The Effects of Silver Nanoparticles (AgNPs) on Thermophilic Bacteria: Antibacterial, Morphological, Physiological and Biochemical Investigations.

Since thermophilic microorganisms are valuable sources of thermostable enzymes, it is essential to recognize the potential toxicity of silver nanoparticles used in diverse industrial sectors. Thermophilic bacteria Geobacillus vulcani 2Cx, Bacillus licheniformis 3CA, Paenibacillus macerans 3CA1, Anoxybacillus ayderensis FMB1, and Bacillus paralicheniformis FMB2-1 were selected, and their MIC and MBC values were assessed by treatment with AgNPs in a range of 62.5-1500 µg mL-1. The growth inhibition curves showed that the G. vulcani 2Cx, and B. paralicheniformis FMB2-1 strains were more sensitive to AgNPs, demonstrating a reduction in population by 71.1% and 31.7% at 62.5 µg mL-1 and by 82.9% and 72.8% at 250 µg mL-1, respectively. TEM and FT-IR analysis revealed that AgNPs caused structural damage, cytoplasmic leakage, and disruption of cellular integrity. Furthermore, cell viability showed a significant decrease alongside an increase in superoxide radical (SOR; O2-) production. ß-galactosidase biosynthesis decreased to 28.8% level at 500 µg mL-1 AgNPs for G. vulcani 2Cx, 32.2% at 250 µg mL-1 for A. ayderensis FMB1, and 38.8% only at 62.5 µg mL-1, but it was completely inhibited at 500 µg mL-1 for B. licheniformis 3CA. Moreover, B. paralicheniformis FMB2-1 showed a significant decrease to 11.2% at 125 µg mL-1. This study is the first to reveal the toxic effects of AgNPs on thermophilic bacteria.

Acinetobacter mesopotamicus sp. nov., Petroleum-degrading Bacterium, Isolated from Petroleum-Contaminated Soil in Diyarbakir, in the Southeast of Turkey.

A new petroleum-degrading bacterium, designated strain GC2T, was isolated from Bozkus 1 petroleum station in Diyarbakir, located in the southeast of Turkey. Cells were Gram-negative staining, aerobic, coccoid-rods, non-motile, non-spore-forming. The bacterium was found to degrade 100% of n-alkanes ranging from C11 to C34 presented in the 1% crude oil after incubation of 7 days. The membrane phospholipids were 1,2 diacylglycero-3-phosphorylethanolamine (PEA), phosphatidylglycerol (PG), dipalmitoyl-sn-glycerol 1- phosphocholine (PC1), 1,2 dipalmitoyl-sn-glycero-3-phosphocholine monohydrate (PC3), cardiolipin also called diphosphatidylglycerol (CL) and l-α- phosphatidic acid, dipalmitoyl (AP); predominant respiratory ubiquinone was Q-8 and C16:0, C18:1ω9c and C16:1 were the major cellular fatty acids. The 16S rRNA sequence analysis revealed that the strain GC2T was a member of genus Acinetobacter and was most closely related to Acinetobacter lwoffii DSM 2403 T (99.79%), Acinetobacter pseudolwoffii ANC 5318 T (98.83%) and Acinetobacter harbinensis HITLi 7 T (98.14%). The rpoB and gyrB gene sequence analysis confirmed that the strain GC2T was a member of genus Acinetobacter and that the closest relative was Acinetobacter lwoffii DSM 2403 T (99.08% and 100% similarity, respectively). DNA-DNA hybridization values between GC2T and its closest relatives ranged from 65.6% (with A. lwoffii) to 5.1% (with A. venetianus). The whole genome sequence of strain GC2T was obtained. The DNA G + C content of this strain was determined to be 42.9 mol %. ANI indexes, in silico estimations of DDH values and wet lab DDH values demonstrated that strain GC2T represents an independent genomospecies. On the basis of phenotypic characteristics, chemotaxonomic, phylogenetic data and DNA-DNA hybridization and whole genome analysis, we propose to assign strain GC2T as a new species of the genus Acinetobacter, for which the name Acinetobacter mesopotamicus sp. nov. is proposed. The type strain of this species is GC2T (DSM 26953 T = JCM 31073 T). The whole genome of strain GC2T has been deposited at DDBJ/ENA/GenBank under the accession JAALFF010000000.

Effects of endosulfan, thiamethoxam, and indoxacarb in combination with atrazine on multi-biomarkers in Gammarus kischineffensis.

Studies addressing the toxicity of pesticides towards non-target organisms focus on the median lethal concentration and biochemical response of individual pesticides. However, when determining environmental risks, it is important to test the combined effects of pesticides, such as insecticides and herbicides, which are frequently used together in agricultural areas. Here we aimed to investigate the toxic effects of the combined use of the herbicide atrazine and the insecticides, endosulfan, indoxacarb, and thiamethoxam on Gammarus kischineffensis. To do this, we tested the activities of oxidative stress, detoxification, and neurotoxicity biomarkers. Compared to atrazine alone, we detected higher glutathione-S-transferase, catalase and superoxide dismutase activities (oxidative stress biomarkers) when atrazine was combined with either endosulfan or indoxacarb. However, higher IBR values were determined in organisms where pesticide mixtures were used according to individual use. Based on these results, mixtures of atrazine and other pesticides may cause synergistic effects and may be evidence of increased toxicity and oxidative stress.

