Journal Archive

Johnson Matthey Technol. Rev., 2015, 59, (2), 109
doi: 10.1595/205651315X686741

Annihilation of Extremely Halophilic Archaea in Hide Preservation Salt Using Alternating Electric Current

Electrochemical disinfection of hide brine liquors for use in the leather industry

    • By Yasar Birbir
    • Department of Electric and Electronic Engineering, Faculty of Technology, Marmara University, Goztepe, Istanbul 34722, Turkey
    • Sema Anik
    • Institute of Pure and Applied Sciences, Marmara University, Goztepe, Istanbul 34722, Turkey
    • Meral Birbir* and Pinar Caglayan
    • Division of Plant Diseases and Microbiology, Department of Biology, Faculty of Arts and Sciences, Marmara University, Goztepe, Istanbul 34722, Turkey

*Email: mbirbir@marmara.edu.tr

Article Synopsis

Salt contains extremely halophilic archaea and these microorganisms degrade leather quality. The aim of this study is to find an effective treatment system to kill these microorganisms in salt used in hide brine curing. Ten salt samples were obtained from Tuz Lake, Turkey, and the total cell counts of extremely halophilic archaea, proteolytic and lipolytic extremely halophilic archaea were determined. Two sets of experiments were designed to detect the inactivation impact of alternating electric current on extremely halophilic archaea. In the first experiment, 2 A alternating electric current was applied for 25 min to the salt samples dissolved in liquid medium. In the second experiment, 2 A alternating electric current was applied for 25 min to the isolates of proteolytic extremely halophilic archaea, lipolytic extremely halophilic archaea, both proteolytic and lipolytic extremely halophilic archaea, and a mixed culture of these isolates. The extremely halophilic archaea in salt (102–104 colony forming units (CFU) g–1) was annihilated in 1 min via alternating electric current and a 5 min treatment with the current was enough to destroy extremely halophilic archaeal isolates (106 CFU ml–1) obtained from salt samples. This electric treatment was found fairly effective to kill proteolytic and lipolytic extremely halophilic archaea in salt used for preservation of hide.

1. Introduction

The leather industry is an important sector in the world economy. Almost 23 billion square feet of leather is produced annually (1, 2) and the total value of this is estimated to be more than US$100 billion (3). Most of the global earnings are accrued from footwear sales. This sector represents 60% of the industry's total income, with annual production of 13 million pairs of shoes (4, 5). Because many consumers can perceive when leather products are manufactured from an inferior hide grade, producers are perforce obliged to dispose of damaged hides. Ignorance of effective preservation techniques therefore causes important economic losses in the industry.

Studies have shown that hides contain extensive populations of Gram-positive and Gram-negative bacteria which may be either resident or transient bacteria. As soon as the animal is slaughtered, these bacteria can grow on the raw hides and degrade the hide quality (6–8). A mixture of salt and boric acid is commonly used to prevent the growth of these microorganisms and preserve the hides. If hides are preserved with only crude solar salt, they can be contaminated by different species of extremely halophilic archaea (9–12). These microorganisms are easily detected on hides as they are revealed by red to orange pigments. Extremely halophilic archaea produce red, orange and pink coloured colonies because of the presence of C50 carotenoids (9–12). Red discolorations on the salted hides, caused by microorganisms, have been scrutinised by researchers since 1929; even at that early date, proteolytic activity of halophilic microorganisms was recognised (13). In addition, occurrence of red colouration produced by extreme halophiles has been noted in the Great Salt Lake (14), Dead Sea (15), Lake Magadi (16) and Tuz Lake (11). Salt lakes and solar saltern crystalliser ponds contain 107–108 CFU of extremely halophilic archaea per ml (9). In previous studies, salt samples which were obtained from Tuz Lake in Turkey contained 104–107 CFU g–1 extremely halophilic archaea (11). In another study, the viable cell number of extremely halophilic archaea in water samples collected from Tuz Lake was found to be 1.38 × 107 CFU ml–1 (17).

