Abstract
D.Tech. (Biotechnology)
Hot-spring environments are valuable resources for novel and useful bacteria in biotechnology.
In South Africa, hot springs in Limpopo Province have been investigated by metagenomics but
not by culture-based methods. This study was performed in order to describe the diversity of
cultured bacteria from hot springs, and to comment on their relevance to public health and
antibiotic resistance. Furthermore, potential bacterial candidates were screened for
characteristics associated with wastewater (WW) bioremediation and applications.
Cultured bacteria were identified by a combination of tools using the 16S rDNA sequence by
comparison with public databases, percent guanine-cytosine content, amplified rDNA restriction
analysis (ARDRA) and phylogeny. Resistance against ten antibiotics (carbenicillin, gentamicin,
kanamycin, streptomycin, tetracycline, chloramphenicol, ceftriaxone, co-trimoxazole, nalidixic
acid and norfloxacin) and eight heavy metal ions (Al, Cr, Cu, Fe, Hg, Mn, Ni and Pb) were
tested using disk diffusion assays. Isolates were screened for production of enzymes and
biophysical characteristics (bioflocculant and biosurfactant activities, biosorption,
bioassimilation, anti-biofilm and antimicrobial properties) and selected cell free culture
supernatants (CFCS) were processed by tandem LC-MS for identification of the bioactive
molecules. CFCS were exposed to pollutants (pigmented food, textile dyes, polyaromatic
hydrocarbons) and food WW samples to establish whether there was a reduction in turbidity or
phenol levels.
Forty-three (43) Gram-positive isolates were of the phylum Firmicutes with the majority of the
genus Bacillus (n = 31). Different species were identified as Anoxybacillus flavithermus,
Anoxybacillus rupiensis, Bacillus subtilis, Bacillus licheniformis and Brevibacillus spp. Single
isolates of Gram-positive Kocuria sp. and Arthrobacter sp., and Gram-negative Cupriavidus sp.,
Ralstonia sp., Cronobacter sp., Tepidimonas sp., Hafnia sp. and Sphingomonas sp. were
identified, previously reported to be emerging opportunistic pathogens, and an absence of
Legionella sp. was reported. Low levels of antibiotic resistance (AR) were reported. Only 2.5%
(n = 40) of the isolates were multiple antibiotic resistant against >3 antibiotics, while 37.5%
were found for both resistance against one or two antibiotics. Resistance was found against
ceftriaxone (52.5%), nalidixic acid (37.5%) and carbenicillin (22.5%). All 29 isolates tested
heavy metals were tolerant to ≥2 heavy-metal salts. No association was observed between
antibiotic resistance and heavy-metal tolerance.
Amylase and protease positive isolates, and isolates that could discolorize bromothymol blue
were detected by conventional agar plate assays. The CFCS of A. rupiensis 19S was selected for...