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.

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.

Comparison of µ-ATR-FTIR spectroscopy and py-GCMS as identification tools for microplastic particles and fibers isolated from river sediments.

In recent years, many studies on the analysis of microplastics (MP) in environmental samples have been published. These studies are hardly comparable due to different sampling, sample preparation, as well as identification and quantification techniques. Here, MP identification is one of the crucial pitfalls. Visual identification approaches using morphological criteria alone often lead to significant errors, being especially true for MP fibers. Reliable, chemical structure-based identification methods are indispensable. In this context, the frequently used vibrational spectroscopic techniques but also thermoanalytical methods are established. However, no critical comparison of these fundamentally different approaches has ever been carried out with regard to analyzing MP in environmental samples. In this blind study, we investigated 27 single MP particles and fibers of unknown material isolated from river sediments. Successively micro-attenuated total reflection Fourier transform infrared spectroscopy (µ-ATR-FTIR) and pyrolysis gas chromatography-mass spectrometry (py-GCMS) in combination with thermochemolysis were applied. Both methods differentiated between plastic vs. non-plastic in the same way in 26 cases, with 19 particles and fibers (22 after re-evaluation) identified as the same polymer type. To illustrate the different approaches and emphasize the complementarity of their information content, we exemplarily provide a detailed comparison of four particles and three fibers and a critical discussion of advantages and disadvantages of both methods.

Success of chemolithoautotrophic SUP05 and Sulfurimonas GD17 cells in pelagic Baltic Sea redox zones is facilitated by their lifestyles as K- and r-strategists.

Chemolithoautotrophic sulfur-oxidizing and denitrifying Gamma- (particularly the SUP05 cluster) and Epsilonproteobacteria (predominantly Sulfurimonas subgroup GD17) are assumed to compete for substrates (electron donors and acceptors) in marine pelagic redox gradients. To elucidate their ecological niche separation we performed 34 S0 , 15 NO3- and H13 CO3- stable-isotope incubations with water samples from Baltic Sea suboxic, chemocline and sulfidic zones followed by combined phylogenetic staining and high-resolution secondary ion mass spectrometry of single cells. SUP05 cells were small-sized (0.06-0.09 µm3 ) and most abundant in low-sulfidic to suboxic zones, whereas Sulfurimonas GD17 cells were significantly larger (0.26-0.61 µm3 ) and most abundant at the chemocline and below. Together, SUP05 and GD17 cells accumulated up to 48% of the labelled substrates but calculation of cell volume-specific rates revealed that GD17 cells incorporated labelled substrates significantly faster throughout the redox zone, thereby potentially outcompeting SUP05 especially at high substrate concentrations. Thus, in synopsis with earlier described features of SUP05/GD17 we conclude that their spatially overlapping association in stratified sulfidic zones is facilitated by their different lifestyles: whereas SUP05 cells are streamlined, non-motile K-strategists adapted to low substrate concentrations, GD17 cells are motile r-strategists well adapted to fluctuating substrate and redox conditions.

Microplastics alter composition of fungal communities in aquatic ecosystems.

Despite increasing concerns about microplastic (MP) pollution in aquatic ecosystems, there is insufficient knowledge on how MP affect fungal communities. In this study, we explored the diversity and community composition of fungi attached to polyethylene (PE) and polystyrene (PS) particles incubated in different aquatic systems in north-east Germany: the Baltic Sea, the River Warnow and a wastewater treatment plant. Based on next generation 18S rRNA gene sequencing, 347 different operational taxonomic units assigned to 81 fungal taxa were identified on PE and PS. The MP-associated communities were distinct from fungal communities in the surrounding water and on the natural substrate wood. They also differed significantly among sampling locations, pointing towards a substrate and location specific fungal colonization. Members of Chytridiomycota, Cryptomycota and Ascomycota dominated the fungal assemblages, suggesting that both parasitic and saprophytic fungi thrive in MP biofilms. Thus, considering the worldwide increasing accumulation of plastic particles as well as the substantial vector potential of MP, especially these fungal taxa might benefit from MP pollution in the aquatic environment with yet unknown impacts on their worldwide distribution, as well as biodiversity and food web dynamics at large.

