A selective medium for recovery and enumeration of endolithic bacteria

The study of lithic microbial communities, inhabiting rock substrates has been gathering momentum due to a growing attention of their wide importance as model systems in ecological studies and for their community structure. It is generally accepted that the success of cultivation-based technique is primarily based on suitable culture medium for isolation. The media available for enumeration and recovery of endolithic bacteria are mainly specific to particular type of rock which may not be suitable to isolate endolithic bacterial community from diverse lithobiontic niches. In this study, a new unoptimized medium was formulated, designated LM10 (unoptimized) for enumeration and recovery of endolithic bacteria by addition and/or omission of media components to the basal medium R2G, which was selected after experimental evaluation of five different existing media. The endolithic bacterial count in LM10 medium (unoptimized) was significantly higher than the R2G medium (t=-12.57, p<0.0001). The culture and nutritional parameters associated with unoptimized LM10 medium were optimized using statistical approach to maximize the recovery and enumeration of endolithic bacteria. The first phase of the study comprised of a Plackett-Burman (PB) design experiment conducted to screen thirteen medium components and two culture parameters as variables with effect on bacterial enumeration and recovery. Out of these, Yeast extract, Casein hydrolysate, Glucose, Starch and Sodium thiosulphate were found to be significantly affecting the bacterial count (p<0.05) based on PB design. On keeping rest of the media components and culture conditions at fixed value as per the PB design analyses (p>0.05 and coefficients), further optimization was carried out for significant factors using Box-Behnken design (BBD) of response surface methodology (RSM). Optimized media components obtained by BBD were Yeast extract, Casein hydrolysate, Glucose and Starch in 0.05g/l each and Sodium thiosulphate in 0.047g/l concentrations. The composition of optimized LM10 medium formulated (per litre) is 0.05g Yeast extract, 0.05g Casein hydrolysate, 0.05g Glucose, 0.05g Starch, 0.01g K2HPO4, 0.02g Sodium pyruvate, 0.2g MgSO4, 0.001g FeSO4·7H2O, 0.285g NH4Cl, 0.039g CaCl2·2H2O, 0.047g Na2S2O3·5H2O, 0.002g NaHCO3 and 11g Gellan gum (pH=7.4). Validation of optimized LM10 medium using nine different rock samples from Meghalaya clearly indicated that optimized LM10 medium was better suited for higher recovery and enumeration of endolithic bacteria under both aerobic and anaerobic conditions.