Salt collected from Tuz Lake, located in Central Anatolia, Turkey, is used to preserve hides in the Turkish leather industry. If the salt obtained from Tuz Lake is directly used in hide preservation, it may contaminate hides with halophiles. During long storage or overseas transportation of the salted hides, viable cell numbers of extremely halophilic archaea on the hides can multiply excessively. In an earlier study, extremely halophilic archaea were observed in considerably high numbers (103–108 CFU g–1) on 36 salt-pack cured hides. Extremely halophilic archaeal counts were fairly high in 72% of the hides (105–107 CFU g–1) as a consequence of inadequate salt-pack curing method. 94% and 81% of the samples contained proteolytic extremely halophilic archaea (102–106 CFU g–1) and lipolytic extremely halophilic archaea (102–106 CFU g–1), respectively (18). These findings were consistent with previous studies' reports of the extremely halophilic archaeal counts (105–108 CFU g–1) on the brine cured hides (19, 20).

The destructive effect of extremely halophilic archaea on salted hides has been observed. It was detected that proteolytic extremely halophilic archaea, originating in unprocessed solar salt, digested the grain surface of the hide and caused a complete disruption of collagen fibers (21). Furthermore, it was noted that proteolytic and lipolytic extremely halophilic archaea produced sponge-like vesicles within the hide and light stains on the suede surface of finished double-face leathers (22, 23). Therefore, inadequately preserved hides and skins may adversely affect final leather quality.

Due to these destructive effects of extremely halophilic archaea on the salted hides, an effective, easy and inexpensive method should be applied to eradicate these archaea in salt. Thus, using sterile salt in hide preservation will increase leather quality and prevent substantial economical losses in the leather industry.

1.1 Use of Electric Current

Electric current application is one of these methods. This treatment system has been used by several researchers to inactivate different types of microorganisms found in synthetic urine (24), water (25), fresh orange juice (26), activated sludge and biofilms (27), effluent seawater (28), soak liquors (29), brine solution (30, 31), on beef surfaces (32) and chicken legs (33). In those experiments, scientists usually used direct electric current to eliminate diverse bacterial populations.

In addition to direct electric current, alternating electric current was also used to inactivate bacteria in phosphate buffer solution (34), seawater (35) and natural waters (36). Researchers mentioned that alternating electric current treatment has advantages over direct electric current (35). The investigators found that application of 1 A direct electric current or 1 A alternating electric current for 100 ms were sufficient to kill Vibrio parahaemolyticus in seawater. They concluded that 1 A alternating electric current application at 5 Hz generated less chlorine concentration (<5 mg l–1) in seawater than when the seawater was treated with direct electric current (approximately 35 mg l–1). It was revealed that this reduced production of chlorine was due to the bidirectional flow of alternating current, resulting in less electrolysis of the test medium. It was also emphasised that chlorine gas produced in direct electric current treatment may cause serious health problems in humans (35). To overcome this adverse effect of chlorine gas, the researchers suggested using alternating electric current to kill bacteria in natural waters (36) and industrial applications (35).

The mechanism of bacterial inactivation by alternating electric current has been explained as a byproduct of chlorine (36, 37) and hydrogen peroxide (34, 35, 38) produced during electrolysis. Previous experiments demonstrated that electric field and current caused cell membrane damage in the form of pores that opened on the membrane following electrical treatment (39, 40). Chloride and hydrogen peroxide, produced during electrolysis, may enter easily through these pores, thus hastening the inactivation process (39, 40). In addition, researchers observed that application of alternating electric current on Escherichia coli cells damaged their cell membranes, thereby causing the leakage of intracellular components, DNA-related materials and ninhydrin-positive materials from the cell (41). Moreover, alternating electric current affected E. coli cells by increasing the negative charge at the surface and decreasing their respiratory rate (42). Furthermore, the amount of phosphatidylethanolamine in the cell membrane of E. coli was reduced by electric current treatment (43).