Purification and Characterization of Thermostable and Detergent-Stable α-Amylase from Anoxybacillus sp. AH1.

A thermostable and detergent-stable α-amylase from a newly isolated Anoxybacillus sp. AH1 was purified and characterized. Maximum enzyme production (1874.8 U/mL) was obtained at 24 h of incubation. The amylase was purified by using Sephadex G-75 gel filtration, after which an 18-fold increase in specific activity and a yield of 9% were achieved. The molecular mass of the purified enzyme was estimated at 85 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature values of the enzyme were 7.0 and 60 °C, respectively. The enzyme was highly stable in the presence of 30% glycerol, retaining 85% of its original activity at 60 °C within 120 min. Km and vmax values were 0.102 µmol and 0.929 µmol/min, respectively, using Lineweaver-Burk plot. The enzyme activity was increased by various detergents, but it was significantly inhibited in the presence of urea. Mg2+ and Ca2+ also significantly activated α-amylase, while Zn2+, Cu2+ and metal ion chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline (phen) greatly inhibited the enzyme activity. α-Amylase activity was enhanced by ß-mercaptoethanol (ß-ME) and dithiothreitol (DTT) to a great extent, but inhibited by p-chloromercuribenzoic acid (PCMB). Iodoacetamide (IAA) and N-ethylmaleimide (NEM) had a slight, whereas phenylmethylsulfonyl fluoride (PMSF) had a strong inhibitory effect on the amylase activity.

Extremely low-frequency magnetic field induces manganese accumulation in brain, kidney and liver of rats.

The aim of the present study was to determine the effects of extremely low-frequency magnetic field (ELF-MF) on accumulation of manganese (Mn) in the kidney, liver and brain of rats. A total of 40 rats were randomly divided into eight groups. Four control groups received 0, 3.75, 15 and 60 mg Mn per kg body weight orally every 2 days for 45 days, respectively. The remaining four groups received same concentrations of Mn and were also exposed to ELF-MF (1.5 mT; 50 Hz) for 4 h for 5 days a week during 45 days. Following the last exposure, kidney, liver and brain were taken from all rats and they were analyzed for Mn accumulation levels using an inductively coupled plasma-optical emission spectrometer. In result of the current study, we observed that Mn levels in brain, kidney and liver were higher in Mn groups than in control groups. Mn levels in brain, kidney and liver were also higher in Mn plus ELF-MF groups than in Mn groups. In conclusion, result of the current study showed that the ELF-MF induced manganese accumulation in kidney, liver and brain of rats.

Cd, Cu, Ni, Mn and Zn resistance and bioaccumulation by thermophilic bacteria, Geobacillus toebii subsp. decanicus and Geobacillus thermoleovorans subsp. stromboliensis.

Bioaccumulation and heavy metal resistance of Cd(2+), Cu(2+), Ni(2+), Zn(2+) and Mn(2+) ions by thermophilic Geobacillus toebii subsp. decanicus and Geobacillus thermoleovorans subsp. stromboliensis were investigated. The metal resistance from the most resistant to the most sensitive was found as Mn > Ni > Cu > Zn > Cd for both Geobacillus thermoleovorans subsp. stromboliensis and Geobacillus toebii subsp. decanicus. It was determined that the highest metal bioaccumulation was performed by Geobacillus toebii subsp. decanicus for Zn (36,496 µg/g dry weight cell), and the lowest metal bioaccumulation was performed by Geobacillus toebii subsp. decanicus for Ni (660.3 µg/g dry weight cell). Moreover, the dead cells were found to biosorbe more metal in their membranes compared to the live cells. In the presence of 7.32 mg/l Cd concentration, the levels of Cd absorbed in live and dead cell membranes were found as 17.44 and 46.2 mg/g membrane, respectively.

The effects of long-term exposure to extremely low-frequency magnetic fields on bone formation in ovariectomized rats.

