Flow cytometry for immediate follow-up of drinking water networks after maintenance.

Drinking water networks need maintenance every once in a while, either planned interventions or emergency repairs. When this involves opening of the water pipes, precautionary measures need to be taken to avoid contamination of the drinking water at all time. Drinking water suppliers routinely apply plating for faecal indicator organisms as quality control in such a situation. However, this takes at least 21 h of waiting time, which can be crucial when dealing with major supply pipes. A combination of flow cytometric (FCM) bacterial cell counts with FCM fingerprinting techniques is proposed in this study as a fast and sensitive additional technique. In three full scale situations, major supply pipes with 400-1050 mm diameter were emptied for maintenance, shock-chlorinated and flushed with large amounts of clean drinking water before taking back in operation. FCM measurements of the discharged flushing water revealed fast lowering and stabilizing bacterial concentrations once flushing is initiated. Immediate comparison with clean reference drinking water used for flushing was done, and the moment when both waters had similar bacterial concentrations was considered as the endpoint of the necessary flushing works. This was usually after 2-4 h of flushing. FCM fingerprinting, based on both bacteria and FCM background, was used as additional method to verify how similar flushing and reference samples were and yielded similar results. The FCM approved samples were several hours later approved as well by the drinking water supplier after plating and incubation for total Coliforms and Enterococci. These were used as decisive control to set the pipes back in operation. FCM proved to be a more conservative test than plating, yet it yielded immediate results. Application of these FCM methods can therefore avoid long unnecessary waiting times and large drinking water losses.

The more, the merrier: heterotroph richness stimulates methanotrophic activity.

Although microorganisms coexist in the same environment, it is still unclear how their interaction regulates ecosystem functioning. Using a methanotroph as a model microorganism, we determined how methane oxidation responds to heterotroph diversity. Artificial communities comprising of a methanotroph and increasing heterotroph richness, while holding equal starting cell numbers were assembled. We considered methane oxidation rate as a functional response variable. Our results showed a significant increase of methane oxidation with increasing heterotroph richness, suggesting a complex interaction in the cocultures leading to a stimulation of methanotrophic activity. Therefore, not only is the methanotroph diversity directly correlated to methanotrophic activity for some methanotroph groups as shown before, but also the richness of heterotroph interacting partners is relevant to enhance methane oxidation too. In this unprecedented study, we provide direct evidence showing how heterotroph richness exerts a response in methanotroph-heterotroph interaction, resulting in increased methanotrophic activity. Our study has broad implications in how methanotroph and heterotroph interact to regulate methane oxidation, and is particularly relevant in methane-driven ecosystems.

Synthetic microbial ecosystems: an exciting tool to understand and apply microbial communities.

Many microbial ecologists have described the composition of microbial communities in a plenitude of environments, which has greatly improved our basic understanding of microorganisms and ecosystems. However, the factors and processes that influence the behaviour and functionality of an ecosystem largely remain black boxes when using conventional approaches. Therefore, synthetic microbial ecology has gained a lot of interest in the last few years. Because of their reduced complexity and increased controllability, synthetic communities are often preferred over complex communities to examine ecological theories. They limit the factors that influence the microbial community to a minimum, allowing their management and identifying specific community responses. However, besides their use for basic research, synthetic ecosystems also found their way towards different applications, like industrial fermentation and bioremediation. Here, we review why and how synthetic microbial communities are applied for research purposes and for which applications they have been and could be successfully used.

Bacterial invasion potential in water is determined by nutrient availability and the indigenous community.

