Combined TCBS and CHROMagar Analyses Allow for Basic Identification of Vibrio vulnificus within a 48 h Incubation Period in the Coastal Baltic Sea.

With rising infection rates in recent years, Vibrio vulnificus poses an increasing threat to public safety in the coastal brackish Baltic Sea. It is therefore important to monitor this organism and assess the V. vulnificus infection risk on a more regular basis. However, as the coastline of the Baltic Sea is 8000 km long and shared by nine nations, a convenient, fast, inexpensive, yet efficient V. vulnificus identification method is essential. We evaluated the effectiveness of a two-step agar-based approach consisting of successive Vibrio isolation and cultivation on thiosulphate-citrate-bile salt sucrose (TCBS) agar and CHROMagar™ Vibrio for V. vulnificus in comparison with V. cholerae, V. parahaemolyticus, and V. alginolyticus. Our study contains isolates from water and sediment across a broad expanse of the Baltic Sea including 13 locations and two different summers, the time of year during which Vibrio infections are usually much more frequent. Confirmation of isolate species identity was carried out using molecular analyses. The two-step agar plating method performed well across different locations and timeframes in correctly identifying V. vulnificus by more than 80%, but the sensitivity in other Vibrio species varied. Thus, our approach yielded promising results as a potential tool for early V. vulnificus detection across a broad timeframe and transect of the Baltic Sea and potentially other brackish environments.

Combined effects of temperature and emersion-immersion cycles on metabolism and bioenergetics of the Pacific oyster Crassostrea (Magallana) gigas.

Life on tidal coasts presents physiological major challenges for sessile species. Fluctuations in oxygen and temperature can affect bioenergetics and modulate metabolism and redox balance, but their combined effects are not well understood. We investigated the effects of intermittent hypoxia (12h/12h) in combination with different temperature regimes (normal (15 °C), elevated (30 °C) and fluctuating (15 °C water/30 °C air)) on the Pacific oyster Crassostrea (Magallana) gigas. Fluctuating temperature led to energetic costly metabolic rearrangements and accumulation of proteins in oyster tissues. Elevated temperature led to high (60%) mortality and oxidative damage in survivors. Normal temperature had no major negative effects but caused metabolic shifts. Our study shows high plasticity of oyster metabolism in response to oxygen and temperature fluctuations and indicates that metabolic adjustments to oxygen deficiency are strongly modulated by the ambient temperature. Co-exposure to constant elevated temperature and intermittent hypoxia demonstrates the limits of this adaptive metabolic plasticity.

Functional responses of key marine bacteria to environmental change - toward genetic counselling for coastal waters.

Coastal ecosystems deteriorate globally due to human-induced stress factors, like nutrient loading and pollution. Bacteria are critical to marine ecosystems, e.g., by regulating nutrient cycles, synthesizing vitamins, or degrading pollutants, thereby providing essential ecosystem services ultimately affecting economic activities. Yet, until now bacteria are overlooked both as mediators and indicators of ecosystem health, mainly due to methodological limitations in assessing bacterial ecosystem functions. However, these limitations are largely overcome by the advances in molecular biology and bioinformatics methods for characterizing the genetics that underlie functional traits of key bacterial populations - "key" in providing important ecosystem services, being abundant, or by possessing high metabolic rates. It is therefore timely to analyze and define the functional responses of bacteria to human-induced effects on coastal ecosystem health. We posit that categorizing the responses of key marine bacterial populations to changes in environmental conditions through modern microbial oceanography methods will allow establishing the nascent field of genetic counselling for our coastal waters. This requires systematic field studies of linkages between functional traits of key bacterial populations and their ecosystem functions in coastal seas, complemented with systematic experimental analyses of the responses to different stressors. Research and training in environmental management along with dissemination of results and dialogue with societal actors are equally important to ensure the role of bacteria is understood as fundamentally important for coastal ecosystems. Using the responses of microorganisms as a tool to develop genetic counselling for coastal ecosystems can ultimately allow for integrating bacteria as indicators of environmental change.

Year-Long Microbial Succession on Microplastics in Wastewater: Chaotic Dynamics Outweigh Preferential Growth.

