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.

Putative degraders of low-density polyethylene-derived compounds are ubiquitous members of plastic-associated bacterial communities in the marine environment.

It remains unknown whether and to what extent marine prokaryotic communities are capable of degrading plastic in the ocean. To address this knowledge gap, we combined enrichment experiments employing low-density polyethylene (LDPE) as the sole carbon source with a comparison of bacterial communities on plastic debris in the Pacific, the North Atlantic and the northern Adriatic Sea. A total of 35 operational taxonomic units (OTUs) were enriched in the LDPE-laboratory incubations after 1 year, of which 20 were present with relative abundances > 0.5% in at least one plastic sample collected from the environment. From these, OTUs classified as Cognatiyoonia, Psychrobacter, Roseovarius and Roseobacter were found in the communities of plastics collected at all oceanic sites. Additionally, OTUs classified as Roseobacter, Pseudophaeobacter, Phaeobacter, Marinovum and Cognatiyoonia, also enriched in the LDPE-laboratory incubations, were enriched on LDPE communities compared to the ones associated to glass and polypropylene in in-situ incubations in the northern Adriatic Sea after 1 month of incubation. Some of these enriched OTUs were also related to known alkane and hydrocarbon degraders. Collectively, these results demonstrate that there are prokaryotes capable of surviving with LDPE as the sole carbon source living on plastics in relatively high abundances in different water masses of the global ocean.

A Low-Diversity Microbiota Inhabits Extreme Terrestrial Basaltic Terrains and Their Fumaroles: Implications for the Exploration of Mars.

A major objective in the exploration of Mars is to test the hypothesis that the planet hosted life. Even in the absence of life, the mapping of habitable and uninhabitable environments is an essential task in developing a complete understanding of the geological and aqueous history of Mars and, as a consequence, understanding what factors caused Earth to take a different trajectory of biological potential. We carried out the aseptic collection of samples and comparison of the bacterial and archaeal communities associated with basaltic fumaroles and rocks of varying weathering states in Hawai'i to test four hypotheses concerning the diversity of life in these environments. Using high-throughput sequencing, we found that all these materials are inhabited by a low-diversity biota. Multivariate analyses of bacterial community data showed a clear separation between sites that have active fumaroles and other sites that comprised relict fumaroles, unaltered, and syn-emplacement basalts. Contrary to our hypothesis that high water flow environments, such as fumaroles with active mineral leaching, would be sites of high biological diversity, alpha diversity was lower in active fumaroles compared to relict or nonfumarolic sites, potentially due to high-temperature constraints on microbial diversity in fumarolic sites. A comparison of these data with communities inhabiting unaltered and weathered basaltic rocks in Idaho suggests that bacterial taxon composition of basaltic materials varies between sites, although the archaeal communities were similar in Hawai'i and Idaho. The taxa present in both sites suggest that most of them obtain organic carbon compounds from the atmosphere and from phototrophs and that some of them, including archaeal taxa, cycle fixed nitrogen. The low diversity shows that, on Earth, extreme basaltic terrains are environments on the edge of sustaining life with implications for the biological potential of similar environments on Mars and their exploration by robots and humans.

Biodegradability standards for carrier bags and plastic films in aquatic environments: a critical review.

Plastic litter is encountered in aquatic ecosystems across the globe, including polar environments and the deep sea. To mitigate the adverse societal and ecological impacts of this waste, there has been debate on whether 'biodegradable' materials should be granted exemptions from plastic bag bans and levies. However, great care must be exercised when attempting to define this term, due to the broad and complex range of physical and chemical conditions encountered within natural ecosystems. Here, we review existing international industry standards and regional test methods for evaluating the biodegradability of plastics within aquatic environments (wastewater, unmanaged freshwater and marine habitats). We argue that current standards and test methods are insufficient in their ability to realistically predict the biodegradability of carrier bags in these environments, due to several shortcomings in experimental procedures and a paucity of information in the scientific literature. Moreover, existing biodegradability standards and test methods for aquatic environments do not involve toxicity testing or account for the potentially adverse ecological impacts of carrier bags, plastic additives, polymer degradation products or small (microscopic) plastic particles that can arise via fragmentation. Successfully addressing these knowledge gaps is a key requirement for developing new biodegradability standard(s) for lightweight carrier bags.

