Tepidibacillus marianensis sp. nov., a novel heterotrophic iron-reducing bacterium isolated from Mariana Trench sediment.

A novel chemoheterotrophic iron-reducing micro-organism, designated as strain LSZ-M11000T, was isolated from sediment of the Marianas Trench. Phylogenetic analysis based on the 16S rRNA gene revealed that strain LSZ-M11000T belonged to genus Tepidibacillus, with 97 % identity to that of Tepidibacillus fermentans STGHT, a mesophilic bacterium isolated from the Severo-Stavropolskoye underground gas storage facility in Russia. The polar lipid profile of strain LSZ-M11000T consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, as well as other unidentified phospholipids and lipids. The major fatty acids were C16 : 0 (28.4 %), C18 : 0 (15.8 %), iso-C15 : 0 (12.9 %), and anteiso-C15 : 0 (12.0 %). Strain LSZ-M11000T had no menaquinone. Genome sequencing revealed that the genome size of strain LSZ-M11000T was 2.97 Mb and the DNA G+C content was 37.9 mol%. The average nucleotide identity values between strain LSZ-M11000T and its close phylogenetic relatives, Tepidibacillus fermentans STGHT and Tepidibacillus decaturensis Z9T, were 76.4 and 72.6 %, respectively. The corresponding DNA-DNA hybridization estimates were 20.9 and 23.4 %, respectively. Cells of strain LSZ-M11000T were rod-shaped (1.0-1.5×0.3-0.5 µm). Using pyruvate as an electron donor, it was capable of reducing KMnO4, MnO2, As(V), NaNO3, NaNO2, Na2SO4, Na2S2O3, and K2Cr2O7. Based on phenotypic, genotypic, and phylogenetic evidence, strain LSZ-M11000T is proposed to be a novel strain of the genus Tepidibacillus, for which the name Tepdibacillus marianensis is proposed. The type strain is LSZ-M11000T (=CCAM 1008T=JCM 39431T).

Tracing Organophosphate Ester Pollutants in Hadal Trenches─Distribution, Possible Origins, and Transport Mechanisms.

Unraveling the mysterious pathways of pollutants to the deepest oceanic realms holds critical importance for assessing the integrity of remote marine ecosystems. This study tracks the transport of pollutants into the depths of the oceans, a key step in protecting the sanctity of these least explored ecosystems. By analyzing hadal trench samples from the Mariana, Mussau, and New Britain trenches, we found the widespread distribution of organophosphate ester (OPE) flame retardants but a complex transport pattern for the OPE in these regions. In the Mariana Trench seawater column, OPE concentrations range between 17.4 and 102 ng L-1, with peaks at depths of 500 and 4000 m, which may be linked to Equatorial Undercurrent and topographic Rossby waves, respectively. Sediments, particularly in Mariana (422 ng g-1 dw), showed high OPE affinity, likely due to organic matter serving as a transport medium, influenced by "solvent switching", "solvent depletion", and "filtering processes". Amphipods in the three trenches had consistent OPE levels (29.1-215 ng g-1 lipid weight), independent of the sediment pollution patterns. The OPEs in these amphipods appeared more linked to surface-dwelling organisms, suggesting the influence of "solvent depletion". This study highlights the need for an improved understanding of deep-sea pollutant sources and transport, urging the establishment of protective measures for these remote marine habitats.

Evidence for Long-Term Anthropogenic Pollution: The Hadal Trench as a Depository and Indicator for Dissemination of Antibiotic Resistance Genes.

