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    Anoxybacillus calidus sp nov., a thermophilic bacterium isolated from soil near a thermal power plant
    (Microbiology Soc, 2014) Cihan, Arzu Coleri; Cokmus, Cumhur; Koc, Melih; Ozcan, Birgul
    A novel thermophilic, Gram-stain-positive, facultatively anaerobic, endospore-forming, motile, rod-shaped bacterium, strain C161ab(T), was isolated from a soil sample collected near Kizildere, Saraykoy-Buharkent power plant in Denizli. The isolate could grow at temperatures between 35 and 70 degrees C (optimum 55 degrees C), at pH 6.5-9.0 (optimum pH 8.0-8.5) and with 0-2.5% NaCl (optimum 0.5 %, w/v). The strain formed cream-coloured, circular colonies and tolerated up to 70 mM boron. Its DNA G+C content was 37.8 mol%. The peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. Strain C161ab(T) contained menaquinones MK-7 (96%) and MK-6 (4%). The major cellular fatty acids were iso-branched fatty acids: iso-C-15:0 (52.2%) and iso-C-17:0 (28.0%) with small amounts of C-16:0 (7.4%). Phylogenetic analysis based on the 16S rRNA gene revealed 94.6-96.8 % sequence similarity with all recognized species of the genus Anoxybacillus. Strain C161ab(T) showed the greatest sequence similarity to Anoxybacillus rupiensis DSM 17127(T) and Anoxybacillus voinovskiensis DSM 17075(T), both had 96.8% similarity to strain C161ab(T), as well as to Anoxybacillus caldiproteolyticus DSM 15730(T) (96.6%). DNA-DNA hybridization revealed low levels of relatedness with the closest relatives of strain C161ab(T), A. rupiensis (21.2%) and A. voinovskiensis (16.5%). On the basis of the results obtained from phenotypic, chemotaxonomic, genomic fingerprinting, phylogenetic and hybridization analyses, the isolate is proposed to represent a novel species, Anoxybacillus calidus sp. nov. (type strain C161ab(T)=DSM 25520(T)=NCIMB 14851(T)).
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    Anoxybacillus salavatliensis sp. nov., an ?-glucosidase producing, thermophilic bacterium isolated from Salavatli, Turkey
    (Wiley, 2011) Cihan, Arzu Coleri; Ozcan, Birgul; Cokmus, Cumhur
    A novel moderately thermophilic, Gram-positive staining, rod-shaped, spore-forming, motile, facultative anaerobic, and a-glucosidase producing strain A343(T), was isolated from a high temperature well-pipeline sediment sample in Salavatli province of Aydin, Turkey. Growth was observed at 37-69 degrees C (optimum 60 degrees C), at pH 5.5-9.5 (optimum 8.0-9.0) and at salinities from 0 to 4.5% (w/v) (optimum 2%). Strain A343(T) was able to grow on a wide range of carbon sources. Gelatin and starch utilization, amylase, catalase and oxidase activities, reduction of nitrate to nitrite were all positive. The G+C content of the genomic DNA was 45.1 mol%. The major menaquinone was MK-7. The dominant cellular fatty acids were: iso-C15:0, C16:0, and iso-C17:0. The phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain A343(T) belonged to the genus Anoxybacillus. The 16S rRNA gene sequence similarity between strain A343(T) and the type strains of recognized Anoxybacillus species was ranged from 95.8 to 99.4%. DNA-DNA hybridization revealed low homology with its closest relative Anoxybacillus kamchatkensis (49.7%). In addition to the total cell protein profiles, the Rep-PCR and the intergenic 16S-23S rRNA gene fingerprinting profiles differentiated strain A343(T) from all of the reference Anoxybacillus species used. Based upon phenotypic, phylogenetic and chemotaxonomic evidence, strain A343(T) was assigned to a new species within the genus Anoxybacillus, A. salavatliensis sp. nov. (The type strain A343(T) = DSM 22626(T) = NCIMB 14579(T)). The 16S rRNA gene nucleotide sequence of strain A343(T) is available in the GenBank database under the accession number - EU326496.
