Monitoring of environmental DNA from nonindigenous species of algae, dinoflagellates and animals in the North East Atlantic.

Monitoring the distribution of marine nonindigenous species is a challenging task. To support this monitoring, we developed and validated the specificity of 12 primer-probe assays for detection of environmental DNA (eDNA) from marine species, all nonindigenous to Europe. The species include sturgeons, a Pacific red algae, oyster thief, a freshwater hydroid from the Black Sea, Chinese mitten crab, Pacific oyster, warty comb jelly, sand gaper, round goby, pink salmon, rainbow trout and North American mud crab. We tested all assays in the laboratory, on DNA extracted from both the target and non-target species to ensure that they only amplified DNA from the intended species. Subsequently, all assays were used to analyse water samples collected at 16 different harbours across two different seasons during 2017. We also included six previously published assays targeting eDNA from goldfish, European carp, two species of dinoflagellates of the genera Karenia and Prorocentrum, two species of the heterokont flagellate genus Pseudochattonella. Conventional monitoring was carried out alongside eDNA sampling but with only one sampling event over the one year. Because eDNA was relatively fast and easy to collect compared to conventional sampling, we sampled eDNA twice during 2017, which showed seasonal changes in the distribution of nonindigenous species. Comparing eDNA levels with salinity gradients did not show any correlation. A significant correlation was observed between number of species detected with conventional monitoring methods and number of species found using eDNA at each location. This supports the use of eDNA for surveillance of the distribution of marine nonindigenous species, where the speed and relative easy sampling in the field combined with fast molecular analysis may provide advantages compared to conventional monitoring methods. Prior validation of assays increases taxonomic precision, and laboratorial setup facilitates analysis of multiple samples simultaneously. The specific eDNA assays presented here can be implemented directly in monitoring programmes across Europe and potentially worldwide to infer a more precise picture of the dynamics in the distribution of marine nonindigenous species.

The diversity of population responses to environmental change.

The current extinction and climate change crises pressure us to predict population dynamics with ever-greater accuracy. Although predictions rest on the well-advanced theory of age-structured populations, two key issues remain poorly explored. Specifically, how the age-dependency in demographic rates and the year-to-year interactions between survival and fecundity affect stochastic population growth rates. We use inference, simulations and mathematical derivations to explore how environmental perturbations determine population growth rates for populations with different age-specific demographic rates and when ages are reduced to stages. We find that stage- vs. age-based models can produce markedly divergent stochastic population growth rates. The differences are most pronounced when there are survival-fecundity-trade-offs, which reduce the variance in the population growth rate. Finally, the expected value and variance of the stochastic growth rates of populations with different age-specific demographic rates can diverge to the extent that, while some populations may thrive, others will inevitably go extinct.

Monitoring of noble, signal and narrow-clawed crayfish using environmental DNA from freshwater samples.

For several hundred years freshwater crayfish (Crustacea-Decapoda-Astacidea) have played an important ecological, cultural and culinary role in Scandinavia. However, many native populations of noble crayfish Astacus astacus have faced major declines during the last century, largely resulting from human assisted expansion of non-indigenous signal crayfish Pacifastacus leniusculus that carry and transmit the crayfish plague pathogen. In Denmark, also the non-indigenous narrow-clawed crayfish Astacus leptodactylus has expanded due to anthropogenic activities. Knowledge about crayfish distribution and early detection of non-indigenous and invasive species are crucial elements in successful conservation of indigenous crayfish. The use of environmental DNA (eDNA) extracted from water samples is a promising new tool for early and non-invasive detection of species in aquatic environments. In the present study, we have developed and tested quantitative PCR (qPCR) assays for species-specific detection and quantification of the three above mentioned crayfish species on the basis of mitochondrial cytochrome oxidase 1 (mtDNA-CO1), including separate assays for two clades of A. leptodactylus. The limit of detection (LOD) was experimentally established as 5 copies/PCR with two different approaches, and the limit of quantification (LOQ) were determined to 5 and 10 copies/PCR, respectively, depending on chosen approach. The assays detected crayfish in natural freshwater ecosystems with known populations of all three species, and show promising potentials for future monitoring of A. astacus, P. leniusculus and A. leptodactylus. However, the assays need further validation with data 1) comparing traditional and eDNA based estimates of abundance, and 2) representing a broader geographical range for the involved crayfish species.