Although studies indicate that direct electric current can annihilate different species of microorganisms in diverse industries (24–33), the inactivation effect of alternating electric current on extremely halophilic archaea in hide curing salt has not yet been evaluated. The alternating current disinfection method was used in the present study precisely because it was proven to release only a low chlorine concentration (35). Therefore, the experiments were designed to evaluate the following: the inactivation impact of 2 A alternating electric current, which produces less chlorine than direct current treatment (35), on extremely halophilic archaea in salt; the inactivation effect of 2 A alternating electric current on the isolates of proteolytic extremely halophilic archaea, lipolytic extremely halophilic archaea, both proteolytic and lipolytic extremely halophilic archaea and a mixed culture of these isolates. The total extremely halophilic archaeal counts, total proteolytic extremely halophilic archaeal counts and total lipolytic extremely halophilic archaeal counts in salt samples were also determined.

2. Experimental procedures

2.1 Determination of the Total Counts of Extremely Halophilic Archaea

Ten salt samples were collected from Tuz Lake in Turkey. A total of 20 g of salt sample was separately placed into an electrolysis cell containing a final volume of 200 ml of 25% NaCl solution in water. Then, this mixture was placed into a shaker incubator (100 rpm) for 4 h at room temperature. A total of 100 μl of the test medium was removed from each electrolysis cell before commencing the experiments. This was diluted to 10-fold (10–1) dilution, 100-fold (10–2) dilution and 1000-fold (10–3) dilution with sterile 25% NaCl solution. The viable extremely halophilic archaeal cell numbers in these samples were determined by the plate count method. To detect the total extremely halophilic, proteolytic and lipolytic extremely halophilic archaeal cell numbers in the salt samples, the direct and the diluted solutions were spread over complex agar medium, gelatin agar and Tween® 80 agar media, respectively, and incubated at 39°C for 30 days. After the incubation period, the colonies on the complex agar surface were counted. To determine protease positive isolates, the colonies on the gelatin agar medium were flooded with Frazier solution. Then, the colonies with clear zones were considered as protease positive, and these colonies were counted (44, 45). The colonies with opaque zones on Tween® 80 agar were considered as lipase positive and were counted (44). Later, viable cell numbers of these archaeal isolates in 1 ml were calculated. Protease and lipase positive colonies with different colours and morphology were selected and streaked out several times to obtain pure cultures on complex agar medium by the streak plate technique.

2.2 The Electrochemical Disinfection System

An electrochemical cell was constructed of a glass beaker containing two internally attached platinum wire electrodes (1 mm in diameter, 80 mm long) submerged in 25% NaCl solution. The distance separating the two electrodes was 40 mm. These electrodes were attached to a regulated alternating current source (Ruhstrat GmbH, Germany) (input = 220 V, frequency = 50 Hz, power = 2250 VA), which had an automatic variable output voltage range of 0–220 V and user-selectable current range of 0–9 A (35, 46).

In the present study, 2 A alternating electric current treatment was applied to two experimental sets. In the first experimental set, consisting of ten tests, 2 A alternating electric current was applied to salt samples mixed separately into 25% NaCl solutions. In the second experimental set, consisting of four tests, 2 A alternating electric current was applied to a mixed culture of proteolytic extremely halophilic archaea, a mixed culture of lipolytic extremely halophilic archaea, a mixed culture of both proteolytic and lipolytic extremely halophilic archaea, plus a mixed culture of these isolates added to 25% NaCl solutions.

2.3 Inactivation of Extremely Halophilic Archaea

As explained above, for the first experimental set, an electrolysis cell containing 200 ml of liquid test medium, 25% NaCl and separate salt samples was used in this experiment. 2 A alternating electric current was applied to the electrolysis cell for 25 min. Aliquots of 100 μl were removed from this medium at intervals of 1, 5, 10, 15, 20 and 25 min during the alternating current treatment. Then, the aliquots were diluted to 10-fold (10–1) dilution, 100-fold (10–2) dilution and 1000-fold (10–3) dilution in sterile physiological saline solution. 100 μl of the direct and diluted solutions were spread over complex agar medium and incubated at 39°C for 30 days. Then, the colonies on the agar surface were counted. The detection limit of this experiment was 10 CFU ml–1.

