Microbial Consortia and Mixed Plastic Waste: Pangenomic Analysis Reveals Potential for Degradation of Multiple Plastic Types via Previously Identified PET Degrading Bacteria.

The global utilization of single-use, non-biodegradable plastics, such as bottles made of polyethylene terephthalate (PET), has contributed to catastrophic levels of plastic pollution. Fortunately, microbial communities are adapting to assimilate plastic waste. Previously, our work showed a full consortium of five bacteria capable of synergistically degrading PET. Using omics approaches, we identified the key genes implicated in PET degradation within the consortium's pangenome and transcriptome. This analysis led to the discovery of a novel PETase, EstB, which has been observed to hydrolyze the oligomer BHET and the polymer PET. Besides the genes implicated in PET degradation, many other biodegradation genes were discovered. Over 200 plastic and plasticizer degradation-related genes were discovered through the Plastic Microbial Biodegradation Database (PMBD). Diverse carbon source utilization was observed by a microbial community-based assay, which, paired with an abundant number of plastic- and plasticizer-degrading enzymes, indicates a promising possibility for mixed plastic degradation. Using RNAseq differential analysis, several genes were predicted to be involved in PET degradation, including aldehyde dehydrogenases and several classes of hydrolases. Active transcription of PET monomer metabolism was also observed, including the generation of polyhydroxyalkanoate (PHA)/polyhydroxybutyrate (PHB) biopolymers. These results present an exciting opportunity for the bio-recycling of mixed plastic waste with upcycling potential.

The metabolic potential of the single cell genomes obtained from the Challenger Deep, Mariana Trench within the candidate superphylum Parcubacteria (OD1).

Candidate phyla (CP) are broad phylogenetic clusters of organisms that lack cultured representatives. Included in this fraction is the candidate Parcubacteria superphylum. Specific characteristics that have been ascribed to the Parcubacteria include reduced genome size, limited metabolic potential and exclusive reliance on fermentation for energy acquisition. The study of new environmental niches, such as the marine versus terrestrial subsurface, often expands the understanding of the genetic potential of taxonomic groups. For this reason, we analyzed 12 Parcubacteria single amplified genomes (SAGs) from sediment samples collected within the Challenger Deep of the Mariana Trench, obtained during the Deepsea Challenge (DSC) Expedition. Many of these SAGs are closely related to environmental sequences obtained from deep-sea environments based on 16S rRNA gene similarity and BLAST matches to predicted proteins. DSC SAGs encode features not previously identified in Parcubacteria obtained from other habitats. These include adaptation to oxidative stress, polysaccharide modification and genes associated with respiratory nitrate reduction. The DSC SAGs are also distinguished by relative greater abundance of genes for nucleotide and amino acid biosynthesis, repair of alkylated DNA and the synthesis of mechanosensitive ion channels. These results present an expanded view of the Parcubacteria, among members residing in an ultra-deep hadal environment.

Single cells within the Puerto Rico trench suggest hadal adaptation of microbial lineages.

Hadal ecosystems are found at a depth of 6,000 m below sea level and below, occupying less than 1% of the total area of the ocean. The microbial communities and metabolic potential in these ecosystems are largely uncharacterized. Here, we present four single amplified genomes (SAGs) obtained from 8,219 m below the sea surface within the hadal ecosystem of the Puerto Rico Trench (PRT). These SAGs are derived from members of deep-sea clades, including the Thaumarchaeota and SAR11 clade, and two are related to previously isolated piezophilic (high-pressure-adapted) microorganisms. In order to identify genes that might play a role in adaptation to deep-sea environments, comparative analyses were performed with genomes from closely related shallow-water microbes. The archaeal SAG possesses genes associated with mixotrophy, including lipoylation and the glycine cleavage pathway. The SAR11 SAG encodes glycolytic enzymes previously reported to be missing from this abundant and cosmopolitan group. The other SAGs, which are related to piezophilic isolates, possess genes that may supplement energy demands through the oxidation of hydrogen or the reduction of nitrous oxide. We found evidence for potential trench-specific gene distributions, as several SAG genes were observed only in a PRT metagenome and not in shallower deep-sea metagenomes. These results illustrate new ecotype features that might perform important roles in the adaptation of microorganisms to life in hadal environments.

Microbiomes of fish, sediment and seagrass suggest connectivity of coral reef microbial populations.

The benthic environments of coral reefs are heavily shaped by physiochemical factors, but also the ecological interactions of the animals and plants in the reef ecosystem. Microbial populations may be shared within the ecosystem of sediments, seagrasses and reef fish. In this study, we hypothesize that coral reef and seagrass environments share members of the microbial community that are rare in some habitats and enriched in others, and that animals may integrate this connectivity. We investigated the potential connectivity between the microbiomes of sediments, seagrass blades and roots (Syringodium isoetifolium), and a seagrass-specialist parrotfish (C. spinidens) guts in reef areas of Fiji. We contrasted these with sediment samples from the Florida Keys, gut samples from surgeonfish (A. nigricauda, Acanthurinae sp. unknown, C. striatus), and ocean water microbiomes from the Atlantic, Pacific and Indian Oceans to test the robustness of our characterizations of microbiome environments. In general, water, sediment and fish gut samples were all distinct microbiomes. Sediment microbiomes were mostly similar between Fiji and Florida, but also showed some regional similarities. In Fiji, we show connectivity of a shared microbiome between seagrass, fish and sediments. Additionally, we identified an environmental reservoir of a surgeonfish symbiont, Epulopiscium. The connection of these ecosystem components suggests that the total microbiome of these environments may vary as their animal inhabitants shift in a changing ocean.

