Thomas Hübschmann

Limits of propidium iodide as a cell viability indicator for environmental bacteria.

Viability measurements of individual bacteria are applied in various scopes of research and industry using approaches where propidium iodide (PI) serves as dead cell indicator. The reliability of PI uptake as a cell viability indicator for dead (PI permeable) and viable (PI impermeable) bacteria was tested using two soil bacteria, the gram−Sphingomonas sp. LB126 and the gram+Mycobacterium frederiksbergense LB501T.

Electroactive mixed culture derived biofilms in microbial bioelectrochemical systems: the role of pH on biofilm formation, performance and composition.

The pH-value played a crucial role for the development and current production of anodic microbial electroactive biofilms. It was demonstrated that only a narrow pH-window, ranging from pH 6 to 9, was suitable for growth and operation of biofilms derived from pH-neutral wastewater. Any stronger deviation from pH neutral conditions led to a substantial decrease in the biofilm performance. Thus, average current densities of 151, 821 and 730 μA cm(-2) were measured for anode biofilms grown and operated at pH 6, 7 and 9 respectively. The microbial diversity of the anode chamber community during the biofilm selection process was studied using the low cost method flow-cytometry. Thereby, it was demonstrated that the pH value as well as the microbial inocula had an impact on the resulting anode community structure. As shown by cyclic voltammetry the electron transfer thermodynamics of the biofilms was strongly depending on the solutions pH-value.

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

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.

Revealing the electrochemically driven selection in natural community derived microbial biofilms using flow-cytometry

In this communication we demonstrate that electrochemically active microbial biofilms and their enrichment at the anode of a microbial bioelectrochemical system (BES) can be quantitatively and easily characterized by flow-cytometry. This analysis revealed that the anodic biofilm of a BES, formed from a highly diverse microbial community and fed with single substrate artificial wastewater, was dominated by only one phylotype.

Correlation of community dynamics and process parameters as a tool for the prediction of the stability of wastewater treatment.

Wastewater treatment often suffers from instabilities and the failure of specific functions such as biological phosphorus removal by polyphosphate accumulating organisms. Since most of the microorganisms involved in water clarification are unknown it is challenging to operate the process accounting for the permanent varying abiotic parameters and the complex composition and unrevealed metabolic capacity of a wastewater microbial community. Fulfilling the demands for water quality irrespective of substrate inflow conditions may emit severe problems if the limited management resources of municipal wastewater treatment plants are regarded. We used flow cytometric analyses of cellular DNA and polyphosphate to create patterns mirroring dynamics in community structure. These patterns were resolved in up to 15 subclusters, the presence and abundances of which correlated with abiotic data. The study used biostatistics to determine the kind and strength of the correlation. Samples investigated were obtained from a primary clarifier and two activated sludge basins. The stability of microbial community structure was found to be high in the basins and low in the primary clarifier. Despite major abiotic changes certain subcommunities were dominantly present (up to 80% stability), whereas others emerged only sporadically (down to 3% stability, both according to equivalence testing). Additionally, subcommunities of diagnostic value were detected showing positive correlation with substrate influxes. For instance blackwater (r(s) = 0.5) and brewery inflow (both r(s) = 0.6) were mirrored by increases in cell abundances in subclusters 1 and 6 as well as 4 and 8, respectively. Phosphate accumulation was obviously positively correlated with nitrate (r(s) = 0.4) and the presence of denitrifying organisms (Rhodacyclaceae). Various other correlations between community structure and abiotic parameters were apparent. The bacterial composition of certain subcommunities was determined by cell sorting and phylogenetic tools like T-RFLP. In essence, we developed a monitoring tool which is quick, cheap and causal in its interpretation. It will make laborious PCR based technique less obligatory as it allows reliable process monitoring and control in wastewater treatment plants.

Phenotypic heterogeneity in metabolic traits among single cells of a rare bacterial species in its natural environment quantified with a combination of flow cell sorting and NanoSIMS

Populations of genetically identical microorganisms residing in the same environment can display marked variability in their phenotypic traits; this phenomenon is termed phenotypic heterogeneity. The relevance of such heterogeneity in natural habitats is unknown, because phenotypic characterization of a sufficient number of single cells of the same species in complex microbial communities is technically difficult. We report a procedure that allows to measure phenotypic heterogeneity in bacterial populations from natural environments, and use it to analyze N2 and CO2 fixation of single cells of the green sulfur bacterium Chlorobium phaeobacteroides from the meromictic lake Lago di Cadagno. We incubated lake water with 15N2 and 13CO2 under in situ conditions with and without NH4+. Subsequently, we used flow cell sorting with auto-fluorescence gating based on a pure culture isolate to concentrate C. phaeobacteroides from its natural abundance of 0.2% to now 26.5% of total bacteria. C. phaeobacteroides cells were identified using catalyzed-reporter deposition fluorescence in situ hybridization (CARD-FISH) targeting the 16S rRNA in the sorted population with a species-specific probe. In a last step, we used nanometer-scale secondary ion mass spectrometry to measure the incorporation 15N and 13C stable isotopes in more than 252 cells. We found that C. phaeobacteroides fixes N2 in the absence of NH4+, but not in the presence of NH4+ as has previously been suggested. N2 and CO2 fixation were heterogeneous among cells and positively correlated indicating that N2 and CO2 fixation activity interact and positively facilitate each other in individual cells. However, because CARD-FISH identification cannot detect genetic variability among cells of the same species, we cannot exclude genetic variability as a source for phenotypic heterogeneity in this natural population. Our study demonstrates the technical feasibility of measuring phenotypic heterogeneity in a rare bacterial species in its natural habitat, thus opening the door to study the occurrence and relevance of phenotypic heterogeneity in nature.