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    Biofilm characteristics and evaluation of the sanitation procedures of thermophilic Aeribacillus pallidus E334 biofilms
    (Taylor & Francis Ltd, 2017) Kilic, Tugba; Karaca, Basar; Ozel, Beste Piril; Ozcan, Birgul; Cokmus, Cumhur; Cihan, Arzu Coleri
    The ability of Aeribacillus pallidus E334 to produce pellicle and form a biofilm was studied. Optimal biofilm formation occurred at 60 degrees C, pH 7.5 and 1.5% NaCl. Extra polymeric substances (EPS) were composed of proteins and eDNA (21.4kb). E334 formed biofilm on many surfaces, but mostly preferred polypropylene and glass. Using CLSM analysis, the network-like structure of the EPS was observed. The A. pallidus biofilm had a novel eDNA content. DNaseI susceptibility (86.8% removal) of eDNA revealed its importance in mature biofilms, but the purified eDNA was resistant to DNaseI, probably due to its extended folding outside the matrix. Among 15 cleaning agents, biofilms could be removed with alkaline protease and sodium dodecyl sulphate (SDS). The removal of cells from polypropylene and biomass on glass was achieved with combined SDS/alkaline protease treatment. Strong A. pallidus biofilms could cause risks for industrial processes and abiotic surfaces must be taken into consideration in terms of sanitation procedures.
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    Characterization of thermostable ?-glucosidases from newly isolated Geobacillus sp A333 and thermophilic bacterium A343
    (Springer, 2009) Cihan, Arzu Coleri; Ozcan, Birgul; Cokmus, Cumhur
    We have partially purified and characterized two new thermostable exo-alpha-1,4-glucosidases (E.C.3.2.1.20) isolated from Geobacillus sp. A333 and thermophilic bacterium A343 strains. A333 alpha-glucosidase showed optimum activity at 60A degrees C, pH 6.8 and had a value of 1.38 K (m) for the pNPG substrate, whereas these results were found to be 65A degrees C, 7.0 and 0.85, respectively for A343 enzyme. Specificity for 20 different substrates and thin layer chromatography studies demonstrated that the A333 enzyme had high transglycosylation activity, and A343 had wide substrate specificity. The substrate specificity of A333 alpha-glucosidase was determined as maltose, dextrin, turanose, maltotriose, maltopentaose, meltotetraose, maltohexaose and phenyl-alpha-d-glycopyranoside. On the other hand, the A343 alpha-glucosidase mostly hydrolyzed dextrin, turanose, maltose, phenyl-alpha-d-glucopyranoside, maltotriose, maltotetraose, maltopentaose, isomaltose, saccharose and kojibiose by acting alpha-1,2, alpha-1,3, alpha-1,4 and alpha-1,6 bonds of these substrates. The relative activites of A333 and A343 enzymes were determined to be 83 and 92% when incubated at 60A degrees C for 5 h whereas, the pH of 50% inactivation at 60A degrees C for 15 h were determined to be pH 4.5/10.0 and pH 5.0/10.0, respectively. In addition, the results not only showed that both of the alpha-glucosidases were stable in a wide range of pH and temperatures, but were also found to be resistant to most of the denaturing agents, inhibitors and metal ions tested. With this study, thermostable exo-alpha-1,4-glucosidases produced by two new thermophilic strains were characterized as having biotechnological potential in transglycosylation reactions and starch hydrolysis processes.
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    CONTROL OF THERMOPHILIC SPORES BY SPORICIDAL AGENTS AND THERMAL INACTIVATION
    (Slovak Univ Agriculture Nitra, 2022) Karakaya, Ayse Busra; Karaca, Basar; Ozcan, Birgul; Cihan, Arzu Coleri
    In this study, the adhesion patterns of thermophilic spores of Geobacillus thermodenitrificans DSM 465(T), Geobacillus thermoglucosidans B84a, Anoxybacillus kamchatkensis subp. asaccharedens F81 and Anoxybacillus flavithermus DSM 2641(T), all of which are biofilm producers in dairy products, were investigated by epifluorescence microscopy on 6 different abiotic surfaces commonly used in the dairy industry. The spores of G. thermodenitrificans DSM 465(T) and A. kamchatkensis subp. asaccharedens F81 were found to adhere mainly to rubber, polycarbonate, PTFE and stainless steel surfaces. In addition, the efficacy of sporicidal agents on the spores of these bacteria was investigated and only peracetic acid, cetylpyridinium chloride and formaldehyde were found to be the most effective of the sporicidal agents tested. Among the sporicidal agents that showed high efficacy against spores, peracetic acid and nitric acid were selected because they had the shortest contact time, low toxicity and cost. Binary combination effects were tested by determining the LD50 values of the selected agents and it was found that there was a synergistic effectbetween these two effective chemicals. In addition, the thermal resistance profiles of planktonic and sessile spores of A. flavithermus DSM 2641(T) and G. thermodenitrificans DSM 465(T) were evaluated.