Analysis of environmental microplastics by vibrational microspectroscopy: FTIR, Raman or both?

The contamination of aquatic ecosystems with microplastics has recently been reported through many studies, and negative impacts on the aquatic biota have been described. For the chemical identification of microplastics, mainly Fourier transform infrared (FTIR) and Raman spectroscopy are used. But up to now, a critical comparison and validation of both spectroscopic methods with respect to microplastics analysis is missing. To close this knowledge gap, we investigated environmental samples by both Raman and FTIR spectroscopy. Firstly, particles and fibres >500 µm extracted from beach sediment samples were analysed by Raman and FTIR microspectroscopic single measurements. Our results illustrate that both methods are in principle suitable to identify microplastics from the environment. However, in some cases, especially for coloured particles, a combination of both spectroscopic methods is necessary for a complete and reliable characterisation of the chemical composition. Secondly, a marine sample containing particles <400 µm was investigated by Raman imaging and FTIR transmission imaging. The results were compared regarding number, size and type of detectable microplastics as well as spectra quality, measurement time and handling. We show that FTIR imaging leads to significant underestimation (about 35 %) of microplastics compared to Raman imaging, especially in the size range <20 µm. However, the measurement time of Raman imaging is considerably higher compared to FTIR imaging. In summary, we propose a further size division within the smaller microplastics fraction into 500-50 µm (rapid and reliable analysis by FTIR imaging) and into 50-1 µm (detailed and more time-consuming analysis by Raman imaging). Graphical Abstract Marine microplastic sample (fraction <400 µm) on a silicon filter (middle) with the corresponding Raman and IR images.

Mercury Emission by the Baltic Sea: A Consequence of Cyanobacterial Activity, Photochemistry, And Low-Light Mercury Transformation.

Marine mercury emission plays an important role in the atmospheric mercury budget. It is caused by the transformation of ionic mercury to volatile elemental mercury (Hg(0)) and the subsequent release of the latter from surface waters. In this study, we investigated mercury transformation using three approaches: incubation experiments, statistical analyses of phytoplankton and Hg(0) data, and microbiological determinations. The incubation experiments revealed that (1) biotic-light-dependent transformation accounted for an average of 30% of the total natural transformation, (2) photochemistry also contributed 30%, such that its contribution was less important than previously considered, and (3) low-light production accounted for 40%. Field experiments and the microbiological investigations suggested the cyanobacterial genera Synechococcus and Aphanizomenon as the main transformers of mercury. On the basis of the rough balance of biotic mercury transformation with mercury emission during summer, on average, only a small portion of a few percent of the mixed layer participates in active transformation.

Identification of microplastics by FTIR and Raman microscopy: a novel silicon filter substrate opens the important spectral range below 1300 cm(-1) for FTIR transmission measurements.

The presence of microplastics in aquatic ecosystems is a topical problem and leads to the need of appropriate and reliable analytical methods to distinctly identify and to quantify these particles in environmental samples. As an example transmission, Fourier transform infrared (FTIR) imaging can be used to analyze samples directly on filters without any visual presorting, when the environmental sample was afore extracted, purified, and filtered. However, this analytical approach is strongly restricted by the limited IR transparency of conventional filter materials. Within this study, we describe a novel silicon (Si) filter substrate produced by photolithographic microstructuring, which guarantees sufficient transparency for the broad mid-infrared region of 4000-600 cm(-1). This filter type features holes with a diameter of 10 µm and exhibits adequate mechanical stability. Furthermore, it will be shown that our Si filter substrate allows a distinct identification of the most common microplastics, polyethylene (PE), and polypropylene (PP), in the characteristic fingerprint region (1400-600 cm(-1)). Moreover, using the Si filter substrate, a differentiation of microparticles of polyesters having quite similar chemical structure, like polyethylene terephthalate (PET) and polybutylene terephthalate (PBT), is now possible, which facilitates a visualization of their distribution within a microplastic sample by FTIR imaging. Finally, this Si filter can also be used as substrate for Raman microscopy-a second complementary spectroscopic technique-to identify microplastic samples.