2.4 Inactivation of Proteolytic and Lipolytic Extremely Halophilic Archaea

For the second experimental set, protease and lipase positive extremely halophilic archaea were isolated from the salt samples obtained from Tuz Lake. Two isolates of protease positive (TLPI1 and TLPI2), two isolates of lipase positive (TLLI1 and TLLI2), two isolates of both protease and lipase positive (TLPLI1 and TLPLI2) and a mixed culture of these extremely halophilic isolates (TLPI1, TLPI2, TLLI1, TLLI2, TLPLI1 and TLPLI2) were selected as test isolates to use in alternating electric current treatment experiments. Each of these isolates was cultivated for 15 days at 39°C in a shaker incubator (Edmund Bühler, Germany) at 100 rpm in a liquid complex medium. After the incubation period, each of these extremely halophilic archaeal isolates was diluted in 25% NaCl solution to a final population density of 107 CFU ml–1. Next, the aforementioned isolate groups were prepared. 20 ml of protease positive (TLPI1 and TLPI2), lipase positive (TLLI1 and TLLI2), both protease and lipase positive (TLPLI1 and TLPLI2) and the mixed culture (TLPI1, TLPI2, TLLI1, TLLI2, TLPLI1 and TLPLI2) were separately placed into an electrolysis cell containing 180 ml of 25% NaCl solution. Later, 2 A alternating electric current was applied to each electrolysis cell for 25 min. A 100 μl quantity of the test medium was removed from the electrolysis cell at intervals of 1, 5, 10, 15, 20 and 25 min. Each of the test media was plated onto complex agar medium both directly and after serial dilutions, then incubated for 30 days at 39°C. After the incubation period, colonies on the agar surface were counted and CFU of extremely halophilic archaea in 1 ml was calculated. The detection limit of this experiment was 10 CFU ml–1.

The log10 reduction factor (RF) for each treatment time was calculated according to the following formula:

RF = log10 nb – log10 na

where nb is the initial number of viable cells (CFU ml–1) in the inoculum of the first experiment (salt samples dissolved in 200 ml of liquid medium containing 25% NaCl) or in the inoculum of the second experiment (test isolates found in 200 ml of liquid medium containing 25% NaCl); and na is the number of viable cells (CFU ml–1) in the inoculum (salt samples or test isolates) after treatment with 2 A alternating electric current.

The pH and temperature of all samples were measured at each of the aforementioned intervals using a pH meter (Professional Meter PP-50, Sartorius AG, Germany). The temperatures of all test media were adjusted to 25°C before application of alternating electric current treatment.

3. Results and Discussion

Extremely halophilic archaea, proteolytic and lipolytic extremely halophilic archaea were detected in all salt samples (Table I). Although salt samples mostly contained between 103 and 104 CFU of extremely halophilic archaea per gram, proteolytic and lipolytic extremely halophilic archaeal numbers were between 101 and 103 CFU g–1 in salt samples (Table I). These findings were consistent with previous experiments (18, 30). In those studies, 80 salt samples used in hide preservation were examined for extremely halophilic archaea. It was found that all salt samples contained 102–105 CFU g–1 of extremely halophilic archaea (18, 30). Moreover, all salt samples contained proteolytic and lipolytic extremely halophilic archaea (102–104 CFU g–1) (18, 30).

Table I

Numbers, Log10 Values and Reduction Factors of Extremely Halophilic Archaea, Proteolytic and Lipolytic Extremely Halophilic Archaea in Salt Samples Dissolved Separately in the Brine Solutions Treated by 2 A Alternating Electric Current within 1 min