The effects of long-term extremely low-frequency magnetic field (ELF-MF) exposure on bone formation and biochemical markers were investigated in ovariectomized rats. Sixty mature female Sprague-Dawley rats were randomly divided into four different groups (n = 15): (i) unexposed control (CTL); (ii) ovariectomized only (OVX); (iii) non-ovariectomized, exposed (SHAM + ELF-MF); and (iv) ovariectomized, exposed (OVX + ELF-MF). The third and fourth groups were exposed to 1.5 mT ELF-MF for 4 h a day for 6 months. Bone mineral density (BMD) was determined using dual energy X-ray absorption (DEXA) measurements. The formation and resorption of bone were evaluated using bone-specific alkaline phosphatase (BAP), osteocalcin, osteoprotogerin, and N-telopeptide. After 6 months of ELF-MF therapy, BMD values were significantly lower in the OVX group and higher in the OVX + ELF-MF and SHAM + ELF-MF groups than they were before therapy (P < 0.001). Although there was no significant difference in BMD values among the groups before therapy, the BMD values increased significantly after 6 months in the OVX + ELF-MF and SHAM + ELF-MF groups and were reduced in the OVX group compared to the CTL group (P < 0.001). The concentrations of BAP, osteocalcin, osteoprotogerin, and N-telopeptide in the three experimental groups also changed in a significant way compared to the CTL group. The results of the present study suggest that osteoporosis can be inhibited by ELF-MF stimulation treatments. It was also concluded that ELF-MF may be useful in the prevention of osteoporosis in ovariectomized rats.

Geobacillus subterraneus subsp. aromaticivorans subsp. nov., a novel thermophilic and alkaliphilic bacterium isolated from a hot spring in Sirnak, Turkey.

A new thermophilic spore-forming strain Ge1(T) was isolated from the Guclukonak hot spring in Sirnak, Turkey. The strain was identified by using a polyphasic taxonomic approach. Strain Ge1(T) was Gram-positive, spore-forming, alkaliphilic rod-shaped, motile, occurring in pairs or filamentous. Growth was observed between 30 and 65°C (optimum 60°C) and at pH 5.5-10.0 (optimum pH 9.0). It was capable of utilizing starch, growth was observed at 0-3% NaCl (w/v) and was positive for catalase and urease. The major cellular fatty acids were iso-C(15:0) and iso-C(17:0), and the predominant lipoquinone found was menaquinone MK7 type. The DNA G+C content of the genomic DNA of strain Ge1(T) was 52.0%. Comparative 16S rRNA gene sequence studies showed that the isolate belonged to the genus Geobacillus. The DNA-DNA hybridization mean values between the representative strain Ge1(T) and the closely related species G. subterraneus, G. thermodenitrificans, G. thermocatenulatus, G. vulcani and G. thermoleovorans were 69.3%, 57%, 37%, 27% and 26%, respectively. The results of DNA-DNA hybridization, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain Ge1(T). Based on these results, we propose assigning a novel subspecies of Geobacillus subterraneus, to be named as Geobacillus subterraneus subsp. aromaticivorans subsp. nov. with the type strain Ge1(T) (DSM 23066 (T)= CIP 110341(T)).

Production and characterization of partially purified extracellular thermostable α-amylase by Bacillus subtilis in submerged fermentation (SmF).

A Bacillus strain was isolated from soil samples from the campus area of Dicle University. Based on 16S ribosomal RNA sequencing, the microorganism was closely related to Bacillus subtilis. Effects of different culture medium, incubation time, carbon and nitrogen sources, and various starches, flours, and chemicals on α-amylase production were examined. Maximum enzyme production (7516 U/mL) was obtained in a basal medium A containing 0.05% Tween 40 in 24 h. Partially purified enzyme showed maximum activity at 60 °C with an optimum pH of 6.0. The effects of 0.2% detergents (sodium dodecyl sulfate [SDS], CHAPS [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate], and commercial detergent Omo Matic) on partially purified enzyme activity over a period of time (15-150 min) were examined and the order of inhibition effect from the most to the least was found as SDS > Omo Matic > CHAPS. Different metal ions inhibited α-amylase activity at low concentrations (1.5 mM). Co²âº was a mild inhibitor and Hg²âº and Cd²âº were potent inhibitors, whereas Ca²âº and Mg²âº increased the enzyme activity. At 20 mM, Ca²âº enhanced enzyme activity, and different Ca²âº concentrations (10-300 mM) were studied.

Anoxybacillus kamchatkensis subsp. asaccharedens subsp. nov., a thermophilic bacterium isolated from a hot spring in Batman.

A new thermophilic spore-forming strain KG8(T) was isolated from the mud of Taslidere hot spring in Batman. Strain KG8(T) was aerobe, Gram-positive, rod-shaped, motile, occurring in pairs or filamentous. Growth was observed from 35-65 degrees C (optimum 55 degrees C) and at pH 5.5-9.5 (optimum pH 7.5). It was capable of utilizing starch, growth was observed until 3% NaCl (w/v) and it was positive for nitrate reduction. On the basis of 16S rRNA gene sequence similarity, strain KG8(T) was shown to be related most closely to Anoxybacillus species. Chemotaxonomic data (major isoprenoid quinone-menaquinone-7; major fatty acid-iso-C15:0 and iso-C17:0) supported the affiliation of strain KG8(T) to the genus Anoxybacillus. The results of DNA-DNA hybridization, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain KG8(T). Based on these results we propose assigning a novel subspecies of Anoxybacillus kamchatkensis, to be named Anoxybacillus kamchatkensis subsp. asaccharedens subsp. nov. with the type strain KG8(T) (DSM 18475(T)=CIP 109280(T)).