Molecular Identification and Characterisation of Acid Tolerant Microorganisms Isolated from Rivelin and Limb Valleys

The Rivelin and Limb Valleys in Sheffield have a long history of Industrial activity and were chosen as acidic environments. The first aim was to analyse the microbial diversity present in the collected water samples from both sites using batch cultures. Two bacterial strains (Bacillus cereus and Micrococcus luteus) and two yeast strains (Aureobasidium pullulans and Debaryomyces hansenii) were successfully isolated and identified using 16S and 18S rDNA molecular identification techniques. Physiological characterisations were carried out on all four strains to examine their response to different pH values and high salinity. On the basis of these results, D. hansenii and M. luteus were chosen for further study based on their growth at high salinity at pH 3. Further physiological studies showed that D. hansenii was well adapted to grow at different, extreme conditions in M9 minimal and rich YPD media, while M. luteus required rich LB medium to successfully adapt to combined acid and salt stress. NMR spectroscopy showed when subject to high salinities, M. luteus accumulated betaine as the main compatible solute while D. hansenii accumulated glycerol. Most importantly, glycine betaine was identified as an additional compatible solute in D. hansenii. This is the first report of glycine betaine acting as a compatible solute in a yeast cell. D. hansenii was shown to maintain an internal pH of 6.7 when grown in pH 3 medium and unlike most acid tolerant microorganisms, their membrane potential remained negative when grown at pH 3. pH shock experiments (varying external pH between 3 and 7) suggested that it takes longer than 30 minutes for the D. hansenii cells to return their internal pH to pre-stress levels.