Medium-chain fatty acid esters are effective even in azole-resistant Malassezia pachydermatis.

BACKGROUND: Malassezia (M.) pachydermatis as a frequent reason for dermatological consultation in dogs and cats was recently shown to be lipid-dependent, too. Lipolytic activity is a prerequisite for activating antimicrobial effectivity of fatty acid esters. OBJECTIVES: It was therefore of interest whether it is possible to induce this mechanism in M. pachydermatis and to identify possible differences between minimal and strong lipid-dependent strains. METHODS: In an agar dilution test, the minimal inhibitory concentrations of six fatty acid esters were determined for seventeen M. pachydermatis strains. GC analysis of parent compounds and liberated fatty acids was used to quantify ester cleavage. RESULTS: Hydrolysis was observed in all test strains in a homogenous manner but was dependent on the chemical structure. Lowest MICs (500 ppm after 14 days of incubation) were obtained applying glyceryl monocaprylate and 3-hydroxylpropyl caprylate, while the corresponding esters of undecylenic acid showed nearly twice the value. As shown by GC analysis with the reference strains CBS 1879 and CBS 1892 and 3-hydroxypropyl caprylate, hydrolysis and caprylic acid formation starts immediately and was dependent on yeast density. Furthermore, nine azole-resistant strains isolated from dogs with treatment failures showed MIC values comparable to the other strains and no resistance to monohydric fatty acid esters. CONCLUSIONS: Medium-chain fatty acid esters may represent a new therapeutic option for veterinary use even in azole-resistant strains. The in vivo verification in M. pachydermatis-associated dermatitis in dogs and cats will be the next step for the successful development of new therapeutics.

[Malassezia spp.: interactions with topically applied lipids-a review : Malassezia and topically applied lipids]. / Malassezia spp.: Wechselwirkungen mit Lipidbestandteilen von Topika ­ eine Übersicht : Malassezia und Topika.

Lipophilic Malassezia yeasts are an important part of the human resident skin flora, especially in seborrheic areas. Besides mutualistic interactions with the host they are also linked to diseases although the specific causes are not yet comprehensively understood. The amount of available lipids on the skin correlates with the Malassezia density and also with the occurrence of certain diseases like tinea versicolor. Here, the naturally produced lipids of the sebaceous glands play a role. Hardly studied thus far is the impact of topically applied lipids. Here, growth promotion as well as inhibition of Malassezia cells as well as the production of new metabolites through ester cleavage are possible. One example is the release of antimicrobial fatty acids from hydroxypropyl caprylate through the action of Malassezia lipases. This "self-kill" principle results in the reduction of the amount of Malassezia cells and can be applied as new therapy option for dandruff treatment. A better understanding of the interaction between topica and Malassezia would increase their skin tolerance and open new therapy options.

Label-Free Four-Dimensional Visualization of Anaerobically Growing Electroactive Biofilms.

Light sheet fluorescence microscopy (LSFM) allows nondestructive, label-free and in vivo imaging of large specimen, even at nontransparent surfaces. We show that LSFM can be applied for label-free analyses of prokaryotes on the example of electroactive biofilms. Biofilm growth is linked to the production of current serving as measure of metabolic activity in vivo by monitoring with high spatial and temporal resolution. After 35 h of exponential growth, a homogeneous biofilm with a thickness of 9 µm was formed. This was followed by a stratification of the biofilm including the formation of 3D structures over the next 100 h. Light reflection was sufficient to visualize the biofilm structure and development over time and the terminal morphology was confirmed using fluorescence staining. This proof of concept on using LSFM for investigation of biofilms opens the door for its application in the entire field of microbial ecology. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

Medium-chain fatty acid esters-Optimising their efficacy as anti-Malassezia agents.