Microplastics are a globally-ubiquitous aquatic pollutant and have been heavily studied over the last decade. Of particular interest are the interactions between microplastics and microorganisms, especially the pursuit to discover a plastic-specific biome, the so-called plastisphere. To follow this up, a year-long microcosm experimental setup was deployed to expose five different microplastic types (and silica beads control) to activated aerobic wastewater in controlled conditions, with microbial communities being measured four times over the course of the year using 16S rDNA (bacterial) and ITS (fungal) amplicon sequencing. The biofilm community shows no evidence of a specific plastisphere, even after a year of incubation. Indeed, the microbial communities (particularly bacterial) show a clear trend of increasing dissimilarity between plastic types as time increases. Despite little evidence for a plastic-specific community, there was a slight grouping observed for polyolefins (PE and PP) in 6-12-month biofilms. Additionally, an OTU assigned to the genus Devosia was identified on many plastics, increasing over time while showing no growth on silicate (natural particle) controls, suggesting this could be either a slow-growing plastic-specific taxon or a symbiont to such. Both substrate-associated findings were only possible to observe in samples incubated for 6-12 months, which highlights the importance of studying long-term microbial community dynamics on plastic surfaces.

Agricultural application of microplastic-rich sewage sludge leads to further uncontrolled contamination.

Municipal sewage sludge has been shown to be high in microplastics (MP) and is applied to agricultural land as fertiliser in many countries. The authors recently proposed in a viewpoint article that MP applied to land in this way may well contaminate other areas in an uncontrolled way. This study examined experimental plots with known history of application of sewage sludge. Results showed that 44% of the MP load found on sludge-applied land was found on nearby land never directly applied with sludge. Examination of polymer type compositions demonstrated marked similarity between the two fields indicating the sludge-applied field was a source of contamination for surrounding areas. Furthermore, MP was detected at a depth of 60-90 cm in the sludge-applied soil indicating that MP may also penetrate deep enough to reach agricultural drainage systems, although this effect is slight (1.6% of surface load). These results show that application of municipal sewage sludge on agricultural land can lead to further uncontrolled contamination, paving the way for future research to improve understanding of the extents of such effects on real farms to better inform future agricultural policy.

Acclimation of Nodularia spumigena CCY9414 to inorganic phosphate limitation - Identification of the P-limitation stimulon via RNA-seq.

Nodularia spumigena is a toxic, filamentous cyanobacterium capable of fixing atmospheric N2, which is often dominating cyanobacterial bloom events in the Baltic Sea and other brackish water systems worldwide. Increasing phosphate limitation has been considered as one environmental factor promoting cyanobacterial mass developments. In the present study, we analyzed the response of N. spumigena strain CCY9414 toward strong phosphate limitation. Growth of the strain was diminished under P-deplete conditions; however, filaments contained more polyphosphate under P-deplete compared to P-replete conditions. Using RNA-seq, gene expression was compared in N. spumigena CCY9414 after 7 and 14 days in P-deplete and P-replete conditions, respectively. After 7 days, 112 genes were significantly up-regulated in P-deplete filaments, among them was a high proportion of genes encoding proteins related to P-homeostasis such as transport systems for different P species. Many of these genes became also up-regulated after 14 days compared to 7 days in filaments grown under P-replete conditions, which was consistent with the almost complete consumption of dissolved P in these cultures after 14 days. In addition to genes directly related to P starvation, genes encoding proteins for bioactive compound synthesis, gas vesicles formation, or sugar catabolism were stimulated under P-deplete conditions. Collectively, our data describe an experimentally validated P-stimulon in N. spumigena CCY9414 and provide the indication that severe P limitation could indeed support bloom formation by this filamentous strain.

Machine Learning Predicts the Presence of 2,4,6-Trinitrotoluene in Sediments of a Baltic Sea Munitions Dumpsite Using Microbial Community Compositions.