Microbial toxicity of methyl tert-butyl ether (MTBE) determined with fluorescent and luminescent bioassays.

The inhibitory effects of the fuel additive methyl tert-butyl ether (MTBE) and potential degradation products tert-butanol (TBA) and formaldehyde was examined using mixed microbial biomass, and six strains of bioluminescent bacteria and yeast. The purpose was to assess microbial toxicity with quantitative bioluminescent and fluorescent endpoints, and to identify sensitive proxies suitable for monitoring MTBE contamination. Bioluminescent Aliivibrio fischeri DSM 7151 (formerly Vibrio fischeri) appeared highly sensitive to MTBE exposure, and was a superior test organisms compared to lux-tagged Escherichia coli DH5α, Pseudomonas fluorescens DF57-40E7 and Saccharomyces cerevisiae BLYR. EC10 and EC50 for acute MTBE toxicity in A. fischeri were 1.1 and 10.9 mg L(-1), respectively. Long term (24h) MTBE exposure resulted in EC10 values of 0.01 mg L(-1). TBA was significantly less toxic with EC10 and EC50 for acute and chronic toxicity >1000 mg L(-1). Inhibition of bioluminescence was generally a more sensitive endpoint for MTBE toxicity than measuring intracellular ATP levels and heterotrophic CO2 assimilation. A weak estrogenic response was detected for MTBE at concentrations ⩾ 3.7 g L(-1) using an estrogen inducible bioluminescent yeast strain (S. cerevisiae BLYES). Microbial hydrolytic enzyme activity in groundwater was affected by MTBE with EC10 values of 0.5-787 mg L(-1), and EC50 values of 59-3073 for alkaline phosphatase, arylsulfatase, beta-1,4-glucanase, N-acetyl-beta-d-glucosaminidase, and leucine-aminopeptidase. Microbial alkaline phosphatase and beta-1,4-glucanase activity were most sensitive to MTBE exposure with EC50 ⩽ 64.8 mg L(-1). The study suggests that bioassays with luminescent A. fischeri, and fluorescent assays targeting hydrolytic enzyme activity are good candidates for monitoring microbial MTBE toxicity in contaminated water.

Isotope array analysis of Rhodocyclales uncovers functional redundancy and versatility in an activated sludge.

Extensive physiological analyses of different microbial community members in many samples are difficult because of the restricted number of target populations that can be investigated in reasonable time by standard substrate-mediated isotope-labeling techniques. The diversity and ecophysiology of Rhodocyclales in activated sludge from a full-scale wastewater treatment plant were analyzed following a holistic strategy based on the isotope array approach, which allows for a parallel functional probing of different phylogenetic groups. Initial diagnostic microarray, comparative 16S rRNA gene sequence, and quantitative fluorescence in situ hybridization surveys indicated the presence of a diverse community, consisting of an estimated number of 27 operational taxonomic units that grouped in at least seven main Rhodocyclales lineages. Substrate utilization profiles of probe-defined populations were determined by radioactive isotope array analysis and microautoradiography-fluorescence in situ hybridization of activated sludge samples that were briefly exposed to different substrates under oxic and anoxic, nitrate-reducing conditions. Most detected Rhodocyclales groups were actively involved in nitrogen transformation, but varied in their consumption of propionate, butyrate, or toluene, and thus in their ability to use different carbon sources in activated sludge. This indicates that the functional redundancy of nitrate reduction and the functional versatility of substrate usage are important factors governing niche overlap and differentiation of diverse Rhodocyclales members in this activated sludge.

Method for measuring substrate preferences by individual members of microbial consortia proposed for bioaugmentation.

