Isolation and characterization of extremely halotolerant Bacillus species from Dead Sea black mud and determination of their antimicrobial and hydrolytic activities

This is the first study that investigated the isolation of extremely halotolerant Bacillus species from Dead Sea black mud. Nine isolates obtained from black mud were considered to be extremely halotolerant Bacillus based on morphological, physiological, and biochemical properties. Most of their colonies were white to light yellow and circular to irregular. All isolates were Gram-positive rod-shaped endospore-forming bacteria, facultative anaerobes, oxidase negative, catalase positive, mesophilic, extremely halotolerant, reacted positively for tryptophan deaminase and Voges-Proskauer, hydrolyzed gelatin and aesculin, and assimilated potassium gluconate. Most of the isolates were found to hydrolyze o-nitrophenyl-beta-D-galactoside (ONPG) and p-nitrophenyl-β-D-galactopyranoside (PNPG) as well as arginine, and assimilate D-mannose, N-acetylglucosamine, D-maltose, and malic acid. All isolates were considered to be nitrate reducers, six of them were nitrite producers and three were N2 producers, suggesting that they may play an important role in nitrification-denitrification processes and in the nitrogen cycle in soil. Based on 16S rRNA gene sequence analysis, the isolates were found to share very high identities (97-99%) with their closest phylogenetic relative and they were assigned to eight Bacillus species (B. oceanisediminis, B. subtilis, B. firmus, B. paralicheniformis, B. methylotrophicus, B. amyloliquefaciens, B. sonorensis, and B. malikii). Interestingly, several enzymatic activities were detected from nonhemolytic isolates DSM2 and DSM7 that were identified as B. paralicheniformis. It was found that only DSM2 isolate produced promising antimicrobial activities. Its aqueous extract showed the highest significant antifungal activity. Whereas, n-butanol and methanol extracts showed significant antibacterial and antifungal activities against human skin pathogens and against other frequent human pathogens.