Journal of Electrostatics 66 (2008) 388–394

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Journal of Electrostatics
journal homepage: www.elsevier.com/locate/elstat

Direct electric current utilization in destruction of extremely

halophilic bacteria in salt that is used in brine curing of hides
Yasar Birbir a, Derya Degirmenci b, Meral Birbir c, *
a _
Technical Education Faculty, Department of Electrical Education, Marmara University, Göztepe, Istanbul 34722, Turkey
b _
Institute for Graduate Studies in Pure and Applied Sciences, Marmara University, Göztepe, Istanbul 34722, Turkey
c _
Science and Letter Faculty, Department of Biology, Marmara University, Göztepe, Istanbul 34722, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: Hydrolytic enzymes, which are produced by extremely halophilic bacteria in salt, may cause serious
Received 17 April 2006 damage on salted hides and may result in significant economic losses. Therefore, to prevent halobacterial
Received in revised form 5 March 2008 damage on hides, the antibacterial effects of different levels of direct electric current on extremely
Accepted 21 March 2008
halophilic bacteria found in salt were examined in brine solution containing 20% NaCl and salt samples,
Available online 8 May 2008
liquid Brown media containing 20% NaCl, organic substances and salt samples, liquid Brown media
separately inoculated with lipase, and protease producing extremely halophilic strains. Direct electric
Keywords:
current (0.1 A, 0.2 A, 0.3 A and 0.4 A) inactivated extremely halophilic bacteria in salt, which was dis-
Low-level direct electric current
Extremely halophilic bacteria
solved in the brine solution within 15 min, 10 min, 5 min and 3 min, respectively. Although 0.5 A direct
Salt electric current inactivated extremely halophilic bacteria in the salt samples (104–105 CFU/g), which were
Hide industry dissolved in the brine solution within 1 min but treatment with 0.5 A direct electric current for 15 min
was necessary to inactivate the extremely halophilic bacterial population in the salt samples dissolved in
the liquid Brown media. Also, 0.5 A direct electric current inactivated lipase and protease producing
extremely halophilic bacteria (105–106 CFU/mL), which were grown separately in the liquid Brown media
within 10 min. Voltage levels; 2.86–3.38 V did not change in all the brine solutions containing salt
samples during electrical treatment, but the voltage levels in all the liquid Brown media containing both
salt samples and protease-plus-lipase producing extremely halophilic bacteria decreased proportionally
to the halobacterial population until the entire halobacterial population was inactivated. When the
halobacterial population in the liquid Brown media was completely destroyed by the direct electric
current, the voltage levels started to increase. This important clue might be used to predict inactivation
time of all of the halobacterial populations grown in the brine solutions containing organic substances in
the hide industry. It was also detected that the temperatures and pH values of the test media changed
during the treatment. The maximum temperature rise was 9  C and the pH increased by 5. These results
showed that applying a 0.5 A direct current is a very effective method to kill extremely halophilic bac-
terial populations in salt used in hide preservation.
Ó 2008 Elsevier B.V. All rights reserved.

1. Introduction
Microorganisms found on animal skins or hides are derived
from many sources, such as air, water, soil, manure, and extraneous
filth. While the animal is alive, most of these organisms have little
effect on the skin. But after the animal has been flayed, these or-
ganisms find themselves in a perfect medium for growth and al-
most immediately start multiplying at an excessively enormous
rate [1]. One of the most important factors affecting leather quality
is microbial spoilage [2,3]. Bacteria require moisture for active
* Corresponding author.
E-mail address: mbirbir@marmara.edu.tr (M. Birbir).
growth and one of the simplest ways of controlling them is to re-
duce the moisture content of the hides or skins. Air drying, salt pack
curing, and brine curing are all methods by which to reduce the
moisture content of the hides. These methods are used in the hide
industry to prevent microbial growth and damage to the hide
during their storage and shipment [2,4,5]. However, halophilic
bacteria found in salt can contaminate raw hides and skins during
the salting process. Earlier works done on extremely halophilic
Archaea demonstrated that these microorganisms were almost
universally present on brine-cured hides processed in different
countries [6–8].
It has been reported that the majority of bacteria isolated from
skins and hides have proteolytic and lipolytic activities [2]. Under
unsuitable transportation and storage conditions, these

0304-3886/$ – see front matter Ó 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.elstat.2008.03.004
 Y. Birbir et al. / Journal of Electrostatics 66 (2008) 388–394 389

