Two thermophilic bacteria (SK3-4 and DT3-1) were isolated from the Sungai Klah (SK) and Dusun Tua (DT) hot springs in Malaysia. The cells from both strains were rod-shaped, stained Gram positive and formed endospores. The optimal growth of both strains was observed at 55 degrees C and pH 7. Strain DT3-1 exhibited a higher tolerance to chloramphenicol (100 microg ml(-1)) but showed a lower tolerance to sodium chloride (2%, w/v) compared to strain SK3-4. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both strains belong to the genus Anoxybacillus. High concentrations of 15:0 iso in the fatty acid profiles support the conclusion that both strains belong to the genus Anoxybacillus and exhibit unique fatty acid compositions and percentages compared to other Anoxybacillus species. The DNA G + C contents were 42.0 mol% and 41.8 mol% for strains SK3-4 and DT3-1, respectively. Strains SK3-4 and DT3-1 were able to degrade pullulan and to produce maltotriose and glucose, respectively, as their main end products. Based on phenotypic and chemotaxonomic characteristics, 16S rRNA gene sequences, and the DNA G + C content, we propose that strains SK3-4 and DT3-1 are new pullulan-degrading Anoxybacillus strains.
A thermophilic bacterium, Bacillus sp. strain L2 was isolated from a hot spring in Perak, Malaysia. An extracellular lipase activity was detected through plate and broth assays at 70 degrees C after 28 h of incubation. The L2 lipase production was growth dependent as revealed by a number of factors affecting the secretion of extracelullar lipase. As for nutritional factors, casamino acids, trehalose, Ca(2+) and Tween 60 were found to be more effective for lipase production. The optimum physical condition for L2 lipase production was obtained at 70 degrees C after 28 h of cultivation time, at pH 7.0, 150 rpm of agitation rate and 1% of starting inoculum size. The activity staining of crude L2 lipase revealed a clearing zone at 39 kDa.
Geobacillus thermoleovorans CCB_US3_UF5 is a thermophilic bacterium isolated from a hot spring in Malaysia. Here, we report the complete genome of G. thermoleovorans CCB_US3_UF5, which shows high similarity to the genome of Geobacillus kaustophilus HTA 426 in terms of synteny and orthologous genes.
Thermus sp. strain CCB_US3_UF1, a thermophilic bacterium, has been isolated from a hot spring in Malaysia. Here, we present the complete genome sequence of Thermus sp. CCB_US3_UF1.
The ability of thermophilic microorganisms and their enzymes to decompose biomass have attracted attention due to their quick reaction time, thermostability, and decreased risk of contamination. Exploitation of efficient thermostable glycoside hydrolases (GHs) could accelerate the industrialization of biofuels and biochemicals. However, the full spectrum of thermophiles and their enzymes that are important for biomass degradation at high temperatures have not yet been thoroughly studied. We examined a Malaysian Y-shaped Sungai Klah hot spring located within a wooded area. The fallen foliage that formed a thick layer of biomass bed under the heated water of the Y-shaped Sungai Klah hot spring was an ideal environment for the discovery and analysis of microbial biomass decay communities. We sequenced the hypervariable regions of bacterial and archaeal 16S rRNA genes using total community DNA extracted from the hot spring. Data suggested that 25 phyla, 58 classes, 110 orders, 171 families, and 328 genera inhabited this hot spring. Among the detected genera, members of Acidimicrobium, Aeropyrum, Caldilinea, Caldisphaera, Chloracidobacterium, Chloroflexus, Desulfurobacterium, Fervidobacterium, Geobacillus, Meiothermus, Melioribacter, Methanothermococcus, Methanotorris, Roseiflexus, Thermoanaerobacter, Thermoanaerobacterium, Thermoanaerobaculum, and Thermosipho were the main thermophiles containing various GHs that play an important role in cellulose and hemicellulose breakdown. Collectively, the results suggest that the microbial community in this hot spring represents a good source for isolating efficient biomass degrading thermophiles and thermozymes.
Several recent studies have shown the presence of genes for the key enzyme associated with archaeal methane/alkane metabolism, methyl-coenzyme M reductase (Mcr), in metagenome-assembled genomes (MAGs) divergent to existing archaeal lineages. Here, we study the mcr-containing archaeal MAGs from several hot springs, which reveal further expansion in the diversity of archaeal organisms performing methane/alkane metabolism. Significantly, an MAG basal to organisms from the phylum Thaumarchaeota that contains mcr genes, but not those for ammonia oxidation or aerobic metabolism, is identified. Together, our phylogenetic analyses and ancestral state reconstructions suggest a mostly vertical evolution of mcrABG genes among methanogens and methanotrophs, along with frequent horizontal gene transfer of mcr genes between alkanotrophs. Analysis of all mcr-containing archaeal MAGs/genomes suggests a hydrothermal origin for these microorganisms based on optimal growth temperature predictions. These results also suggest methane/alkane oxidation or methanogenesis at high temperature likely existed in a common archaeal ancestor.
Species of Anoxybacillus are widespread in geothermal springs, manure, and milk-processing plants. The genus is composed of 22 species and two subspecies, but the relationship between its lifestyle and genome is little understood. In this study, two high-quality draft genomes were generated from Anoxybacillus spp. SK3-4 and DT3-1, isolated from Malaysian hot springs. De novo assembly and annotation were performed, followed by comparative genome analysis with the complete genome of Anoxybacillus flavithermus WK1 and two additional draft genomes, of A. flavithermus TNO-09.006 and A. kamchatkensis G10. The genomes of Anoxybacillus spp. are among the smaller of the family Bacillaceae. Despite having smaller genomes, their essential genes related to lifestyle adaptations at elevated temperature, extreme pH, and protection against ultraviolet are complete. Due to the presence of various competence proteins, Anoxybacillus spp. SK3-4 and DT3-1 are able to take up foreign DNA fragments, and some of these transferred genes are important for the survival of the cells. The analysis of intact putative prophage genomes shows that they are highly diversified. Based on the genome analysis using SEED, many of the annotated sequences are involved in carbohydrate metabolism. The presence of glycosyl hydrolases among the Anoxybacillus spp. was compared, and the potential applications of these unexplored enzymes are suggested here. This is the first study that compares Anoxybacillus genomes from the aspect of lifestyle adaptations, the capacity for horizontal gene transfer, and carbohydrate metabolism.
Bacillus strain NS 8, a lipase-producing bacterium isolated from a Malaysian hot spring, is able to tolerate a broad range of temperature and pH, which makes it beneficial for this study. It generated PCR products with molecular weight of 1,532 bp, and the 16S rRNA sequence analysis identified it as Bacillus subtilis with accession number AB110598. It showed a 71% similarity index with B. subtilis using Biolog Microstation System. Its lipase production was optimized using a shake flask system by changing the physical (agitation speed, pH and temperature) and nutritional (nitrogen, carbon and minerals) factors. The most suitable combination of the basal medium for lipase production was 2.5% olive oil (carbon), 1.5% peptone (nitrogen), 0.1% MgSO(4) (mineral) at an optimum temperature of 50°C, pH 7.5 and 150 rpm agitation, giving an enzyme yield of 4.23 U/ml. Statistical optimization using response surface methodology was carried out. An optimum lipase production of 5.67 U/ml was achieved when olive oil concentration of 3%, peptone 2%, MgSO(4)·7H(2)O 0.2% and an agitation rate of 200 rpm were combined. Lipase production was further carried out inside a 2-liter bioreactor, which yielded an enzyme activity of 14.5 U/ml after 15 h of incubation.
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.