Knowledge of the distribution and dissemination of antibiotic resistance genes (ARGs) is essential for understanding anthropogenic impacts on natural ecosystems. The transportation of ARGs via aquatic environments is significant and has received great attention, but whether there has been anthropogenic ARG pollution to the hadal ocean ecosystem has not been well explored. For investigating ecological health concerns, we profiled the ARG occurrence in sediments of the Mariana Trench (MT) (10 890 m), the deepest region of the ocean. Metagenomic-based ARG profiles showed a sudden increase of abundance and diversity in the surface layer of MT sediments reaching 2.73 × 10-2 copy/cell and 81 subtypes, and a high percentage of ∼63.6% anthropogenic pollution sources was predicted by the Bayesian-modeling classification method. These together suggested that ARG accumulation and anthropogenic impacts have already permeated into the bottom of the deepest corner on the earth. Moreover, six ARG-carrying draft genomes were retrieved using a metagenomic binning strategy, one of which assigned as Streptococcus was identified as a potential bacterial host to contribute to the ARG accumulation in MT, carrying ermF, tetM, tetQ, cfxA2, PBP-2X, and PBP-1A. We propose that the MT ecosystem needs further long-term monitoring for the assessment of human impacts, and our identified three biomarkers (cfxA2, ermF, and mefA) could be used for the rapid monitoring of anthropogenic pollution. Together our findings imply that anthropogenic pollution has penetrated into the deepest region of the ocean and urge for better pollution control to reduce the risk of ARG dissemination to prevent the consistent accumulation and potential threat to the natural environment.

Halomonas piezotolerans sp. nov., a multiple-stress-tolerant bacterium isolated from a deep-sea sediment sample of the New Britain Trench.

A piezotolerant, H2O2-tolerant, heavy-metal-tolerant, slightly halophilic bacterium (strain NBT06E8T) was isolated from a deep-sea sediment sample collected from the New Britain Trench at depth of 8900 m. The strain was aerobic, motile, Gram-stain-negative, rod-shaped, oxidase-positive and catalase-positive. Growth of the strain was observed at 4-45 °C (optimum, 30 °C), at pH 5-11 (optimum, pH 8-9) and in 0.5-21 % (w/v) NaCl (optimum, 3-7 %). The optimum pressure for growth was 0.1-30 MPa with tolerance up to 60 MPa. Under optimum growth conditions, the strain could tolerate 15 mM H2O2. Resuls of 16S rRNA gene sequence analysis showed that strain NBT06E8T is closely related to Halomonas aquamarina DSM 30161T (99.5%), Halomonas meridiana DSM 5425T (99.43%) and Halomonas axialensis Althf1T (99.35%). The digital DNA-DNA hybridization values between strain NBT06E8T and the three related type strains, H. aquamarina, H. meridiana and H. axialensis, were 30.5±2.4 %, 30.7±2.5% and 31.5±2.5 %, respectively. The average nucleotide identity values between strain NBT06E8T and the three related type strains were 86.26, 86.26 and 83.63 %, respectively. The major fatty acids were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and C16 : 0. The predominant respiratory quinone detected was ubiquinone-9 (Q-9). Based on its phenotypic and phylogenetic characteristics, we conclude that strain NBT06E8T represents a novel species of the genus Halomonas, for which the name Halomonas piezotolerans sp. nov. is proposed (type strain NBT06E8T= MCCC 1K04228T=KCTC 72680T).

Muricauda hadalis sp. nov., a novel piezophile isolated from hadopelagic water of the Mariana Trench and reclassification of Muricauda antarctica as a later heterotypic synonym of Muricauda teanensis.

A novel marine Gram-stain-negative, non-motile, aerobic and rod-shaped bacterium, designated as strain MT-229T, was isolated from the deep seawater in the Mariana Trench and characterized phylogenetically and phenotypically. Bacterial optimal growth occurred at 30 °C (ranging 10-40 °C), pH 6 (ranging 3-11) and with 11 % (w/v) NaCl (ranging 0-17 %). Strain MT-229T was a piezophile, growing optimally at 20 MPa (range 0.1-70 MPa). The nearest phylogenetic neighbours were Muricauda antarctica CGMCC 1.2174T and Muricauda taeanensis JCM 17757T with 16S rRNA gene similarity of 98.7 %. The sole respiratory quinone was menaquinone-6 (MK-6). The major polar lipids were phosphatidylethanolamine (PE), two unidentified aminolipids (AL) and ten unidentified lipids. The major fatty acids of strain MT-229T were iso-C15 : 0, iso-C17 : 0 3-OH and iso-C15 : 1 G. The G+C content of the genomic DNA was 45.6 mol%. The combined genotypic and phenotypic data indicated that strain MT-229T represents a novel species of the genus Muricauda, for which the name Muricauda hadalis sp. nov. is proposed, with the type strain MT-229T (=DSM 109894T=MCCC 1K04201T). In addition, the whole-genome-based comparisons revealed that the type strains of Muricauda antarctica and Muricauda teanensis belong to a single species. It is, therefore, proposed that M. antarctica be recognized as a heterotypic synonym of M. teanensis.

