Susanne Günther

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.

Dynamics of Polyphosphate-Accumulating Bacteria in Wastewater Treatment Plant Microbial Communities Detected via DAPI (4′,6′-Diamidino-2-Phenylindole) and Tetracycline Labeling

ABSTRACT Wastewater treatment plants with enhanced biological phosphorus removal represent a state-of-the-art technology. Nevertheless, the process of phosphate removal is prone to occasional failure. One reason is the lack of knowledge about the structure and function of the bacterial communities involved. Most of the bacteria are still not cultivable, and their functions during the wastewater treatment process are therefore unknown or subject of speculation. Here, flow cytometry was used to identify bacteria capable of polyphosphate accumulation within highly diverse communities. A novel fluorescent staining technique for the quantitative detection of polyphosphate granules on the cellular level was developed. It uses the bright green fluorescence of the antibiotic tetracycline when it complexes the divalent cations acting as a countercharge in polyphosphate granules. The dynamics of cellular DNA contents and cell sizes as growth indicators were determined in parallel to detect the most active polyphosphate-accumulating individuals/subcommunities and to determine their phylogenetic affiliation upon cell sorting. Phylotypes known as polyphosphate-accumulating organisms, such as a “Candidatus Accumulibacter”-like phylotype, were found, as well as members of the genera Pseudomonas and Tetrasphaera. The new method allows fast and convenient monitoring of the growth and polyphosphate accumulation dynamics of not-yet-cultivated bacteria in wastewater bacterial communities.

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.

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.

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.

Non-random distribution of macromolecules as driving forces for phenotypic variation.

Clonal populations employ many strategies of diversification to deal with constraints. All these strategies result in the generation of different phenotypes with diverse functions. Events like cell division are major sources of phenotypic variability due to the unequal partitioning of cellular components. In this review we concentrate on passive and active mechanisms cells employ to distribute macromolecules between their offspring. Different types of segregation are described, addressing both metabolically pertinent molecules such as PHA/PHB or polyphosphates, and components that adversely affect cells by promoting aging, such as damaged protein complexes or extrachromosomal rDNA circles. We also refer to mechanisms generating plasmid copy number (PCN) variation between cells in a population, and how elaborate partitioning systems counteract partitioning errors and ensure equal distribution. Finally, we demonstrate how simple differences in chromosomal copy number determine the fate of a cell, in this case the effect of gene dosage on the onset of sporulation in Bacillus subtilis or on a functional trait in Sinorhizobium meliloti.

Species-sorting and mass-transfer paradigms control managed natural metacommunities

A complex microbial system consisting of six different interconnected localities was thoroughly investigated at full scale for over a year. The metacommunity concept originating from macro-ecology was used to uncover mechanisms of community assembly by observing microbial interrelationships in and between the different localities via correlation and network analysis. The individual-based observation approach was applied using high-throughput microbial community cytometry in addition to next generation sequencing. We found robust α-diversity values for each of the six localities and high β-diversity values despite directed connectivity between localities, classifying for endpoint assembly of organisms in each locality. Endpoint characteristics were based on subcommunities with high cell numbers whereas those with lower cell numbers were involved in dispersal. Perturbation caused abiotic parameters to alter local community assembly with especially the rare cells announcing community restructuration processes. The mass-effect paradigm as part of the metacommunity concept was identified by an increase in interlocality biotic correlations under perturbation which, however, did not unbalance the predominant species-sorting paradigm in the studied full scale metacommunity. Data as generated in this study might contribute to the development of individual-based models for controlling managed multispecies natural systems in future.

Biodiversity of Polyphosphate Accumulating Bacteria in Eight WWTPs with Different Modes of Operation

AbstractEnhanced biological phosphorous removal (EBPR) from wastewater has been successfully used for more than three decades and is considered to be an environmentally friendly wastewater-treatment process. Biologically, this process is realized by incorporation of phosphate as polyphosphate (polyP) granules in activated sludge bacteria. Important groups of bacteria responsible for P removal have been identified, but the full microbial diversity involved in this process is still unknown. This paper reports on the microbial composition of activated sludge communities in eight wastewater-treatment plants (WWTPs) with different sizes and modes of operation. The polyphosphate accumulating organisms (PAOs) within this complex biocenosis were identified by fluorescent dye staining and classified by in situ hybridization techniques. Of the bacteria in the aerobic basin, 5–13% contained polyP granules. In addition, flow cytometry was used to quantify PAOs after tetracycline staining and to separate these cells. ...

Estimating the Energy Content of Wastewater Using Combustion Calorimetry and Different Drying Processes

The energy content of wastewater is routinely assessed by chemical oxygen demand (COD) measurements that only provide an incomplete picture and the data fundament of other energy parameters remains scarce. The heat of combustion (∆CH) of raw wastewater from a municipal wastewater treatment plant (WWTP) was assessed using oven drying method (∆CH = −6.8±4.3 kJ L−1, n = 20) and freeze drying method (∆CH = −20.2±9.7 kJ L−1, n = 6) illustrating the substantial loss during the oven drying approach. Normalizing ∆CH to COD of raw wastewater yielded −6.2±3.5 kJ gCOD−1 for oven-dried samples (n = 14) and −13.0±1.6 kJ gCOD−1 for freeze-dried samples (n = 3). A subsequent correlation analysis with further chemical wastewater parameters revealed a dependency of ∆CHvol on COD, TOC, C:N ratio and total sulfur content. To verify these correlations, wastewater of a second WWTP was sampled and analyzed. Only COD and TOC were in accordance with the data set from the first WWTP representing potential predictors for the chemical energy stored in wastewater for comparable WWTPs. Unfortunately, during the most practical method (oven drying), a certain loss of volatile compounds is inevitable so that the derived ∆CHvol systematically underestimates the total energetic potential of wastewater. Nevertheless, this work expands the, so far, little data fundament on the energy resource wastewater and implies the requirement for further long-term studies on different sites and different wastewater types with a highly standardized sample treatment protocol.

Facilitated gate setting by sequential dot plot scanning.

Microbial communities comprising thousands of unknown organisms can be studied flow cytometrically by applying just one fluorescent parameter and using scatter characteristics of cells. Resulting 2D‐plots need to represent high numbers of cells to detect the many subcommunities, even rare ones that might be present in the sample. Evaluation of such data can be faulty and subjective due to the low number of parameters available for data discrimination and the high numbers of overlaying events. Here, we describe a procedure that helps to evaluate such data using facilitated gate setting by sequential dot‐plot scanning.