Genome and physiology of a model Epsilonproteobacterium responsible for sulfide detoxification in marine oxygen depletion zones.

Eutrophication and global climate change lead to expansion of hypoxia in the ocean, often accompanied by the production of hydrogen sulfide, which is toxic to higher organisms. Chemoautotrophic bacteria are thought to buffer against increased sulfide concentrations by oxidizing hydrogen sulfide before its diffusion to oxygenated surface waters. Model organisms from such environments have not been readily available, which has contributed to a poor understanding of these microbes. We present here a detailed study of "Sulfurimonas gotlandica" str. GD1, an Epsilonproteobacterium isolated from the Baltic Sea oxic-anoxic interface, where it plays a key role in nitrogen and sulfur cycling. Whole-genome analysis and laboratory experiments revealed a high metabolic flexibility, suggesting a considerable capacity for adaptation to variable redox conditions. S. gotlandica str. GD1 was shown to grow chemolithoautotrophically by coupling denitrification with oxidation of reduced sulfur compounds and dark CO(2) fixation. Metabolic versatility was further suggested by the use of a range of different electron donors and acceptors and organic carbon sources. The number of genes involved in signal transduction and metabolic pathways exceeds those of other Epsilonproteobacteria. Oxygen tolerance and environmental-sensing systems combined with chemotactic responses enable this organism to thrive successfully in marine oxygen-depletion zones. We propose that S. gotlandica str. GD1 will serve as a model organism in investigations that will lead to a better understanding how members of the Epsilonproteobacteria are able to cope with water column anoxia and the role these microorganisms play in the detoxification of sulfidic waters.

Distribution of acI-Actinorhodopsin genes in Baltic Sea salinity gradients indicates adaptation of facultative freshwater photoheterotrophs to brackish waters.

Knowledge on Actinobacteria rhodopsin gene (actR) diversity and spatial distribution is scarce. The Baltic Sea is characterized by strong salinity gradients leading to the coexistence of marine and freshwater bacteria and hence is an ideal study area to elucidate the dispersion and phylogenetic affiliation of actR in dependence on salinity. ActR DGGE fingerprints in summer 2008 revealed between 3 and 19 distinct bands within a salinity range of 2.4-27 PSU. Environmental actR clone sequences were obtained from stations distributed along the whole salinity gradient. Overall, 20 different actR sequence groups (operational taxonomic units) were found, with up to 11 different ones per station. Phylogenetically, the actR sequences were predominantly (80%) affiliated with freshwater acI-Actinobacteria whose 16S rRNA gene accounted for 2-33% of total 16S rRNA genes in both the Bothnian Sea and central Baltic Sea. However, at salinities above 14 PSU, acI-16S rRNA gene accounted for less than 1%. In contrast, the diversity of actR remained high. Changes in actR gene diversity were significantly correlated with salinity, oxygen, silica or abundance of Synechococcus sp. Our results demonstrate a wide distribution of freshwater actR along the Baltic Sea salinity gradient indicating that some freshwater Actinobacteria might have adapted to higher salinities.

N and O isotope fractionation in nitrate during chemolithoautotrophic denitrification by Sulfurimonas gotlandica.

