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Proteolytic and lipolytic extremely halophilic archaea found in curing salt may contaminate skins during the brine curing process and damage skin structure. In the present study, three proteolytic and lipolytic extremely halophilic archaea were isolated from deteriorated salted sheepskins and characterised using conventional and molecular methods. Each test strain (Haloarcula salaria AT1, Halobacterium salinarum 22T6, Haloarcula tradensis 7T3), a mixed culture of these strains and the mixed culture treated with 1.5 A direct current (DC) were used for brine curing processes of fresh sheepskins and examined during 47 days of storage to evaluate the degree of destruction wreaked by these microorganisms. Both organoleptic properties and scanning electron microscopy (SEM) images of sheepskins proved that each separate test strain and the mixed culture caused serious damage. However, the mixed culture of strains treated with electric current did not damage sheepskin structure. Therefore, we highly recommend sterilisation of brine using DC to prevent archaeal damage on cured hides and skins in the leather industry.

Bacteria forms biofilm to be resistant to antibacterial agents and other unfavorable environment as compared to planktonic bacterial cells. Due to resistance of bacterial biofilms to commonly used antimicrobial agents and adverse effects of these biofilms in different industries, potential natural compounds which can inhibit bacterial biofilms have attracted more attention in recent years. Lichens are known to have unique secondary metabolites with various biological activities including anti-biofilm properties. Therefore, Bacillus toyonensis, Bacillus mojavensis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, Bacillus cereus, and Bacillus licheniformis, isolated from soak liquor samples in the previous study, were tested for their ability to form biofilm in this study. Biofilm-forming Bacillus species were detected as B. subtilis, B. amyloliquefaciens, and B. velezensis. The anti-biofilm effect of the acetone extracts of Usnea sp. was evaluated at various concentrations against these biofilm-forming isolates. The anti-biofilm effect of acetone extracts of Usnea sp. against B. subtilis and B. amyloliquefaciens was observed at the concentration of 5 µg/mL by inhibition ratios of 62.75% and 72.72%, respectively. In addition, biofilm formation of B. velezensis was inhibited by the treatment with 1.25 µg/mL extracts at a 62.69% inhibition rate. Biofilm formations of B. amyloliquefaciens and B. velezensis were also suppressed by the extracts at varying percentages of inhibition ranging between 10.11-43.69% and 21.25-46.35%, respectively. This study may provide an alternative approach to overcome the biofilm formation and bacterial resistance to the antibacterial agents in the leather industry.

Prokaryotic communities and physico-chemical characteristics of 30 brine samples from the thalassohaline Tuz Lake (Salt Lake), Deep Zone, Kayacik, Kaldirim, and Yavsan salterns (Turkey) were analyzed using 16S rRNA amplicon sequencing and standard methods, respectively. Archaea (98.41% of reads) was found to dominate in these habitats in contrast to the domain Bacteria (1.38% of reads). Representatives of the phylum Euryarchaeota were detected as the most predominant, while 59.48% and 1.32% of reads, respectively, were assigned to 18 archaeal genera, 19 bacterial genera, 10 archaeal genera, and one bacterial genus that were determined to be present, with more than 1% sequences in the samples. They were the archaeal genera Haloquadratum, Haloarcula, Halorhabdus, Natronomonas, Halosimplex, Halomicrobium, Halorubrum, Halonotius, Halolamina, Halobacterium, and Salinibacter within the domain Bacteria. The genera Haloquadratum and Halorhabdus were found in all sampling sites. While Haloquadratum, Haloarcula, and Halorhabdus were the most abundant genera, two uncultured Tuz Lake Halobacteria (TLHs) 1 and 2 were detected in high abundance, and an additional uncultured haloarchaeal TLH-3 was found as a minor abundant uncultured taxon. Their future isolation in pure culture would permit us to expand our knowledge on hypersaline thalassohaline habitats, as well as their ecological role and biomedical and biotechnological potential applications.