In drinking water (DW) and the distribution systems, bacterial growth and biofilm formation have to be controlled both for limiting taste or odour development and preventing clogging or biocorrosion problems. After a contamination with undesired bacteria, factors like nutrient availability and temperature will influence the survival of these invaders. Understanding the conditions enabling invaders to proliferate is essential for a holistic approach towards microbial risk assessment in DW. Pseudomonas putida was used as a model invader because this easy-growing bacterium can use a wide range of substrates. Invasion experiments in oligo- to eutrophic waters showed the requirement of both a carbon and phosphate source for survival of P. putida in DW. Addition of C, N and P enabled P. putida to grow in DW from 5.80 × 10(4) to 1.84 × 10(8) cells mL(-1) and survive for at least 12 days. However, in surface water with similar nutrient concentrations, P. putida did not survive, indicating the concomitant importance of the present indigenous microbial community of the specific water sample. Either extensive carbon or phosphate limitation can be used in water treatment design in order to obtain a DW which is not susceptible for unwanted bacterial growth.

Biofilm-grown Burkholderia cepacia complex cells survive antibiotic treatment by avoiding production of reactive oxygen species.

The presence of persister cells has been proposed as a factor in biofilm resilience. In the present study we investigated whether persister cells are present in Burkholderia cepacia complex (Bcc) biofilms, what the molecular basis of antimicrobial tolerance in Bcc persisters is, and how persisters can be eradicated from Bcc biofilms. After treatment of Bcc biofilms with high concentrations of various antibiotics often a small subpopulation survived. To investigate the molecular mechanism of tolerance in this subpopulation, Burkholderia cenocepacia biofilms were treated with 1024 µg/ml of tobramycin. Using ROS-specific staining and flow cytometry, we showed that tobramycin increased ROS production in treated sessile cells. However, approximately 0.1% of all sessile cells survived the treatment. A transcriptome analysis showed that several genes from the tricarboxylic acid cycle and genes involved in the electron transport chain were downregulated. In contrast, genes from the glyoxylate shunt were upregulated. These data indicate that protection against ROS is important for the survival of persisters. To confirm this, we determined the number of persisters in biofilms formed by catalase mutants. The persister fraction in ΔkatA and ΔkatB biofilms was significantly reduced, confirming the role of ROS detoxification in persister survival. Pretreatment of B. cenocepacia biofilms with itaconate, an inhibitor of isocitrate lyase (ICL), the first enzyme in the glyoxylate shunt, reduced the persister fraction approx. 10-fold when the biofilms were subsequently treated with tobramycin. In conclusion, most Bcc biofilms contain a significant fraction of persisters that survive treatment with high doses of tobramycin. The surviving persister cells downregulate the TCA cycle to avoid production of ROS and at the same time activate an alternative pathway, the glyoxylate shunt. This pathway may present a novel target for combination therapy.

Environmental conditions and community evenness determine the outcome of biological invasion.

Biological invasion is widely studied, however, conclusions on the outcome of this process mainly originate from observations in systems that leave a large number of experimental variables uncontrolled. Here using a fully controlled system consisting of assembled bacterial communities, we evaluate the degree of invasion and the effect on the community functionality in relation to the initial community evenness under specific environmental stressors. We show that evenness influences the level of invasion and that the introduced species can promote functionality under stress. The evenness-invasibility relationship is negative in the absence and neutral in the presence of stress. Under these conditions, the introduced species is able to maintain the functionality of uneven communities. These results indicate that communities, initially having the same genetic background, in the presence of the same invader, react in a different way with respect to invasibility and functionality depending on specific environmental conditions and community evenness.

Comparing metabolic functionalities, community structures, and dynamics of herbicide-degrading communities cultivated with different substrate concentrations.

Two 4-chloro-2-methylphenoxyacetic acid (MCPA)-degrading enrichment cultures selected from an aquifer on low (0.1 mg liter(-1)) or high (25 mg liter(-1)) MCPA concentrations were compared in terms of metabolic activity, community composition, population growth, and single cell physiology. Different community compositions and major shifts in community structure following exposure to different MCPA concentrations were observed using both 16S rRNA gene denaturing gradient gel electrophoresis fingerprinting and pyrosequencing. The communities also differed in their MCPA-mineralizing activities. The enrichments selected on low concentrations mineralized MCPA with shorter lag phases than those selected on high concentrations. Flow cytometry measurements revealed that mineralization led to cell growth. The presence of low-nucleic acid-content bacteria (LNA bacteria) was correlated with mineralization activity in cultures selected on low herbicide concentrations. This suggests that LNA bacteria may play a role in degradation of low herbicide concentrations in aquifers impacted by agriculture. This study shows that subpopulations of herbicide-degrading bacteria that are adapted to different pesticide concentrations can coexist in the same environment and that using a low herbicide concentration enables enrichment of apparently oligotrophic subpopulations.

