Improved Most-Probable-Number Method To Detect Sulfate-Reducing Bacteria with Natural Media and a Radiotracer

A greatly improved most-probable-number (MPN) method for selective enumeration of sulfate-reducing bacteria (SRB) is described. The method is based on the use of natural media and radiolabeled sulfate ((35)SO(4)(2−)). The natural media used consisted of anaerobically prepared sterilized sludge or sediment slurries obtained from sampling sites. The densities of SRB in sediment samples from Kysing Fjord (Denmark) and activated sludge were determined by using a normal MPN (N-MPN) method with synthetic cultivation media and a tracer MPN (T-MPN) method with natural media. The T-MPN method with natural media always yielded significantly higher (100- to 1,000-fold-higher) MPN values than the N-MPN method with synthetic media. The recovery of SRB from environmental samples was investigated by simultaneously measuring sulfate reduction rates (by a (35)S-radiotracer method) and bacterial counts by using the T-MPN and N-MPN methods, respectively. When bacterial numbers estimated by the T-MPN method with natural media were used, specific sulfate reduction rates (qSO(4)(2−)) of 10(−14) to 10(−13) mol of SO(4)(2−) cell(−1) day(−1) were calculated, which is within the range of qSO(4)(2−) values previously reported for pure cultures of SRB (10(−15) to 10(−14) mol of SO(4)(2−) cell(−1) day(−1)). qSO(4)(2−) values calculated from N-MPN values obtained with synthetic media were several orders of magnitude higher (2 × 10(−10) to 7 × 10(−10) mol of SO(4)(2−) cell(−1) day(−1)), showing that viable counts of SRB were seriously underestimated when standard enumeration media were used. Our results demonstrate that the use of natural media results in significant improvements in estimates of the true numbers of SRB in environmental samples.