Georhizobium profundi gen. nov., sp. nov., a piezotolerant bacterium isolated from a deep-sea sediment sample of the New Britain Trench.

A novel alphaproteobacterium, strain WS11T, was isolated from a deep-sea sediment sample collected from the New Britain Trench. The full-length 16S rRNA gene of strain WS11T had the highest sequence similarity of 97.6 % to Rhizobium subbaraonis JC85T, followed by Mycoplana ramosa DSM 7292T (96.9 %) and Rhizobium azooxidifex Po 20/26T (96.8 %). Phylogenetic analysis of concatenated 16S rRNA, atpD and recA gene sequences showed that strain WS11T was deeply separated from the species within the family Rhizobiaceae. Phylogenomic analysis based on the whole-genome protein sequences showed that strain WS11T formed an independent monophyletic branch in the family Rhizobiaceae, paralleled with the species in the families Brucellaceae and Phyllobacteriaceae within the order Rhizobiales. Cells were Gram-stain-negative, oxidase- and catalase-positive, and aerobic short rods (1.5-2.4×0.9-1.0 µm). Growth was observed at salinities ranging from 0 to 5% (optimum, 1 %), from pH 6.5 to 9 (optimum, pH 7) and at temperatures between 20 and 30 °C (optimum, 28 °C). Strain WS11T was piezotolerant, growing optimally at 0.1 MPa (range 0.1-70 MPa). The main fatty acid was summed feature 8 (C18 : 1 ω7c/C18  : 1 ω 6c). The sole respiratory quinone was ubiquinone-10 (Q-10). The predominant polar lipids were phosphatidylcholine, two unidentified aminophospholipids and an unidentified phospholipid. The genome size was about 4.36 Mbp and the G+C content was 62.3 mol%. The combined genotypic and phenotypic data show that strain WS11T represents a novel species of a novel genus in the family Rhizobiaceae, for which the name Georhizobium profundi gen. nov., sp. nov. is proposed (type strain WS11T=MCCC 1K03498T=KCTC 62439T).

Alteromonas indica Lin et al. 2018 is a later heterotypic synonym of Salinimonas sediminis Cao et al. 2018.

Alteromonas indica IO390401T was compared with Salinimonas sediminis N102T to examine the taxonomic relationship between the two type strains. The 16S rRNA gene sequence of A. indica IO390401T shared high similarity (99.9 %) with that of S. sediminis N102T. The results of phylogenetic analyses based on 16S rRNA gene sequences indicated that the two strains formed a tight cluster within the genus Salinimonas. Whole genomic comparison between the two strains revealed an average nucleotide identity of 99.2 % and a digital DNA-DNA hybridization estimate of 92.6 %, strongly indicating that the two strains represented a single species. In addition, neither strain displayed any striking difference in metabolic, physiological or chemotaxonomic features. Therefore, we propose Alteromonas indica as a later heterotypic synonym of Salinimonas sediminis.

Marinomonas shanghaiensis sp. nov., isolated from the junction between an ocean and a freshwater lake.

A Gram-stain-negative, motile with single polar flagellum, rod-shaped bacterium, designated as strain DSL-35T, was isolated from the location where the ocean and Dishui lake meet at Shanghai on the East China Sea and characterized phylogenetically and phenotypically. Optimal growth occurred at 35 °C (range, 4-40 °C), pH 8 pH 5-11) and with 3-4 % (w/v) NaCl (0-12 %). Phylogenetic analysis based on its 16S rRNA gene sequence showed that strain DSL-35T was related to members of the genus Marinomonas and shared the highest sequence identities with Marinomonasarctica 328T (98.0 %), Marinomonashwangdonensis HDW-15T (97.5 %) and Marinomonasrhizomae IVIA-Po-145T (97.2 %). The 16S rRNA gene sequence identities between strain DSL-35T and other members of the genus Marinomonas were below 96.8 %. The digital DNA-DNA hybridization values between strain DSL-35T and the three type strains, Marinomonas. arctica 328T, M. rhizomae HDW-15T and M. rhizomae IVIA-Po-145T, were 30.9±2.4 %, 21.7±2.2% and 22±2.3 %, respectively. The average nucleotide identity values between strain DSL-35T and the three type strains were 87.6 %, 84.6 and 84.2 %, respectively. The predominant ubiquinone was Q-8. The major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The predominant cellular fatty acids of strain DSL-35T were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c; 40.0 %), C16 : 0 (22.5 %), summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c; 11.2 %), summed feature 2 (C14 : 0 3-OH and/or iso I C16 : 1; 7.2 %), C14 : 0 (6.8 %) and C12 : 0 (5.2 %). The G+C content of the genomic DNA was 44.5 mol%. The combined genotypic and phenotypic data indicated that strain DSL-35T represents a novel species of the genus Marinomonas, for which the name Marinomonas shanghaiensis sp. nov. is proposed, with the type strain DSL-35T (=KCTC 62646T=MCCC 1K03535T).

Paracoccus sediminilitoris sp. nov., isolated from a tidal flat sediment.

A novel marine Gram-stain-negative, non-spore-forming, motile, aerobic, coccoid or ovoid bacterium, designated as strain DSL-16T, was isolated from a tidal flat sediment on the East China Sea and characterized phylogenetically and phenotypically. Optimal growth of the strain occurred at 35 °C (range 4-40 °C), at pH 6 (range 5-11) and with 4 % (w/v) NaCl (range 1-14 %). The nearest phylogenetic neighbour was Paracoccusseriniphilus DSM 14827T (98.2 % 16S rRNA gene sequence similarity). The digital DNA-DNA hybridization value between strain DSL-16T and P. seriniphilus DSM 14827T was 19.5±2.2 %. The average nucleotide identity value between strain DSL-16T and P. seriniphilus DSM 14827T was 83.6 %. The sole respiratory ubiquinone was Q-10. The major polar lipids were phosphatidylmonomethylethanolamine (PME), phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), diphosphatidyglycerol (DPG) and glycolipid (GL). The predominant cellular fatty acids of strain DSL-16T were C18 : 1ω7c, C18 : 0 and 11-methyl C18 : 1ω7c. The G+C content of the genomic DNA was 64.5 mol%. The combined genotypic and phenotypic data indicated that strain DSL-16T represents a novel species of the genus Paracoccus, for which the name Paracoccus sediminilitoris sp. nov. is proposed. The type strain is DSL-16T (=KCTC 62644T=MCCC 1K03534T).

Marinobacter profundi sp. nov., a slightly halophilic bacterium isolated from a deep-sea sediment sample of the New Britain Trench.

A piezotolerant, cold-adapted, slightly halophilic bacterium, designated strain PWS21T, was isolated from a deep-sea sediment sample collected from the New Britain Trench. Cells were observed to be Gram-stain negative, rod-shaped, oxidase- and catalase-positive. Growth of the strain was observed at 4-45 °C (optimum 37 °C), at pH 5.0-9.0 (optimum 7.0) and in 0.5-20% (w/v) NaCl (optimum 3-4%). The optimum pressure for growth was 0.1 MPa (megapascal) with tolerance up to 70 MPa. 16S rRNA gene sequence analysis showed that strain PWS21T is closely related to Marinobacter guineae M3BT (98.4%) and Marinobacter lipolyticus SM19T (98.2%). Multilocus sequence analysis (MLSA) based on sequences of housekeeping genes gyrB, recA, atpD, rpoB and rpoD indicates that strain PWS21T represents a distinct evolutionary lineage within the genus Marinobacter. Furthermore, strain PWS21T showed low ANI and diDDH values to the closely related species. The principal fatty acids were identified as C12:0, C12:0 3-OH, C16:1ω9c, C16:0 and C18:1ω9c. Ubiquinone-9 was identified as the major respiratory quinone. The polar lipids were identified as phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), aminophospholipid (APL), two unidentified lipids and an unidentified phospholipid (PL). The G + C content of the genomic DNA was determined to be 60.3 mol%. On the basis of phenotypic, chemotaxonomic and molecular data, we conclude that strain PWS21T represents a novel species of the genus Marinobacter, for which the name Marinobacter profundi sp. nov. is proposed (type strain PWS21T = KCTC 52990T = MCCC 1K03345T).

Salinimonas sediminis sp. nov., a piezophilic bacterium isolated from a deep-sea sediment sample from the New Britain Trench.

A cold-adapted, piezophilic, slightly halophilic bacterium, designated as N102T, was isolated from a deep-sea (4700 m) sediment sample collected from the New Britain Trench. Strain N102T was Gram-stain-negative, rod-shaped, oxidase- and catalase-positive, and grew optimally at 28 °C (range, 4-40 °C), pH 7.0-7.5 (range, 6.0-9.0) and 3-4 %(w/v) NaCl (range, 2-15 %). The optimum pressure for growth was 10 MPa with tolerance up to 70 MPa. 16S rRNA gene sequence analysis showed that strain N102T was most closely related to Alteromonas addita R10SW13T (97.2 %), Alteromonas stellipolaris LMG 21861T (97.1 %), Alteromonas gracilis 9a2T (97.1 %), Salinimonas lutimaris DPSR-4T (96.1 %) and Salinimonas chungwhensis BH030046T (95.4 %). Phylogenetic analyses based on 16S rRNA gene, gyrB gene and whole-genome sequences placed strain N102T within the genus Salinimonas. Genomic comparisons based on average nucleotide identity and tetranucleotide signature frequencies corroborated the results of the phylogenetic analyses. The principal fatty acids were summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), C16 : 0 and summed feature 8 (C18 : 1ω7c/C18 : 1ω6c). The major respiratory quinone was ubiquinone 8. The predominant polar lipids were phosphatidylethanolamine, phosphatidylglycerol and an unidentified phospholipid. The G+C content of the genomic DNA was 48.2 mol%. On the basis of phenotypic, chemotaxonomic and molecular data, we conclude that strain N102T represents a novel species of the genus Salinimonas, for which the name Salinimonassediminis sp. nov. is proposed (type strain N102T=MCCC 1K03497T=KCTC 62440T).

Diversity of Total Bacterial Communities and Chemoautotrophic Populations in Sulfur-Rich Sediments of Shallow-Water Hydrothermal Vents off Kueishan Island, Taiwan.

Shallow-water hydrothermal vents (HTVs) are an ecologically important habitat with a geographic origin similar to that of deep-sea HTVs. Studies on shallow-water HTVs have not only facilitated understanding of the influences of vents on local ecosystems but also helped to extend the knowledge on deep-sea vents. In this study, the diversity of bacterial communities in the sediments of shallow-water HTVs off Kueishan Island, Taiwan, was investigated by examining the 16S ribosomal RNA gene as well as key functional genes involved in chemoautotrophic carbon fixation (aclB, cbbL and cbbM). In the vent area, Sulfurovum and Sulfurimonas of Epsilonproteobacteria appeared to dominate the benthic bacterial community. Results of aclB gene analysis also suggested involvement of these bacteria in carbon fixation using the reductive tricarboxylic acid (rTCA) cycle. Analysis of the cbbM gene showed that Alphaproteobacterial members such as the purple non-sulfur bacteria were the major chemoautotrophic bacteria involving in carbon fixation via the Calvin-Benson-Bassham (CBB) cycle. However, they only accounted for <2% of the total bacterial community in the vent area. These findings suggest that the rTCA cycle is the major chemoautotrophic carbon fixation pathway in sediments of the shallow-water HTVs off Kueishan Island.

Tracking the relative concentration between Bacteroidales DNA markers and culturable Escherichia coli in fecally polluted subtropical seawater: potential use in differentiating fresh and aged pollution.

Routine water quality monitoring practices based on the enumeration of culturable Escherichia coli provides no information about the source or age of fecal pollution. An emerging strategy is to use culturable E. coli and the DNA markers of Bacteroidales complementarily for microbial source tracking. In this study, we consistently observed in seawater microcosms of 3 different conditions that culturable E. coli decayed faster (T99 = 1.14 - 4.29 days) than Bacteroidales DNA markers did (T99 = 1.81 - 200.23 days). Concomitantly, the relative concentration between Bacteroidales DNA markers and culturable E. coli increased over time in all treatments. Particularly, the increase during the early stage of the experiments (before T99 of E. coli was reached) was faster than during the later stage (after T99 of E. coli was attained). We propose that the tracking of the relative concentration between Bacteroidales DNA markers and culturable E. coli provides an opportunity to differentiate a pollution that is relatively fresh from one that has aged. This method, upon further investigation and validation, could be useful in episodic pollution events where the surge of E. coli concentration causes noncompliance to the single sample maximum criterion that mandates high frequency follow-up monitoring.