BACKGROUND: For fatty acid esters of monohydric alcohols, cleavage by exo-enzymes of Malassezia (M.) spp. and release of fatty acids with antimicrobial activity have been shown recently. On skin surface, this selective activation of antimicrobial activity might result in a 'self-kill' targeted locally at the site with the highest M. density. OBJECTIVES: As for the disadvantage of strong odour, use of these esters for topical therapy is limited to low concentrations. Therefore, cleavage was also tested for monoesters of octanoic and undec-10-enoic acid with the bihydric alcohol propane-1,3-diol or the trihydric glycerol. METHODS: In an agar dilution test, the minimal inhibitory concentrations of these compounds were determined for M. furfur, M. globosa, M. sympodialis and M. restricta, respectively. GC analysis of parent compounds and liberated fatty acids was used to reveal ester cleavage. RESULTS: Ester cleavage started immediately. MICs for the test compounds ranged between ~1000-8000 ppm after 14 days of incubation. 1,3-propanediol esters, especially 3-hydroxypropyl octanoate and 3-hydroxypropyl undecylenate were most effective, binary combinations exerted synergistic effects. CONCLUSIONS: The new substances are advantageous in terms of odour and substantivity and have also beneficial skin caring properties if not hydrolysed by M. spp. As a different panel of hydrolases of each single M. species is responsible for variation in efficacy among the test substances, tailored products to treat preferentially single species or blends with a broader effectivity can be designed. In vivo verification will be the next step for the successful development of this new therapeutical concept for M.-associated diseases.

Flow cytometric quantification, sorting and sequencing of methanogenic archaea based on F420 autofluorescence.

BACKGROUND: The widely established production of CH4 from renewable biomass in industrial scale anaerobic reactors may play a major role in the future energy supply. It relies on methanogenic archaea as key organisms which represent the bottleneck in the process. The quantitative analysis of these organisms can help to maximize process performance, uncover disturbances before failure, and may ultimately lead to community-based process control schemes. Existing qPCR and fluorescence microscopy-based methods are very attractive but can be cost-intensive and laborious. RESULTS: In this study we present an autofluorescence-based, flow cytometric method for the fast low-cost quantification of methanogenic archaea in complex microbial communities and crude substrates. The method was applied to a methanogenic enrichment culture (MEC) and digester samples (DS). The methanogenic archaea were quantified using the distinct fluorescence of their cofactor F420 in a range from 3.7 × 108 (± 3.3 × 106) cells mL-1 and 1.8 x 109 (± 1.1 × 108) cells mL-1. We evaluated different fixation methods and tested the sample stability. Stable abundance and fluorescence intensity were recorded up to 26 days during aerobic storage in PBS at 6 °C. The discrimination of the whole microbial community from the ubiquitous particle noise was facilitated by SYBR Green I staining and enabled calculation of relative abundances of methanogenic archaea of up to 9.64 ± 0.23% in the MEC and up to 4.43 ± 0.74% in the DS. The metaprofiling of the mcrA gene reinforced the results. CONCLUSIONS: The presented method allows for fast and reliable quantification of methanogenic archaea in microbial communities under authentic digester conditions and can thus be useful for process monitoring and control in biogas digesters.

CHIC-an automated approach for the detection of dynamic variations in complex microbial communities.

Altering environmental conditions change structures of microbial communities. These effects have an impact on the single-cell level and can be sensitively detected using community flow cytometry. However, although highly accurate, microbial monitoring campaigns are still rarely performed applying this technique. One reason is the limited access to pattern analysis approaches for the evaluation of microbial cytometric data. In this article, a new analyzing tool, Cytometric Histogram Image Comparison (CHIC), is presented, which realizes trend interpretation of variations in microbial community structures (i) without any previous definition of gates, by working (ii) person independent, and (iii) with low computational demand. Various factors influencing a sensitive determination of changes in community structures were tested. The sensitivity of this technique was found to discriminate down to 0.5% internal variation. The final protocol was exemplarily applied to a complex microbial community dataset, and correlations to experimental variation were successfully shown.

Monitoring functions in managed microbial systems by cytometric bar coding.