Methanogens Within a High Salinity Oil Reservoir From the Gulf of Mexico.

Oil reservoirs contain microbial populations that are both autochthonously and allochthonously introduced by industrial development. These microbial populations are greatly influenced by external factors including, but not limited to, salinity and temperature. In this study, we used metagenomics to examine the microbial populations within five wells of the same hydrocarbon reservoir system in the Gulf of Mexico. These elevated salinity (149-181 ppt salinity, 4-5× salinity of seawater) reservoirs have limited taxonomic and functional microbial diversity dominated by methanogens, Halanaerobium and other Firmicutes lineages, and contained less abundant lineages such as Deltaproteobacteria. Metagenome assembled genomes (MAGs) were generated and analyzed from the various wells. Methanogen MAGs were closely related to Methanohalophilus euhalobius, a known methylotrophic methanogen from a high salinity oil environment. Based on metabolic reconstruction of genomes, the Halanaerobium perform glycine betaine fermentation, potentially produced by the methanogens. Industrial introduction of methanol to prevent methane hydrate formation to this environment is likely to be consumed by these methanogens. As such, this subsurface oil population may represent influences from industrial processes.

Environmental Consortium Containing Pseudomonas and Bacillus Species Synergistically Degrades Polyethylene Terephthalate Plastic.

Plastics, such as polyethylene terephthalate (PET) from water bottles, are polluting our oceans, cities, and soils. While a number of Pseudomonas species have been described that degrade aliphatic polyesters, such as polyethylene (PE) and polyurethane (PUR), few from this genus that degrade the semiaromatic polymer PET have been reported. In this study, plastic-degrading bacteria were isolated from petroleum-polluted soils and screened for lipase activity that has been associated with PET degradation. Strains and consortia of bacteria were grown in a liquid carbon-free basal medium (LCFBM) with PET as the sole carbon source. We monitored several key physical and chemical properties, including bacterial growth and modification of the plastic surface, using scanning electron microscopy (SEM) and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy. We detected by-products of hydrolysis of PET using 1H-nuclear magnetic resonance (1H NMR) analysis, consistent with the ATR-FTIR data. The full consortium of five strains containing Pseudomonas and Bacillus species grew synergistically in the presence of PET and the cleavage product bis(2-hydroxyethyl) terephthalic acid (BHET) as sole sources of carbon. Secreted enzymes extracted from the full consortium were capable of fully converting BHET to the metabolically usable monomers terephthalic acid (TPA) and ethylene glycol. Draft genomes provided evidence for mixed enzymatic capabilities between the strains for metabolic degradation of TPA and ethylene glycol, the building blocks of PET polymers, indicating cooperation and ability to cross-feed in a limited nutrient environment with PET as the sole carbon source. The use of bacterial consortia for the biodegradation of PET may provide a partial solution to widespread planetary plastic accumulation.IMPORTANCE While several research groups are utilizing purified enzymes to break down postconsumer PET to the monomers TPA and ethylene glycol to produce new PET products, here, we present a group of five soil bacteria in culture that are able to partially degrade this polymer. To date, mixed Pseudomonas spp. and Bacillus spp. biodegradation of PET has not been described, and this work highlights the possibility of using bacterial consortia to biodegrade or potentially to biorecycle PET plastic waste.

Novel clostridial lineages recovered from metagenomes of a hot oil reservoir.

Oil reservoirs have been shown to house numerous microbial lineages that differ based on the in-situ pH, salinity and temperature of the subsurface environment. Lineages of Firmicutes, including Clostridiales, have been frequently detected in oil reservoirs, but are typically not considered impactful or relevant due to their spore-forming nature. Here we show, using metagenomics, a high temperature oil reservoir of marine salinity contains a microbial population that is predominantly from within the Order Clostridiales. These organisms form an oil-reservoir specific clade based on the phylogenies of both 16S rRNA genes and ribosomal proteins, which we propose to name UPetromonas tenebris, meaning they are single-celled organisms from dark rocks. Metagenome-assembled genomes (MAGs) of these Petromonas sp. were obtained and used to determine that these populations, while capable of spore-formation, were also likely replicating in situ in the reservoir. We compared these MAGs to closely related genomes and show that these subsurface Clostridiales differ, from the surface derived genomes, showing signatures of the ability to degrade plant-related compounds, whereas subsurface genomes only show the ability to process simple sugars. The estimation of in-situ replication from genomic data suggest that UPetromonas tenebris lineages are functional in-situ and may be specifically adapted to inhabit oil reservoirs.

Metabolic potentials of archaeal lineages resolved from metagenomes of deep Costa Rica sediments.

Numerous archaeal lineages are known to inhabit marine subsurface sediments, although their distributions, metabolic capacities, and interspecies interactions are still not well understood. Abundant and diverse archaea were recently reported in Costa Rica (CR) margin subseafloor sediments recovered during IODP Expedition 334. Here, we recover metagenome-assembled genomes (MAGs) of archaea from the CR margin and compare them to their relatives from shallower settings. We describe 31 MAGs of six different archaeal lineages (Lokiarchaeota, Thorarchaeota, Heimdallarchaeota, Bathyarcheota, Thermoplasmatales, and Hadesarchaea) and thoroughly analyze representative MAGs from the phyla Lokiarchaeota and Bathyarchaeota. Our analysis suggests the potential capability of Lokiarchaeota members to anaerobically degrade aliphatic and aromatic hydrocarbons. We show it is genetically possible and energetically feasible for Lokiarchaeota to degrade benzoate if they associate with organisms using nitrate, nitrite, and sulfite as electron acceptors, which suggests a possibility of syntrophic relationships between Lokiarchaeota and nitrite and sulfite reducing bacteria. The novel Bathyarchaeota lineage possesses an incomplete methanogenesis pathway lacking the methyl coenzyme M reductase complex and encodes a noncanonical acetogenic pathway potentially coupling methylotrophy to acetogenesis via the methyl branch of Wood-Ljungdahl pathway. These metabolic characteristics suggest the potential of this Bathyarchaeota lineage to be a transition between methanogenic and acetogenic Bathyarchaeota lineages. This work expands our knowledge about the metabolic functional repertoire of marine benthic archaea.

The distribution and relative ecological roles of autotrophic and heterotrophic diazotrophs in the McMurdo Dry Valleys, Antarctica.

The McMurdo Dry Valleys (MDV) in Antarctica harbor a diverse assemblage of mat-forming diazotrophic cyanobacteria that play a key role in nitrogen cycling. Prior research showed that heterotrophic diazotrophs also make a substantial contribution to nitrogen fixation in MDV. The goals of this study were to survey autotrophic and heterotrophic diazotrophs across the MDV to investigate factors that regulate the distribution and relative ecological roles of each group. Results indicated that diazotrophs were present only in samples with mats, suggesting a metabolic coupling between autotrophic and heterotrophic diazotrophs. Analysis of 16S rRNA and nifH gene sequences also showed that diazotrophs were significantly correlated to the broader bacterial community, while co-occurrence network analysis revealed potential interspecific interactions. Consistent with previous studies, heterotrophic diazotrophs in MDV were diverse, but largely limited to lakes and their outlet streams, or other environments protected from desiccation. Despite the limited distribution, heterotrophic diazotrophs may make a substantial contribution to the nitrogen budget of MDV due to larger surface area and longer residence times of lakes. This work contributes to our understanding of key drivers of bacterial community structure in polar deserts and informs future efforts to investigate the contribution of nitrogen fixation to MDV ecosystems.

Draft Genome Sequences of Five Environmental Bacterial Isolates That Degrade Polyethylene Terephthalate Plastic.

Here, we report the annotated draft genome sequences of three Pseudomonas spp. and two Bacillus spp. that, as consortia, degrade polyethylene terephthalate plastic. Improved microbial degradation of plastic waste could help reduce the billions of metric tons of these materials that currently exist in our environment.

Deep Subsurface Life from North Pond: Enrichment, Isolation, Characterization and Genomes of Heterotrophic Bacteria.

Studies of subsurface microorganisms have yielded few environmentally relevant isolates for laboratory studies. In order to address this lack of cultivated microorganisms, we initiated several enrichments on sediment and underlying basalt samples from North Pond, a sediment basin ringed by basalt outcrops underlying an oligotrophic water-column west of the Mid-Atlantic Ridge at 22°N. In contrast to anoxic enrichments, growth was observed in aerobic, heterotrophic enrichments from sediment of IODP Hole U1382B at 4 and 68 m below seafloor (mbsf). These sediment depths, respectively, correspond to the fringes of oxygen penetration from overlying seawater in the top of the sediment column and upward migration of oxygen from oxic seawater from the basalt aquifer below the sediment. Here we report the enrichment, isolation, initial characterization and genomes of three isolated aerobic heterotrophs from North Pond sediments; an Arthrobacter species from 4 mbsf, and Paracoccus and Pseudomonas species from 68 mbsf. These cultivated bacteria are represented in the amplicon 16S rRNA gene libraries created from whole sediments, albeit at low (up to 2%) relative abundance. We provide genomic evidence from our isolates demonstrating that the Arthrobacter and Pseudomonas isolates have the potential to respire nitrate and oxygen, though dissimilatory nitrate reduction could not be confirmed in laboratory cultures. The cultures from this study represent members of abundant phyla, as determined by amplicon sequencing of environmental DNA extracts, and allow for further studies into geochemical factors impacting life in the deep subsurface.