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    The genetic diversity of genus Bacillus and the related genera revealed by 16s rRNA gene sequences and ARDRA analyses isolated from geothermal regions of Turkey
    (2012) Cihan, Arzu Coleri; Tekin, Nilgun; Ozcan, Birgul; Cokmus, Cumhur
    Previously isolated 115 endospore-forming bacilli were basically grouped according to their temperature requirements for growth: the thermophiles (74%), the facultative thermophiles (14%) and the mesophiles (12%). These isolates were taken into 16S rRNA gene sequence analyses, and they were clustered among the 7 genera: Anoxybacillus, Aeribacillus, Bacillus, Brevibacillus, Geobacillus, Paenibacillus, and Thermoactinomycetes. Of these bacilli, only the thirty two isolates belonging to genera Bacillus (16), Brevibacillus (13), Paenibacillus (1) and Thermoactinomycetes (2) were selected and presented in this paper. The comparative sequence analyses revealed that the similarity values were ranged as 91.4-100%, 91.8- 99.2%, 92.6- 99.8% and 90.7 - 99.8% between the isolates and the related type strains from these four genera, respectively. Twenty nine of them were found to be related with the validly published type strains. The most abundant species was B. thermoruber with 9 isolates followed by B. pumilus (6), B. lichenformis (3), B. subtilis (3), B. agri (3), B. smithii (2), T. vulgaris (2) and finally P. barengoltzii (1). In addition, isolates of A391a, B51a and D295 were proposed as novel species as their 16S rRNA gene sequences displayed similarities ? 97% to their closely related type strains. The AluI-, HaeIII- and TaqI-ARDRA results were in congruence with the 16S rRNA gene sequence analyses. The ARDRA results allowed us to differentiate these isolates, and their discriminative restriction fragments were able to be determined. Some of their phenotypic characters and their amylase, chitinase and protease production were also studied and biotechnologically valuable enzyme producing isolates were introduced in order to use in further studies.
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    THE GENETIC DIVERSITY OF GENUS BACILLUS AND THE RELATED GENERA REVEALED BY 16S rRNA GENE SEQUENCES AND ARDRA ANALYSES ISOLATED FROM GEOTHERMAL REGIONS OF TURKEY
    (Springer, 2012) Cihan, Arzu Coleri; Tekin, Nilgun; Ozcan, Birgul; Cokmus, Cumhur
    Previously isolated 115 endospore-forming bacilli were basically grouped according to their temperature requirements for growth: the thermophiles (74%), the facultative thermophiles (14%) and the mesophiles (12%). These isolates were taken into 16S rRNA gene sequence analyses, and they were clustered among the 7 genera: Anoxybacillus, Aeribacillus, Bacillus, Brevibacillus, Geobacillus, Paenibacillus, and Thermoactinomycetes. Of these bacilli, only the thirty two isolates belonging to genera Bacillus (16), Brevibacillus (13), Paenibacillus (1) and Thermoactinomycetes (2) were selected and presented in this paper. The comparative sequence analyses revealed that the similarity values were ranged as 91.4-100 %, 91.8- 99.2 %, 92.6- 99.8 % and 90.7 - 99.8 % between the isolates and the related type strains from these four genera, respectively. Twenty nine of them were found to be related with the validly published type strains. The most abundant species was B. thermoruber with 9 isolates followed by B. pumilus (6), B. lichenformis (3), B. subtilis (3), B. agri (3), B. smithii (2), T. vulgaris (2) and finally P. barengoltzii (1). In addition, isolates of A391a, B51a and D295 were proposed as novel species as their 16S rRNA gene sequences displayed similarities <= 97% to their closely related type strains. The AluI-, HaeIII-and TaqI-ARDRA results were in congruence with the 16S rRNA gene sequence analyses. The ARDRA results allowed us to differentiate these isolates, and their discriminative restriction fragments were able to be determined. Some of their phenotypic characters and their amylase, chitinase and protease production were also studied and biotechnologically valuable enzyme producing isolates were introduced in order to use in further studies.
