Recyclable Multifunctional Nanocomposites Based on Carbon Nanotube Reinforced Vitrimers with Shape Memory and Joule Heating Capabilities.

The present study focuses on the multifunctional capabilities of carbon nanotube (CNT)-reinforced vitrimers. More specifically, the thermomechanical properties, the Joule effect heating capabilities, the electrical conductivity, the shape memory, and the chemical recycling capacity are explored as a function of the CNT content and the NH2/epoxy ratio. It is observed that the electrical conductivity increases with the CNT content due to a higher number of electrical pathways, while the effect of the NH2/epoxy ratio is not as prevalent. Moreover, the Tg of the material decreases when increasing the NH2/epoxy ratio due to the lower cross-link density, whereas the effect of the CNTs is more complex, in some cases promoting a steric hindrance. The results of Joule heating tests prove the suitability of the proposed materials for resistive heating, reaching average temperatures above 200 °C when applying 100 V for the most electrically conductive samples. Shape memory behavior shows an outstanding shape fixity ratio in every case (around 100%) and a higher shape recovery ratio (95% for the best-tested condition) when decreasing the NH2/epoxy ratio and increasing the CNT content, as both hinder the rearrangement of the dynamic bonds. Finally, the results of the recyclability tests show the ability to regain the nanoreinforcement for their further use. Therefore, from a multifunctional analysis, it can be stated that the proposed materials present promising properties for a wide range of applications, such as Anti-icing and De-icing Systems (ADIS), Joule heating devices for comfort or thermotherapy, or self-deployable structures, among others.

Determination of Haematological Reference Values for Tucúquere (Bubo magellanicus) Habiting in Central Chile.

Tucuquere (Bubo magellanicus) is an owl inhabiting Chile, which is classified as a species of agricultural interest, but its current conservation status is not well defined. The determination of previously unknown haematological ranges via laboratory techniques for species analysis is of great importance in the search for diagnoses in clinical veterinary work. Thirty-three samples from healthy tucuquere were obtained from zoos and rehabilitation centres in central Chile and analysed using standard laboratory techniques to obtain reference values that were determined according to the standard recommendations for animal species. The mean, standard deviation, maximum, and minimum reference limits were calculated at 95% and 97.5% error. These results differ from those of other studies, especially in terms of the white blood cells because most of the values were lower than those described for the species. These results could be explained by the habitat and location from which the samples were taken, and they corroborate the need to obtain reference values for different geographical areas. This study provides useful haematological values for use in conservation medicine and zoos and highlights the importance of carrying out these studies in different geographical areas for species of ecological interest.

Novel and Accessible Physical Recycling for Expanded Polystyrene Waste with the Use of Acetone as a Solvent and Additive Manufacturing (Direct Ink-Write 3D Printing).

The current high production of plastics has prompted the exploration of alternative pathways to facilitate recycling, aiming for a progressively sustainable society. This paper presents an alternative and affordable technology for treating waste expanded polystyrene (EPS) mixed with acetone in a 100:1 volume ratio to be used as 3D printing ink for Direct Ink Write technology. In order to optimize the printing parameters, a comprehensive study was conducted, evaluating different needle diameters, printing speeds, and bed temperature values to achieve homogenous pieces and a highly repeatable 3D printing process. Results showed that the main optimum printing parameters were using needles with diameters of 14 to 16 G and printing speeds ranging from 2 to 12 mm/s, which were found to yield the most uniform ribbons. Increasing the bed temperature, despite favoring acetone evaporation, led to the generation of more heterogeneous structures due to void growth inside the printed ribbons. Thus, employing room temperature for the bed proved to be the optimal value. Lastly, a comparative study between the starting material and the EPS after the printing process was conducted using FTIR-ATR and GPC analyses, ensuring the preservation of the original polymer's integrity during physical recycling.

Use of Biomarkers to Improve 28-Day Mortality Stratification in Patients with Sepsis and SOFA ≤ 6.

Early diagnosis and appropriate treatments are crucial to reducing mortality risk in septic patients. Low SOFA scores and current biomarkers may not adequately discern patients that could develop severe organ dysfunction or have an elevated mortality risk. The aim of this prospective observational study was to evaluate the predictive value of the biomarkers mid-regional pro-adrenomedullin (MR-proADM), procalcitonin (PCT), C-reactive protein (CRP), and lactate for 28-day mortality in patients with sepsis, and patients with a SOFA score ≤6. 284 were included, with a 28-day all-cause mortality of 8.45% (n = 24). Non-survivors were older (p = 0.003), required mechanical ventilation (p = 0.04), were ventilated for longer (p = 0.02), and had higher APACHE II (p = 0.015) and SOFA (p = 0.027) scores. Lactate showed the highest predictive ability for all-cause 28-day mortality, with an area under the receiver-operating characteristic curve (AUROC) of 0.67 (0.55-0.79). The AUROC for all-cause 28-day mortality in patients with community-acquired infection was 0.69 (0.57-0.84) for SOFA and 0.70 (0.58-0.82) for MR-proADM. A 2.1 nmol/L cut-off point for this biomarker in this subgroup of patients discerned, with 100% sensibility, survivors from non-survivors at 28 days. In patients with community-acquired sepsis and initial SOFA score ≤ 6, MR-proADM could help identify patients at risk of 28-day mortality.

Revisiting Microbial Diversity in Hypersaline Microbial Mats from Guerrero Negro for a Better Understanding of Methanogenic Archaeal Communities.

Knowledge regarding the diversity of methanogenic archaeal communities in hypersaline environments is limited because of the lack of efficient cultivation efforts as well as their low abundance and metabolic activities. In this study, we explored the microbial communities in hypersaline microbial mats. Bioinformatic analyses showed significant differences among the archaeal community structures for each studied site. Taxonomic assignment based on 16S rRNA and methyl coenzyme-M reductase (mcrA) gene sequences, as well as metagenomic analysis, corroborated the presence of Methanosarcinales. Furthermore, this study also provided evidence for the presence of Methanobacteriales, Methanomicrobiales, Methanomassiliicoccales, Candidatus Methanofastidiosales, Methanocellales, Methanococcales and Methanopyrales, although some of these were found in extremely low relative abundances. Several mcrA environmental sequences were significantly different from those previously reported and did not match with any known methanogenic archaea, suggesting the presence of specific environmental clusters of methanogenic archaea in Guerrero Negro. Based on functional inference and the detection of specific genes in the metagenome, we hypothesised that all four methanogenic pathways were able to occur in these environments. This study allowed the detection of extremely low-abundance methanogenic archaea, which were highly diverse and with unknown physiology, evidencing the presence of all methanogenic metabolic pathways rather than the sheer existence of exclusively methylotrophic methanogenic archaea in hypersaline environments.

Postoperative Acute Kidney Injury With Dual NSAID Use After Outpatient Primary Total Joint Arthroplasty.

BACKGROUND: NSAIDs have been shown to be highly effective analgesic agents in the postoperative period. NSAIDs do have several potential adverse effects, including kidney injury (AKI). Little is known about AKI in the outpatient total joint arthroplasty (TJA) setting, where patient labs are not closely monitored. The objective of this study was to evaluate the renal safety of combined use of ibuprofen for pain control and aspirin for deep vein thrombosis chemoprophylaxis after outpatient primary TJA. METHODS: Patients undergoing primary total hip or total knee arthroplasty between January 2020 and July 2020 at a single center were included for analysis. All included patients were discharged on a standard regimen including aspirin 81 mg twice a day and ibuprofen 600 mg three times a day. Patients were ordered a serum creatinine test at 2 and 4 weeks postoperatively. Patients with postoperative acute kidney injury were identified per Acute Kidney Injury Network criteria. RESULTS: Between January 23, 2020, and August 30, 2020, 113 patients were included in this study, of whom creatinine levels were measured in 103 patients (90.3%) at the 2-week postoperative time point, 58 patients (50.9%) at the 4-week time point, and 48 (42.1%) at combined 2- and 4-week time points. Three patients (2.9%) were found to have an AKI. CONCLUSION: This study found a rate of AKI of 2.9% with the use of dual NSAID therapy postoperatively after primary TJA. All cases occurred at 2 weeks postoperatively and saw spontaneous resolution.

Veronia nyctiphanis gen. nov., sp. nov., Isolated from the Stomach of the Euphausiid Nyctiphanes simplex (Hansen, 1911) in the Gulf of California, and Reclassification of Enterovibrio pacificus as Veronia pacifica comb. nov.

The bacterial strain 42Xb2 T was isolated from a female adult krill Nyctiphanes simplex infected with the apostome parasitoid ciliate Pseudocollinia brintoni in January 2007 in the Gulf of California. The strain has the morphological, phenotypic, and molecular characteristics of the bacteria of the family Vibrionaceae. The 16S rRNA gene sequence has a similarity of 97.7% with Enterovibrio pacificus SW014 T and 96.1% similarity with Enterovibrio norvegicus LMG 19839 T. A phylogenomic and a multilocus sequence analyses placed this strain close to the genera Enterovibrio, Grimontia, and Salinivibrio, but clearly forming a separate branch from these bacterial genera. Genomic analyses presented further support this result. A novel genus Veronia gen. nov. and a species Veronia nyctiphanis sp. nov. is here described with CAIM 600 T (= DSM 24592 T = CECT 7578 T) as the type strain. Morphological, physiological, and genetic evidence presented here support the unification of Enterovibrio pacificus and Veronia nyctiphanis in the new genus Veronia. Enterovibrio pacificus is reclassified as Veronia pacifica. V. pacifica is assigned as the type species of the new genus Veronia.Genome Sequencing Data The GenBank/EMBL/DDBJ accession numbers for the genome sequence of Veronia nyctiphanis CAIM 600 T is PEIB01 and of Enterovibrio pacificus CAIM 1920 T is LYBM01. The 16S rRNA gene sequence of V. nyctiphanis CAIM 600 T is JX129353.

Complex Geometry Strain Sensors Based on 3D Printed Nanocomposites: Spring, Three-Column Device and Footstep-Sensing Platform.

Electromechanical sensing devices, based on resins doped with carbon nanotubes, were developed by digital light processing (DLP) 3D printing technology in order to increase design freedom and identify new future and innovative applications. The analysis of electromechanical properties was carried out on specific sensors manufactured by DLP 3D printing technology with complex geometries: a spring, a three-column device and a footstep-sensing platform based on the three-column device. All of them show a great sensitivity of the measured electrical resistance to the applied load and high cyclic reproducibility, demonstrating their versatility and applicability to be implemented in numerous items in our daily lives or in industrial devices. Different types of carbon nanotubes-single-walled, double-walled and multi-walled CNTs (SWCNTs, DWCNTs, MWCNTs)-were used to evaluate the effect of their morphology on electrical and electromechanical performance. SWCNT- and DWCNT-doped nanocomposites presented a higher Tg compared with MWCNT-doped nanocomposites due to a lower UV light shielding effect. This phenomenon also justifies the decrease of nanocomposite Tg with the increase of CNT content in every case. The electromechanical analysis reveals that SWCNT- and DWCNT-doped nanocomposites show a higher electromechanical performance than nanocomposites doped with MWCNTs, with a slight increment of strain sensitivity in tensile conditions, but also a significant strain sensitivity gain at bending conditions.

Intestinal Microbiota Analyses of Litopenaeus vannamei During a Case of Atypical Massive Mortality in Northwestern Mexico.

This study investigated the intestinal microbial community structure of Litopenaeus vannamei at six different stages during shrimp farming. Our goal was to elucidate the bacterial profile and the changes in the relative abundance of taxa during an atypical massive mortality event in Sonora, Mexico. High-throughput sequencing of the 16S rRNA gene and denaturing gradient gel electrophoresis showed that Vibrionaceae was persistent with high relative abundances in the intestine from cultivated shrimp during all the studied stages. The massive mortality observed at day 63 could be related to an overabundance of different Operational Taxonomic Units (OTUs) of Vibrio, Shewanella and Clostridium. Principal coordinate analysis (PCoA) showed variations in microbial structure at different culture times. These findings suggest that OTUs of different taxa contributed to the community switch from healthy to diseased individuals, questioning the hypothesis that single bacterial species is the cause of disease outbreaks. This study provided data to improve the understanding of disease outbreaks during shrimp farming.

Mechanical and Strain-Sensing Capabilities of Carbon Nanotube Reinforced Composites by Digital Light Processing 3D Printing Technology.

Mechanical and strain sensing capabilities of carbon nanotube (CNT) reinforced composites manufactured by digital light processing (DLP) 3D printing technology have been studied. Both CNT content and a post-curing treatment effects have been analyzed. It has been observed that post-curing treatment has a significant influence on mechanical properties, with an increase of Young's modulus and glass transition temperature whereas their effect in electrical properties is not so important. Furthermore, the strain sensing tests show a linear response of electrical resistance with applied strain, with higher values of sensitivity when decreasing CNT content due to a higher interparticle distance. Moreover, the electrical sensitivity of bending tests is significantly lower than in tensile ones due to the compression subjected face effect. Therefore, the good gauge factor values (around 2-3) and the high linear response proves the applicability of the proposed nanocomposites in structural health monitoring applications.

Biomarkers and clinical scores to aid the identification of disease severity and intensive care requirement following activation of an in-hospital sepsis code.

BACKGROUND: Few validated biomarker or clinical score combinations exist which can discriminate between cases of infection and other non-infectious conditions following activation of an in-hospital sepsis code, as well as provide an accurate severity assessment of the corresponding host response. This study aimed to identify suitable blood biomarker (MR-proADM, PCT, CRP and lactate) or clinical score (SOFA and APACHE II) combinations to address this unmet clinical need. METHODS: A prospective, observational study of patients activating the Vall d'Hebron University Hospital sepsis code (ISC) within the emergency department (ED), hospital wards and intensive care unit (ICU). Area under the receiver operating characteristic (AUROC) curves, logistic and Cox regression analysis were used to assess performance. RESULTS: 148 patients fulfilled the Vall d'Hebron ISC criteria, of which 130 (87.8%) were retrospectively found to have a confirmed diagnosis of infection. Both PCT and MR-proADM had a moderate-to-high performance in discriminating between infected and non-infected patients following ISC activation, although the optimal PCT cut-off varied significantly across departments. Similarly, MR-proADM and SOFA performed well in predicting 28- and 90-day mortality within the total infected patient population, as well as within patients presenting with a community-acquired infection or following a medical emergency or prior surgical procedure. Importantly, MR-proADM also showed a high association with the requirement for ICU admission after ED presentation [OR (95% CI) 8.18 (1.75-28.33)] or during treatment on the ward [OR (95% CI) 3.64 (1.43-9.29)], although the predictive performance of all biomarkers and clinical scores diminished between both settings. CONCLUSIONS: Results suggest that the individual use of PCT and MR-proADM might help to accurately identify patients with infection and assess the overall severity of the host response, respectively. In addition, the use of MR-proADM could accurately identify patients requiring admission onto the ICU, irrespective of whether patients presented to the ED or were undergoing treatment on the ward. Initial measurement of both biomarkers might therefore facilitate early treatment strategies following activation of an in-hospital sepsis code.

Characterizing the Use of Expectations in Orthopedic Surgery Research: A Scoping Review.

OBJECTIVE: Orthopedic literature on expectations is limited by lack of uniformity in how expectations are defined, conceptualized, and measured. Within this scoping review, we present a conceptual framework for understanding the construct of expectations and its derivatives (ie, expectation, expectancy, expectancies, etc) in studies of elective surgical orthopedic procedures. We also utilize this framework to map the current orthopedic surgery literature on expectations, highlighting its strengths, weaknesses, and gaps in its knowledge base. METHODS: We included articles that mentioned both expectations and one or more of 10 selected surgeries in their title or abstract. We focused on representative elective orthopedic surgeries that covered the body's major regions. We operationalized each study's individual expectation items into one of the expectation concept types within the presented conceptual model. We also extracted the name and type of expectations measurement tool used, whose expectations were measured, and descriptive information (eg, surgery type, publication date, country of origin, and study type). RESULTS: Ninety studies were included, with 70% published after 2008. A total of 64% investigated total knee and total hip arthroplasty, whereas 90% of studies investigated the expectations of patients, 5.6% investigated the expectations of physicians, and 4.4% investigated both. Of all studies, 72% utilized either study-specific instruments with close-ended, Likert-type response formats or modifications of existing expectations questionnaires. Most studies focused on desirable, nontimeline-related, treatment outcome expectations. Many studies aggregated multiple expectation results into a single score. CONCLUSION: Adopting the standardized framework for expectations presented in this study will foster clearer communication and permit researchers to aggregate results across studies.

A value-based care analysis of magnetic resonance imaging in patients with suspected rotator cuff tendinopathy and the implicated role of conservative management.

BACKGROUND: Magnetic resonance imaging (MRI) is often used to evaluate the integrity of the rotator cuff in patients with suspected full-thickness rotator cuff tears or other cuff tendinopathies. The value of advanced imaging value comes into question when it is used as the initial musculoskeletal imaging test before a trial of conservative therapy in patients with atraumatic shoulder pain, minimal to no strength deficits on examination, and suspected cuff tendinopathy. METHODS: A prospective study of a group of patients suspected to have cuff tendinopathy based on clinical findings was performed. Every patient underwent MRI and was offered an initial trial of conservative management. Patients had an average follow-up of 28.3 ± 5.3 months after imaging to determine whether surgery was performed. RESULTS: A total of 51 patients were included in this study. Of this cohort, 46 (90.2%) patients did not go on to surgical intervention, whereas 5 (9.8%) patients did at an average 68.3 days after imaging. These results suggest that over 90.2% of patients (46 of 51) had premature MRI, posing an unnecessary economic burden of $181,619 in advanced imaging charges. CONCLUSIONS: The use of MRI before a trial of conservative management in patients with atraumatic shoulder pain, minimal to no strength deficits on physical examination, and suspected cuff tendinopathy other than full-thickness tears provides negative value in the management of these patients, at both the individual and population level.

Racial and ethnic differences in the experience and treatment of noncancer pain.

The burden of pain is unequal across racial and ethnic groups. In addition to racial and ethnic differences in the experience of pain, there are racial and ethnic disparities in the assessment and treatment of pain. In this article, we provide a nonexhaustive review of the biopsychosocial mechanistic factors contributing to racial and ethnic differences in both the experience and treatment of pain. Using a modified version of the Socioecological Model, we focus on patient-, provider- and system-level factors including coping, perceived bias and discrimination, patient preferences, expectations, patient/provider communication, treatment outcomes and healthcare access. In conclusion, we provide psychosocial factors influencing racial and ethnic differences in pain and highlight future research targets and possible solutions to reduce these disparities.

Bacterial and archaeal profiling of hypersaline microbial mats and endoevaporites, under natural conditions and methanogenic microcosm experiments.

Bacterial and archaeal community structure of five microbial communities, developing at different salinities in Baja California Sur, Mexico, were characterized by 16S rRNA sequencing. The response of the microbial community to artificial changes in salinity-sulfate concentrations and to addition of trimethylamine was also evaluated in microcosm experiments. Ordination analyses of the microbial community structure showed that microbial composition was distinctive for each hypersaline site. Members of bacteria were dominated by Bacteroidetes and Proteobacteria phyla, while Halobacteria of the Euryarchaeota phylum was the most represented class of archaea for all the environmental samples. At a higher phylogenetic resolution, methanogenic communities were dominated by members of the Methanosarcinales, Methanobacteriales and Methanococcales orders. Incubation experiments showed that putative hydrogenotrophic methanogens of the Methanomicrobiales increased in abundance only under lowest salinity and sulfate concentrations. Trimethylamine addition effectively increased the abundance of methylotrophic members from the Methanosarcinales, but also increased the relative abundance of the Thermoplasmata class, suggesting the potential capability of these microorganisms to use trimethylamine in hypersaline environments. These results contribute to the knowledge of microbial diversity in hypersaline environments from Baja California Sur, Mexico, and expand upon the available information for uncultured methanogenic archaea in these ecosystems.

Screening of polyhydroxyalkanoate-producing bacteria and PhaC-encoding genes in two hypersaline microbial mats from Guerrero Negro, Baja California Sur, Mexico.

Hypersaline microbial mats develop through seasonal and diel fluctuations, as well as under several physicochemical variables. Hence, resident microorganisms commonly employ strategies such as the synthesis of polyhydroxyalkanoates (PHAs) in order to resist changing and stressful conditions. However, the knowledge of bacterial PHA production in hypersaline microbial mats has been limited to date, particularly in regard to medium-chain length PHAs (mcl-PHAs), which have biotechnological applications due to their plastic properties. The aim of this study was to obtain evidence for PHA production in two hypersaline microbial mats of Guerrero Negro, Mexico by searching for PHA granules and PHA synthase genes in isolated bacterial strains and environmental samples. Six PHA-producing strains were identified by 16S rRNA gene sequencing; three of them corresponded to a Halomonas sp. In addition, Paracoccus sp., Planomicrobium sp. and Staphylococcus sp. were also identified as PHA producers. Presumptive PHA granules and PHA synthases genes were detected in both sampling sites. Moreover, phylogenetic analysis showed that most of the phylotypes were distantly related to putative PhaC synthases class I sequences belonging to members of the classes Alphaproteobacteria and Gammaproteobacteria distributed within eight families, with higher abundances corresponding mainly to Rhodobacteraceae and Rhodospirillaceae. This analysis also showed that PhaC synthases class II sequences were closely related to those of Pseudomonas putida, suggesting the presence of this group, which is probably involved in the production of mcl-PHA in the mats. According to our state of knowledge, this study reports for the first time the occurrence of phaC and phaC1 sequences in hypersaline microbial mats, suggesting that these ecosystems may be a novel source for the isolation of short- and medium-chain length PHA producers.

Histophagous ciliate Pseudocollinia brintoni and bacterial assemblage interaction with krill Nyctiphanes simplex. I. Transmission process.

Histophagous ciliates of the genus Pseudocollinia cause epizootic events that kill adult female krill (Euphausiacea), but their mode of transmission is unknown. We compared 16S rRNA sequences of bacterial strains isolated from stomachs of healthy krill Nyctiphanes simplex specimens with sequences of bacterial isolates and sequences of natural bacterial communities from the hemocoel of N. simplex specimens infected with P. brintoni to determine possible transmission pathways. All P. brintoni endoparasitic life stages and the transmission tomite stage (outside the host) were associated with bacterial assemblages. 16S rRNA sequences from isolated bacterial strains showed that Photobacterium spp. and Pseudoalteromonas spp. were dominant members of the bacterial assemblages during all life phases of P. brintoni and potential pathobionts. They were apparently unaffected by the krill's immune system or the histophagous activity of P. brintoni. However, other bacterial strains were found only in certain P. brintoni life phases, indicating that as the infection progressed, microhabitat conditions and microbial interactions may have become unfavorable for some strains of bacteria. Trophic infection is the most parsimonious explanation for how P. brintoni infects krill. We estimated N. simplex vulnerability to P. brintoni infection during more than three-fourths of their life span, infecting mostly adult females. The ciliates have relatively high prevalence levels (albeit at <10% of sampled stations) and a short life cycle (estimated <7 d). Histophagous ciliate-krill interactions may occur in other krill species, particularly those that form dense swarms and attain high population densities that potentially enhance trophic transmission and allow completion of the Pseudocollinia spp. life cycle.

Histophagous ciliate Pseudocollinia brintoni and bacterial assemblage interaction with krill Nyctiphanes simplex. II. Host responses.

Unlike decapod crustaceans of commercial interest, the krill defense system and its response to parasites and pathogens is virtually unknown. Histophagous ciliates of the genus Pseudocollinia interact with at least 7 krill species in the northeastern Pacific. