BB-12 has a great potential for humidity sensing device at room temperature.