The UK Centre for Astrobiology: A Virtual Astrobiology Centre. Accomplishments and Lessons Learned, 2011-2016.

The UK Centre for Astrobiology (UKCA) was set up in 2011 as a virtual center to contribute to astrobiology research, education, and outreach. After 5 years, we describe this center and its work in each of these areas. Its research has focused on studying life in extreme environments, the limits of life on Earth, and implications for habitability elsewhere. Among its research infrastructure projects, UKCA has assembled an underground astrobiology laboratory that has hosted a deep subsurface planetary analog program, and it has developed new flow-through systems to study extraterrestrial aqueous environments. UKCA has used this research backdrop to develop education programs in astrobiology, including a massive open online course in astrobiology that has attracted over 120,000 students, a teacher training program, and an initiative to take astrobiology into prisons. In this paper, we review these activities and others with a particular focus on providing lessons to others who may consider setting up an astrobiology center, institute, or science facility. We discuss experience in integrating astrobiology research into teaching and education activities. Key Words: Astrobiology-Centre-Education-Subsurface-Analog research. Astrobiology 18, 224-243.

Vibrational Spectroscopy for Imaging Single Microbial Cells in Complex Biological Samples.

Vibrational spectroscopy is increasingly used for the rapid and non-destructive imaging of environmental and medical samples. Both Raman and Fourier-transform infrared (FT-IR) imaging have been applied to obtain detailed information on the chemical composition of biological materials, ranging from single microbial cells to tissues. Due to its compatibility with methods such as stable isotope labeling for the monitoring of cellular activities, vibrational spectroscopy also holds considerable power as a tool in microbial ecology. Chemical imaging of undisturbed biological systems (such as live cells in their native habitats) presents unique challenges due to the physical and chemical complexity of the samples, potential for spectral interference, and frequent need for real-time measurements. This Mini Review provides a critical synthesis of recent applications of Raman and FT-IR spectroscopy for characterizing complex biological samples, with a focus on developments in single-cell imaging. We also discuss how new spectroscopic methods could be used to overcome current limitations of single-cell analyses. Given the inherent complementarity of Raman and FT-IR spectroscopic methods, we discuss how combining these approaches could enable us to obtain new insights into biological activities either in situ or under conditions that simulate selected properties of the natural environment.

Astrobiology as a framework for investigating antibiotic susceptibility: a study of Halomonas hydrothermalis.

Physical and chemical boundaries for microbial multiplication on Earth are strongly influenced by interactions between environmental extremes. However, little is known about how interactions between multiple stress parameters affect the sensitivity of microorganisms to antibiotics. Here, we assessed how 12 distinct permutations of salinity, availability of an essential nutrient (iron) and atmospheric composition (aerobic or microaerobic) affect the susceptibility of a polyextremotolerant bacterium, Halomonas hydrothermalis, to ampicillin, kanamycin and ofloxacin. While salinity had a significant impact on sensitivity to all three antibiotics (as shown by turbidimetric analyses), the nature of this impact was modified by iron availability and the ambient gas composition, with differing effects observed for each compound. These two parameters were found to be of particular importance when considered in combination and, in the case of ampicillin, had a stronger combined influence on antibiotic tolerance than salinity. Our data show how investigating microbial responses to multiple extremes, which are more representative of natural habitats than single extremes, can improve our understanding of the effects of antimicrobial compounds and suggest how studies of habitability, motivated by the desire to map the limits of life, can be used to systematically assess the effectiveness of antibiotics.

AstRoMap European Astrobiology Roadmap.

The European AstRoMap project (supported by the European Commission Seventh Framework Programme) surveyed the state of the art of astrobiology in Europe and beyond and produced the first European roadmap for astrobiology research. In the context of this roadmap, astrobiology is understood as the study of the origin, evolution, and distribution of life in the context of cosmic evolution; this includes habitability in the Solar System and beyond. The AstRoMap Roadmap identifies five research topics, specifies several key scientific objectives for each topic, and suggests ways to achieve all the objectives. The five AstRoMap Research Topics are • Research Topic 1: Origin and Evolution of Planetary Systems • Research Topic 2: Origins of Organic Compounds in Space • Research Topic 3: Rock-Water-Carbon Interactions, Organic Synthesis on Earth, and Steps to Life • Research Topic 4: Life and Habitability • Research Topic 5: Biosignatures as Facilitating Life Detection It is strongly recommended that steps be taken towards the definition and implementation of a European Astrobiology Platform (or Institute) to streamline and optimize the scientific return by using a coordinated infrastructure and funding system.