Cytometric monitoring of microbial community dynamics can be used to estimate stability of technical microbial processes like biogas production by analysis of segregated cell abundance changes. In this study, structure variations of a biogas community were cytometrically recorded over 9 months and found to be of diagnostic value for process details. The reactor regime was intentionally disturbed with regard to substrate overload or H(2)S accumulation. A single-cell based approach called cytometric bar coding (CyBar) for fast identification of reactive subcommunities was used. Functionality of specific subcommunities was uncovered by processing CyBar data with abiotic reactor parameters using Spearman's correlation coefficient. Twenty subcommunities showed a discrete and divergent behavior. For example, a 4-fold substrate overload increased the cell number of two acidogenic index subcommunities to 176 and 193% within three days. Supplementary analyses were done using DNA fingerprinting, cloning, and sequencing. Bioreactor perturbations were shown to create cell abundance changes in subcommunities rather than variations in their phylogenetic composition. The used workflow and macros are ready-to-use tools and allow on-site monitoring and interpretation of variation in microbial community functions within a few hours.

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.

Identification of Clostridium cochlearium as an electroactive microorganism from the mouse gut microbiome.

Microbial electroactivity, the metabolically relevant transfer of electrons between microorganisms and solid conductors, was first discovered for now well characterized model organisms from hypoxic or anaerobic water or sediment samples. Recent findings indicate that the metabolic trait of electroactivity might as well be important within the microbiome of the mammalian gut. Based on a pre-selection from the mouse intestinal bacterial collection five microorganisms originating from diverse parts of the gut were screened for electroactivity. As there is no marker gene for electroactivity, the ability to synthesize cytochromes and metabolize redox-mediators was studied in-silico. Clostridium cochlearium showed highest electroactivity and Lactobacillus reuteri as well as Staphylococcus xylosus show putative electroactivity, as well. The maximum current density of C. cochlearium of 0.53 ±â€¯0.02 mA cm-2 after only 5.2 h of incubation was clearly linked to growth and glucose consumption. Cyclic voltammetric analysis on C. cochlearium revealed a formal potential of the extracellular electron transfer (EET) site of +0.22 ±â€¯0.05 V versus Ag/AgCl sat. KCl (and + 0.42 V versus SHE) and indicates that EET is not based on biofilm formation, but the involvement of either redox-active molecules or planktonic cells. The potential of the gut as habitat for electroactives and their physiological role are discussed.

Trophic networks improve the performance of microbial anodes treating wastewater.

Microbial anodes represent a distinct ecological niche that is characterized mainly by the terminal electron acceptor, i.e., the anode potential, and the substrate, i.e., the electron source. Here, we determine the performance and the biofilm community of anode microbiomes while using substrates of increasing complexity (organic acids or organic acids and sugar or real domestic wastewater) to mimic different, practically relevant, trophic levels. α-Diversity values increased with substrate complexity. In addition, the higher abundance value of Deltaproteobacteria in the biofilms corresponds to higher reactor performance (i.e., COD removal, current density, and Coulombic efficiency). In reactors exploiting real wastewater, the diversity of the planktonic microorganisms was only little affected. Microbiome network analysis revealed two important clusters for reactor performance as well as performance-independent pathogen-containing clusters. Interestingly, Geobacter was not found to be integrated in the network underlining its outstanding individual ecological role in line with its importance for the efficiency of the electron harvest for all reactors. The microbiome analysis of different trophic levels and their temporal development from initial colonization to stable treatment demonstrate important principles for the implementation of microbial anodes for wastewater treatment.

Fatty acid composition of serum lipid classes in mice following allergic sensitisation with or without dietary docosahexaenoic acid-enriched fish oil substitution.

Dietary fatty acids have been shown to influence allergic sensitisation. Both n-3 and n-6 PUFA are involved in targeted mediation of inflammatory responses during allergic sensitisation and manifestation of atopic diseases. In the present experiments we investigated whether supplementation of DHA-enriched fish oil partly substituting dietary sunflower-seed oil, in comparison with sunflower-seed oil, supplemented to mice influences fatty acid composition of serum lipid classes. The effects of the two different diets were also investigated depending on allergic sensitisation. Supplementation of DHA and EPA in doses of 2 and 0.12 % (w/w) to non-sensitised and sensitised mice resulted in significantly increased percentile contributions of DHA to all lipid classes. In contrast, serum values of the n-6 PUFA arachidonic acid (AA) were significantly lower, both in non-sensitised and sensitised mice fed the DHA-enriched diet. The fatty acid composition of serum lipids also reflected allergic sensitisation: the EPA:AA ratio in TAG, cholesteryl esters and phospholipids in non-supplemented animals fell to 23, 29 and 29 % respectively of the original value after allergic sensitisation, whereas it decreased to 70, 80 and 76 % respectively only in the animals supplemented with DHA. In summary, allergic sensitisation alone decreased significantly the EPA:AA ratios in serum TAG, while concomitant supplementation of DHA-enriched fish oil ameliorated this decrease. We postulate from the present results that the amelioration of the severity of allergic sensitisation after DHA supplementation may be linked to altered ratios of the eicosanoid precursors EPA and AA as well as DHA needed for further metabolic activation to pro- or anti-inflammatory bioactive lipids.