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    Geobacillus thermodenitrificans subsp calidus, subsp nov., a thermophilic and ?-glucosidase producing bacterium isolated from Kizilcahamam, Turkey
    (Microbiol Res Foundation, 2011) Cihan, Arzu Coleri; Ozcan, Birgul; Tekin, Nilgun; Cokmus, Cumhur
    An alpha-glucosidase producing, thermophilic, facultatively anaerobic, and endospore-forming, motile, rod-shaped bacterial strain F84b(T) was isolated from a high temperature well-pipeline sediment sample in Kizilcahamam, Turkey. The growth occurred at temperatures, pH and salinities ranging from 45 to 69 degrees C (optimum 60 degrees C), 7.0 to 8.5 (optimum 8.0) and 0 to 5% (w/v) (optimum 3.5%), respectively. Strain F84b(T) was able to grow on a wide range of carbon sources. Starch and tyrosine utilization, amylase, catalase and oxidase activities, nitrate reduction, and gas production from nitrate were all positive. The G+C content of the genomic DNA was 49.6 mol%. The menaquinone content was MK-7. The dominant cellular fatty acids were iso-C17:0, iso-C15:0, and C16:0. In phylogenetic analysis of 16S rRNA gene sequence, strain F84b(T) showed high sequence similarity to Geobacillus thermodenitrificans (99.8%) and to Geobacillus subterraneus (99.3%) with DNA hybridization values of 74.3% and 29.1%, respectively. In addition, the Rep-PCR and the intergenic 16S-23S rRNA gene fingerprinting profiles differentiated strain F84b(T) from the Geobacillus species studied. The results obtained from the physiological and biochemical characters, the menaquinone contents, the borderline DNA-DNA hybridization homology, and the genomic fingerprinting patterns had allowed phenotypic, chemotaxonomic and genotypic differentiation of strain F84b(T) from G. thermodenitrificans. Therefore, strain F84b(T) is assigned to be a new subspecies of G. thermodenitrificans, for which the name Geobacillus thermodenitrificans subsp. calidus, subsp. nov. is proposed (The type strain F84b(T) = DSM 22629(T) = NCIMB 14582(T)).
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    Phylogenetic diversity of isolates belonging to genera Geobacillus and Aeribacillus isolated from different geothermal regions of Turkey
    (Springer, 2011) Cihan, Arzu Coleri; Ozcan, Birgul; Tekin, Nilgun; Cokmus, Cumhur
    The phylogenetic diversity of 31 thermophilic bacilli belonging to genera Geobacillus and Aeribacillus were investigated which were isolated from various geothermal sites of Turkey. Twenty-seven of these isolates were found to be belonged within the genus Geobacillus, whereas 4 of them were identified as Aeribacillus pallidus. The comparative 16S rRNA gene sequence analyses revealed that the A. pallidus isolates displayed sequence similarity values from 98.0 to 99.6% to their closest relative. Furthermore, Geobacillus isolates showed sequence similarity values from 88.9 to 99.8% with the reference type strains. According to the phylogenetic analysis, isolates belonging to genus Geobacillus were diverged into nine clusters and among these isolates, 19 of them were identified as strains related to G. caldoproteolyticus, G. thermodenitrificans, G. stearothermophilus, G. thermoglucosidasius and G. toebii with the most abundant 13 isolates from G. caldoproteolyticus. Four of the Geobacillus isolates were named as unidentified mix group, as they found to be genetically very homogenous like their closely related type species: G. thermoleovorans, G. vulcani, G. lituanicus, G. kaustophilus, G. caldovelox, G. caldotenax, and G. uralicus. Moreover, the sequence comparisons of E173a, E265, C161ab and A142 isolates demonstrated that they represented novel species among genus Geobacillus as they shared lower than 96.7% sequence similarity to all the described type species. The AluI-, HaeIII- and TaqI-ARDRA results were in congruence with the 16S rRNA gene sequence analyses. By ARDRA results, the isolates were able to be differentiated and clustered, the discriminative restriction fragments of these isolates and type species were determined and the novelty of E173, E265, C161ab and A142 isolates could be displayed. Some differentiating phenotypic characters and the ability of amylase, glucosidase and protease production of these bacilli were also studied and biotechnologically valuable thermostable enzyme producing isolates were introduced in order to use in further studies.
