Resistance, removal, and bioaccumulation of Ni (II) and Co (II) and their impacts on antioxidant enzymes of Anoxybacillus mongoliensis.

In this study, it was hypothesis that A. mongoliensis could be used as bioindicator for Ni (II) and Co (II). Thus, Ni (II) and Co (II) resistance, removal, bioaccumulation, and the impacts of them on antioxidant enzyme systems of thermophilic Anoxybacillus mongoliensis were investigated in details. The bioaccumulation of Ni (II) and Co (II) on the cell membrane of thermophilic A. mongoliensis, variations on surface macrostructure and functionality by FT-IR and SEM, and determination of antioxidant enzyme activities were also tested. The highest bioaccumulation values of Co (II) and Ni (II) were detected as 102.0 mg metal/g of dry bacteria at 10 mg/L for the 12th h and 90.4 mg metal/g of dry bacteria for the 24th h, respectively, and the highest Ni (II) and Co (II) cell membrane bioaccumulation capacities of A. mongoliensis were determined as 268.5 and 274.9 mg metal/g wet membrane, respectively at the 24th h. In addition, increasing on SOD and CAT activities were observed on depend of concentration of Ni (II) and Co (II) with respect to control. The antioxidant enzyme activity results also indicated that A. mongoliensis might be used as a bioindicator for Ni (II) and Co (II) pollution in environmental water specimens.

Low frequency ultrasound inactivation of thermophilic bacilli (Geobacillus spp. and Anoxybacillus flavithermus) in the presence of sodium hydroxide and hydrogen peroxide.

The vegetative cells and spores of Geobacillus spp. and Anoxybacillus flavithermus were subjected to 20 kHz ultrasound with a power ∼8 W. Ultrasonication had considerable effect on vegetative cells (5-log reduction in Geobacillus spp. and 1.6-log reduction in A.flavithermus). TEM imaging of the ultrasonicated vegetative cells showed an extensive damage both internally and externally. However, spores showed high resistance towards ultrasound treatment in the absence of NaOH and H2O2, although the outer layers such as the exosporium and the outer coat layer were disrupted, resulting in the reduced resistance of spores towards sonication. The combination of 0.12 M NaOH and 10 min ultrasonication inactivated 6 log spores of Geobacillus spp. A 7 log spore reduction of A.flavithermus was achieved by combining 0.17 M NaOH with 10 min ultrasonication. Ultrasonication combined with 1% H2O2 inactivated ∼7 log Geobacillus spp. spores in 6 min and ∼7 log A.flavithermus spores in 3 min. These ultrasound treatments in the presence of NaOH and H2O2 are synergistic as they showed a greater spore reduction when compared to NaOH combined with high temperature (85 °C), where only 1 and 3 log reduction was achieved in Geobacillus spp. and A.flavithermus spores, respectively.

Global transcriptomic response of Anoxybacillus sp. SK 3-4 to aluminum exposure.

Anoxybacillus sp. SK 3-4 is a Gram-positive, rod-shaped bacterium and a member of family Bacillaceae. We had previously reported that the strain is an aluminum resistant thermophilic bacterium. This is the first report to provide a detailed analysis of the global transcriptional response of Anoxybacillus when the cells were exposed to 600 mg L-1 of aluminum. The transcriptome was sequenced using Illumina MiSeq sequencer. Total of 708 genes were differentially expressed (fold change >2.00) with 316 genes were up-regulated while 347 genes were down-regulated, in comparing to control with no aluminum added in the culture. Based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the majority of genes encoding for cell metabolism such as glycolysis, sulfur metabolism, cysteine and methionine metabolism were up-regulated; while most of the gene associated with tricarboxylic acid cycle (TCA cycle) and valine, leucine and isoleucine metabolism were down-regulated. In addition, a significant number of the genes encoding ABC transporters, metal ions transporters, and some stress response proteins were also differentially expressed following aluminum exposure. The findings provide further insight and help us to understand on the resistance of Anoxybacillus sp. SK 3-4 toward aluminium.

Changes in Sodium, Calcium, and Magnesium Ion Concentrations That Inhibit Geobacillus Biofilms Have No Effect on Anoxybacillus flavithermus Biofilms.