Personalized microbiome dynamics - Cytometric fingerprints for routine diagnostics.

Microbiomes convoy human life in countless ways. They are an essential part of the human body and interact with its host in countless ways. Currently, extensive microbiome analyses assessing the microbiomes' composition and functions based on sequencing information are still far away from being routine analyses due to the complexity of applied techniques and data analysis, their time demand as well as high costs. With the growing demand for on-time community assessment and monitoring of its dynamic behavior with high resolution, alternative high-throughput methods such as microbial community flow cytometry come into focus. Our flow cytometric approach provides single-cell based high-dimensional data by using only three parameters but for every cell in a system which is enough to characterize whole communities' attributes with high acuity over time. To interpret such complex cytometric time-series data, novel concepts are required. We provide a workflow which is applicable for easy-to-use handling and measurement of microbiomes. Drawing inspiration from macro-ecology, in which a rich set of concepts has been developed for describing population dynamics, we interpret huge sets of community single cell data in an intuitive and actionable way using a series of bioinformatics tools which we either developed or adapted from sequence based evaluation approaches for the interpretation of single cell data. The developed evaluation pipeline tests for e.g. ecological measures such as community assembly, functioning, and evolution. We also addressed the meta-community-concept which is a well acknowledged idea in macro-ecology on how interconnected communities perform. The last concept discusses stability which is a metrics of paramount importance. A fast quantification of stability properties may not only detect disturbances and their impact on the organisms but also allow for on-time microbiome treatment. The workflow's immanent ability to support high temporal sample densities below bacterial generation times provides new insight into the ecology of microbiomes and may also provide access to community control for microbiome based health management. The future developments will facilitate cytometric fingerprinting for human routine diagnostics to be as simple and meaningful as a blood count today.

Microbial ecology-based engineering of Microbial Electrochemical Technologies.

Microbial ecology is devoted to the understanding of dynamics, activity and interaction of microorganisms in natural and technical ecosystems. Bioelectrochemical systems represent important technical ecosystems, where microbial ecology is of highest importance for their function. However, whereas aspects of, for example, materials and reactor engineering are commonly perceived as highly relevant, the study and engineering of microbial ecology are significantly underrepresented in bioelectrochemical systems. This shortfall may be assigned to a deficit on knowledge and power of these methods as well as the prerequisites for their thorough application. This article discusses not only the importance of microbial ecology for microbial electrochemical technologies but also shows which information can be derived for a knowledge-driven engineering. Instead of providing a comprehensive list of techniques from which it is hard to judge the applicability and value of information for a respective one, this review illustrates the suitability of selected techniques on a case study. Thereby, best practice for different research questions is provided and a set of key questions for experimental design, data acquisition and analysis is suggested.

Microbial electricity driven anoxic ammonium removal.

Removal of nitrogen, mainly in form of ammonium (NH4+), in wastewater treatment plants (WWTPs) is a highly energy demanding process, mainly due to aeration. It causes costs of about half a million Euros per year in an average European WWTP. Alternative, more economical technologies for the removal of nitrogen compounds from wastewater are required. This study proves the complete anoxic conversion of ammonium (NH4+) to dinitrogen gas (N2) in continuously operated bioelectrochemical systems at the litre-scale. The removal rate is comparable to conventional WWTPs with 35 ± 10 g N m-3 d-1 with low accumulation of NO2-, NO3-, N2O. In contrast to classical aerobic nitrification, the energy consumption is considerable lower (1.16 ± 0.21 kWh kg-1 N, being more than 35 times less than for the conventional wastewater treatment). Biotic and abiotic control experiments confirmed that the anoxic nitrification was an electrochemical biological process mainly performed by Nitrosomonas with hydroxylamine as the main substrate (mid-point potential, Eox = +0.67 ± 0.08 V vs. SHE). This article proves the technical feasibility and reduction of costs for ammonium removal from wastewater, investigates the underlying mechanisms and discusses future engineering needs.