Rock geochemistry induces stress and starvation responses in the bacterial proteome.

Interactions between microorganisms and rocks play an important role in Earth system processes. However, little is known about the molecular capabilities microorganisms require to live in rocky environments. Using a quantitative label-free proteomics approach, we show that a model bacterium (Cupriavidus metallidurans CH34) can use volcanic rock to satisfy some elemental requirements, resulting in increased rates of cell division in both magnesium- and iron-limited media. However, the rocks also introduced multiple new stresses via chemical changes associated with pH, elemental leaching and surface adsorption of nutrients that were reflected in the proteome. For example, the loss of bioavailable phosphorus was observed and resulted in the upregulation of diverse phosphate limitation proteins, which facilitate increase phosphate uptake and scavenging within the cell. Our results revealed that despite the provision of essential elements, rock chemistry drives complex metabolic reorganization within rock-dwelling organisms, requiring tight regulation of cellular processes at the protein level. This study advances our ability to identify key microbial responses that enable life to persist in rock environments.

Aerobically respiring prokaryotic strains exhibit a broader temperature-pH-salinity space for cell division than anaerobically respiring and fermentative strains.

Biological processes on the Earth operate within a parameter space that is constrained by physical and chemical extremes. Aerobic respiration can result in adenosine triphosphate yields up to over an order of magnitude higher than those attained anaerobically and, under certain conditions, may enable microbial multiplication over a broader range of extremes than other modes of catabolism. We employed growth data published for 241 prokaryotic strains to compare temperature, pH and salinity values for cell division between aerobically and anaerobically metabolizing taxa. Isolates employing oxygen as the terminal electron acceptor exhibited a considerably more extensive three-dimensional phase space for cell division (90% of the total volume) than taxa using other inorganic substrates or organic compounds as the electron acceptor (15% and 28% of the total volume, respectively), with all groups differing in their growth characteristics. Understanding the mechanistic basis of these differences will require integration of research into microbial ecology, physiology and energetics, with a focus on global-scale processes. Critical knowledge gaps include the combined impacts of diverse stress parameters on Gibbs energy yields and rates of microbial activity, interactions between cellular energetics and adaptations to extremes, and relating laboratory-based data to in situ limits for cell division.

tRNA-dependent alanylation of diacylglycerol and phosphatidylglycerol in Corynebacterium glutamicum.

Aminoacyl-phosphatidylglycerol synthases (aaPGSs) are membrane proteins that utilize aminoacylated tRNAs to modify membrane lipids with amino acids. Aminoacylation of membrane lipids alters the biochemical properties of the cytoplasmic membrane and enables bacteria to adapt to changes in environmental conditions. aaPGSs utilize alanine, lysine and arginine as modifying amino acids, and the primary lipid recipients have heretofore been defined as phosphatidylglycerol (PG) and cardiolipin. Here we identify a new pathway for lipid aminoacylation, conserved in many Actinobacteria, which results in formation of Ala-PG and a novel alanylated lipid, Alanyl-diacylglycerol (Ala-DAG). Ala-DAG formation in Corynebacterium glutamicum is dependent on the activity of an aaPGS homolog, whereas formation of Ala-PG requires the same enzyme acting in concert with a putative esterase encoded upstream. The presence of alanylated lipids is sufficient to enhance the bacterial fitness of C. glutamicum cultured in the presence of certain antimicrobial agents, and elucidation of this system expands the known repertoire of membrane lipids acting as substrates for amino acid modification in bacterial cells.

Identification and Quantification of Microplastics in Wastewater Using Focal Plane Array-Based Reflectance Micro-FT-IR Imaging.