This study investigated the effects of varied sodium, calcium, and magnesium concentrations in specialty milk formulations on biofilm formation by Geobacillus spp. and Anoxybacillus flavithermus. The numbers of attached viable cells (log CFU per square centimeter) after 6 to 18 h of biofilm formation by three dairy-derived strains of Geobacillus and three dairy-derived strains of A. flavithermus were compared in two commercial milk formulations. Milk formulation B had relatively high sodium and low calcium and magnesium concentrations compared with those of milk formulation A, but the two formulations had comparable fat, protein, and lactose concentrations. Biofilm formation by the three Geobacillus isolates was up to 4 log CFU cm(-2) lower in milk formulation B than in milk formulation A after 6 to 18 h, and the difference was often significant (P ≤ 0.05). However, no significant differences (P ≤ 0.05) were found when biofilm formations by the three A. flavithermus isolates were compared in milk formulations A and B. Supplementation of milk formulation A with 100 mM NaCl significantly decreased (P ≤ 0.05) Geobacillus biofilm formation after 6 to 10 h. Furthermore, supplementation of milk formulation B with 2 mM CaCl2 or 2 mM MgCl2 significantly increased (P ≤ 0.05) Geobacillus biofilm formation after 10 to 18 h. It was concluded that relatively high free Na(+) and low free Ca(2+) and Mg(2+) concentrations in milk formulations are collectively required to inhibit biofilm formation by Geobacillus spp., whereas biofilm formation by A. flavithermus is not impacted by typical cation concentration differences of milk formulations.

Characterization of aluminum resistant Anoxybacillus sp. SK 3-4 isolated from a hot spring.

The Anoxybacillus sp. SK 3-4, previously isolated from a hot spring, was screened for its heavy metals resistance (Al(3+), Mn(2+), Cu(2+), Co(2+), Zn(2+), and Ni(2+)) and the strain was found to be most resistant to aluminum. Significant growth of the strain was observed when it was grown in medium containing aluminum (200 mg L(-1)-800 mg L(-1)) with relative growth rates ranging between 77% and 100%. A gene encoding the aluminum resistance protein (accession number: WP_021095658.1) was found in genome of strain SK 3-4, which revealed high sequence identity (>95%) to its homologues from Anoxybacillus species. Sequence comparisons with two functionally characterized aluminum resistance proteins, namely G2alt and ALU1-P, showed 97% and 81% of sequence identity, respectively. Four putative metal binding sites were detected in SK 3-4 aluminum resistance protein and G2alt at same amino acid residue positions of 186, 195, 198, and 201. Strain SK 3-4 was found to be able to remove aluminum from aqueous solution. This study demonstrated that Anoxybacillus sp. SK 3-4 could be applied in the treatment of aluminum contaminated wastewater.

Influence of cations on growth of thermophilic Geobacillus spp. and Anoxybacillus flavithermus in planktonic culture.

Free ions of Na(+), K(+), Ca(2+), and Mg(2+) influenced the optical density of planktonic cultures of thermophilic bacilli. Anoxybacillus flavithermus E16 and Geobacillus sp. strain F75 (milk powder manufacturing plant isolates) and A. flavithermus DSM 2641 and G. thermoleovorans DSM 5366 were studied. Ca(2+) and Mg(2+) were associated with increases in optical density more so than Na(+) and K(+). Overall, it appeared that Ca(2+) and/or Mg(2+) was required for the production of protein in thermophilic bacilli, as shown by results obtained with A. flavithermus E16, which was selected for further study.

Isolation and characterization of a novel organic solvent-tolerant Anoxybacillus sp. PGDY12, a thermophilic Gram-positive bacterium.

AIMS: To isolate and characterize new bacteria capable of tolerating high concentrations of organic solvents at high temperature. METHODS AND RESULTS: A solvent-tolerant, thermophilic bacterium was isolated from hot spring samples at 55°C. The strain PGDY12 was characterized as a Gram-positive bacterium. It was able to tolerate 100% solvents, such as toluene, benzene and p-xylene on plate overlay and high concentrations of these solvents in liquid cultures. A comparison of growth showed that 0·2% (v/v) benzene and 0·15% (v/v) p-xylene were capable of enhancing the final cell yields. Transmission electron micrographs showed the incrassation of electron-transparent intracellular material and the distorted cytoplasm in case of the cells grown in toluene. A phylogenetic analysis based on 16S rRNA sequence data indicated that the strain PGDY12 was member of the genus Anoxybacillus. CONCLUSIONS: The thermophilic, Gram-positive Anoxybacillus sp. PGDY12 exhibited a unique and remarkable ability to tolerate solvents at 55°C. SIGNIFICANCE AND IMPACT OF THE STUDY: The solvent tolerance properties are less known in thermophilic bacteria. The Anoxybacillus sp. PGDY12 is the first strictly thermophilic bacterium able to tolerate a broad range of solvents. This strain is a promising candidate for use as a high temperature biocatalyst in the biotechnological applications.