Study of Electrochemical Reduction of CO2 for Future Use in Secondary Microbial Electrochemical Technologies.

The fluctuation and decentralization of renewable energy have triggered the search for respective energy storage and utilization. At the same time, a sustainable bioeconomy calls for the exploitation of CO2 as feedstock. Secondary microbial electrochemical technologies (METs) allow both challenges to be tackled because the electrochemical reduction of CO2 can be coupled with microbial synthesis. Because this combination creates special challenges, the electrochemical reduction of CO2 was investigated under conditions allowing microbial conversions, that is, for their future use in secondary METs. A reproducible electrodeposition procedure of In on a graphite backbone allowed a systematic study of formate production from CO2 with a high number of replicates. Coulomb efficiencies and formate production rates of up to 64.6±6.8 % and 0.013±0.002 mmolformate h-1 cm-2 , respectively, were achieved. Electrode redeposition, reusability, and long-term performance were investigated. Furthermore, the effect of components used in microbial media, that is, yeast extract, trace elements, and phosphate salts, on the electrode performance was addressed. The results demonstrate that the integration of electrochemical reduction of CO2 in secondary METs can become technologically relevant.

Predicting and experimental evaluating bio-electrochemical synthesis - A case study with Clostridium kluyveri.

Microbial electrosynthesis is a highly promising application of microbial electrochemical technologies for the sustainable production of organic compounds. At the same time a multitude of questions need to be answered and challenges to be met. Central for its further development is using appropriate electroactive microorganisms and their efficient extracellular electron transfer (EET) as well as wiring of the metabolism to EET. Among others, Clostridia are believed to represent electroactive microbes being highly promising for microbial electrosynthesis. We investigated the potential steps and challenges for the bio-electrochemical fermentation (electro-fermentation) of mid-chain organic acids using Clostridium kluyveri. Starting from a metabolic model the potential limitations of the metabolism as well as beneficial scenarios for electrochemical stimulation were identified and experimentally investigated. C. kluyveri was shown to not be able to exchange electrons with an electrode directly. Therefore, exogenous mediators (2-hydroxy-1,4-naphthoquinone, potassium ferrocyanide, neutral red, methyl viologen, methylene blue, and the macrocyclic cobalt hexaamine [Co(trans-diammac)]3+) were tested for their toxicity and electro-fermentations were performed in 1L bioreactors covering 38 biotic and 8 abiotic runs. When using C. kluyveri and mediators, maximum absolute current densities higher than the abiotic controls were detected for all runs. At the same time, no significant impact on the cell metabolism (product formation, carbon recovery, growth rate) was found. From this observation, we deduce general potential limitations of electro-fermentations with C. kluyveri and discuss strategies to successfully overcome them.

Enhancing methane production from food waste fermentate using biochar: the added value of electrochemical testing in pre-selecting the most effective type of biochar.