Chemolithoautotrophic denitrification is an important mechanism of nitrogen loss in the water column of euxinic basins, but its isotope fractionation factor is not known. Sulfurimonas gotlandica GD1(T), a recently isolated bacterial key player in Baltic Sea pelagic redoxcline processes, was used to determine the isotope fractionation of nitrogen and oxygen in nitrate during denitrification. Under anoxic conditions, nitrate reduction was accompanied by nitrogen and oxygen isotope fractionation of 23.8 ± 2.5‰ and 11.7 ± 1.1‰, respectively. The isotope effect for nitrogen was in the range determined for heterotrophic denitrification, with only the absence of stirring resulting in a significant decrease of the fractionation factor. The relative increase in δ(18)ONO3 to δ(15)NNO3 did not follow the 1:1 relationship characteristic of heterotrophic, marine denitrification. Instead, δ(18)ONO3 increased slower than δ(15)NNO3, with a conserved ratio of 0.5:1. This result suggests that the periplasmic nitrate reductase (Nap) of S. gotlandica strain GD1(T) fractionates the N and O in nitrate differently than the membrane-bound nitrate reductase (Nar), which is generally prevalent among heterotrophic denitrifiers and is considered as the dominant driver for the observed isotope fractionation. Hence in the Baltic Sea redoxcline, other, as yet-unidentified factors likely explain the low apparent fractionation.

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.

Impact of protist grazing on a key bacterial group for biogeochemical cycling in Baltic Sea pelagic oxic/anoxic interfaces.

Barrier zones between oxic and anoxic water masses (redoxclines) host highly active prokaryotic communities with important roles in biogeochemical cycling. In Baltic Sea pelagic redoxclines, Epsilonproteobacteria of the genus Sulfurimonas (subgroup GD17) have been shown to dominate chemoautotrophic denitrification. However, little is known on the loss processes affecting this prokaryotic group. In the present study, the protist grazing impact on the Sulfurimonas subgroup GD17 was determined for suboxic and oxygen/hydrogen sulphide interface depths of Baltic Sea redoxclines, using predator exclusion assays and bacterial amendment with the cultured representative 'Sulfurimonas gotlandica' strain GD1. Additionally, the principal bacterivores were identified by RNA-Stable Isotope Probing (RNA-SIP). The natural Sulfurimonas subgroup GD17 population grew strongly under oxygen/hydrogen sulphide interface conditions (doubling time: 1-1.5 days), but protist grazing could consume the complete new cell production per day. In suboxic samples, little or no growth of Sulfurimonas subgroup GD17 was observed. RNA-SIP identified five active grazers, belonging to typical redoxcline ciliates (Oligohymenophorea, Prostomatea) and globally widespread marine flagellate groups (MAST-4, Chrysophyta, Cercozoa). Overall, we demonstrate for the first time that protist grazing can control the growth, and potentially the vertical distribution, of a chemolithoautotrophic key-player of oxic/anoxic interfaces.

Chemolithoautotrophic denitrification of epsilonproteobacteria in marine pelagic redox gradients.

Pelagic marine oxygen-depleted zones often exhibit a redox gradient, caused by oxygen depletion due to biological demand exceeding ventilation, and the accumulation of reduced chemical species, such as hydrogen sulfide. These redox gradients harbour a distinct assemblage of epsilonproteobacteria capable of fixing carbon dioxide autotrophically in the dark and potentially of utilizing hydrogen sulfide chemolithotrophically by oxidation with nitrate. Together, these two processes are referred to as chemolithoautotrophic denitrification. The focus of this study was the recently isolated and cultivated representative strain of pelagic epsilonproteobacteria, 'Sulfurimonas gotlandica' strain GD1, specifically dark carbon dioxide fixation and its substrate turnovers during chemolithotrophic denitrification. By connecting these processes stoichiometrically and comparing the results with those obtained for dark carbon dioxide fixation and nutrient concentrations measured in pelagic redox gradients of the Baltic Sea, we were able to estimate the role of chemolithoautotrophic denitrification in the environment. Evidence is provided for a defined zone where chemolithoautotrophic denitrification of these epsilonproteobacteria allows the complete removal of nitrate and hydrogen sulfide from the water column. This water layer is roughly equivalent in thickness to the average overlapping region of the two substrates, but slightly larger. Such a difference may be explained by a variety of reasons, including, e.g. utilization of substrates present at concentrations below the detection limit, alternative usage of other substrates as thiosulfate or nitrous oxide, or comparable activities of other microbes. However, the combined results of in vitro and in situ studies strongly suggest that epsilonproteobacteria are primarily responsible for hydrogen sulfide and nitrate removal from pelagic Baltic Sea redox gradients.

