Mfabeni Peatland is one of the oldest active peatland regions in Southern Africa (Grundling et al., 2013). It is a biologically diverse ecosystem that supports a wide range of plant and animal life.
Microorganisms contribute significantly to the productivity of wetland systems and play an important role in the cycling of carbon and other nutrients (Gorham et al., 2001). Conditions within Mfabeni peatland are moderately acidic and it experiences fluctuating salinity levels due to seasonal water fluctuations and water infiltration from surrounding costal dune systems (Grundling et al., 2013; Naidoo, 2017). Physico-chemical conditions such as these impacts bacterial community structure and functionality. It stands to reason that microbes adapted to this environment would also produce bioactive compounds that tolerate the prevailing conditions. Since the diversity and functioning of microbes within this ecosystem is largely unexplored it represents an untapped source of microbial diversity with potential biotechnological applications.
A recent study found that genotypically distinct populations of aerobic endospore-forming bacteria (AEFB) were prevalent within a sediment core sample taken from Mfabeni peatland (Naidoo, 2017). Members of the AEFB group are recognized for their ability to produce compounds that are of biotechnological interest (Wipat and Harwood, 1999; Mandic-Mulec and Prosser 2011). In this study, AEFB isolates from Mfabeni peatland were screened for biosurfactant activity. Increasingly, biosurfactants are being recognized for their industrial and environmental applications and have gained favour over synthetic surfactants because of their ecological acceptability (Muthusamy et al., 2008; Mulligan, 2009; Banat et al., 2010; Ławniczak et al., 2013). The purpose of the study, therefore, was to select promising biosurfactant producing isolates for further characterization.
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Isolates were screened for their ability to produce biosurfactants using the oil spreading assay, hemolysis assay, and drop collapse assay. Isolates which demonstrated biosurfactant activity were subjected to different environmental parameters i.e. temperature, pH, and salinity to determine their effect on biosurfactant activity. Surface tension measurement and emulsification (E24) index were determined for selected isolates that showed promising biosurfactant activity levels. These isolates were further identified through 16S rRNA gene sequencing and the biosurfactant compound(s) extracted and characterized through acid precipitation, TLC, and UPLC ESI-TOF MS.
From this study it was established that:
• Most of the AEFB isolates screened demonstrated biosurfactant activity. This result was not unexpected since biosurfactant production contributes to an organism’s ecological fitness and can play an important role in motility, biofilm formation, antagonism and nutrient solubilization.
• The three assays used for the preliminary screening were found to be relatively simple to use and did not require any special equipment. Of the three, the hemolysis assay was found to be the least sensitive, whereas the oil spreading assay was judged to be the best because it is a semi-quantitative method, which gives a direct correlation between the concentration of the biosurfactant produced and the extent of the oil displacement.
• The physicochemical parameters (viz., temperature, pH, and salinity) tested had varying effects on the biosurfactant activity. Most of the isolates were able to maintain their activity over the different temperature ranges tested. Thirty-three percent of the isolates showed highest activity at temperature 55°C. For salinity, varied results were observed.
At pH 3 and 5.5, the negative control exhibited oil displacement. Therefore, the high biosurfactant activity showed by the isolates at low pH had to be viewed with caution and could not be recorded as significant.
• Eight isolates (viz., SAB19, SAB42, SAC15, SAC18, SAD5, SAD17, SAD18, and SAD23) showed significantly higher levels of biosurfactant activity for each of the environmental parameters tested and were selected for further evaluation and characterization. Surface
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tension measurements showed that the isolates were able to reduce surface tension of TSB medium from 57.3 mN/m to between 30.6 mN/m to 44.7 mN/m. Emulsification (E24) efficiency tests using sunflower seed oil and paraffin oil ranged from 19.5% up to 61.85%.
Interestingly, isolates which exhibited the highest emulsification stability (viz., SAB19, SAB42) showed the least capacity for lowering surface tension. This finding is not out of place as it has been reported that surface activity of isolates does not necessarily correlate with their emulsification capacity (Walter et al., 2010).
• All of the selected isolates produced lipopeptide compounds when cultured on Landy medium. Biosurfactant compounds were successfully extracted from each culture medium using an acid precipitation step. UPLC ESI-TOF MS anlysis of extracts revealed that all of the Isolates produced surfactin homologs as well as a hydrophobic compound (m/z 1326.1) that was putatively assigned as a precursor of the antibiotic Plantazolicin (PZN). A number of isolates also produced homologs of iturin/bacillomycin and/or fengycin lipopeptides.
• REP-PCR and 16S rRNA gene sequence analysis were found to be effective tools in distinguishing and identifying the genetic diversity amongst the AEFB isolates. Several groups of closely related strains were distinguished. Taxonomic classification revealed that the isolates could be separated into two genera namely Bacillus and Brevibacillus.
The assignment of strains at species level proved to be difficult due to matches having high levels of similarity (≥99%) to several closely related species. The Bacillus spp. strains fell within the “B.amyloliquefaciens Operational Group”, a sub-group within the clade that makes up the B. subtilis complex of related taxa. The Brevibacillus spp. strains matched closely to strains of Brev. brevis and Brev. formosus. For future studies a Multi-locus sequence typing (MLST) approach targeting various housekeeping genes could be used to characterize isolates to species level.
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