Bacteria are ubiquitous and live in complex microbial communities. Due to differences in physiological properties and niche preferences among community members, microbial communities respond in specific ways to environmental drivers, potentially resulting in distinct microbial fingerprints for a given environmental state. As proof of the principle, our goal was to assess the opportunities and limitations of machine learning to detect microbial fingerprints indicating the presence of the munition compound 2,4,6-trinitrotoluene (TNT) in southwestern Baltic Sea sediments. Over 40 environmental variables including grain size distribution, elemental composition, and concentration of munition compounds (mostly at pmol⋅g-1 levels) from 150 sediments collected at the near-to-shore munition dumpsite Kolberger Heide by the German city of Kiel were combined with 16S rRNA gene amplicon sequencing libraries. Prediction was achieved using Random Forests (RFs); the robustness of predictions was validated using Artificial Neural Networks (ANN). To facilitate machine learning with microbiome data we developed the R package phyloseq2ML. Using the most classification-relevant 25 bacterial genera exclusively, potentially representing a TNT-indicative fingerprint, TNT was predicted correctly with up to 81.5% balanced accuracy. False positive classifications indicated that this approach also has the potential to identify samples where the original TNT contamination was no longer detectable. The fact that TNT presence was not among the main drivers of the microbial community composition demonstrates the sensitivity of the approach. Moreover, environmental variables resulted in poorer prediction rates than using microbial fingerprints. Our results suggest that microbial communities can predict even minor influencing factors in complex environments, demonstrating the potential of this approach for the discovery of contamination events over an integrated period of time. Proven for a distinct environment future studies should assess the ability of this approach for environmental monitoring in general.

Emission, Transport, and Deposition of visible Plastics in an Estuary and the Baltic Sea-a Monitoring and Modeling Approach.

AIM: was to assess whether a comprehensive approach linking existing knowledge with monitoring and modeling can provide an improved insight into coastal and marine plastics pollution. We focused on large micro- and mesoplastic (1-25 mm) and selected macroplastic items. Emission calculations, samplings in the Warnow river and estuary (water body and bottom sediments) and a flood accumulation zone monitoring served as basis for model simulations on transport and behavior in the entire Baltic Sea. Considered were the most important pathways, sewage overflow and stormwater. The coastline monitoring together with calculations allowed estimating plastics emissions for Rostock city and the Warnow catchment. Average concentrations at the Warnow river mouth were 0.016 particles/m³ and in the estuary 0.14 particles/m³ (300 µm net). The estuary and nearby Baltic Sea beaches were hot-spots for plastic accumulation with 6-31 particles/m². With increasing distance from the estuary, the concentrations dropped to 0.3 particles/m². This spatial pattern, the plastic pollution gradients and the observed annual accumulation values were consistent with the model results. Indicator items for sewer overflow and stormwater emissions exist, but were only found at low numbers in the environment. The considered visible plastics alone can hardly serve as indicator for microplastic pollution (<1 mm). The use of up-scaled emission data as input for Baltic Sea model simulations provided information on large scale emission, transport and deposition patterns of visible plastics. The results underline the importance of plastic retention in rivers and estuaries.

Identification and quantification of microplastic particles in drinking water treatment sludge as an integrative approach to determine microplastic abundance in a freshwater river.

Microplastic (MP) has been detected ubiquitously in freshwater systems. Until now MP sampling, however, is predominantly based on short-term net or pumping and filtration systems which can only provide snapshots of MP abundance; especially in flowing water bodies. To improve representativeness in the determination of MP occurrences in these aquatic compartments, an integrative approach that covers larger water volumes for a longer period of time is required. In this regard, surface water supplied drinking water treatment plants (DWTPs) represent an opportunity. In DWTPs, suspended solids from thousands of cubic metres of raw water are continuously removed over several hours and enriched in coagulation/flocculation and filtration processes. Our hypothesis was that MP is also removed to a full extent, like suspended solids, and that an integrative approach for identification and quantification in raw water can be derived from the analysis of MP in the treatment sludge. To prove this hypothesis, treatment sludge from a riverside DWTP (Warnow river, North-Eastern Germany) was analysed for MP > 50 µm. A sample purification protocol overcoming potential matrix effects caused by coagulants and flocculants was developed and validated. MP was analysed using micro-Raman spectroscopy. MP occurrence determined for the Warnow river was compared with in situ reference sampling using an established pumping and filtration system at relatively stable flow conditions. As result, the number of MP particles derived from treatment sludge was extrapolated to 196 ± 42 m-3 for the Warnow river and is statistically insignificantly different from 233 ± 36 m-3 identified by conventional water sampling. In addition, the polymer distribution and particles shape indicated the validity of the integrative concept. Consequently, the determination of MP abundance for freshwater systems based on DWTP treatment sludge represents an adequate method to estimate MP concentrations in flowing waters in an integrative way.