In this study we used the assimilation of isotope labeled CO(2) to measure the substrate preferences by two different bioaugmentation mixtures proposed for bioremediation of diesel oil contamination. All active microorganisms assimilate CO(2) in various carboxylation processes involved in growth. The CO(2) assimilation by the two mixtures was measured upon addition of glucose, diesel oil or specific compounds present in diesel oil (naphthalene, toluene, hexadecane, and octane). It was shown that within short term incubations with diesel oil (<5 h), one bioaugmentation mixture was superior to the other regarding the assimilation of CO(2). This observation was confirmed in a labor-intensive long term microcosm study (60 days). The applied method open various possibilities for fast pre-testing of substrate-preferences by microbial-bioaugmentation mixtures without microcosm experiments, on-site tests, and complicated chemical analysis. This study also demonstrates the possibility to obtain further information on the substrate preferences at a single cell level of phylogenetically defined microbial subgroups in bioaugmentation mixtures, based on combined analyses of microautoradiography and fluorescence in situ hybridization.

Degradation of organic pollutants by methane grown microbial consortia.

Microbial consortia were enriched from various environmental samples with methane as the sole carbon and energy source. Selected consortia that showed a capacity for co-oxidation of naphthalene were screened for their ability to degrade methyl-tert-butyl-ether (MTBE), phthalic acid esters (PAE), benzene, xylene and toluene (BTX). MTBE was not removed within 24 h by any of the consortia examined. One consortium enriched from activated sludge ("AAE-A2"), degraded PAE, including (butyl-benzyl)phthalate (BBP), and di-(butyl)phthalate (DBP). PAE have not previously been described as substrates for methanotrophic consortia. The apparent Km and Vmax for DBP degradation by AAE-A2 at 20 degrees C was 3.1 +/- 1.2 mg l(-1) and 8.7 +/- 1.1 mg DBP (g protein x h)(-1), respectively. AAE-A2 also showed fast degradation of BTX (230 +/- 30 nmol benzene (mg protein x h)(-1) at 20 degrees C). Additionally, AAE-A2 degraded benzene continuously for 2 weeks. In contrast, a pure culture of the methanotroph Methylosinus trichosporium OB3b ceased benzene degradation after only 2 days. Experiments with methane mono-oxygenase inhibitors or competitive substrates suggested that BTX degradation was carried out by methane-oxidizing bacteria in the consortium, whereas the degradation of PAE was carried out by non-methanotrophic bacteria co-existing with methanotrophs. The composition of the consortium (AAE-A2) based on polar lipid fatty acid (PLFA) profiles showed dominance of type II methanotrophs (83-92% of biomass). Phylogeny based on a 16S-rRNA gene clone library revealed that the dominating methanotrophs belonged to Methylosinus/Methylocystis spp. and that members of at least 4 different non-methanotrophic genera were present (Pseudomonas, Flavobacterium, Janthinobacterium and Rubivivax).

Isotope labeling and microautoradiography of active heterotrophic bacteria on the basis of assimilation of 14CO(2).

Most heterotrophic bacteria assimilate CO(2) in various carboxylation reactions during biosynthesis. In this study, assimilation of (14)CO(2) by heterotrophic bacteria was used for isotope labeling of active microorganisms in pure cultures and environmental samples. Labeled cells were visualized by microautoradiography (MAR) combined with fluorescence in situ hybridization (FISH) to obtain simultaneous information about activity and identity. Cultures of Escherichia coli and Pseudomonas putida assimilated sufficient (14)CO(2) during growth on various organic substrates to obtain positive MAR signals. The MAR signals were comparable with the traditional MAR approach based on uptake of (14)C-labeled organic substrates. Experiments with E. coli showed that (14)CO(2) was assimilated during both fermentation and aerobic and anaerobic respiration. The new MAR approach, HetCO(2)-MAR, was evaluated by targeting metabolic active filamentous bacteria, including "Candidatus Microthrix parvicella" in activated sludge. "Ca. Microthrix parvicella" was able to take up oleic acid under anaerobic conditions, as shown by the traditional MAR approach with [(14)C]oleic acid. However, the new HetCO(2)-MAR approach indicated that "Ca. Microthrix parvicella," did not significantly grow on oleic acid under anaerobic conditions with or without addition of NO(2)(-), whereas the addition of O(2) or NO(3)(-) initiated growth, as indicated by detectable (14)CO(2) assimilation. This is a metabolic feature that has not been described previously for filamentous bacteria. Such information could not have been derived by using the traditional MAR procedure, whereas the new HetCO(2)-MAR approach differentiates better between substrate uptake and substrate metabolism that result in growth. The HetCO(2)-MAR results were supported by stable isotope analysis of (13)C-labeled phospholipid fatty acids from activated sludge incubated under aerobic and anaerobic conditions in the presence of (13)CO(2). In conclusion, the novel HetCO(2)-MAR approach expands the possibility for studies of the ecophysiology of uncultivated microorganisms.