BACKGROUND: Recent studies have suggested that addition of electrically conductive biochar particles is an effective strategy to improve the methanogenic conversion of waste organic substrates, by promoting syntrophic associations between acetogenic and methanogenic organisms based on interspecies electron transfer processes. However, the underlying fundamentals of the process are still largely speculative and, therefore, a priori identification, screening, and even design of suitable biochar materials for a given biotechnological process are not yet possible. RESULTS: Here, three charcoal-like products (i.e., biochars) obtained from the pyrolysis of different lignocellulosic materials, (i.e., wheat bran pellets, coppiced woodlands, and orchard pruning) were tested for their capacity to enhance methane production from a food waste fermentate. In all biochar-supplemented (25 g/L) batch experiments, the complete methanogenic conversion of fermentate volatile fatty acids proceeded at a rate that was up to 5 times higher than that observed in the unamended (or sand-supplemented) controls. Fluorescent in situ hybridization analysis coupled with confocal laser scanning microscopy revealed an intimate association between archaea and bacteria around the biochar particles and provided a clear indication that biochar also shaped the composition of the microbial consortium. Based on the application of a suite of physico-chemical and electrochemical characterization techniques, we demonstrated that the positive effect of biochar is directly related to the electron-donating capacity (EDC) of the material, but is independent of its bulk electrical conductivity and specific surface area. The latter properties were all previously hypothesized to play a major role in the biochar-mediated interspecies electron transfer process in methanogenic consortia. CONCLUSIONS: Collectively, these results of this study suggest that for biochar addition in anaerobic digester operation, the screening and identification of the most suitable biochar material should be based on EDC determination, via simple electrochemical tests.

Dynamics in the microbial cytome-single cell analytics in natural systems.

Natural microbial systems are highly dynamic due to the short generation times of the comprised organisms and their rapid and distinct reactions to changing environments. Microbial flow cytometry approaches are capable techniques for following such community dynamics in a fast and inexpensive way. Newly developed bioinformatics tools not only enable quantification of single cell dynamics, they also make nearly on-line evaluation of community attributes possible, enable interpretation of community trends, and reveal possible constraints that influence community structure and function. Microbial flow cytometry is poised to make the microbial cytome accessible for ambitious ecosystem studies. Functions of cells within the cytome can be determined either by cell sorting in combination with other omics-approaches of choice or by simple correlation analyses.

Microbiomes in bioenergy production: from analysis to management.

Currently, bioreactors exploiting natural microbial communities, that is, microbiomes, for bioenergy production are almost exclusively operated based on bulk parameters and empirical expert knowledge. The microbiome of these bioreactors often remains a "black box", that is, its composition and function are only analyzed retrospectively (mostly in a case of failure). Here, on-time microbiome analysis can allow a proactive process management. However, today's sophisticated molecular ecology methods appear inaccessible for the routine analysis of reactor microbiomes in bioenergy production. This review analyzes the requirements of methods for routine microbiome diagnostics. Especially the ability of current molecular and cell based methods to derive structure-function-relationships, that is, correlations between the microbial community structure and dynamics and the reactor performance, are emphasized and key-criteria for routine on-site monitoring are defined. Finally, a critical assessment of selected methods for microbiome monitoring is performed focusing on (i) the production of biogas in anaerobic digesters and (ii) the production of the biofuel precursor n-butyrate.

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

Optical characteristics of individual bacterial cells of natural communities can be measured with flow cytometry (FCM) in high throughput. The resulting data are visualized in cytometric histograms. These histograms represent individual cytometric fingerprints of microbial communities, e.g., at certain time points or microenvironmental conditions. Up to now four tools for analyzing the variation in these cytometric fingerprints are available but have not yet been systematically compared regarding application: Dalmatian Plot, Cytometric Histogram Image Comparison (CHIC), Cytometric Barcoding (CyBar), and FlowFP. In this article these tools were evaluated concerning (i) the required experience of the operator in handling cytometric data sets, (ii) the detection level of changes, (iii) time demand for analysis, and (iv) software requirements. As an illustrative example, FCM was used to characterize the microbial community structure of electroactive microbial biofilms. Their cytometric fingerprints were determined, analyzed with all four tools, and correlated to experimental and functional parameters. The source of inoculum (four different types of wastewater samples) showed the strongest influence on the microbial community structure and biofilm performance while the choice of substrate (acetate or lactate) had no significant effect in the present study. All four evaluation tools were found suitable to monitor structural changes of natural microbial communities. The Dalmatian Plot was shown to be most sensitive to operator impact but nevertheless provided an overview on community shifts. CHIC, CyBar, and FlowFP showed less operator dependence and gave highly resolved information on community structure variation on different detection levels. In conclusion, experimental and productivity parameters correlated with the biofilm structures and practical process integration details were available from cytometric fingerprint analysis.