Cytometric fingerprinting for analyzing microbial intracommunity structure variation and identifying subcommunity function

Abstract

Functions of complex natural microbial communities are realized by single cells that contribute differently to the overall performance of a community. Usually, molecular and, more recently, deep-sequencing techniques are used for detailed but resource-consuming phylogenetic or functional analyses of microbial communities. Here we present a method for analyzing dynamic community structures that rapidly detects functional (rather than phylogenetic) coherent subcommunities by monitoring changes in cell-specific and abiotic microenvironmental parameters. The protocol involves the use of flow cytometry to analyze elastic light scattering and fluorescent cell labeling, with subsequent determination of cell gate abundance and finally the creation of a cytometric community fingerprint. Abiotic parameter analysis data are correlated with the dynamic cytometric fingerprint to obtain a time-bound functional heat map. The map facilitates the identification of activity hot spots in communities, which can be further resolved by subsequent cell sorting of key subcommunities and concurrent phylogenetic analysis (terminal restriction fragment length polymorphism, tRFLP). The cytometric fingerprint information is based on gate template settings and the functional heat maps are created using an R script. Cytometric fingerprinting and evaluation can be accomplished in 1 d, and additional subcommunity composition information can be obtained in a further 6 d.

Molecular Biomonitoring of Microbial Communities in Tannery Wastewater Treatment Plant for the Removal of Retanning Chemicals

“…The genus Deinococcus was more abundant in the aerobic reactor than in any other part of the treatment system. Members of the class Betaproteobacteria such as the genera Burkholderia, Rhodocyclus, and Nitrosomonas were identiied from the aerobic system and are inferred to be involved in ammonia oxidation and aromatic compound degradation [36,37].…”

“…Average characteristics of the inluent and eluent wastewaters of the integrated treatment system at the time of sludge sampling (concentrations are in mg/l, except for pH). The phyla Bacteroidetes, Cyanobacteria, Actinobacteria, and Chlorolexi follow the next levels of abundance in the anaerobic reactor, with members identiied in the degradation on both priority nutrients and synthetic aromatic compounds [36,37].…”

“…were processed for taxonomic identiication of the diferent subcommunities using T-RFLP and clone library-based 16S rRNA sequence analysis described in Koch et al [36]. Correlation analyses between the abiotic parameters and the gated subcommunities were done by Spearman's rankorder correlation coeicient using the program R (htp://CRAN.R-project.org) Version 2.14.0.…”

Extracellular electron transfer of biocathodes: Revealing the potentials for nitrate and nitrite reduction of denitrifying microbiomes dominated by Thiobacillus sp.

“…PCR amplification of 16S rDNA and functional denitrification genes was done according to [16] and [17].Representative samples were further cloned and sequenced.…”

Cytometric fingerprints: evaluation of new tools for analyzing microbial community dynamics

“…Therefore, the resulting cytometric fingerprint based on FSC and the DAPI-DNA fluorescence in a 2D histogram (Figure 1C) represents the microbial community structure at the point of measurement. This fingerprint is (almost) unique by the number of cell clusters, the position of these clusters in the histogram, and the numbers of cells within each cluster (Koch et al, 2013c). …”