Salt sample Exposure time, min Extremely halophilic archaea Proteolytic extremely halophilic archaea Lipolytic extremely halophilic archaea
Number, CFU ml–1 Log10 values Reduction factor Number, CFU ml–1 Log10 values Reduction factor Number, CFU ml–1 Log10 values Reduction factor
1 0 2.8 × 104 4.4 – 1.9 × 102 2.3 – 3.4 × 102 2.5 –
1 <10 <1 >3.4 <10 <1 >1.3 <10 <1 >1.5
2 0 4.1 × 104 4.6 – 6.8 × 103 3.8 – 9.0 × 103 3.9 –
1 <10 <1 >3.6 <10 <1 >2.8 <10 <1 >2.9
3 0 1.4 × 104 4.1 – 1.3 × 103 3.1 – 1.9 × 103 3.2 –
1 <10 <1 >3.1 <10 <1 >2.1 <10 <1 >2.2
4 0 1.4 × 103 3.1 – 1.0 × 102 2.0 – 4.0 × 102 2.6 –
1 <10 <1 >2.1 <10 <1 >1.0 <10 <1 >1.6
5 0 2.0 × 103 2.3 – 1.6 × 102 2.2 – 9.0 × 102 2.9 –
1 <10 <1 >1.3 <10 <1 >1.2 <10 <1 >1.9
6 0 3.4 × 102 2.5 – 1.0 × 101 1.0 – 2.0 × 101 1.3 –
1 <10 <1 >1.5 <10 <1 –a <10 <1 >0.3
7 0 9.7 × 103 3.9 – 3.4 × 102 2.5 – 6.5 × 102 2.8 –
1 <10 <1 >2.9 <10 <1 >1.5 <10 <1 >1.8
8 0 1.2 × 104 4.0 – 1.1 × 103 3.0 – 2.8 × 103 3.4 –
1 <10 <1 >3.0 <10 <1 >2.0 <10 <1 >2.4
9 0 8.1 × 103 3.9 – 4.0 × 101 1.6 – 1.1 × 102 2.0 –
1 <10 <1 >2.9 <10 <1 >0.6 <10 <1 >1.0
10 0 8.7 × 103 3.9 – 1.0 × 101 1.0 – 1.0 × 102 2.0 –
1 <10 <1 >2.9 <10 <1 –a <10 <1 >1.0

aThe number was not detected because it was under the detection limit

In this study, two sets of experiments were conducted to examine the inactivation effect of alternating electric current on extremely halophilic archaea. In the first experiment, extremely halophilic archaea, proteolytic and lipolytic extremely halophilic archaea in salt samples were killed in 1 min via 2 A alternating electric current treatment. There was a sharp decrease in the numbers of extremely halophilic archaea, proteolytic and lipolytic extremely halophilic archaea in the salt samples after 1 min of 2 A alternating electric current treatment (Table I).

Temperature values of the test media did not change when the extremely halophilic archaea in the salt samples were killed. The temperature of all test media containing salt samples was 25°C before the experiment and remained the same after 1 min of electric treatment. At the end of experiment (after 25 min), the temperature of these media reached 35°C. The pH of the test media did not change during the experiment. Voltage levels of the test media containing the salt samples varied according to the samples and the duration of electric current treatment. However, all voltage levels decreased at the end of these experiments (Table II).

Table II

Values of Temperature, Voltage and pH in the Liquid Test Media Containing Salt Samples Treated by 2 A Alternating Electric Current Over 25 min

Salt sample 1 2 3 4 5
Exposure time, min °Cb Vc pH °C V pH °C V pH °C V pH °C V pH
0 25 – 7.57 25 – 7.73 25 – 7.52 25 – 7.09 25 – 8.03
1 25 4.9 7.57 25 5.0 7.73 25 5.0 7.52 25 7.0 7.09 25 7.0 8.03
5 27 4.9 7.57 27 4.9 7.73 27 4.8 7.52 27 6.9 7.09 27 6.9 8.03
10 29 4.8 7.57 29 4.8 7.73 29 4.7 7.52 29 6.8 7.09 29 6.8 8.03
15 31 4.7 7.57 31 4.7 7.73 31 4.6 7.52 31 6.8 7.09 31 6.7 8.03
20 33 4.6 7.57 33 4.6 7.73 33 4.6 7.52 33 6.6 7.09 33 6.6 8.03
25 35 4.6 7.57 35 4.5 7.73 35 4.5 7.52 35 6.1 7.09 35 6.4 8.03

