Approaches for diversity analysis of cultivable and non-cultivable bacteriain real soil

Abstract

Until recently, investigators had no idea how accurately cultivated microorganisms represented the overall microbial diversity. The cultivation-dependent approach is limited by the fact that the overwhelming majority of microorganisms present in soil cannot be cultivated under laboratory conditions. The development of molecular phylogenetics has recently enabled characterization of naturally occurring microbial biota without cultivation. There is a vast amount of information held within the genomes of cultivable and non-cultivable microorganisms, and new methods based on analysis of DNA allow to investigate this potential. In this work we show some aspects, advantages and disadvantages of classical and new approaches in taxonomical and functional description of bacteria present in natural microbial assemblages on the example of cultivable bacteria isolated from rhizosphere of plants, tobacco and black nightshade, planted in PCB contaminated soil. Biochemical analysis of isolates showed 8 different bacterial species. This identification was compared by discrimination using MALDI-TOF mass spectrometry and identity evaluation after sequencing of 16S rDNA. Six strains from original number of 8 were positively identified after 16S rDNA sequencing and their phylogenetic relations were compared. These analyses confirmed closed relations of all species (two of isolates exhibited the same characteristics and were discriminated as the same species <I>Pseudomonas stutzeri</I>) and also of <I>Burkholderia xenovorans</I> LB 400, a well-known PCB degrader. Nevertheless, only two isolates gave a positive reaction after amplification of the biphenyl dioxygenase gene and exhibited potential to degrade PCB. These results indicate that only a subset of the recovered molecular information, derived from active population based on molecular and functional analysis is relevant to microbial ecology.

Characterization of the microbiota in the guts of Triatoma brasiliensis and Triatoma pseudomaculata infected by Trypanosoma cruzi in natural conditions using culture independent methods

“…Most are based on culture-dependent methods [ 20 - 22 ]. Approaches based on microbiota cultivation in controlled conditions are estimated to allow the recovery of a limited number of bacterial species in nature [ 23 , 24 ]. Therefore, this method is expected to introduce a bias in the estimation of the real species composition of microbial communities because of the changes induced by the culture process itself [ 25 , 26 ].…”

Comparative analysis of airborne bacteria and fungi in two salt mines in Poland

“…In the salt mine “Polkowice–Sieroszowice,” the air samples were taken from the places of salt mining, ventilation inlet and outlet and miners’ chamber. The culture method, used in this research, has many limitations mentioned by different authors (e.g., Štursa et al 2009 ; Vartoukian et al 2010 ; Rastogi and Sani 2011 ) and the most important of them is that it underestimates the real amount of microbes in the tested samples because a vast majority of microorganisms (> 99%) are non-cultivable and they could be detected only by molecular methods. In case of cultivable microbes, the single colonies visible on plates can be clumps of bacterial and fungal cells and the ones with different nutritional and environmental requirements will not grow together (on common medium and growth conditions).…”

Bacterial strains isolated from PCB‐contaminated sediments and their use for bioaugmentation strategy in microcosms

“… isolated and identified P. aeruginosa and P. stutzeri , respectively, from contaminated soil and confirmed the presence of the bphA gene; Stursa et al . described S. maltophilia as bacteria capable of utilizing PCBs and identified it in the rhizospehere of black nightshade; Ilori et al . proved the ability of A. xylosoxidans to degrade 2,5‐dichlorobenzoate and 2‐chlorobenzoate as the sole carbon and energy source and to effectively degrade chlorinated biphenyls; Ilori et al .…”