A PCR-MPN BASED QUANTITATIVE APPROACH TO ENUMERATE NITRIFYING BACTERIA IN ZEOPONIC SUBSTRATES

Self-sustaining, regenerative life-support systems are required for long duration missions to the Moon and Mars. Improved activity of nitrifying bacteria to convert NH4+ to NO3- has been shown to promote plant growth in zeoponic substrates. Due to physiological characteristics, such as slow growth and low yield, nitrifying bacteria are not easily enumerated by traditional microbiological techniques. A method for rapid detection and enumeration of a commercial inoculum of nitrifying bacteria in a zeoponic substrate was developed using a polymerase chain reaction (PCR)-most probable number (MPN) approach. Samples from four-week laboratory incubation studies were processed to extract their total microbial community DNA and the sequences specific to 16s rRNA of Nitrobacter spp. were PCR amplified. The detection limit of the methodology was 2,000 Nitrobacter cells per assay. The quantitative assay demonstrated that the zeoponic substrate was capable of supporting 105 to 106 MPN Nitrobacter cells per gram of substrate. The PCR-MPN method can be an effective and rapid approach to enumerate nitrifying bacteria in zeoponic substrates.

[1]  G. Soulas,et al.  DNA Extraction from Soils: Old Bias for New Microbial Diversity Analysis Methods , 2001, Applied and Environmental Microbiology.

[2]  E. Wellington,et al.  Comparison of different methods for the isolation and purification of total community DNA from soil. , 1999, Journal of microbiological methods.

[3]  V. Ramisse,et al.  Quantification of Bias Related to the Extraction of DNA Directly from Soils , 1999, Applied and Environmental Microbiology.

[4]  M. Penninckx,et al.  Nitrification and Autotrophic Nitrifying Bacteria in a Hydrocarbon-Polluted Soil , 1999, Applied and Environmental Microbiology.

[5]  D. Ming,et al.  Nutrient-substituted hydroxyapatites: synthesis and characterization. , 1999, Soil Science Society of America journal. Soil Science Society of America.

[6]  V. Degrange,et al.  Detection and counting of Nitrobacter populations in soil by PCR , 1995, Applied and environmental microbiology.

[7]  P. Normand,et al.  Characterization of natural populations of Nitrobacter spp. using PCR/RFLP analysis of the ribosomal intergenic spacer , 1992, Archives of Microbiology.