bTemperature, cVoltage

Table II Continued

Salt sample 6 7 8 9 10
Exposure time, min °C V pH °C V pH °C V pH °C V pH °C V pH
0 25 – 7.35 25 – 7.74 25 – 8.24 25 – 7.44 25 – 7.40
1 25 7.7 7.35 25 7.6 7.74 25 7.5 8.24 25 5.8 7.44 25 5.8 7.40
5 27 7.5 7.35 27 7.4 7.74 27 7.3 8.24 27 5.7 7.44 27 5.7 7.40
10 29 7.3 7.35 29 7.2 7.74 29 7.2 8.24 29 5.6 7.44 29 5.5 7.40
15 31 7.0 7.35 31 7.0 7.74 31 7.0 8.24 31 5.5 7.44 31 5.2 7.40
20 33 6.8 7.35 33 6.8 7.74 33 6.8 8.24 33 5.2 7.44 33 5.1 7.40
25 35 6.6 7.35 35 6.6 7.74 35 6.7 8.24 35 5.1 7.44 35 5.0 7.40

The inactivation effect of alternating electric current on Gram-negative bacteria has been examined in previous studies (34–36, 47, 48). E. coli cells in the test medium containing nutrient broth, peptone and NaCl were killed by 110 mA alternating electric current in 10 s (36). E. coli cells suspended in a phosphate buffer solution were also killed by 300 mA cm–2 alternating electric current (34). V. parahaemolyticus in effluent seawater was killed by 3 A alternating electric current treatment in 30 ms (35). In an earlier study, both E. coli ATCC 25922 (resistant to ampicillin (10 μg ml–1) and amoxicillin-clavulanic acid (30 μg ml–1)) and fecal E. coli MAAG 1405 (resistant to ampicillin (10 μg ml–1) and amoxicillin-clavulanic acid (30 μg ml–1), imipenem (10 μg ml–1), cefuroxime (30 μg ml–1), cefotaxime (30 μg ml–1) were killed within 1–3 min in media containing marine waters (1 A), 5–10 min in Ayamama River water (1 A), 5 min in Sarisu River water (0.5 A) and 20–35 min in lauryl sulfate broth (1 A) by applying alternating electric current. Log10 reduction factors of E. coli ATCC 25922 and fecal E. coli MAAG 1405 were between 3.47 and 3.99 in the different water samples when these bacteria were completely inactivated. The pH of all water samples was between 6 and 7 and remained constant during alternating current treatment (47). Furthermore, in another study 1.5 A alternating electric current was used to kill Enterobacter cloacae, Pseudomonas luteola and Vibrio fluvialis, as well as a mixed population of these Gram-negative bacteria isolated from hides. 15 min exposure to 1.5 A alternating electric current inactivated Enterobacter cloacae, Pseudomonas luteola and Vibrio fluvialis in the brine solution containing 25% NaCl. Moreover, exposure to 1.5 A alternating electric current for 15 min followed by 1.5 A direct current for 1 min inactivated the mixed population of these bacteria in the brine solution. The maximum temperature rise was 6°C (48).

In the second experiment, the mixed culture of protease positive isolates (TLPI1 and TLPI2), mixed culture of lipase positive isolates (TLLI1 and TLLI2), mixed culture of both proteolytic and lipolytic isolates (TLPLI1 and TLPLI2) and mixed culture of all test isolates (TLPI1, TLPI2, TLLI1, TLLI2, TLPLI1 and TLPLI2) were prepared as four different treatment groups. In our previous study we detected proteolytic and lipolytic extremely halophilic archaea between 102–106 CFU g–1 in 36 salted hides (18). Hence, high populations of proteolytic and lipolytic extremely halophilic archaeal isolates (106 CFU ml–1) were used to examine the inactivation effect of alternating electric current. 2 A alternating electric current was applied to the mixed cultures of these isolates in the test media containing 25% NaCl for 1, 5, 10, 15, 20 and 25 minutes.