Resilience of sulfate-reducing granular sludge against temperature, pH, oxygen, nitrite, and free nitrous acid.

Sulfate-reducing granular sludge has recently been developed and characterized in detail as part of the development of the sulfate reduction, autotrophic denitrification, nitrification integrated (SANI) process. However, information regarding temperature of granules to environmental fluctuation is lacking, an aspect that is important in dealing with real wastewater. A comprehensive assessment of sulfate-reducing granular sludge performance under various environmental conditions was thus conducted in this study, including temperature, pH, oxygen, nitrite, and free nitrous acid (FNA) as possible encountering conditions in the removal of organics and/or nitrate. Specific chemical oxygen demand removal rate of the granules was determined to be reduced by 65 % when the temperature varied between 10-15 °C, reduced by 70 % when dissolved oxygen (DO) was 0.5 mg/L or greater, and at least, reduced by 75 % when nitrite was 30 mg N/L or above. Nevertheless, the sludge activity recovered by 82, 100, and 86 % from exposure to high oxygen and nitrite and low temperature levels, respectively. Combined inhibition of nitrite and FNA on the sludge is strong and complex, while FNA alone reduced cell viability from 60 to 40 % when its concentration increased to 2.3 mg N/L. The present study demonstrates that sulfate-reducing bacteria (SRB) granules possess high resilience against varying environmental conditions, showing the high application potential of sulfate-reducing granular sludge in dealing with brackish and saline industrial or domestic wastewaters.

Differential utility of the Bacteroidales DNA and RNA markers in the tiered approach for microbial source tracking in subtropical seawater.

Source tracking of fecal pollution is an emerging component in water quality monitoring. It may be implemented in a tiered approach involving Escherichia coli and/or Enterococcus spp. as the standard fecal indicator bacteria (FIB) and the 16S rRNA gene markers of Bacteroidales as source identifiers. The relative population dynamics of the source identifiers and the FIB may strongly influence the implementation of such approach. Currently, the relative performance of DNA and RNA as detection targets of Bacteroidales markers in the tiered approach is not known. We compared the decay of the DNA and RNA of the total (AllBac) and ruminant specific (CF128) Bacteroidales markers with those of the FIB in seawater spiked with cattle feces. Four treatments of light and oxygen availability simulating the subtropical seawater of Hong Kong were tested. All Bacteroidales markers decayed significantly slower than the FIB in all treatments. Nonetheless, the concentrations of the DNA and RNA markers and E. coli correlated significantly in normoxic seawater independent of light availability, and in hypoxic seawater only under light. In hypoxic seawater without light, the concentrations of RNA but not DNA markers correlated with that of E. coli. Generally, the correlations between Enterococcus spp. and Bacteroidales were insignificant. These results suggest that either DNA or RNA markers may complement E. coli in the tiered approach for normoxic or hypoxic seawater under light. When light is absent, either DNA or RNA markers may serve for normoxic seawater, but only the RNA markers are suitable for hypoxic seawater.

Determining the abundance, composition and spatial distribution of organohalogens in marine sediments using combustion-ion chromatography.

Understanding the distribution of halogenated organic compounds (HOCs) in marine sediments is essential for understanding the marine carbon and halogen cycling, and also important for assessing the ecosystem health. In this study, a method based on combustion-ion chromatography was developed for determination of the composition and abundance of HOCs in marine sediments. The method showed high accuracy, precision and reproducibility in determining the content of adsorbable organic halogens (AOX), including fluorine, chlorine and bromine (AOF, AOCl, AOBr) and the corresponding insoluble organic halogens (IOF, IOCl, IOBr, IOX), as well as total organic halogen contents (TOX). Application of the method in coastal and deep-sea sediments revealed high ratios of organic halogens in the organic carbon pool of marine sediments, suggesting that organic halogen compounds represent an important yet previously overlooked stock of carbon and energy in marine sediments. Both the TOX and the proportion of organohalogens in organic carbon (X:C ratio) showed an increasing trend from the coast to the deep-sea sediments, indicating an increased significance of HOCs in deep-sea environments. The developed method and the findings of this study lay the foundation for further studies on biogeochemical cycling of HOCs in the ocean.