Aim: This study aimed to isolate moderately halophilic bacteria from salted goat skins, to characterize these microorganisms and to determine their industrially important enzymes such as amylase, catalase, oxidase, caseinase, cellulase, DNase, lipase, lecithinase, protease, pullulanase, urease, phospholipase, xylanase and β-galactosidase. Methods: Enzymes of these bacteria, isolated from skin samples belonging to eight countries and identified using phenotypic and genotypic methods, were examined in agar media. Results: Thirty-nine isolates were fairly similar to species of genera Staphylococcus, Bacillus, Salinicoccus, Gracilibacillus, Chromohalobacter and Halomonas. Various carbon sources were utilized, and all isolates produced enzyme. Enzyme-producing species were Staphylococcus saprophyticus subsp. saprophyticus, Staphylococcus arlettae, Bacillus pumilus, Gracilibacillus dipsosauri, Salinicoccus roseus, Bacillus licheniformis, Chromohalobacter beijerinckii, Staphylococcus xylosus, Halomonas eurihalina, Staphylococcus equorum subsp. equorum, Halomonas zhanjiangensis, Halomonas venusta and Chromohalobacter canadensis. Fairly high percentage of isolates produced protease (87%) and catalase (100%). While more than 50% of isolates produced lipase (64%), β-galactosidase (59%) and oxidase (56%), less than 50% of isolates produced urease (46%), caseinase (28%), amylase (26%), lecithinase (8%) and cellulase (5%). Conclusion: We detected that moderately halophilic bacteria on skins produced important enzymes, which may be used in diverse industrial applications in leather, feed, detergent, paper, food, chemical, medical, pharmaceutical, textile industries.

A strain of Haloarcula hispanica isolated from Tuzkoy salt mine, Turkey exhibited extracellular lipolytic activity. Important parameters such as carbon sources and salt concentration for lipase production were investigated. Optimal conditions for the enzyme production from Haloarcula hispanica 2TK2 were determined. It was observed that the lipolytic activity of Haloarcula hispanica was stimulated by some of the carbon sources. The high lipase acitivity values were obtained in the presence of 2% (v/v) walnut oil (6.16 U/ml), 1% (v/v) fish oil (5.07 U/ml), 1% (v/v) olive oil (4.52 U/ml) and 1% (w/v) stearic acid (4.88 U/ml) at 4M NaCl concentration. Lipase was partially purified by ammonium sulfate precipitation and ultrafiltration. Optimal temperature and pH values were determined as 45°C and 8.0, respectively. Lipase activity decreased with the increasing salt concentration, but 85% activity of the enzyme was maintained at 5M NaCl concentration. The enzyme preserved 41% of its relative activity at 90°C. The partially purified lipase maintained its activity in the presence of surfactants such as Triton X-100 and SDS. Therefore, the lipase which is an extremozyme may have potential applications especially in detergent industry.

Salt contains extremely halophilic archaea and these microorganisms degrade leather quality. The aim of this study is to fi nd 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 fi rst 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.

Inactivation effect of an electric current treatment combining both 188 μA/m 2 DC and 251 μA/m 2 AC on Gram negative, Gram positive bacteria and their mixed culture, which were isolated from hides, was separately analyzed. This electric treatment, applied for 16 min was found to be fairly effective in reducing the bacterial cell count (7.10–7.48 log 10 colony forming units/mL) to a relatively low level (4.52–4.90 log 10 colony forming units/mL). At this level bacterial damage to the hides is reduced in soak liquors. Cheap and efficient electric current may be applied to rapidly reduce the destructive bacterial populations in main hide-soak liquors.

Inactivation of bacteria by electric currents has received much attention due to its high efficiency, low cost, and ease of application. Therefore, the antibacterial effect of passing 2 A direct electric current for 50 min on mesophilic bacteria in both first and main hide-soak liquors obtained from different tanneries in Tuzla-Istanbul, Turkiye was examined. The mesophilic bacterial numbers in the main hide-soak liquors were higher than that of the first hide-soak liquors. The total numbers of gram-negative bacteria (101–104 CFU/mL) and halotolerant bacteria (103–104 CFU/mL), in both first and main hide-soak liquors, were almost the same. The numbers of total of proteolytic and lipolytic mesophilic bacteria and the total of proteolytic and lipolytic halotolerant bacteria in the main hide-soak liquors were higher than that of the first hide-soak liquors. Mesophilic bacteria in the first (seven samples) and main (three samples) hide-soak liquors were inactivated after 20–30 min of electrical treatment. In the other four main hide-soak liquors, the inactivation occurred after 50 min of treatment. The most important result of this experiment is that there was a correlation between the voltage level and the numbers of bacteria in the electrolysis cell. The bacterial populations in the hide-soak liquors decreased proportionally to the voltage level until all of the bacterial population was inactivated. After the bacteria in the hide-soak liquors were completely destroyed by direct electric current, the voltage level started to increase. This important clue might be used to predict the inactivation time of all of the bacterial population in hide-soak liquors in the hide industry. It is suggested that the bacterial inactivation in hide-soak liquors by electrical treatment might be used in tanneries, which do not have a waste treatment system and discharge their effluents directly into environment.

Hydrolytic enzymes, which are produced by extremely halophilic bacteria in salt, may cause serious damage on salted hides and may result in significant economic losses. Therefore, to prevent halobacterial damage on hides, the antibacterial effects of different levels of direct electric current on extremely halophilic bacteria found in salt were examined in brine solution containing 20% NaCl and salt samples, 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 current (0.1 A, 0.2 A, 0.3 A and 0.4 A) inactivated extremely halophilic bacteria in salt, which was dissolved in the brine solution within 15 min, 10 min, 5 min and 3 min, respectively. Although 0.5 A direct electric current inactivated extremely halophilic bacteria in the salt samples (104–105 CFU/g), which were 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 bacterial populations in salt used in hide preservation.

Tuz Lake is a hypersaline lake located in Central Anatolia, Turkey. The lake and its salterns, Kaldirim and Kayacik, are the major sources of solar salt for industrial applications in Turkey, especially in the food and leather industries. Use of the crude solar salt often results in microbial deterioration of the products. We therefore initiated a thorough characterization of the microbial communities in Tuz Lake and its adjacent salterns, and we present here the results of investigations on diversity of extremely halophilic Archaea. Twenty-seven colonies of aerobic red or pink Archaea (family Halobacteriaceae) were selected according to colony shape, size, consistency and pigmentation, and characterized according to their phenotypic characteristics, polar lipid contents, and antibiotic sensitivities. Furthermore, 16S rRNA genes of the isolates were screened by DGGE analysis and partially sequenced. Phylogenetic analysis showed that most isolates belonged to the genera Haloarcula, Halorubrum and Halobacterium. Haloarcula was found to be dominant both in Tuz Lake and in the saltern samples. Halorubrum species were isolated from Tuz Lake and from the Kaldirim saltern, and Halobacterium species were recovered from Tuz Lake and from the Kayacik saltern. All strains showed various activities of hydrolytic enzymes (proteases, amylases, cellulases, and others), activities which are responsible for the detrimental effects of the crude salt in food and leather products.

The presence of proteolytic and lipolytic extremely halophilic bacteria on brine-cured hides reduces the value of the hide as a raw material for leather manufacturing. Every brine curing raceway that produces hide contains a significant number of extremely halophilic bacteria that contaminate almost every hide. The usual brine raceway treatments used to control bacteria during curing do not control halophilic bacteria. The antibacterial effects of passing 0.5-A low level, direct electric current for 30 min on different species of protease, lipase, both protease and lipase producing extremely halophilic bacteria, as well as a mixed population of extremely halophilic bacteria, were examined in liquid media containing organic substances and 25% NaCl. Although the antibacterial effects varied with different extremely halophilic bacterial species, the application of current demonstrated an antibacterial effect within 20 min of treatment of all species tested. A mixed culture of extremely halophilic bacteria was killed within 10 min. It has been demonstrated that treatment time as short as 5 min is sufficient for the total inactivation of protease producing extremely halophilic bacteria of 10 7 CFU/mL while lipase or lipase and protease producing extremely halophilic bacteria were inactivated within 20 min. It was also found that the temperature and pH of the test medium increased during treatment. The maximum temperature rise was 9 °C, and the pH increased by 4.

Seven extremely halophilic strains were isolated from the Ayvalik Saltern in the north-eastern part of Turkey. Chemical analyses of the brine and salt samples were performed to measure their salt content, hardness and pH. Isolated strains were tested for their antibiotic sensitivities; cell and colony morphologies; hydrolysis of casein, starch, gelatin, Tween 20 and Tween 80; and oxidase and catalase activity. All strains were found to belong to the domain Archaea. Characterization of polar lipids by thin layer chromatography indicated that all isolates contained phytanyl diether derivatives of phosphatidylglycerol (PG), the methyl ester of phosphatidyl glycerophosphate (PGP-Me), and phosphatidylglycerosulphate (PGS). Four isolates had triglycosyl diether (TGD-2) as glycolipid, and the other three contained a sulphated diglycosyl diether instead. All isolates were examined for the presence of plasmids by agarose gel electrophoresis. Four strains were found to harbour plasmids ranging in size from 13.8 to 15.3 kbp. Correlation between the protein profiles in SDS–PAGE and the phenotypic properties of the strains was poor. The data presented here provide the first published account of the microbiota of the Ayvalik saltern, which provides a large part of the salt produced in Turkey.

Tuzkoy is a large and unpolluted salt mine in Central Anatolia, Turkey. High quality salt in this mine may be supplied to the table salt market after a simple process instead of complicated refining. However, the extremely halophilic microbial content of the salt has not yet been investigated which may result in detrimental effects in industrial processes such the food and leather industries, if unprocessed salt is directly used. Therefore, to identify and characterize the microbial contaminants in Tuzkoy salt, chemical and microbial analyses were conducted on salt crystal samples collected from three different locations of the mine. Generally 105–106 colony forming units of extremely halophilic microorganisms were detected per gram of salt samples. Twelve colonies were selected randomly for further characterization. Phenotypic characterization, lipid analysis, antibiotic susceptibility tests and positive PCR amplification results with Archaea-specific primers confirmed that all strains were halophilic Archaea belong to family Halobacteriaceae. According to denaturing gradient gel electrophoresis (DGGE) results, 10 comparatively different strains were selected for DNA sequencing. DNA sequences and phylogenetic analyses revealed that the isolated strains were mainly species of the genera Halobacterium, Haloarcula, Natrinema and Halorubrum. In addition enzymatic activity tests were also conducted to evaluate the salt quality for industrial applications. Results of gelatinase, caseinase, amylase, cellulase and lipase activity tests revealed that the isolated strains produced hydrolytic enzymes, which could cause deterioration in salt-treated food and hide. It may be pointed out that cellulase activity in halophilic Archaea has not been reported previously. Moreover, β-galactosidase activity has been reported in some Haloferax and Halorubrum species but not in the genus Halobacterium.

The inhibitory effect of calcium propionate, on growth of Aspergillus flavus, Aspergillus versicolor, Aspergillus niger, Penicillium citrinum, Penicillium vernicosum, Cladosporium macrocarpum, Fusarium semitectum, Mucor racemosus and Rhizopus oligosporus was evaluated at pH 5, 6 and 7 on Potato Dextrose Agar. The highest inhibition of the mold species was seen at pH 5 on the medium. The inhibitory effect of calcium propionate on the mold colonies increased significantly when the concentration of calcium propionate was raised from 1g/l to 2g/l. The inhibitory effect of calcium propionate was the highest at a concentration of 3g/to 4g/l. 1g/l, 2.5g/l and 4 g/l of calcium propionate were added to sterilized ground corn with 14% humidity to determine the effect of calcium propionate on the different molds growth at 25°C. The colony counts of mold species decreased from 106 cfu/ml to 105 cfu/ml at concentrations of 2.5 g/l and 4 g/l calcium propionate in PDA at pH 5. All concentrations of calcium propi...

