Identification, Characterization, and Hydrolase Producing Performance of Thermophilic Bacteria: Geothermal Hot Springs in Eastern and Southeastern Anatolia Region of Turkey

In the last twenty years, researchers have increasingly focused on the rich microorganism-based diversity of natural hot spring resources to explore the benefits of thermophiles in industrial and biotechnological fields. For this purpose, a total of 83 thermophilic bacilli were isolated from 7 different geothermal hot springs (at temperatures ranging between 40 and 85 ◦ C) located in the east and Southeastern of Turkey. These isolates were identified by different methods such as physiological, morphological, biochemical, molecular properties. According to 16S rRNA gene sequence analysis, 5 different isolates ( Bacillus coagulans , Bacillus licheniformis , Bacillus subtilis , Bacillus thuringiensis , and Geobacillus kaustophilus ) were identified. It was observed that B. licheniformis and B. subtilis were the most species obtained from the researched hot spring sources. Phylogenetic relationships of isolates were evaluated with the help of a phylogenetic tree. The conditions of bacterial isolates to synthesize various hydrolytic enzymes such as protease, cellulase, lipase, and amylase were investigated. When the potential of isolates to produce hydrolytic enzymes was examined, protease 73 (88%), cellulase 34 (41%), lipase 69 (83%), and amylase 68 (82%) were detected. All isolates have at least one or more extracellular protease, cellulase, amylase, or lipase activity. Besides, 32.8% (27) of bacterial isolates were able to synthesize all of the hydrolytic enzymes.

Biofilm-Forming Ability and Effect of Sanitation Agents on Biofilm-Control of Thermophile Geobacillus sp. D413 and Geobacillus toebii E134

Geobacillus sp. D413 and Geobacillus toebii E134 are aerobic, non-pathogenic, endospore-forming, obligately thermophilic bacilli. Gram-positive thermophilic bacilli can produce heat-resistant spores. The bacteria are indicator organisms for assessing the manufacturing process’s hygiene and are capable of forming biofilms on surfaces used in industrial sectors. The present study aimed to determine the biofilm-forming properties of Geobacillus isolates and how to eliminate this formation with sanitation agents. According to the results, extracellular DNA (eDNA) was interestingly not affected by the DNase I, RNase A, and proteinase K. However, the genomic DNA (gDNA) was degraded by only DNase I. It seemed that the eDNA had resistance to DNase I when purified. It is considered that the enzymes could not reach the target eDNA. Moreover, the eDNA resistance may result from the conserved folded structure of eDNA after purification. Another assumption is that the eDNA might be protected by other extracellular polymeric substances (EPS) and/or extracellular membrane vesicles (EVs) structures. On the contrary, DNase I reduced unpurified eDNA (mature biofilms). Biofilm formation on surfaces used in industrial areas was investigated in this work: the D413 and E134 isolates adhered to all surfaces. Various sanitation agents could control biofilms of Geobacillus isolates. The best results were provided by nisin for D413 (80%) and α-amylase for E134 (98%). This paper suggests that sanitation agents could be a solution to control biofilm structures of thermophilic bacilli.

Molecular and Genomic Characterization of PFAB2: A Non-virulent Bacillus anthracis Strain Isolated from an Indian Hot Spring

Background: Thermophilic bacilli in both aerobic or facultative anaerobic forms have been isolated for over a hundred years from different mesophilic or thermophilic environments as they are potential source of bioactive secondary metabolites. But the taxonomic resolution in the Bacillus genus at species or at strain level is very challenging for the insufficient divergence of the 16S rRNA genes. One such recurring problem is among Bacillus anthracis, B. cereus and B. thuringiensis. The disease-causing B. anthracis strains have their characteristic virulence factors coded in two wellknown plasmids, namely pXO1 (toxin genes) and pXO2 (capsule genes). Objective: The present study aimed at the molecular and genomic characterization of a recently reported thermophilic and environmental isolate of B. anthracis, strain PFAB2. Methods: We performed comparative genomics between the PFAB2 genome and different strains of B. anthracis, along with closely related B. cereus strains. Results: The pangenomic analysis suggests that the PFAB2 genome harbors no complete prophage genes. Cluster analysis of Bray-Kurtis similarity resemblance matrix revealed that gene content of PFAB2 is more closely related to other environmental strains of B. anthracis. The secretome analysis and the in vitro and in vivo pathogenesis experiments corroborate the avirulent phenotype of this strain. The most probable explanation for this phenotype is the apparent absence of plasmids harboring genes for capsule biosynthesis and toxins secretion in the draft genome. Additional features of PFAB2 are good spore-forming and germinating capabilities and rapid replication ability. Conclusion: The high replication rate in a wide range of temperatures and culture media, the nonpathogenicity, the good spore forming capability and its genomic similarity to the Ames strain together make PFAB2 an interesting model strain for the study of the pathogenic evolution of B. anthracis.