Microplastics (<5 mm) have been documented in environmental samples on a global scale. While these pollutants may enter aquatic environments via wastewater treatment facilities, the abundance of microplastics in these matrices has not been investigated. Although efficient methods for the analysis of microplastics in sediment samples and marine organisms have been published, no methods have been developed for detecting these pollutants within organic-rich wastewater samples. In addition, there is no standardized method for analyzing microplastics isolated from environmental samples. In many cases, part of the identification protocol relies on visual selection before analysis, which is open to bias. In order to address this, a new method for the analysis of microplastics in wastewater was developed. A pretreatment step using 30% hydrogen peroxide (H2O2) was employed to remove biogenic material, and focal plane array (FPA)-based reflectance micro-Fourier-transform (FT-IR) imaging was shown to successfully image and identify different microplastic types (polyethylene, polypropylene, nylon-6, polyvinyl chloride, polystyrene). Microplastic-spiked wastewater samples were used to validate the methodology, resulting in a robust protocol which was nonselective and reproducible (the overall success identification rate was 98.33%). The use of FPA-based micro-FT-IR spectroscopy also provides a considerable reduction in analysis time compared with previous methods, since samples that could take several days to be mapped using a single-element detector can now be imaged in less than 9 h (circular filter with a diameter of 47 mm). This method for identifying and quantifying microplastics in wastewater is likely to provide an essential tool for further research into the pathways by which microplastics enter the environment.

Reduction of the temperature sensitivity of Halomonas hydrothermalis by iron starvation combined with microaerobic conditions.

The limits to biological processes on Earth are determined by physicochemical parameters, such as extremes of temperature and low water availability. Research into microbial extremophiles has enhanced our understanding of the biophysical boundaries which define the biosphere. However, there remains a paucity of information on the degree to which rates of microbial multiplication within extreme environments are determined by the availability of specific chemical elements. Here, we show that iron availability and the composition of the gaseous phase (aerobic versus microaerobic) determine the susceptibility of a marine bacterium, Halomonas hydrothermalis, to suboptimal and elevated temperature and salinity by impacting rates of cell division (but not viability). In particular, iron starvation combined with microaerobic conditions (5% [vol/vol] O2, 10% [vol/vol] CO2, reduced pH) reduced sensitivity to temperature across the 13°C range tested. These data demonstrate that nutrient limitation interacts with physicochemical parameters to determine biological permissiveness for extreme environments. The interplay between resource availability and stress tolerance, therefore, may shape the distribution and ecology of microorganisms within Earth's biosphere.

Rapid bacterial colonization of low-density polyethylene microplastics in coastal sediment microcosms.

BACKGROUND: Synthetic microplastics (≤5-mm fragments) are emerging environmental contaminants that have been found to accumulate within coastal marine sediments worldwide. The ecological impacts and fate of microplastic debris are only beginning to be revealed, with previous research into these topics having primarily focused on higher organisms and/or pelagic environments. Despite recent research into plastic-associated microorganisms in seawater, the microbial colonization of microplastics in benthic habitats has not been studied. Therefore, we employed a 14-day microcosm experiment to investigate bacterial colonization of low-density polyethylene (LDPE) microplastics within three types of coastal marine sediment from Spurn Point, Humber Estuary, U.K. RESULTS: Bacterial attachment onto LDPE within sediments was demonstrated by scanning electron microscopy and catalyzed reporter deposition fluorescence in situ hybridisation (CARD-FISH). Log-fold increases in the abundance of 16S rRNA genes from LDPE-associated bacteria occurred within 7 days with 16S rRNA gene numbers on LDPE surfaces differing significantly across sediment types, as shown by quantitative PCR. Terminal-restriction fragment length polymorphism (T-RFLP) analysis demonstrated rapid selection of LDPE-associated bacterial assemblages whose structure and composition differed significantly from those in surrounding sediments. Additionally, T-RFLP analysis revealed successional convergence of the LDPE-associated communities from the different sediments over the 14-day experiment. Sequencing of cloned 16S rRNA genes demonstrated that these communities were dominated after 14 days by the genera Arcobacter and Colwellia (totalling 84-93% of sequences). Attachment by Colwellia spp. onto LDPE within sediments was confirmed by CARD-FISH. CONCLUSIONS: These results demonstrate that bacteria within coastal marine sediments can rapidly colonize LDPE microplastics, with evidence for the successional formation of plastisphere-specific bacterial assemblages. Although the taxonomic compositions of these assemblages are likely to differ between marine sediments and the water column, both Arcobacter and Colwellia spp. have previously been affiliated with the degradation of hydrocarbon contaminants within low-temperature marine environments. Since hydrocarbon-degrading bacteria have also been discovered on plastic fragments in seawater, our data suggest that recruitment of hydrocarbonoclastic bacteria on microplastics is likely to represent a shared feature between both benthic and pelagic marine habitats.