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    Rapid detection of Geobacillus and Anoxybacillus species by quantitative qPCR (qPCR) in commercial dairy products
    (Wiley, 2022) Karaca, Basar; Karakaya, Ayse Busra; Ozcan, Birgul; Cihan, Arzu Coleri
    Thermophilic spore-forming bacteria, especially Anoxybacillus and Geobacillus species, are a major problem for dairy industry. The presence of these thermophilic bacilli in the dairy products is considered a poor hygiene indicator during product processing. The commonly preferred culture-dependent detection methods are time consuming. Therefore, the development of rapid and reliable molecular approaches for the detection of these problematic microorganisms in food processing is essential. In this context, specific primers for the gene (spo0A) that initiates sporulation were designed for Anoxybacillus kamchatkensis subsp. asaccharedens (F81) and Geobacillus thermodenitrificans (DSM 465(T)), which have been shown to be strong biofilm producers in dairy products. For this purpose, a quantitative polymerase chain reaction (qPCR) assay was developed. The qPCR standard curves obtained with these primers allowed a total viable cell count in the range of 10(1)-10(8) CFU/ml for G. thermodenitrificans, while the spore count was in the range of 10(3)-10(8) CFU/ml. The primer developed for A. kamchatkensis subsp. asaccharedens was able to detect viable total cells and spores with much higher sensitivity (10(1)-10(8) CFU/ml viable total cells, 10(2)-10(8) CFU/ml spores). The primers developed in this study allowed separate detection of Anoxybacillus and Geobacillus in dual culture experiments. The primers and method developed in this study can also be used for rapid detection of Anoxybacillus and Geobacillus contamination in mixed cultures.
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    Thermolongibacillus altinsuensis gen. nov., sp nov and Thermolongibacillus kozakliensis sp nov., aerobic, thermophilic, long bacilli isolated from hot springs
    (Microbiology Soc, 2014) Cihan, Arzu Coleri; Koc, Melih; Ozcan, Birgul; Tekin, Nilgun; Cokmus, Cumhur
    Two novel endospore-forming, aerobic bacilli, strains E173a(T) and E265(T), were isolated from soil and sediment samples from Kozakli and Altinsu hot springs, Nevsehir (Turkey). Their young cells in the exponential phase of growth were motile, Gram-stain-positive, straight rods, 0.6-1.1x3.0-8.0 mu m in size, but they became strikingly long, approximately 0.6-1.2 by 9.0-35.0 mu m, after the stationary phase of growth. Cells varied in tests for oxidase, and had a weakly positive reaction for catalase. Both strains could grow between 40 and 70 degrees C, with optimal growth at 60 degrees C (E173a(T)) and 55 degrees C (E265(T)). Growth occurred within the range pH 5.0-11.0 with optimal growth at pH 9.0 (E173a(T)) and pH 8.5 (E265(T)). Strain E173a(T) grew within a salinity range from 0 to 1.5 % (w/v) NaCl with optimal growth at 0.5%, while strain E265(T) grew within the range 0-5.0% (w/v), with an optimum at 3.0%. The new isolates differed from each other in some phenotypic and chemotaxonomic characters as well as repetitive extragenic palindromic element PCR (rep-PCR) fingerprints. 16S rRNA gene sequence similarities suggested distant relationships with other members of the family Bacillaceae (<95.8%), although the two strains showed 97.5% sequence similarity between them, and had 55% relatedness by DNA-DNA hybridization. The DNA G+C contents were 44.8 (E173a(T)) and 43.5 mol% (E265(T)). Moreover, the chemotaxonomic data of E173a(T) and E265(T) [presence of low amounts of meso-diaminopimelic acid, A1 gamma to A1 gamma' cross-linkage types in peptidoglycan, fatty acids including iso-C-15:0 (>60%), iso-C-17:0 and C-16:0] supported the consideration of these isolates as members of a novel genus. Based upon phenotypic, phylogenetic and chemotaxonomic characteristics, it is proposed that new isolates represent a novel genus, Thermolongibacillus gen. nov., with two novel species: Thermolongibacillus altinsuensis sp. nov. (type strain E265(T)=DSM 24979(T)=NCIMB 14850(T)) and Thermolongibacillus kozakliensis sp. nov. (type strain E173a(T)=DSM 24978(T)=NCIMB 14849(T)).