As indicated in Table III, the archaeal cell counts in all test media also decreased during the electric treatments. The mixed culture of protease positive isolates dramatically decreased during 1 min of electric treatment. Likewise, lipase positive isolates, both proteolytic and lipolytic isolates, and the mixed culture of all isolates also decreased sharply. After 1 min alternating electric treatment, the numbers of protease positive isolates, the numbers of lipase positive isolates, the numbers of both proteolytic and lipolytic isolates, and mixed culture of all isolates decreased 1.0 log10, 3.5 log10, 2.1 log10, and 3.2 log10, respectively. In this experiment, within 5 min of 2 A alternating electric current application, all archaeal cells were annihilated in each treatment (Table III).

Table III

Values of Temperature, Voltage and pH, Total Counts, Log10 Values and Reduction Factors of the Test Isolates in the Brine Media Treated by 2 A Alternating Electric Current Over 25 min

Exposure time, min Mixed culture of proteolytic extremely halophilic isolates (TLPI1 and TLPI2) Mixed culture of extremely halophilic lipolytic isolates (TLLI1 and TLLI2)
°C V pH Number, CFU ml–1 Log10 values Reduction factor °C V pH Number, CFU ml–1 Log10 values Reduction factor
0 25 – 7.0 6.3 × 106 6.8 – 25 – 7.0 5.9 × 106 6.8 –
1 25 5.6 7.0 1.0 × 101 1 5.8 25 5.8 7.0 3.5 × 103 3.5 3.3
5 27 5.3 7.0 <10 <1 >5.8 27 5.7 7.0 <10 <1 >5.8
10 29 5.1 7.0 <10 <1 >5.8 29 5.4 7.0 <10 <10 >5.8
15 31 4.9 7.0 <10 <1 >5.8 31 5.2 7.0 <10 <10 >5.8
20 33 4.7 7.0 <10 <1 >5.8 33 5.1 7.0 <10 <10 >5.8
25 35 4.6 7.0 <10 <1 >5.8 35 5.0 7.0 <10 <10 >5.8

Table III Continued

Exposure time, min Mixed culture of extremely halophilic both proteolytic and lipolytic
isolates (TLPLI1 and TLPLI2)		 Mixed culture of all extremely halophilic archaeal isolates (TLPI1, TLPI2,
TLLI1, TLLI2, TLPLI1 and TLPLI2)
°C V pH Number, CFU ml–1 Log10 values Reduction factor °C V pH Number, CFU ml–1 Log10 values Reduction factor
0 25 – 7.0 5.7 × 106 6.7 – 25 – 7.0 4.9 × 106 6.7 –
1 25 5.9 7.0 1.2 × 102 2.1 4.6 25 5.7 7.0 1.6 × 103 3.2 3.5
5 27 5.8 7.0 <10 <1 >5.7 27 5.6 7.0 <10 <1 >5.7
10 29 5.5 7.0 <10 <1 >5.7 29 5.4 7.0 <10 <1 >5.7
15 31 5.3 7.0 <10 <1 >5.7 31 5.2 7.0 <10 <1 >5.7
20 33 5.2 7.0 <10 <1 >5.7 33 5.1 7.0 <10 <1 >5.7
25 35 5.1 7.0 <10 <1 >5.7 35 5.0 7.0 <10 <1 >5.7

In the second experiment set, temperature values of the test media increased slightly when the test isolates were inactivated. All voltage levels decreased slightly when the test isolates were inactivated. At the end of the experiment, temperature values and voltage levels of the test media were measured respectively at 35°C and between 4.6–5.1 V (Table III).

4. Recommendations

The authors of this study highly recommend this easy, effective, economical, fast, broad-spectrum activity system to prevent archaeal damage on brine cured hides. Although there are a few studies which explain the mechanism of bacterial inactivation by alternating electric current (34–38, 41–43), to our knowledge there is no study of the mechanism of haloarchaeal inactivation by alternating electric current. The mechanism of haloarchaeal inactivation by alternating electric current requires elucidation. This will necessitate additional studies for determination of disinfection byproducts arising after alternating electric current treatment. Also, the morphological and physiological effects of the treatment on the cells should be examined in order to clarify the inactivation mechanism of this process. With improved understanding of the uses of alternating electric treatment for haloarchaeal inactivation, this cheap and practical application is expected to prove itself useful as science and as a boon to the leather industry.The knowledge gap will be reduced further as the mechanisms of haloarchaeal inactivation by alternating electric current are analysed in forthcoming experiments.