microorganisms can grow on hides and skins and subsequently Table 1

begin the degenerative process [9,10]. Halophilic Archaea can cause Brown medium

discoloration of the flesh side of the skin or hide. This condition is Sodium chloride (g) 250.0
referred to as ‘‘red heat’’ in the hide industry. This discoloration is Yeast extract (g) 5.0
due to the massive growth of halophilic Archaea, which produce Sodium citrate (g) 3.0
Magnesium sulfate$7H2O (g) 20.0
red to orange colored carotenoids called bacterioruberins and Potassium chloride (g) 2.0
inducible pigments involved in energy metabolisms [11]. In addi- Distilled water (mL) 1000.0
tion, hair slips or pinpricks can be seen on hides that have been
inadequately salt packed or brine-cured. Proteolytic halophilic
bacteria in salt may grow in the hair follicles of the hide and cause described procedures. Proteolytic and lipolytic activities were
the degradation of the entire follicle leaving a hole in the grain [2]. detected at the media containing 2% gelatin (w/v) and 1% Tween 80
Furthermore, microorganisms grown on hides may cause uneven (v/v), respectively [6,27].
dyeing in leathers [12].
Researchers stated that the source of halophilic bacterial
2.2. Electrical treatment
contamination on brine-cured hides was the salt obtained from
natural salterns or salt lakes [8]. In Türkiye salt, which is used in
The electrolysis cell used was a glass beaker having 2 internally
hide preservation, is obtained from natural salt sources, such as salt
attached platinum wire electrodes immersed in 200 mL of the brine
lakes, salterns of lake, and sea or salt mines. A survey of 94 halo-
solution. The 2 electrodes were 1 mm in diameter and 80 mm in
philic strains from Tuz Lake, Kaldırım saltern, Kayacık saltern, and
length, at a separation of 40 mm. The electrodes were connected to
Tuzköy salt mine showed that most of these strains were able to
a regulated dc current source (Kenwood, PD56-10AD, Japan), which
digest collagen and lipid [13]. If the unprocessed salt is used directly
had an automatic variable output voltage range of 0–60 V and user-
in the brine curing of the hides, these strains will digest the colla-
selectable current range of 0–6 A (Fig. 1)[14,28]. Each test was
gen of the hide and lower the value of the leather. Therefore, before
repeated 2 times using 200 mL of the brine solutions. Different
using the salt in hide preservation, especially proteolytic and li-
direct electric current levels, extremely halophilic bacterial species,
polytic extremely halophilic bacterial strains should be inactivated
and test solutions were used in this experiment to compare
to prevent hide damage.
inactivation time of extremely halophilic bacteria in salt samples.
Many methods, such as heat sterilization, radiation sterilization,
Hence, 10 salt samples, which were dissolved in the sterile brine
filter sterilization, treatment with antimicrobial agents [11], and
solution containing 20% NaCl, were treated by 5 different direct
electrical treatments [14–16] are used to reduce bacterial contam-
electric current levels (0.1 A, 0.2 A, 0.3 A, 0.4 A and 0.5 A) within
ination in many industrial applications. Microorganisms found in
20 min. In addition, to examine the antibacterial effects of direct
seawater [14], milk [15–18], synthetic urine [19], salt solutions [18],
electric currents on different extremely halophilic bacterial
on human skin [20], on the surface of catheters [21], in activated
populations in salt samples, 40 salt samples dissolved in the sterile
sludge and biofilms [22], in effluent seawater [23], and recirculated
brine solution containing 20% NaCl were treated by a 0.5 A direct
brine [24] were inactivated by different levels of electric current.
electric current within 20 min. Also, the antibacterial effect of
While there are many studies on the inactivation of different
a 0.5 A direct electric current in the presence of an organic
bacterial species by electric currents in a variety of applications, the
substance was examined by using 5 salt samples dissolved in
antibacterial effect of low-level electric current on extremely hal-
Brown media containing 20% NaCl for6 h at 25  C. Furthermore, 2-
ophilic bacteria, in salt dissolved in brine solution containing 20%
lipase and 2-protease producing extremely halobacterial strains,
NaCl or organic substances, has not been reported yet. The present
grown separately in liquid Brown media containing 20% NaCl for 7
study was conducted in order to assess the total extremely halo-
days at 40  C, were treated by a 0.5 A direct electric current within
philic bacterial population, proteolytic, and the lipolytic extremely
20 min. A 100 mL quantity of the test solutions was removed from
halophilic bacterial populations in salt, which are used in the hide
the electrolysis cell at intervals of 1 min, 3 min, 5 min, 10 min,
industry and to inactivate these microorganisms by different levels
15 min and 20 min. Each of the test solutions was plated onto
of direct electric current.
Brown media, both directly and after serial dilutions. The CFU of the
extremely halophilic bacteria were counted after 2 months of
2. Experimental methods
incubation at 40  C. The Log10-reduction factor (RF) for each
treatment time was calculated according to the following formula:
2.1. Microbiological analyses
RF ¼ Log10 nc  Log10 nd
A total of 40 salt samples, which are used in hide preservation,
_
were collected from different tanneries in the Tuzla–Istanbul where nc is the initial number of viable cells (CFU) in the inoculums
Leather Organized Tannery Region in Türkiye. The moisture content in the test solutions and nd is the number of viable cells (CFU) in the
of all of the salt samples was determined as described earlier [25]. inoculums after treatment with direct electric currents.
Twenty grams of salt were placed in a flask containing 180 mL of The pH and temperature values of all salt samples were
20% NaCl containing sterile saline solution that was then placed in determined as described earlier at each of the above mentioned
a shaking incubator for 4 h at 25  C. Spread plate technique was intervals using a pH meter [25]. Range, mode, mean and median
used to determine the total numbers of extremely halophilic values of voltage, temperature, and RF of all of the bacteria were
bacteria, proteolytic, and lipolytic extremely halophilic bacteria in calculated according to standard statistical methods [29]. All direct
the salt samples [26]. A 0.1 mL of direct salt solution and serial electric current treatments in this work were conducted at room
dilutions of the solution were spread onto the surface of Brown temperature, and all of the temperatures of the test solutions were
media containing 25% NaCl (Table 1). After 6 weeks of incubation at 20  C before the treatment.
40  C, colonies were counted. All experiments were duplicated. The
pH and temperature of all salt solutions were measured as de- 3. Results and discussion
scribed earlier using a pH meter (Sartorius Professional Meter PT-10
P, Goettingen, Germany) [10]. Proteolytic and lipolytic activities of The moisture contents of salt samples were low, in the range of
extremely halophilic bacteria were tested using previously 0.55–4.97%, and changed according to the samples (Table 2). The
390 Y. Birbir et al. / Journal of Electrostatics 66 (2008) 388–394