The use of liposomes to differentiate between the effects of nickel accumulation and altered food quality in Daphnia magna exposed to dietary nickel.

A potential drawback of traditional dietary metal toxicity studies is that it is difficult to distinguish between the direct toxicity of the metal and indirect effects caused by altered concentrations of essential nutrients in the metal-contaminated diet. In previous studies it has become clear that this can hamper the study of the real impact of dietary metal exposure and also complicates the analysis of the mechanisms of dietary metal toxicity in filter-feeding freshwater invertebrates like Daphnia magna. This problem has been partly circumvented by the production of liposomes, since these vectors are invulnerable to metal-induced food quality shifts and as such can be applied to study the mechanisms of dietary metal toxicity without the confounding effect of nutritional quality shifts. The aim of current study was to evaluate if there is relevance for dietary Ni toxicity under natural exposures, i.e., when D. magna is exposed to dietary Ni via living algae, and secondly, to quantify how nutritional quality shifts contribute to the toxic effects that are observed when algae are used as contaminated food vectors. For this aim, liposomes were prepared by the hydration of phosphatidylcholine in media containing 0 (control), 10, 50, 100 and 500 mg Ni/L. The liposome particles were then mixed with uncontaminated green algae in a 1/10 ratio (on a dry wt basis) to make up diets with constant nutrient quality and varying Ni contents (i.e., 1.2 µg Ni/g dry wt in the control and 18.7, 140.3, 165.0 and 501.6 µg Ni/g dry wt in the Ni-contaminated diet, respectively). A second food type was prepared on the basis of a 1/10 mixture (on a dry weight basis) of control liposomes and Ni-contaminated algae, representing a diet that differed in Ni content (i.e., 1.2, 26.8, 84.7, 262.3 and 742.7 µg Ni/g dry wt) and concentrations of essential nutrients (in terms of P and omega 3 poly-unsaturated fatty acids like eicosapentaenoic acid and α-linolenic acid). Both diets were then simultaneously fed to D. magna during a 21-day chronic bioassay, using reproduction, growth, survival, ingestion rate and Ni bioaccumulation as endpoints. Ni delivered by liposomes caused a significant inhibition of reproduction and growth when the metal accumulated to minimum levels of 11.9 and 20.0 µg Ni/g dry wt after 7 and 14 days, respectively. Using algae as Ni vector, similar effects of dietary Ni exposure occurred when algae had been pre-exposed to concentrations of at least 133 µg/L of bioavailable Ni (i.e., Ni2+), which is similar to the reproductive EC50 of waterborne Ni exposure for D. magna (115 µg Ni2+/L). While this may have some consequences for predicting chronic Ni toxicity in this range of Ni concentrations with the biotic ligand model--which could be further improved by including the dietary toxicity pathway in this model, the occurrence of such high concentrations in the field is very rare. Hence, there seems to be very little environmental relevance for dietary Ni toxicity to D. magna. Finally, besides the direct effects of Ni there was no evidence that nutritional quality shifts could have affected daphnids' growth, but it is very likely that the impairment of reproduction at toxic exposure levels of Ni was also partly the result of reduced fatty acid levels.

Flow cytometry for fast microbial community fingerprinting.

Characterizing the microbial community of water is important in different domains, ranging from food and beverage production to wastewater treatment. Conventional methods, such as heterotrophic plate count, selective plating and molecular techniques, are time consuming and labor intensive. A flow cytometry based approach was developed for a fast and objective comparison of microbial communities based on the distribution of cellular features from single cells within these communities. The method consists of two main parts, firstly the generation of fingerprint data by flow cytometry and secondly a novel statistical pipeline for the analysis of flow cytometric data. The combined method was shown to be useful for the discrimination and classification of different brands of drinking water. It was also successfully applied to detect changes in microbial community composition of drinking water caused by changing environmental factors. Generally, the method can be used as a fast fingerprinting method of microbial communities in aquatic samples and as a tool to detect shifts within these communities.

