A novel culture-independent method to search unknown dominant bacterial groups and its application to microbial analysis of membrane bioreactors

As we could not get numerical information for unknown unculturable microorganisms through conventional culture-independent analysis methods such as next-generation sequencing, or real time PCR, we developed an original culture-independent method, and searched the numerically dominant bacteria in three industrial membrane bioreactors for livestock farms. Although Actinobacteria was the numerically dominant phylum (9.3x105MPN/mL) on 6/August/2014 in the MBR of A farm, when a bacteria with the same genotype to Arthrobacter sp. (AF197047; 4.3x105MPN/mL), and those similar to Burkholderia sp. (AB299593; 4.3x105MPN/mL) were the numerically dominant, after about 13 months (24/October/2015) a number of the Arthrobacter genotype increased to 930x105 MPN (230 times) and become dominant, and those similar to the Microbacterium sp. (AM403628) increased to 92x105MPN, while that of the Burkholderia genotype disappeared. In the other MBR of B farm, bacteria having a similar genotype to Enshifer sp. (AB195268, CP000738), or Shinorhizobium sp. (AF227755, AB195268), or Mesorhizobium sp. (BA000012, Mso.tians29), or Agrobacterium vitis (D12795) was dominant on 18/August/2015 (24x105 MPN) and 30/August/2015 (15.5x105 MPN). In the other MBR of C farm (9/October/2015), bacteria having a similar genotype to uncultured Betaproteobacteria (AY921864) was dominant (430x105MPN), followed by uncultured bacterium (74x105MPN ; AM268745), and Mycobacteriaceae (AB298730), or Propionibacteriaceae (AB298731) (7.4 x105MPN). There was no common bacterial groups among tested three MBRs. Present results indicated that different kinds of homogeneous bacteria were numerically dominant in the three tested membrane bioreactors, where their numbers and ratios were varied with the duration of the driving periods. IMPORTANCEAlthough the conventional molecular-based culture independent methods have been used in place of traditional culture-based methods for microbiological research and expanded information of unculturable low-abundance bacterial groups, not all of them were always highly active in the environment and it was difficult to search for microorganisms among them which were highly active and play an important role in the environment. As numerical data of each bacteria might become an important index to know their activity in environment, we had created a novel culture-independent enumeration method for numerically dominant unidentified bacteria. Through the method, we found that different kinds of homogeneous bacteria were numerically dominant in the three tested membrane bioreactors, whose numbers were high enough to affect the performance of the reactor as a single strain. The method was found useful to specify and trace unknown numerically dominant bacterial groups in a culture independent manner.