Use of heterotrophic CO2 assimilation as a measure of metabolic activity in planktonic and sessile bacteria.

We have examined whether assimilation of CO2 can be used as a measure of metabolic activity in planktonic and sessile heterotrophic bacteria. CO2 assimilation by environmental samples and pure cultures of heterotrophic bacteria was studied using 14CO2 and 13CO2 as tracers. Heterotrophic growth on complex organic substrates resulted in assimilation of CO2 into cell biomass by activated sludge, drinking water biofilm, and pure cultures of Escherichia coli ATCC 25922, Es. coli ATCC 13706, Rhodococcus ruber, Burkholderia sp., Bacillus circulans, Pseudomonas putida, Pseudomonas stutzeri, and Pseudomonas aeruginosa. Analysis of 13C-labelled phospholipid fatty acids (PLFAs) confirmed that heterotrophic bacteria may assimilate 13CO2 into cell macromolecules such as membrane lipids. All major PLFAs extracted from activated sludge and drinking water biofilm samples were enriched in 13C after incubation with CO2. Between 1.4% and 6.5% of the biomass produced by cultures of P. putida and a drinking water biofilm during growth in complex media was apparently derived from assimilation of CO2. Resting cells assimilated less CO2 compared to actively growing cells, and CO2 assimilation activity correlated with the amount of biomass produced during heterotrophic growth. The 14CO2 assimilation assay was evaluated as a tool to examine inhibitory effects of biocides on planktonic and sessile heterotrophs (biofilms). On the basis of 14CO2 assimilation activity, the minimum inhibitory concentration (MIC) of benzalkonium chloride was estimated to 21.1 and 127.2 mg l(-1) for planktonic and biofilm samples, respectively. The results indicate that assimilation of isotopically labelled CO2 can be used as a relatively simple measure of metabolic activity in heterotrophic bacteria. CO2 assimilation assays may be used to study the effects of antimicrobial agents on growth and survival of planktonic and sessile heterotrophic organisms.

The isotope array, a new tool that employs substrate-mediated labeling of rRNA for determination of microbial community structure and function.

A new microarray method, the isotope array approach, for identifying microorganisms which consume a (14)C-labeled substrate within complex microbial communities was developed. Experiments were performed with a small microarray consisting of oligonucleotide probes targeting the 16S rRNA of ammonia-oxidizing bacteria (AOB). Total RNA was extracted from a pure culture of Nitrosomonas eutropha grown in the presence of [(14)C]bicarbonate. After fluorescence labeling of the RNA and microarray hybridization, scanning of all probe spots for fluorescence and radioactivity revealed that specific signals were obtained and that the incorporation of (14)C into rRNA could be detected unambiguously. Subsequently, we were able to demonstrate the suitability of the isotope array approach for monitoring community composition and CO(2) fixation activity of AOB in two nitrifying activated-sludge samples which were incubated with [(14)C]bicarbonate for up to 26 h. AOB community structure in the activated-sludge samples, as predicted by the microarray hybridization pattern, was confirmed by quantitative fluorescence in situ hybridization (FISH) and comparative amoA sequence analyses. CO(2) fixation activities of the AOB populations within the complex activated-sludge communities were detectable on the microarray by (14)C incorporation and were confirmed independently by combining FISH and microautoradiography. AOB rRNA from activated sludge incubated with radioactive bicarbonate in the presence of allylthiourea as an inhibitor of AOB activity showed no incorporation of (14)C and thus was not detectable on the radioactivity scans of the microarray. These results suggest that the isotope array can be used in a PCR-independent manner to exploit the high parallelism and discriminatory power of microarrays for the direct identification of microorganisms which consume a specific substrate in the environment.