Fixation procedures for flow cytometric analysis of environmental bacteria

Analysis of environmental bacteria on the single cell level often requires fixation to store the cells and to keep them in a state as near life-like as possible. Fixation procedures should furthermore counteract the increase of autofluorescence, cell clogging, and distortion of surface characteristics. Additionally, they should meet the specific fixation demands of both aerobically and anaerobically grown bacteria. A fixation method was developed based on metal solutions in combination with sodium azide. The fixation efficiencies of aluminium, barium, bismuth, cobalt, molybdenum, nickel, and tungsten salts were evaluated by flow cytometric measurement of the DNA contents as a bacterial population/community stability marker. Statistical equivalence testing was involved to permit highly reliable flow cytometric pattern evaluation. Investigations were carried out with pure cultures representing environmentally important metabolic and respiratory pathways as controls and with activated sludge as an example for highly diverse bacterial communities. A mixture of 5 mM barium chloride and nickel chloride, each and 10% sodium azide was found to be a suitable fixative for all tested bacteria. The described method provided good sample stability for at least 9 days.

Evaluation of the Effects of Low Oxygen Concentration on Stable Isotope Fractionation during Aerobic MTBE Biodegradation

Laboratory experiments were performed with two aerobic MTBE degrading strains ( Methylibium sp. PM1 and Aquincola tertiaricarbonaris L108) in order to determine whether conditions of low oxygen availability, typically found in fuel-contaminated aquifers, can influence stable isotope fractionation of MTBE. Although single carbon and hydrogen enrichment factors of the two strains were not significantly or were only slightly (L108) affected by low oxygen concentrations (fully oxic incubation with initial 21% O2 in the headspace tested versus hypoxic conditions always <2% O2), the experiments showed indirect effects caused by competition interactions in mixed cultures. In a mixed culture of PM1 and L108 under oxic and even more so under hypoxic conditions, the total observed carbon isotope enrichment factor was significantly reduced, while hydrogen fractionation was not detectable. This indicates that the low fractionating model strain L108 is more competitive in degrading MTBE compared to strain PM1. Consistently, higher oxygen affinities during MTBE degradation were observed for strain L108. These first studies, conducted with resting cells, may explain the low isotope fractionation observed in some field studies that are not necessarily related to a lack of biodegradation.

Monitoring of population dynamics of Corynebacterium glutamicum by multiparameter flow cytometry

Phenotypic variation of microbial populations is a well‐known phenomenon and may have significant impact on the success of industrial bioprocesses. Flow cytometry (FC) and the large repertoire of fluorescent dyes bring the high‐throughput analysis of multiple parameters in single bacterial cells into reach. In this study, we evaluated a set of different fluorescent dyes for suitability in FC single cell analysis of the biotechnological platform organism Corynebacterium glutamicum. Already simple scattering properties of C. glutamicum cells in the flow cytometer were shown to provide valuable information on the growth activity of analysed cells. Furthermore, we used DAPI staining for a FC‐based determination of the DNA content of C. glutamicum cells grown on standard minimal or complex media. Characteristic DNA patterns were observed mirroring the typical uncoupled DNA synthesis in the logarithmic (log) growth phase and are in agreement with a symmetric type of cell division of C. glutamicum. Application of the fluorescent dyes Syto 9, propidium iodide, and DiOC2(3) allowed the identification of subpopulations with reduced viability and membrane potential within early log and stationary phase populations. The presented data highlight the potential of FC‐based analyses for online monitoring of C. glutamicum bioprocesses and provide a first reference for future applications and protocols.

Resolution of natural microbial community dynamics by community fingerprinting, flow cytometry, and trend interpretation analysis.

Natural microbial communities generally have an unknown structure and composition because of their still not yet cultivable members. Therefore, understanding the relationships among the bacterial members, prediction of their behaviour, and controlling their functions are difficult and often only partly successful endeavours to date. This study aims to test a new idea that allows to follow community dynamics on the basis of a simple concept. Terminal restriction fragment length polymorphism (T-RFLP) analysis of bacterial 16S ribosomal RNA genes was used to describe a community profile that we define as composition of a community. Flow cytometry and analysis of DNA contents and forward scatter characteristics of the single cells were used to describe a community profile, which we define as structure of a community. Both approaches were brought together by a non-metric multidimensional scaling (n-MDS) for trend interpretation of changes in the complex community data sets. This was done on the basis of a graphical evaluation of the cytometric data, leading to the newly developed Dalmatian plot tool, which gave an unexpected insight into the dynamics of the unknown bacterial members of the investigated natural microbial community. The approach presented here was compared with other techniques described in the literature. The microbial community investigated in this study was obtained from a BTEX contaminated anoxic aquifer. The indigenous bacteria were allowed to colonise in situ microcosms consisting of activated carbon. These microcosms were amended with benzene and one of the electron acceptors nitrate, sulphate or ferric iron to stimulate microbial growth. The data obtained in this study indicated that the composition (via T-RFLP) and structure (via flow cytometry) of the natural bacterial community were influenced by the hydro-geochemical conditions in the test site, but also by the supplied electron acceptors, which led to distinct shifts in relative abundances of specific community members. It was concluded that engineered environments can be successfully monitored by single cell analytics in combination with established molecular tools and sophisticated statistical analyses, a combination that holds great promise for studying and monitoring natural microbial community behaviour.