Fig. 1. Schematic diagram of electrolysis cell system for brine solutions.

moisture content of 35% and 62.5% of the salt samples were 0.55–
1.75% and 2.24–3.94%, respectively. The moisture content of only 1
salt sample was 4.97%. These results agreed with previous research
results. The moisture contents of the salt samples obtained from
Tuz Lake, Kaldırım saltern, Kayacık saltern, and Tuzköy salt mine
were 0.32–7.56, 0.78–3.29, 0.49–1.54 and 0.068–0.72, respectively
[25,30,31].
In this study, the pH values of all of the salt samples (pH 6) were
lower than the other salt samples examined in our previous studies.
The pH values of salt samples obtained from Tuz Lake, Kaldırım
saltern, Kayacık saltern, and Tuzköy salt mine were 8.09–8.43,
8.35–8.76, 8.21–8.72 and 8.00–8.29, respectively [25,30,31].
Mean value of the total extremely halophilic bacteria in 40 salt
samples was found as 8.3  104 CFU/g (Table 2). Our previous re-
search results confirmed these results also. The salt taken from the
Tuz Lake, Kaldırım saltern, Kayacık saltern, and Tuzköy salt mine in
Türkiye contained 104–107 CFU, 105–107 CFU, 105 CFU, and 105–
106 CFU of extremely halophilic bacteria per gram, respectively
[25,30,31]. These experiments indicated that the salt samples
contained a significant number of extremely halophilic bacteria.
Proteolytic and lipolytic extremely halophilic bacterial numbers
in these salt samples were commonly between 102 CFU/g and
103 CFU/g. While 7.5% of the salt samples contained 104 CFU of
proteolytic extremely halophilic bacteria per gram, 17.5% salt
samples contained 104 CFU of lipolytic extremely halophilic bac-
teria per gram. Mean values of proteolytic and lipolytic extremely
bacteria in 40 salt samples were found as 2.79  103 CFU/g and
8.95  103 CFU/g, respectively (Table 2).
Fifty, 20, 9, and 15 extremely halophilic bacteria were isolated
from Tuz Lake, Kaldırım saltern, Kayacık saltern, and Tuzköy salt
mine, respectively. Eighty percent and 90% of Tuz Lake strains, 70%
and 35% of Kaldırım saltern strains, 78% and 33% of Kayacık saltern
Table 2
Range, mode, mean and median values of moisture contents, total extremely halo-
philic, proteolytic extremely halophilic, and lipolytic extremely halophilic bacterial
numbers in 40 salt samples
Moisture Extremely Proteolytic
content (%) halophilic extremely
bacteria halophilic
bacteria
Range 4.42 5.99  105 3.69  104
Mode 0.89 1  104 1  102
2.48 2  104
Mean 2.44 8.32  104 2.79  103
Median 2.48 3.5  104 2.5  102
strains, and 67% and 100% of Tuzköy salt mine strains showed
proteolytic and lipolytic activities [13].
Extremely halophilic Archaeal numbers in salt may increase
when they find an ideal environment for growth. Salted hides
containing blood, dirt, and dung are an ideal growth medium for
halophiles. In our earlier research on halophiles we showed that
brine-cured hides had 105–108 CFU of extremely halophilic bacteria
per gram [6].
One hundred and thirty one brine-cured hides processed in the
United States were tested for extremely halophilic bacteria and 98%
of them contained these microorganisms. A total of 332 extremely
halophilic bacteria strains were isolated, and 94% of these strains
were protease positive [6]. Moreover, 35 salt-cured French and
Russian hides were tested for extremely halophilic microorgan-
isms, and 91% of them contained these microorganisms [7]. From
these hides, 85 extremely halophilic strains were isolated and 67%
of the strains were protease positive.
In Ref. [12] researchers found that salt-cured sheepskins con-
tained 106–107 CFU of extremely halophilic bacteria per gram.
While 53–74% of extremely halophilic bacteria showed proteolytic
activity, 47–62% of extremely halophilic bacteria showed lipolytic
activity.
If the hide is treated with salt as free as possible from extremely
halophilic bacteria and is stored and transported at cold tempera-
tures, the halophilic bacteria left in the salt will not matter. How-
ever, if the temperature of the hide store rises too much or if the
atmosphere gets too moist, the growth of extremely halophilic
bacteria will be encouraged.
When the salt containing proteolytic extremely halophilic
bacteria are used in hide preservation, these microorganisms can
potentially digest the grain surface of the hide under extended
storage conditions at elevated temperatures [3]. These lower the
value of the leather and represent a huge loss to the Turkish leather
industry. It was demonstrated that extremely halophilic Archaea
damaged the grain of brine-cured hide within 7 weeks at a tem-
perature of 41  C. This damage was easily observed by the naked
eye, and scanning electron microscopy clearly showed that the
damage done by halophilic microorganisms resembled sueded
grain [3]. Furthermore, researchers mentioned that defects on the
Lipolytic appearance, such as that of light stains have been found on
extremely
halophilic
the suede surface of finished double-face leathers as a result of
bacteria the microorganisms’ activities on salted sheepskins, and that these
8.3  104 defects correspond in proportion to 35% of the total double-face
1  102 amount [12].
In addition, researchers [32] followed the sequence of events
8.95  103 leading to the appearance of red heat on salt-cured hides within 2 h
1.55  103
exposure. Scanning electron microscopy study showed that
Table 3
Range, mode, mean and median values of temperature, voltage, pH of electrical treatment systems, and RF of inactivated extremely halophilic bacteria in 10 salt samples dissolved separately in the brine solution containing 20% NaCl
by different levels of electric current within 20 min