Liposomes as an alternative delivery system for investigating dietary metal toxicity to Daphnia magna.

Dietary metal toxicity studies with invertebrates such as Daphnia magna are often performed using metal-contaminated algae as a food source. A drawback of this approach is that it is difficult to distinguish between the direct toxicity of the metal and indirect effects caused by a reduced essential nutrient content in the metal-contaminated diet, due to prior exposure of the algae to the metal. This hampers the study of the mechanisms of dietary metal toxicity in filter-feeding freshwater invertebrates. The aim of the present study was to develop a technique for producing metal-contaminated liposomes as an alternative delivery system of dietary metals. These liposomes are not vulnerable to metal-induced shifts in nutrient quality. Liposomes were prepared by the hydration of phosphatidylcholine in media containing either 0 (control) or 50mg Ni/L. The liposomes had average diameters of 19.31 (control) and 10.48 µm (Ni-laden), i.e., a size appropriate for ingestion by D. magna. The liposome particles were then mixed with uncontaminated green algae in a 1/10 ratio (on a dry wt. basis) to make up two diets that differed in Ni content (i.e., 2.0 µg Ni/g dry wt. in the control and 144.2 µg Ni/g dry wt. in the Ni-contaminated diet, respectively). This diet was then fed to D. magna during a 21-day chronic bioassay. The experiment showed that the Ni content and the size distribution of the liposomes were stable for at least 7 days. Also the use of phosphatidylcholine as a liposome component did not affect the reproduction of the daphnids. Exposure to increased level of dietary Ni resulted in 100% mortality after 14 days of exposure and in an increased whole-body Ni concentration in D. magna of 14.9 and 20.4 µg Ni/g dry wt. after 7 and 14 days of exposure, respectively. The Ni-exposed daphnids also exhibited a reduced size (i.e., 30% smaller than the control) after 7 days and a completely halted growth between day 7 and day 14. In terms of reproduction, the size of the first brood (number of juveniles) of the Ni-exposed daphnids was significantly reduced (by 85%) compared to the control. None of the Ni-exposed individuals were able to produce a second brood before dying. The algal ingestion rate - after correction for the indirect effect of a reduced size - was increased (by 68%) by dietary Ni after 6 days of exposure compared to the control, but was severely reduced (by 80% compared to the control) after 13 days. These data suggest that an inhibition of the ingestion process may have contributed to the observed effects of dietary Ni on growth and reproduction beyond 6 days of exposure, although the involvement of other mechanisms cannot be excluded. The mechanism(s) which led to the reduced growth during the first week of exposure remain unclear, although inhibition of the ingestion process can likely be excluded here as an explanation. Overall, this paper demonstrates, using this new method of delivering dietary Ni via liposome carriers and thus excluding potential diet quality shifts, that dietary Ni can indeed induce toxic effects in D. magna. This method may therefore be a promising tool to help further elucidate the mechanisms of dietary metal toxicity to filter-feeding invertebrates.

Past, present and future applications of flow cytometry in aquatic microbiology.

Flow cytometry (FCM) is rapidly becoming an essential tool in the field of aquatic microbiology. It provides opportunities for microbial analysis at both the community and single-cell levels. Compared with other techniques, FCM facilitates rapid data acquisition and multi-parameter analysis, leading to increased popularity and widespread applications. Here, we briefly review the current status of FCM in the field of microbiology, focusing on its recent applications in aquatic microbiology. These developments range from straightforward total cell counts to community structure analysis, and further extend to physiological analysis at a single-cell level. Exciting future prospects for the applications of FCM in aquatic microbiology are also discussed based on current scientific and practical needs.