Metagenomic characterization of a novel non-ammonia-oxidizing Thaumarchaeota from hadal sediment.

BACKGROUND: The hadal sediment, found at an ocean depth of more than 6000 m, is geographically isolated and under extremely high hydrostatic pressure, resulting in a unique ecosystem. Thaumarchaeota are ubiquitous marine microorganisms predominantly present in hadal environments. While there have been several studies on Thaumarchaeota there, most of them have primarily focused on ammonia-oxidizing archaea (AOA). However, systematic metagenomic research specifically targeting heterotrophic non-AOA Thaumarchaeota is lacking. RESULTS: In this study, we explored the metagenomes of Challenger Deep hadal sediment, focusing on the Thaumarchaeota. Functional analysis of sequence reads revealed the potential contribution of Thaumarchaeota to recalcitrant dissolved organic matter degradation. Metagenome assembly binned one new group of hadal sediment-specific and ubiquitously distributed non-AOA Thaumarchaeota, named Group-3.unk. Pathway reconstruction of this new type of Thaumarchaeota also supports heterotrophic characteristics of Group-3.unk, along with ABC transporters for the uptake of amino acids and carbohydrates and catabolic utilization of these substrates. This new clade of Thaumarchaeota also contains aerobic oxidation of carbon monoxide-related genes. Complete glyoxylate cycle is a distinctive feature of this clade in supplying intermediates of anabolic pathways. The pan-genomic and metabolic analyses of metagenome-assembled genomes belonging to Group-3.unk Thaumarchaeota have highlighted distinctions, including the dihydroxy phthalate decarboxylase gene associated with the degradation of aromatic compounds and the absence of genes related to the synthesis of some types of vitamins compared to AOA. Notably, Group-3.unk shares a common feature with deep ocean AOA, characterized by their high hydrostatic pressure resistance, potentially associated with the presence of V-type ATP and di-myo-inositol phosphate syntheses-related genes. The enrichment of organic matter in hadal sediments might be attributed to the high recruitment of sequence reads of the Group-3.unk clade of heterotrophic Thaumarchaeota in the trench sediment. Evolutionary and genetic dynamic analyses suggest that Group-3 non-AOA consists of mesophilic Thaumarchaeota organisms. These results indicate a potential role in the transition from non-AOA to AOA Thaumarchaeota and from thermophilic to mesophilic Thaumarchaeota, shedding light on recent evolutionary pathways. CONCLUSIONS: One novel clade of heterotrophic non-AOA Thaumarchaeota was identified through metagenome analysis of sediments from Challenger Deep. Our study provides insight into the ecology and genomic characteristics of the new sub-group of heterotrophic non-AOA Thaumarchaeota, thereby extending the knowledge of the evolution of Thaumarchaeota. Video Abstract.

Biogeographic distribution, ecotype partitioning and controlling factors of Chloroflexi in the sediments of six hadal trenches of the Pacific Ocean.

The hadal trenches are "hot spots" for mineralization of organic matter in the deep ocean. Chloroflexi are one of the most dominant and active taxa in trench sediments, serving as important drivers of carbon cycles in hadal trenches. However, current understanding on hadal Chloroflexi is largely restricted to individual trench. This study systematically analyzed the diversity, biogeographic distribution, ecotype partitioning as well as environmental drivers of Chloroflexi in the sediments of hadal trenches, by reanalyzing 16S rRNA gene libraries of 372 samples from 6 trenches around the Pacific Ocean. The results showed that Chloroflexi averagely account for 10.10 % and up to 59.95 % of total microbial communities in the trench sediments. Positive correlations between relative abundance of Chloroflexi and depths down the vertical sediment profiles were observed in all of the sediment cores analyzed, suggesting the increasing significance of Chloroflexi in deeper sediment layers. Overall, trench sediment Chloroflexi were mainly composed of the classes Dehalococcidia, Anaerolineae and JG30-KF-CM66, and four orders i.e. SAR202, Anaerolineales, norank JG30-KF-CM66 and S085, were identified as core taxa that were dominant and prevalent in the hadal trench sediments. A total of 22 subclusters were identified within these core orders, and distinct patterns of ecotype partitioning related with depths down the vertical sediment profiles were observed, suggesting the great diversification of metabolic potentials and environment preference of different Chloroflexi lineages. The spatial distribution of hadal Chloroflexi were found to be significantly related with multiple environmental factors, while depths down the vertical sediment profiles explained the highest proportion of variations. These results provide valuable information for further exploring the roles of Chloroflexi in biogeochemical cycle of the hadal zone, and lay the foundation for understanding the adaptive mechanisms and evolutionary characteristics of microorganisms in hadal trenches.