Cross-Hemisphere Study Reveals Geographically Ubiquitous, Plastic-Specific Bacteria Emerging from the Rare and Unexplored Biosphere.

While it is now appreciated that the millions of tons of plastic pollution travelling through marine systems carry complex communities of microorganisms, it is still unknown to what extent these biofilm communities are specific to the plastic or selected by the surrounding ecosystem. To address this, we characterized and compared the microbial communities of microplastic particles, nonplastic (natural and wax) particles, and the surrounding waters from three marine ecosystems (the Baltic, Sargasso and Mediterranean seas) using high-throughput 16S rRNA gene sequencing. We found that biofilm communities on microplastic and nonplastic particles were highly similar to one another across this broad geographical range. The similar temperature and salinity profiles of the Sargasso and Mediterranean seas, compared to the Baltic Sea, were reflected in the biofilm communities. We identified plastic-specific operational taxonomic units (OTUs) that were not detected on nonplastic particles or in the surrounding waters. Twenty-six of the plastic-specific OTUs were geographically ubiquitous across all sampled locations. These geographically ubiquitous plastic-specific OTUs were mostly low-abundance members of their biofilm communities and often represented uncultured members of marine ecosystems. These results demonstrate the potential for plastics to be a reservoir of rare and understudied microbes, thus warranting further investigations into the dynamics and role of these microbes in marine ecosystems. IMPORTANCE This study represents one of the largest comparisons of biofilms from environmentally sampled plastic and nonplastic particles from aquatic environments. By including particles sampled through three separate campaigns in the Baltic, Sargasso, and Mediterranean seas, we were able to make cross-geographical comparisons and discovered common taxonomical signatures that define the plastic biofilm. For the first time, we identified plastic-specific bacteria that reoccur across marine regions. Our data reveal that plastics have selective properties that repeatedly enrich for similar bacteria regardless of location, potentially shifting aquatic microbial communities in areas with high levels of plastic pollution. Furthermore, we show that bacterial communities on plastic do not appear to be strongly influenced by polymer type, suggesting that other properties, such as the absorption and/or leaching of chemicals from the surface, are likely to be more important in the selection and enrichment of specific microorganisms.

Genomic and proteomic profiles of biofilms on microplastics are decoupled from artificial surface properties.

Microplastics in marine ecosystems are colonized by diverse prokaryotic and eukaryotic communities. How these communities and their functional profiles are shaped by the artificial surfaces remains broadly unknown. In order to close this knowledge gap, we set up an in situ experiment with pellets of the polyolefin polymer polyethylene (PE), the aromatic hydrocarbon polymer polystyrene (PS), and wooden beads along a coastal to estuarine gradient in the Baltic Sea, Germany. We used an integrated metagenomics/metaproteomics approach to evaluate the genomic potential as well as protein expression levels of aquatic plastic biofilms. Our results suggest that material properties had a minor influence on the plastic-associated assemblages, as genomic and proteomic profiles of communities associated with the structurally different polymers PE and PS were highly similar, hence polymer-unspecific. Instead, it seemed that these communities were shaped by biogeographic factors. Wood, on the other hand, induced the formation of substrate-specific biofilms and served as nutrient source itself. Our study indicates that, while PE and PS microplastics may be relevant in the photic zone as opportunistic colonization grounds for phototrophic microorganisms, they appear not to be subject to biodegradation or serve as vectors for pathogenic microorganisms in marine habitats.

High-Throughput Analyses of Microplastic Samples Using Fourier Transform Infrared and Raman Spectrometry.

Determining microplastics in environmental samples quickly and reliably is a challenging task. With a largely automated combination of optical particle analysis, Fourier transform infrared (FT-IR), and Raman microscopy along with spectral database search, particle sizes, particle size distributions, and the type of polymer including particle color can be determined. We present a self-developed, open-source software package for realizing a particle analysis approach with both Raman and FT-IR microspectroscopy. Our software GEPARD (Gepard Enabled PARticle Detection) allows for acquiring an optical image, then detects particles and uses this information to steer the spectroscopic measurement. This ultimately results in a multitude of possibilities for efficiently reviewing, correcting, and reporting all obtained results.

Ecosystem-wide metagenomic binning enables prediction of ecological niches from genomes.