Treatment time (min) 0.1 A RF 4.56b 0.2 A RF 4.56 0.3 A RF 4.56 0.4 A RF 4.56 0.5 A RF 4.56
a     
BE 20 C Voltage pH 6 20 C Voltage pH 6 20 C Voltage pH 6 20 C Voltage pH 6 20 C Voltage pH 6
1 Range 0 0.30 1 1.45 0 0.1 5 2.95 0.5 0.3 5 1.99 1 0.6 2 4.15 1 0.2 2 2.36
Mode 20 2.70 11 0.37 20 3.0 11 – 20 3.0 10 1.37 20.5 3.4 11 – 20 0.3 9 4.00
2.90 3.1 0.4
3.00
Mean 20 2.86 11 0.97 20 3.01 9.9 1.34 20.15 3.09 9.5 2.09 20.4 3.38 10.4 3.69 20.4 3.38 9.3 4.56
Median 20 2.9 11 0.92 20 3.0 10.5 1.24 20 3.1 10 2.06 20.5 3.4 11 3.53 20 3.4 9 4.49

3 Range 0.5 0.3 1 2.4 0 0.1 5 4.67 0.5 0.3 5 2.51 1 0.6 2 2.36 1 0.2 2 2.36

Y. Birbir et al. / Journal of Electrostatics 66 (2008) 388–394

Mode 20 2.70 11 – 20 3.0 11 – 20 3.0 10 3.21 21 3.4 11 4.00 21 0.3 9 4.00
2.90 3.1 4.0 0.4
3.00
Mean 20.2 2.86 11 1.57 20 3.01 9.9 3.01 20.15 3.09 9.5 4.12 20.7 3.38 10.4 4.56 20.65 3.38 9.3 4.56
Median 20 2.9 11 1.84 20 3.0 10.5 2.73 20 3.1 10 4.0 21 3.4 11 4.49 21 3.4 9 4.49

5 Range 1 0.3 1 4.55 0.5 0.1 5 4.37 1 0.3 5 2.36 1.5 0.6 2 2.36 2 0.2 2 2.36
Mode 20.5 2.70 11 – 20 3.0 11 – 20.5 3.0 10 4.00 21 3.4 11 4.00 21 0.3 9 4.00
2.90 20.5 21.0 3.1 0.4
3.00
Mean 20.45 2.86 11 2.96 20.25 3.01 9.9 3.66 20.6 3.09 9.5 4.56 20.75 3.38 10.4 4.56 20.75 3.38 9.3 4.56
Median 20.5 2.9 11 3.0 20.25 3.0 10.5 3.69 20.5 3.1 10 4.49 21 3.4 11 4.49 21 3.4 9 4.49

10 Range 1 0.3 1 4.07 0 0.1 5 2.36 1.5 0.3 5 2.36 1.5 0.6 2 2.36 1.5 0.2 2 2.36
Mode 20.5 2.70 11 4.0 20 3.0 11 4.0 20.5 3.0 10 4.00 21.5 3.4 11 4.00 22 0.3 9 4.00
2.90 21 3.1 0.4
3.00
Mean 20.45 2.86 11 4.09 20 3.01 9.9 4.56 20.75 3.09 9.5 4.56 21.25 3.38 10.4 4.56 21.55 3.38 9.3 4.56
Median 20.5 2.9 11 4.06 20 3.0 10.5 4.49 20.75 3.1 10 4.49 21.5 3.4 11 4.49 22 3.4 9 4.49

15 Range 1 0.3 1 2.36 1 0.1 5 2.36 1.5 0.3 5 2.36 2.5 0.6 2 2.36 2 0.2 2 2.36
Mode 20.5 2.70 11 4.00 20.5 3.0 11 4.0 21 3.0 10 4.00 22 3.4 11 4.00 23 0.3 9 4.00
2.90 3.1 0.4
3.00
Mean 20.5 2.86 11 4.56 20.35 3.01 9.9 4.56 21 3.09 9.5 4.56 21.6 3.38 10.4 4.56 22.3 3.38 9.3 4.56
Median 20.5 2.9 11 4.49 20.5 3.0 10.5 4.49 21 3.1 10 4.49 21.75 3.4 11 4.49 22.5 3.4 9 4.49

20 Range 1 0.3 1 2.36 1 0.1 2 2.36 1 0.3 5 2.36 1.5 0.6 2 2.36 2 0.2 2 2.36
Mode 20.5 2.70 11 4.00 21 3.0 11 4.0 21 3.0 10 4.00 22.5 3.4 11 4.00 24 0.3 9 4.00
2.90 3.1 0.4
3.00
Mean 20.6 2.86 11 4.56 20.75 3.01 10.2 4.56 21.15 3.09 9.5 4.56 22.3 3.38 10.4 4.56 23.5 3.38 9.3 4.56
Median 20.5 2.9 11 4.49 21 3.0 10.5 4.49 21 3.1 10 4.49 22.5 3.4 11 4.49 24 3.4 9 4.49
a
BE: before experiment.
b
Mean of Log10 values of total extremely halophilic bacteria in the brine solutions before the electric treatment.

391
392 Y. Birbir et al. / Journal of Electrostatics 66 (2008) 388–394

damage began at the hide surface before the red heat became current within 1 min but long exposure time (15 min) of electric
visible. They also noticed that the bacteria moved down the hair treatment was necessary to inactivate extremely halophilic bacteria
follicles to attack underlying dermal layers. Damage to underlying in 5 salt samples dissolved in the presence of organic substances
dermal layers became apparent in 3 weeks. It was also mentioned (Table 4). These results were similar to our previous experiment
that halophilic bacteria caused a complete disruption of collagen results [28]. Our previous study on inactivation of halophiles with
fibers and production of sponge like vesicles within the hide. direct electric current showed that 0.5 A low-level direct electric
Considerable attempts have been made to use bactericides current inactivated both lipase and lipase-plus-protease producing
during the brine curing of hides [33–35]. Effective bactericides extremely halophilic bacteria of 107 CFU/mL in presence of organic
[9,35–37], bile salts [38], and natural antimicrobial agent, such as substances within 20 min. While a mixed culture of extremely
halocins produced by extremely halophilic Archaea [39] have been halophilic bacteria was inactivated within 10 min, protease
recommended to prevent halobacterial damage during the brine producing extremely halophilic strains was inactivated within
curing of hides. However, in recent years, the uses of bactericides 5 min [28].
have been questioned, due to their toxicity and bacterial resistance The experiment results showed that when the stored salt is used
with repeated use. In addition, biocides are usually oil emulsions, directly in the brine curing of hides, 1 min exposure 0.5 A direct
which break down in highly polar salt brines, thus becoming electric treatment is enough to kill all extremely halophilic bacterial
uncontrolled applications [4]. Hence, a much more cheap, effective populations in the brine solution. But some countries utilize used
and compatible control system is necessary to inactivate extremely salt, which was swept from hides or skins, and sometimes used salt
halophilic bacteria in brine solutions that will be used in hide is mixed with fresh salt. It must be recognized that used salt may
preservation. contain organic substances and high number of extremely halo-
Direct electric current has been used in many studies to philic bacteria. Direct electric current may be used to kill high
inactivate different species of microorganisms. Nitrifying bacteria populations of extremely halophilic bacteria found in used salt.
in activated sludge and biofilms, consisting of bacteria immobilized Hence, in this experiment, the effect of 0.5 A direct electric current
on polypropylene packing were inactivated by 2.5 A/m2 and 5 A/m2 on lipase and protease producing extremely halophilic bacteria
direct electric current, respectively [22]. (105–106 CFU/mL) grown separately in liquid Brown medium were
In Ref. [40], low densities of both sulfur-oxidizing bacteria (a examined. It was found that the inactivation time of these test
mixed culture) and pure culture of Thiobacillus ferrooxidans, as well strains were the same, which was 10 min (Table 5). If used salt
as heterotrophs (e.g., the acidophilic heterotroph Acidiphilium SJH) containing organic substances and high population of different
were inactivated by direct electric current densities of 200 A/m2 in extremely halophilic bacteria (107 CFU/mL) will be used again in
liquid cultures. brine curing of hide, this brine solution should be treated by 0.5 A
In Ref. [41], suspensions of Escherichia coli, Pseudomonas aeru- direct electric current for 15–20 min to inactivate all of the
ginosa, bacteriophage MS2, and PRD1 at both high (approximately extremely halophilic bacterial contents in it.
1 106 CFU or PFU/mL) and low (approximately 1 103 CFU or PFU/
mL) population densities were treated with currents ranging from
25 mA to 350 mA in 5 s pulses. It was found that the inactivation Table 4
rate of E. coli was 2.1–4.3 times greater than that of M52. Range, mode, mean and median values of temperature, voltage, pH of electrical
treatment systems, and RF of inactivated extremely halophilic bacteria in the 5 salt
Furthermore, researchers reduced the number of Staphylococcus
samples, dissolved separately in the liquid Brown media containing organic sub-
aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella stances, by 0.5 A direct electric current within 20 min
pneumoniae and Proteus mirabilis on intravascular catheters by
Treatment time (min) 20  C Voltage pH 6 RF 5.03b
10 mA direct electric current. They mentioned that bacteria colo-
nizing the surface of catheters were similarly affected by the ap- BEa
plication of a 10 mA electric current [42]. 1 Range 1 0.7 2 2.16
The lethal effects of low-amperage electric treatment on Mode 21 3.7 5 –
6
microorganisms in natural seawater and seawater inoculated with
Mean 20.6 3.9 5.8 1.12
Vibrio parahaemolyticus were investigated. In both cases, bacteria Median 21 3.8 6 0.84
were completely inhibited within 100 ms by a 0.5 A, 12 V direct
3 Range 1.5 0.7 1 3.8
electric current [14].
Mode 22 3.8 6 –
In addition, V. parahaemolyticus in effluent seawater was inac- Mean 21.4 3.88 5.6 3.49
tivated by 3 A alternating current within 30 ms [23]. Median 22 3.8 6 2.87
In Ref. [43] hydatid cyst protoscoleces, which was cultured in 5 Range 3.5 0.7 1 2.37
hydatid cyst fluid, were inactivated by 62.5 mA/cm2, 53.71 mA/cm2 Mode 24 3.9 6 –
and 18.18 mA/cm2 within 1 min, 2 min and 3 min, respectively. Mean 22.7 3.84 5.6 3.77
As applied in previous studies, it is possible to inactivate Median 24 3.9 6 3.17
extremely halophilic bacteria in salt by low-level direct electric 10 Range 4 0.5 1 2.1
current. When the level of direct electric current that was applied Mode – 3.9 6 –
to extremely halophilic bacteria in the brine solution was increased, Mean 24.42 3.86 5.6 4.63
Median 25 3.9 6 5.02
the inactivation time of extremely halophilic bacteria decreased
(Table 3). Range, mode, mean and median values of the reduction 15 Range 5 0.5 1 0.4
Mode – 3.9 6 –
factors (RFs) of the extremely halophilic bacteria are shown in Table
Mean 25.6 3.92 5.6 5.03
3. While extremely halophilic bacterial population in the brine Median 26 3.9 6 5.07
solution was killed by 0.1 A direct electric current within 15 min,
20 Range 6 0.6 1 0.4
0.2 A direct electric current killed these populations within 10 min.
Mode – 3.9 6 –
Although 0.3 A direct electric current inactivated extremely halo- Mean 27 3.94 5.6 5.03
philic population within 5 min, 0.4 A direct electric current inacti- Median 28 3.9 6 5.07
vated these populations within 3 min (Table 3). Furthermore, the a
BE: before experiment.
extremely halophilic bacterial population in all of 40 salt samples b
Mean of Log10 values of total extremely halophilic bacteria in the liquid Brown
dissolved in the brine solution inactivated by 0.5 A direct electric media before the electric treatment.
 Y. Birbir et al. / Journal of Electrostatics 66 (2008) 388–394 393

Table 5
Range, mode, mean, median values of temperature, voltage, pH of electrical treatment systems, and RF of inactivated lipase and protease producing extremely halophilic
bacteria in the liquid Brown media containing organic substances by 0.5 A direct electric current within 20 min

Treatment Lipase producers isolated from salt samples 2 and 5 Protease producers isolated form salt samples 4 and 6
time (min)

BEa 20  C Voltage pH 6 RF 6.3b 20  C Voltage pH 6 RF 5.64c

1 Range 0 0.1 0.5 0.05 1 0.8 0 0.04
Mode 20 – – – – – 7 –
Mean 20 3.85 7.75 0.61 21.5 4.6 7 0.05
Median 20 3.85 7.75 0.61 21.5 4.6 7 0.05

3 Range 0 0.1 0.5 1.04 3 0.8 0.5 2.36

Mode 20 – – – – – – –
Mean 20 3.75 7.75 1.67 22.5 4.5 7.25 2.51
Median 20 3.75 7.75 1.67 22.5 4.5 7.25 2.51

5 Range 0.5 0.1 0.5 3.64 2 0.8 1 3.61

Mode – – – – – – – –
Mean 20.75 3.65 7.75 3.8 24 4.4 7.5 4. 2
Median 20.75 3.65 7.75 3.8 24 4.4 7.5 4.2

10 Range 0 0.1 0 1.36 4.5 0.7 0.5 0.69

Mode 23 – 8 – – – – –
Mean 23 3.85 8 6.3 26.75 4.55 7.75 5.64
Median 23 3.85 8 6.3 26.75 4.55 7.75 5.64

15 Range 1 0.1 0 1.36 4 0.5 0.5 0.69

Mode – – 8 – – – – –
Mean 23.5 4.05 8 6.3 29 4.65 7.75 5.64
Median 23.5 4.05 8 6.3 29 4.65 7.75 5.64

20 Range 0 0.1 0 1.36 2 0.7 0.5 0.69

Mode 25.5 – 8 – – – – –
Mean 25.5 4.05 8 6.3 31 4.75 7.75 5.64
Median 25.5 4.05 8 6.3 31 4.75 7.75 5.64
a
BE: before experiment.
b
Mean of Log10 values of lipase producers in the liquid Brown media before the electric treatment.
c
Mean of Log10 values of protease producers in the liquid Brown media before the electric treatment.

When all extremely halophilic bacteria in the 10 salt samples
were inactivated by 0.1–0.5 A direct electric currents, the mean
values of temperature in the brine solutions were between 20  C
and 20.75  C. The mean values of pH of these samples were
between 9.3 and 11 when these bacteria were inactivated (Table 3).
When all extremely halophilic bacteria in the 5 salt samples
were inactivated (15 min) by 0.5 A direct electric current, the mean
value of the temperature in the liquid Brown media containing
organic substances was 25.6  C (Table 4). While pH values of 2 salt
samples decreased from 6 to 5 and the pH values of 3 salt samples
did not change, the mean value of pH of these samples was 5.6
when these bacteria were inactivated (Table 4).
When all of the lipase and protease producing extremely halo-
philic bacteria in the liquid Brown media containing organic sub-
stances were inactivated (10 min) by 0.5 A direct electric current,
the mean values of temperature of these samples were between
23  C and 26.7  C. The mean values of pH of these samples were
8.0 and 7.75, respectively when these bacteria were inactivated
(Table 5).
The mean values of voltage of all the brine solutions containing
salt samples did not change during electrical treatments and were
between 2.86 and 3.38 (Table 3), but voltage levels in all of the
liquid Brown media, containing both salt samples and protease-
plus-lipase producing extremely halophilic, decreased propor-
tionally to the halobacterial population until all of the halobacterial
populations was inactivated. After the halobacterial population in
the liquid Brown media was completely destroyed by direct electric
current, voltage level started to increase (Tables 4 and 5). This
important clue might be used to predict inactivation time of all
halobacterial populations grown in the presence of organic
substances in the hide industry.
In our previous experiment, the pH of the liquid Brown media
containing lipase producers, protease producers, and lipase-plus-
protease producers was 6.5, while the liquid Brown medium con-
taining a mixed culture of extremely halophilic bacteria was 6.0
before electrical treatment. A rapid increase of pH from 6.0 or 6.5 to
8 or 9 was observed within 1 min of exposure to the electrical
treatment in all of the media tested. The temperature of all of the
tested media increased slowly, reaching a maximum of 28–29  C
after 30 min. Liquid pH in all cases increased to a maximum of 10
after 15 min.
Our previous results showed that most of the extremely halo-
philic bacteria isolated from Tuz Lake and the salterns of Tuz Lake
grow at a pH of 11 [25,30]. Hence, the increase in pH in all tested
Brown liquid media did not cause the inactivation of the extremely
halophilic bacteria.
4. Conclusion
This is the first study in which the electrical inactivation of
different halobacterial populations in salt, which is used in pres-
ervation of hides, has been examined. This treatment system
proved to be a very effective way to inactivate both different species
of extremely halophilic population and lipolytic and proteolytic
extremely halophilic bacteria in salt. The results suggest that the
electrical treatment system can be used as a simple and effective
method for inactivating different halobacterial populations in salt
that is used to cure hides. The use of this technique in the leather
industry may prove to be more secure, simpler, and less expensive
than the use of antibacterial agents. In addition, since many leather
faults originate from proteolytic and lipolytic halobacterial damage
during brine curing of the hides, it is useful to examine the hal-
obacterial population in brine solutions. Proteolytic and lipolytic
bacterial numbers in salt give specific information on salt quality.
Hence, a quality control procedure for salt, which could be utilized
394 Y. Birbir et al. / Journal of Electrostatics 66 (2008) 388–394
by tanners, prior to the brine curing of the hide, would be of great
benefit to the leather industry.
Acknowledgements
We would like to thank the tanneries located in the Tuzla–
_
Istanbul Leather Organized Tannery Region in Türkiye for providing
us with salt crystal samples. We would also like to express our
deepest appreciation to Mrs. Didem Yazı Berber and Ms. Hüsniye
Hacıoğlu for helping us during the experiment.
References
[1] S. Dahl, Prevention of microbiological deterioration of leather, J. Am. Leather
Chem. Assoc. 51 (1956) 103–118.
[2] D.T. Didato, J. Browen, E. Hurlow, Microorganism control during leather
manufacture, in: M.K. Leafe (Ed.), Leather Technologists Pocket Book, The
Society of Leather Technologists and Chemists, East Yorkshire, England, 1999,
pp. 339–352.
[3] D.G. Bailey, M. Birbir, The impact of halophilic organisms on the grain quality
of brine cured hides, J. Am. Leather Chem. Assoc. 91 (1996) 47–51.
[4] W.E. Kallenberger, Halophilic Bacteria in Hide Curing. Ph.D. thesis, Division of
Graduate Studies and Research of the University of Cincinnati, Department of
Basic Science Tanning Research of the Collage of Arts and Science, 1985, pp.
1–79.
[5] M. Birbir, E.W. Kallenberger, A. Ilgaz, D.G. Bailey, Halophilic bacteria isolated
from brine-cured cattle hides, J. Soc. Leather Technol. Chem. 80 (1996) 87–90.
[6] D.G. Bailey, M. Birbir, A study of the extremely halophilic microorganisms
found on commercially brine-cured cattle hides, J. Am. Leather Chem. Assoc.
88 (1993) 285–293.
[7] M. Birbir, Investigation of salted-cured France and Russian hides for halophilic
bacteria, J. Turk. Microbiol. Soc. 27 (1997) 68–73.
[8] W.D. Grant, M. Kamekura, T. J.Mcgenity, A. Ventosa, Class III Halobacteria Class.
Nov. Order I. Halobacteriales, in: D.R. Boone, G.M. Garrity (Eds.), The Archaea
and the Deeply Branching and Phototrophic Bacteria, Bergey’s Manual of
Systematic Bacteriology, vol. 1, Springer-Verlag, New York, 2001, pp. 294–334.
[9] M.R. Lollar, E.W. Kallenberger, Halophilic bacteria thrive in seasonal cycles, J.