Improving the performance of an end-point PCR assay commonly used for the detection of Bacteroidales pertaining to cow feces.

Bacteroidales are normal gut flora of warm-blooded animals. Since each host species carries a different diversity of Bacteroidales, the detection of host-associated gene markers of Bacteroidales has emerged as a promising tool for the tracking of the source of fecal pollution in aquatic ecosystems. To detect cow-associated Bacteroidales, a commonly used method has been an end-point PCR assay with the 16S rRNA genes primers CF128F (cow-associated) and Bac708R (all Bacteroidales). The PCR assay has demonstrated high rates of true-positive detection (i.e., high sensitivity) in all previous studies. However, the assay also had high rates of false-positive detection to the samples of non-target hosts in some cases (i.e., low specificity). In opposite to the reason many investigators have proposed, our results suggested that false detection was not necessarily due to the presence of the target sequence of CF128F in the feces of non-target hosts. Instead, we found sequences of non-target hosts having single internal mismatches with CF128F. Those mismatches were well tolerated in PCR, partly due to the universality of Bac708R. To improve the detection performance, we designed a novel primer CF592R (targeting the same clade of sequences as CF128F) to substitute Bac708R. The use of CF529R alleviated false detection and also led to a tenfold reduction in detection limit in the samples tested, compared to the use of Bac708R. Many other end-point PCR assays that detect the 16S rRNA genes in Bacteroidales also use a host-associated primer to couple with Bac708R, and low specificity or sensitivity has been reported. Based on our findings for CF128F, we suggest that the suitability of Bac708R in those PCR assays needs to be revisited.

Microbial communities and biosynthetic pathways for the production of sulfated polysaccharides in the activated sludge system.

Sulfated polysaccharides (SP) are widely used as industrial additives and pharmaceutical intermediates. As SP can only be extracted from sea algae, making them scarce raw materials. Recently, SP have been detected and extracted from the waste activated sludge of a saline secondary wastewater treatment plant, suggesting that there are alternative primary producers and synthesis pathways of the SP within the biological activated sludge. This study aimed to identify the primary SP producers, the SP biosynthesis pathways as well as the SP production rates in different types of activated sludges cultivated anoxically and/or anaerobically, with and without the presence of sufficient sulfate. The results showed that alternating anaerobic/anoxic conditions in sludge effectively produced the SP by the ordinary heterotrophic organisms (OHOs). The synthesis pathways for the three most common bioactive SP viz. fucoidan, carrageen, and heparin, were identified and elucidated at both the substrate and enzymatic levels. The Western Blot analyses revealed key enzymes for the SP synthesis (e.g., GDP-L-fucose-synthetase, GDP-fucose-pyrophosphorylase, ß-1,4-galactosyltransferase), when sulfate was sufficient (>170 mg S/L) under an alternating anaerobic/anoxic conditions. In contrast, the absence of sulfate suppressed the SP production during the initial step of the SP generation. The synthesis of the SP in the sulfate-reducing (anaerobic) sludge was suppressed by the enzymatic inhibition, when sulfide exceeded 160 mg S/L, due to the competition for energy between the SP synthesis and sulfide detoxification. However, in the case of the sulfide-oxidizing sludge both the organic carbon and metabolism energy deficiencies inhibited the SP production. The findings of this study expand the understandings of the SP synthesis in the activated sludge under different operating conditions, including different sulfate levels.