Two Gram-positive, moderately halophilic bacteria, designated strains 29CMI T and 53CMI, were isolated from salted hides. Both strains were non-motile, strictly aerobic cocci, growing in the presence of 3–25% (w/v) NaCl (optimal growth at 7.5–12.5% [w/v] NaCl), between pH 5.0 and 10.0 (optimal growth at pH 7.5) and at temperatures between 15 and 40 °C (optimal growth at 37 °C). Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that both strains showed a similarity of 98.7% and were closely related to species of the genus Salimicrobium , within the phylum Firmicutes . Strains 29CMI T and 53CMI exhibited 16S rRNA gene sequence similarity values of 97.9–97.6% with Salimicrobium album DSM 20748 T , Salimicrobium halophilum DSM 4771 T , Salimicrobium flavidum ISL-25 T and Salimicrobium luteum BY-5 T . The DNA G+C content was 50.7 mol% and 51.5 mol% for strains 29CMI T and 53CMI, respectively. The DNA–DNA hybridization between both strains was 98%, whereas the values between strain 29CMI T and the species S. album CCM 3517 T , S. luteum BY-5 T , S. flavidum ISL-25 T and S. halophilum CCM 4074 T were 45%, 28%, 15% and 10%, respectively, showing unequivocally that strains 29CMI T and 53CMI constitute a new genospecies. The major cellular fatty acids were anteiso-C 15:0 , anteiso-C 17:0 , iso-C 15:0 and iso-C 14:0 . The main respiratory isoprenoid quinone was MK-7, although small amounts of MK-6 were also found. The polar lipids of the type strain consist of diphosphatidylglycerol, phosphatidylglycerol, one unidentified phospholipid and one glycolipid. The peptidoglycan type is A1γ, with meso -diaminopimelic acid as the diagnostic diamino acid. On the basis of the phylogenetic analysis, and phenotypic, genotypic and chemotaxonomic characteristics, we propose strains 29CMI T and 53CMI as a novel species of the genus Salimicrobium , with the name Salimicrobium salexigens sp. nov. The type strain is 29CMI T (=CECT 7568 T = JCM 16414 T = LMG 25386 T ).

A Gram-positive, moderately halophilic and endospore-forming bacterium, designated strain 18OMT, was isolated from salted animal hides. The cells were rods and produced ellipsoidal endospores at a terminal position. Strain 18OMT was motile, strictly aerobic and grew at 0.5–25 % (w/v) NaCl [optimal growth at 10 % (w/v) NaCl], at between pH 5.0 and 9.0 (optimal growth at pH 7.5) and at temperatures between 15 and 45 °C (optimal growth at 37 °C). Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that strain 18OMT was closely related to species of the genus Thalassobacillus within the phylum Firmicutes. The closest phylogenetic similarity was with Thalassobacillus devorans G-19.1T (98.4 %), Thalassobacillus cyri HS286T (97.9 %) and Thalassobacillus hwangdonensis AD-1T (97.4 %). The major cellular fatty acids were anteiso-C15 : 0 (57.9 %), anteiso-C17 : 0 (14.0 %), iso-C15 : 0 (10.8 %) and iso-C16 : 0 (8.1 %). The respiratory isoprenoid quinones were MK-7 (98.5 %) and MK-6 (1.5 %). The DNA G+C content was 42.9 mol%. These features confirmed the placement of strain 18OMT within the genus Thalassobacillus. The DNA–DNA hybridization values between strain 18OMT and T. devorans G-19.1T, T. cyri HS286T and T. hwangdonensis AD-1T were 49 %, 9 % and 15 %, respectively, showing unequivocally that strain 18OMT constituted a novel genospecies. On the basis of phylogenetic analysis and phenotypic, genotypic and chemotaxonomic characteristics, strain 18OMT is considered to represent a novel species of the genus Thalassobacillus, for which the name Thalassobacillus pellis sp. nov. is proposed. The type strain is 18OMT ( = CECT 7566T = DSM 22784T = JCM 16412T).

Halophilic Archaea are adapted to a life in the extreme conditions and some of them are capable of growth on cellulosic waste as carbon and energy source by producing cellulase enzyme. The production of cellulase using free and immobilized cells of halophilic archaeal strain Haloarcula 2TK2 isolated from Tuzkoy Salt Mine and capable of producing cellulose was studied. The cells were cultured in a liquid medium containing 2.5 M NaCl to obtain the maximum cellulase activity and immobilized on agarose or polyacrylamide or alginate. Optimal salt dependence of free and immobilized cells of Haloarcula 2TK2 was established and the effects of pH and temperature were investigated. Immobilization to Na-alginate enhanced the enzymatic activity of the haloarchaeal cells when compared to free cells and other polymeric supports. From the results obtained it is reasonable to infer that decomposition of plant polymers into simpler end products does occur at high salinities and cellulase producing haloarchael cells may be potentially utilized for the treatment of hypersaline waste water to remove cellulose.