Am. Leather Chem. Assoc. 81 (1986) 248–265.
[10] M. Birbir, A. Ilgaz, Isolation and identification of bacteria adversely affecting
hide and leather quality, J. Soc. Leather Technol. Chem. 80 (1996) 147–153.
[11] T. Madigan, J.M. Martingo, Biology of Microorganisms, 11th ed. Pearson Prentice
Hall, Pearson Education, Inc, Upper Saddle River, NJ, 2006, pp. 419–446.
[12] B.O. Bitlisli, H.A. Karavana, B. Basaran, O. Sarı, I. Yasa, M. Birbir, The effect of
conservation defects on the suede quality of double-face, J. Am. Leather Chem.
Assoc. 99 (2004) 494–501.
[13] M. Birbir, Examination of proteolytic and lipolytic bacteria in salt used in hide
conservation, in: I. National Leather Symposium Proceedings Book, 2004, pp.
39–49.
[14] J.C. Park, M.S. Lee, D.H. Lee, B.J. Park, D.-W. Han, M. Uzawa, K. Takatori,
Inactivation of bacteria in seawater by low-amperage electric current, Appl.
Environ. Microbiol. 69 (2003) 2405–2408.
[15] A.K. Andson, R. Finkelstein, A study of the electropure process of treating milk,
J. Dairy Sci. 2 (1919) 374–406.
[16] J.M. Beattie, F.C. Lewis, The electric current (apart from the heat generated). A
bacteriological agent in the sterilization of milk and other fluids, J. Hyg. 24
(1925) 123–127.
[17] S.C. Prescott, The treatment of milk by an electrical method, Am. J. Public
Health 17 (1927) 221–223.
[18] G. Pareilleux, N. Sicard, Lethal effects of electric current on Escherichia coli,
Appl. Microbiol. 19 (1970) 421–424.
[19] C.P. Davis, N. Wagle, M.D. Anderson, M.M. Warren, Bacterial and fungal killing
by iontophoresis with long-lived electrodes, Antimicrob. Agents Chemther. 35
(1991) 2131–2134.
[20] L. Bolton, B. Foleno, B. Means, S. Petrucelli, Direct-current bactericidal effect on
intact skin, J. Antimicrob. Chemother 18 (1980) 137–141.
[21] W.K. Liu, M.R. Brown, T.S. Elliott, Mechanisms of the bactericidal activity of
low amperage electric current (DC), J. Antimicrob. Chemother 39 (1997)
687–695.
[22] X.G. Li, H.B. Cao, J.C. Wu, K.T. Yu, Inhibition of the metabolism of nitrifying
bacteria by direct electric current, Biotechnol. Lett. 23 (2001) 705–709.
[23] J.C. Park, M.S. Lee, D.W. Han, D.H. Lee, B.J. Park, I.S. Lee, M. Uzawa, M. Aihara,
K. Takatori, Inactivation of Vibrio parahaemolyticus in effluent seawater by
alternating-current treatment, Appl. Environ. Microbiol. 70 (3) (2004) 1833–
1835.
[24] J. Ye, H. Yang, H.-K. Kim, Y. Li, Inactivation of Listeria monocytogenes in
recirculated brine for chilling thermally processed bacon using an electro-
chemical treatment system, J. Food Sci. 66 (2001) 729–733.
[25] M. Birbir, C. Sesal, Extremely halophilic bacterial communities in Sere-
flikochisar Salt Lake in Turkey, Turk. J. Biol. 27 (7) (2003) 7–22.
[26] J.P. Harley, L.M. Prescott, Laboratory Exercises in Microbiology, fifth ed. The
McGraw-Hill Companies, New York, NY, 2002, pp. 93–95, 161–163.
[27] M. Birbir, N. Kalli, C. Johannson, The examination of salt quality of
Şereflikochisar Lake used in Turkish leather industry, J. Soc. Leather Technol.
Chem. 86 (2002) 112–118.
[28] Y. Birbir, M. Birbir, Inactivation of extremely halophilic hide-damaging bac-
teria via low-level direct electric current, J. Electrostat. 64 (2006) 791–795.
[29] D.V. Huntsberger, P. Billingsley, Elements of Statistical Inference, third ed.
Allyn and Bacon, Inc., Boston, 1973, pp. 20–57.
[30] M. Birbir, R. Elevi, C. Sesal, R.H. Vreeland, A. Oren, Characteristics of extremely
halophilic bacteria isolated from the Kaldırım and Kayacık Salterns of
Şereflikoçhisar Salt Lake in Türkiye, in: Proceedings of Halophiles 2001,
International Conference on Halophilic Microorganisms, Sevilla, Spain,
September 2001, pp. 23–27.
[31] M. Birbir, A. Ogan, B. Calli, B. Mertoglu, Enzymatic characteristics of extremely
halophilic archaeal community in Tuzkoy salt mine, Türkiye, World J. Micro-
biol. Biotechnol. 20 (2004) 613–621.
[32] R.H. Vreeland, S. Angelini, D.G. Bailey, Anatomy of halophile induced damage
to brine cured cattle hides, J. Am. Leather Chem. Assoc. 93 (1998) 121–131.
[33] G.W. Vivian, The preservation of hides and skins against bacterial damage,
J. Am. Leather Chem. Assoc. 64 (1969) 489.
[34] M.F. Hendry, D.R. Cooperand, D.R. Woods, The microbiology curing and tan-
ning process, part IV, the laboratory screening of antiseptics, J. Am. Leather
Chem. Assoc. 66 (1971) 31.
[35] M. Birbir, D.G. Bailey, Controlling the growth of extremely halophilic bacteria
on brine cured cattle hides, J. Soc. Leather Technol. Chem. 84 (2001) 201–204.
[36] E.F. Weiss, R.L. Thornton, Growth and Control of Microbiological Activity
during Hide Curing Process, Buckman Laboratories Inc., 1984, p. 18.
[37] J.W. Mitchell, Prevention of bacterial damage on brine cured and fresh cattle
hides, J. Am. Leather Chem. Assoc. 82 (1987) 372.
[38] R.H. Vreeland, D.G. Bailey, R.W. Claunch, Method of Using Bile Salts to Inhibit
Red Heat in Stored Brine-cured Hides and Skins U.S. Patent Number 5,945,027,
Docket Number 22497, Serial Number 8906333, Agricultural Research Service,
1999.
[39] M. Birbir, S. Eryılmaz, A. Ogan, Prevention of halophilic microbial damage on
brine cured hides by extremely halophilic halocin producer strains, J. Soc.
Leather Technol. Chem. 80 (2004) 99–104.
[40] S.A. Jackman, G. Maini, A.K. Sharman, C.J. Knowles, The effects of direct electric
current on the viability and metabolism of acidophilic bacteria, Enzyme
Microb. Technol. 24 (1999) 316–324.
[41] K.P. Drees, M. Abbaszadegan, R.M. Maier, Comparative electrochemical
inactivation of bacteria and bacteriophage, Water Res. 37 (2003) 2291–2300.
[42] W.K. Liu, S.E. Tebbs, P.O. Byrne, T.S. Elliott, The effects of electric current on
bacteria colonizing intravenous catheters, J. Infect. 27 (1993) 261–269.
[43] A. Dalimi, R. Ghasemikhah, B.H. Malayeri, Echinococcus granulosus: lethal
effect of low voltage direct electric current on hydatid cyst protoscoleces, Exp.
Parasitol. 109 (2004) 237–240.