5. Conclusions

The experimental results of this study proved that using 2 A alternating current may effectively kill extremely halophilic archaea, proteolytic extremely halophilic archaea and lipolytic extremely halophilic archaea in a time period between 1 min and 5 min. The extremely halophilic archaeal population in salt samples was killed in 1 min, and the extremely halophilic archaeal isolates were killed in 5 min. This alternating current treatment was found to be fairly effective in killing extremely halophilic archaea in salt. In conclusion, this cheaper and simpler inactivation treatment system compared to antimicrobial agent applications may be used in hide brine solutions to destroy proteolytic and lipolytic extremely halophilic archaea.

Acknowledgements

We thank Dr Martin Louis Duncan, Department of Translation and Interpreting (English Language), Marmara University, for editing this manuscript.

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The Authors

Yasar Birbir received his BS degree from Gazi University, Turkey, and MS and PhD from Marmara University, Turkey. He attended the World Bank Industrial Training Project at Indiana and Purdue Universities, USA, from 1989 to 1990. He worked as a visiting research scientist for fifteen months at the Electrical and Computer Engineering Department of Drexel University, USA, from 1992 to 1993. Currently he is an Associate Professor at the Technology Faculty in the Department of Electrical Engineering at Marmara University. His current interests are power electronic converters and drivers, electromagnetic filtering processes in industry and the application of electric currents and electric field effects for sterilisation of different microorganisms. He has published 24 research articles and graduated 18 masters and one doctoral student. He has presented 39 oral and poster presentations in national and international congress and has completed seven scientific projects.

Sema Anik graduated from the Departments of Biology and Chemistry, Faculty of Arts and Sciences, Marmara University, in 2010. She received her MSc degree in Biology in 2014 from the Institute for Graduate Studies in Pure and Applied Science, Marmara University. She then worked as a researcher in a clinical microbiology laboratory. She has also worked as a biology teacher in a private high school. Her research experience is in controlling the growth of extremely halophilic archaea using electric current applications. She has presented two poster presentations in international congresses.

Meral Birbir received a bachelor's degree in Biology Education in 1985, MS and PhD degrees in 1987 and 1991, respectively from the Department of Biology, Marmara University. She has been working in the Biology Department of Marmara University since 1985. She was a visiting research scientist at the Department of Pathology and Microbiology, Veterinary Medical School, Purdue University, USA, in 1990. She was a research scientist in the Hides and Leather Department of the United States Department of Agriculture (USDA) from 1992 to 1993. Her research interests are in halophilic bacteria, hide microbiology, food microbiology, antimicrobial agents, electric current application and microbiology of hypersaline environments. Professor Birbir especially focuses on halophilic and non-halophilic bacteria that live on salted hides and their control with antimicrobials or electric current. She has published 61 research articles and graduated 32 masters and one doctoral student. She has presented 50 oral and poster presentations in national and international congresses and has completed 25 scientific projects.

Pinar Caglayan graduated from the Department of Biology, Ataturk Faculty of Education, Marmara University, in 2007. She received her MSc degree in Biology in 2010 from the Institute of Pure and Applied Sciences, Marmara University. She was an Erasmus student in the Department of Microbiology and Parasitology, Faculty of Pharmacy, Sevilla University, Spain, from 2008 to 2009. Pinar Caglayan is currently a graduate student (PhD) at Marmara University, Department of Biology. She has been working as a research and teaching assistant at the Division of Plant Diseases and Microbiology, Department of Biology, Faculty of Science and Letters, Marmara University since 2011. Her research interests are moderately halophilic bacteria, extremely halophilic archaea, antimicrobial agents, electric currents and hide microbiology. She has published five research articles and presented 17 poster presentations at international congresses.

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