A conserved hydrolase responsible for the cleavage of aminoacylphosphatidylglycerol in the membrane of Enterococcus faecium.

Aminoacylphosphatidylglycerol synthases (aaPGSs) are enzymes that transfer amino acids from aminoacyl-tRNAs (aa-tRNAs) to phosphatidylglycerol (PG) to form aa-PG in the cytoplasmic membrane of bacteria. aa-PGs provide bacteria with resistance to a range of antimicrobial compounds and stress conditions. Enterococcus faecium encodes a triple-specific aaPGS (RakPGS) that utilizes arginine, alanine, and lysine as substrates. Here we identify a novel hydrolase (AhyD), encoded immediately adjacent to rakPGS in E. faecium, which is responsible for the hydrolysis of aa-PG. The genetic synteny of aaPGS and ahyD is conserved in >60 different bacterial species. Deletion of ahyD in E. faecium resulted in increased formation of Ala-PG and Lys-PG and increased sensitivity to bacitracin. Our results suggest that AhyD and RakPGS act together to maintain optimal levels of aa-PG in the bacterial membrane to confer resistance to certain antimicrobial compounds and stress conditions.

The limits for life under multiple extremes.

Life on Earth is limited by physical and chemical extremes that define the 'habitable space' within which it operates. Aside from its requirement for liquid water, no definite limits have been established for life under any extreme. Here, we employ growth data published for 67 prokaryotic strains to explore the limitations for microbial life under combined extremes of temperature, pH, salt (NaCl) concentrations, and pressure. Our review reveals a fundamental lack of information on the tolerance of microorganisms to multiple extremes that impedes several areas of science, ranging from environmental and industrial microbiology to the search for extraterrestrial life.

The applicability of reflectance micro-Fourier-transform infrared spectroscopy for the detection of synthetic microplastics in marine sediments.

Synthetic microplastics (≤5-mm fragments) are globally distributed contaminants within coastal sediments that may transport organic pollutants and additives into food webs. Although micro-Fourier-transform infrared (micro-FT-IR) spectroscopy represents an ideal method for detecting microplastics in sediments, this technique lacks a standardized operating protocol. Herein, an optimized method for the micro-FT-IR analysis of microplastics in vacuum-filtered sediment retentates was developed. Reflectance micro-FT-IR analyses of polyethylene (PE) were compared with attenuated total reflectance FT-IR (ATR-FT-IR) measurements. Molecular mapping as a precursor to the imaging of microplastics was explored in the presence and absence of 150-µm PE fragments, added to sediment at concentrations of 10, 100, 500 and 1000ppm. Subsequently, polymer spectra were assessed across plastic-spiked sediments from fifteen offshore sites. While all spectra obtained of evenly shaped plastics were typical to PE, reflectance micro-FT-IR measurements of irregularly shaped materials must account for refractive error. Additionally, we provide the first evidence that mapping successfully detects microplastics without their visual selection for characterization, despite this technique relying on spectra from small and spatially separated locations. Flotation of microplastics from sediments only enabled a fragment recovery rate of 61 (±31 S.D.) %. However, mapping 3-mm(2) areas (within 47-mm filters) detected PE at spiking concentrations of 100ppm and above, displaying 69 (±12 S.D.) % of the fragments in these locations. Additionally, mapping detected a potential PE fragment in a non-spiked retentate. These data have important implications for research into the imaging of microplastics. Specifically, the sensitivity and spatial resolution of the present protocol may be improved by visualizing the entire filter with high-throughput detection techniques (e.g., focal plane array-based imaging). Additionally, since micro-FT-IR analyses depend on methods of sample collection, our results emphasize the urgency of developing efficient and reproducible techniques to separate microplastics from sediments.