SUP05 dominates the Gammaproteobacterial sulfur oxidizer assemblages in pelagic redoxclines of the central Baltic and Black Seas.

Gammaproteobacterial sulfur oxidizers (GSOs), particularly SUP05-related sequences, have been found worldwide in numerous oxygen-deficient marine environments. However, knowledge regarding their abundance, distribution, and ecological role is scarce. In this study, on the basis of phylogenetic analyses of 16S rRNA gene sequences originating from a Baltic Sea pelagic redoxcline, the in situ abundances of different GSO subgroups were quantified by CARD-FISH (catalyzed reporter fluorescence in situ hybridization) with oligonucleotide probes developed specifically for this purpose. Additionally, ribulose bisphosphate carboxylase/oxygenase form II (cbbM) gene transcript clone libraries were used to detect potential active chemolithoautotrophic GSOs in the Baltic Sea. Taken together, the results obtained by these two approaches demonstrated the existence of two major phylogenetic subclusters embedded within the GSO, one of them affiliated with sequences of the previously described SUP05 subgroup. CARD-FISH analyses revealed that only SUP05 occurred in relatively high numbers, reaching 10 to 30% of the total prokaryotes around the oxic-anoxic interface, where oxygen and sulfide concentrations are minimal. The applicability of the oligonucleotide probes was confirmed with samples from the Black Sea redoxcline, in which the SUP05 subgroup accounted for 10 to 13% of the total prokaryotic abundance. The cbbM transcripts presumably originating from SUP05 cells support previous evidence for the chemolithoautotrophic activity of this phylogenetic group. Our findings on the vertical distribution and high abundance of SUP05 suggest that this group plays an important role in marine redoxcline biogeochemistry, probably as anaerobic or aerobic sulfur oxidizers.

Sulfurimonas gotlandica sp. nov., a chemoautotrophic and psychrotolerant epsilonproteobacterium isolated from a pelagic redoxcline, and an emended description of the genus Sulfurimonas.

A psychro- and aerotolerant bacterium was isolated from the sulfidic water of a pelagic redox zone of the central Baltic Sea. The slightly curved rod- or spiral-shaped cells were motile by one polar flagellum or two bipolar flagella. Growth was chemolithoautotrophic, with nitrate or nitrite as electron acceptor and either a variety of sulfur species of different oxidation states or hydrogen as electron donor. Although the bacterium was able to utilize organic substances such as acetate, pyruvate, peptone and yeast extract for growth, these compounds yielded considerably lower cell numbers than obtained with reduced sulfur or hydrogen; in addition, bicarbonate supplementation was necessary. The cells also had an absolute requirement for NaCl. Optimal growth occurred at 15 °C and at pH 6.6-8.0. The predominant fatty acid of this organism was 16 : 1ω7c, with 3-OH 14 : 0, 16 : 0, 16 : 1ω5c+t and 18 : 1ω7c present in smaller amounts. The DNA G+C content was 33.6 mol%. As determined in 16S rRNA gene sequence phylogeny analysis, the isolate belongs to the genus Sulfurimonas, within the class Epsilonproteobacteria, with 93.7 to 94.2 % similarity to the other species of the genus Sulfurimonas, Sulfurimonas autotrophica, Sulfurimonas paralvinellae and Sulfurimonas denitrificans. However, the distinct physiological and genotypic differences from these previously described taxa support the description of a novel species, Sulfurimonas gotlandica sp. nov. The type strain is GD1(T) ( = DSM 19862(T) = JCM 16533(T)). Our results also justify an emended description of the genus Sulfurimonas.

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.

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.

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.