The genome encodes the metabolic and functional capabilities of an organism and should be a major determinant of its ecological niche. Yet, it is unknown if the niche can be predicted directly from the genome. Here, we conduct metagenomic binning on 123 water samples spanning major environmental gradients of the Baltic Sea. The resulting 1961 metagenome-assembled genomes represent 352 species-level clusters that correspond to 1/3 of the metagenome sequences of the prokaryotic size-fraction. By using machine-learning, the placement of a genome cluster along various niche gradients (salinity level, depth, size-fraction) could be predicted based solely on its functional genes. The same approach predicted the genomes' placement in a virtual niche-space that captures the highest variation in distribution patterns. The predictions generally outperformed those inferred from phylogenetic information. Our study demonstrates a strong link between genome and ecological niche and provides a conceptual framework for predictive ecology based on genomic data.

When every particle matters: A QuEChERS approach to extract microplastics from environmental samples.

The identification of microplastics (MP), especially small (<500 µm) MP, using automated surface-chemistry approaches requires the best possible reduction of natural particles whilst preserving the integrity of the targeted synthetic polymers particles. In general, both natural and synthetic particles can be highly diverse physically and chemically and MP extraction, particularly from complex matrices such as sediments, sludge and soils, requires efficient method pipelines. Our paper presents a universal framework of modular protocols (presented in a decision tree) that fulfil predefined user requirements (QuEChERS: Quick, Easy, Cheap, Effective, Rugged, Safe) as well as providing best practises for reasonable MP working conditions within a standard laboratory. New procedures and technical innovations for density separation of particle-rich matrices are presented, such as a spiral conveyor developed and validated for MP recovery. In sharing such best-practice protocols, we aim to help in the push towards MP quantification method standardisation. •Publication of protocols of an entire MP extraction (10 µm - 5 mm) pipeline for particle-based analysis of various environmental matrices•Modularity: Optimised quantitative sample preparation adapted to particle sizes and sample matrices•New protocols and technical innovations (e.g. spiral conveyor) optimise MP extraction.

Vibrio Colonization Is Highly Dynamic in Early Microplastic-Associated Biofilms as Well as on Field-Collected Microplastics.

Microplastics are ubiquitous in aquatic ecosystems and provide a habitat for biofilm-forming bacteria. The genus Vibrio, which includes potential pathogens, was detected irregularly on microplastics. Since then, the potential of microplastics to enrich (and serve as a vector for) Vibrio has been widely discussed. We investigated Vibrio abundance and operational taxonomic unit (OTU) composition on polyethylene and polystyrene within the first 10 h of colonization during an in situ incubation experiment, along with those found on particles collected from the Baltic Sea. We used 16S rRNA gene amplicon sequencing and co-occurrence networks to elaborate the role of Vibrio within biofilms. Colonization of plastics with Vibrio was detectable after one hour of incubation; however, Vibrio numbers and composition were very dynamic, with a more stable population at the site with highest nutrients and lowest salinity. Likewise, Vibrio abundances on field-collected particles were variable but correlated with proximity to major cities. Vibrio was poorly connected within biofilm networks. Taken together, this indicates that Vibrio is an early colonizer of plastics, but that the process is undirected and independent of the specific surface. Still, higher nutrients could enhance a faster establishment of Vibrio populations. These parameters should be considered when planning studies investigating Vibrio on microplastics.

Marine Microbial Assemblages on Microplastics: Diversity, Adaptation, and Role in Degradation.

We have known for more than 45 years that microplastics in the ocean are carriers of microbially dominated assemblages. However, only recently has the role of microbial interactions with microplastics in marine ecosystems been investigated in detail. Research in this field has focused on three main areas: (a) the establishment of plastic-specific biofilms (the so-called plastisphere); (b) enrichment of pathogenic bacteria, particularly members of the genus Vibrio, coupled to a vector function of microplastics; and (c) the microbial degradation of microplastics in the marine environment. Nevertheless, the relationships between marine microorganisms and microplastics remain unclear. In this review, we deduce from the current literature, new comparative analyses, and considerations of microbial adaptation concerning plastic degradation that interactions between microorganisms and microplastic particles should have rather limited effects on the ocean ecosystems. The majority of microorganisms growing on microplastics seem to belong to opportunistic colonists that do not distinguish between natural and artificial surfaces. Thus, microplastics do not pose a higher risk than natural particles to higher life forms by potentially harboring pathogenic bacteria. On the other hand, microplastics in the ocean represent recalcitrant substances for microorganisms that are insufficient to support prokaryotic metabolism and will probably not be microbially degraded in any period of time relevant to human society. Because we cannot remove microplastics from the ocean, proactive action regarding research on plastic alternatives and strategies to prevent plastic entering the environment should be taken promptly.

Cultivation and functional characterization of 79 planctomycetes uncovers their unique biology.

When it comes to the discovery and analysis of yet uncharted bacterial traits, pure cultures are essential as only these allow detailed morphological and physiological characterization as well as genetic manipulation. However, microbiologists are struggling to isolate and maintain the majority of bacterial strains, as mimicking their native environmental niches adequately can be a challenging task. Here, we report the diversity-driven cultivation, characterization and genome sequencing of 79 bacterial strains from all major taxonomic clades of the conspicuous bacterial phylum Planctomycetes. The samples were derived from different aquatic environments but close relatives could be isolated from geographically distinct regions and structurally diverse habitats, implying that 'everything is everywhere'. With the discovery of lateral budding in 'Kolteria novifilia' and the capability of the members of the Saltatorellus clade to divide by binary fission as well as budding, we identified previously unknown modes of bacterial cell division. Alongside unobserved aspects of cell signalling and small-molecule production, our findings demonstrate that exploration beyond the well-established model organisms has the potential to increase our knowledge of bacterial diversity. We illustrate how 'microbial dark matter' can be accessed by cultivation techniques, expanding the organismic background for small-molecule research and drug-target detection.

Author Correction: Tracing microplastics in aquatic environments based on sediment analogies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Tracing microplastics in aquatic environments based on sediment analogies.

Microplastics (MP) data collection from the aquatic environment is a challenging endeavour that sets apparent limitations to regional and global MP quantification. Expensive data collection causes small sample sizes and oftentimes existing data sets are compared without accounting for natural variability due to hydrodynamic processes governing the distribution of particles. In Warnow estuarine sediments (Germany) we found significant correlations between high-density polymer size fractions (≥500 mm) and sediment grain size. Among potential predictor variables (source and environmental terms) sediment grain size was the critical proxy for MP abundance. The MP sediment relationship can be explained by the force necessary to start particle transport: at the same level of fluid motion, transported sediment grains and MP particles are offset in size by one to two orders of magnitude. Determining grain-size corrected MP abundances by fractionated granulometric normalisation is recommended as a basis for future MP projections and identification of sinks and sources.

Uneven host cell growth causes lysogenic virus induction in the Baltic Sea.

In the Baltic Sea redoxcline, lysogenic viruses infecting prokaryotes have rarely been detected using the commonly used inducing agent mitomycin C. However, it is well known that not all viruses are induceable by mitomycin C and growing evidence suggests that changes in trophic conditions may trigger the induction of lysogenic viruses. We hypothesized that using antibiotics to simulate a strong change in trophic conditions for antibiotica-resistant cells due to reduced competition for resources might lead to the induction of lysogenic viruses into the lytic cycle within these cells. This hypothesis was tested by incubating prokaryotes obtained throughout the Baltic Sea redoxcline in seawater with substantially reduced numbers of viruses. We used a mixture of the protein synthesis-inhibiting antibiotics streptomycin and erythromycin to induce the desired changes in trophic conditions for resistant cells and at the same time ensuring that no progeny viruses were formed in sensitive cells. No inducible lysogenic viruses could be detected in incubations amended with mitomycin C. Yet, the presence of streptomycin and erythromycin increased virus-induced mortality of prokaryotes by 56-930% compared to controls, resulting in the induction of lysogenic viruses equivalent to 2-14% of in situ prokaryotic abundance. The results indicate the existence of a previously unrecognized induction mechanism for lysogenic viruses in the Baltic Sea redoxcline, as the mode of action distinctly differs between the used antibiotics (no virus production within affected cells) and mitomycin C (lysogenic viruses are produced within affected cells). Obtaining accurate experimental data on levels of lysogeny in prokaryotic host cells remains challenging, as relying on mitomycin C alone may severely underestimate lysogeny.