Meral Birbir

Production of cellulase by immobilized whole cells of Haloarcula

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.

Thalassobacillus pellis sp. nov., a moderately halophilic, Gram-positive bacterium isolated from salted hides

A Gram-positive, moderately halophilic and endospore-forming bacterium, designated strain 18OM(T), was isolated from salted animal hides. The cells were rods and produced ellipsoidal endospores at a terminal position. Strain 18OM(T) 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 18OM(T) was closely related to species of the genus Thalassobacillus within the phylum Firmicutes. The closest phylogenetic similarity was with Thalassobacillus devorans G-19.1(T) (98.4 %), Thalassobacillus cyri HS286(T) (97.9 %) and Thalassobacillus hwangdonensis AD-1(T) (97.4 %). The major cellular fatty acids were anteiso-C(15 : 0) (57.9 %), anteiso-C(17 : 0) (14.0 %), iso-C(15 : 0) (10.8 %) and iso-C(16 : 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 18OM(T) within the genus Thalassobacillus. The DNA-DNA hybridization values between strain 18OM(T) and T. devorans G-19.1(T), T. cyri HS286(T) and T. hwangdonensis AD-1(T) were 49 %, 9 % and 15 %, respectively, showing unequivocally that strain 18OM(T) constituted a novel genospecies. On the basis of phylogenetic analysis and phenotypic, genotypic and chemotaxonomic characteristics, strain 18OM(T) 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 18OM(T) ( = CECT 7566(T) = DSM 22784(T) = JCM 16412(T)).

Salimicrobium salexigens sp. nov., a moderately halophilic bacterium from salted hides

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 50.7mol% and 51.5mol% 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)).

Preparation, characterization, and in vitro evaluation of isoniazid and rifampicin‐loaded archaeosomes

The ability of Archaea to adapt their membrane lipid compositions to extreme environments has brought in archaeosomes into consideration for the development of drug delivery systems overcoming the physical, biological blockades that the body exhibits against drug therapies. In this study, we prepared unilamellar archaeosomes, from the polar lipid fraction extracted from Haloarcula 2TK2 strain, and explored its potential as a drug delivery vehicle. Rifampicin and isoniazid which are conventional drugs in tuberculosis medication were loaded separately and together in the same archaeosome formulation for the benefits of the combined therapy. Particle size and zeta potential of archaeosomes were measured by photon correlation spectroscopy, and the morphology was assessed by with an atomic force microscope. Encapsulation efficiency and loading capacities of the drugs were determined, and in vitro drug releases were monitored spectrophotometrically. Our study demonstrates that rifampicin and isoniazid could be successfully loaded separately and together in archaeosomes with reasonable drug‐loading and desired vesicle‐specific characters. Both of the drugs had greater affinity for archaeosomes than a conventional liposome formulation. The results imply that archaeosomes prepared from extremely halophilic archaeon were compatible with the liposomes for the development of stable and sustained release of antituberculosis drugs.

Enzymatic activity of a novel halotolerant lipase from Haloarcula hispanica 2TK2

Abstract 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.

Detection of industrially potential enzymes of moderately halophilic bacteria on salted goat skins

Abstract 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.

Polyhydroxyalkanoates Production from Renewable and Waste Materials Using Extremophiles/Recombinant Microbes

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters produced by various species of Bacteria and Archaea as reserves of energy and carbon in nutrient poor environments. Being biodegradable and biocompatible, PHAs have found many industrial and medical applications as attractive bio-based alternatives to petroleum-based polymers. To compete with petroleum-based polymers and garner a bigger market share, cost-effective PHA production processes are needed. Therefore, renewable, cheap, sustainable, and readily available carbon sources from industrial wastes and agricultural by-products should be considered in PHA production.

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

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 (10 2 –10 4 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 (10 6 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.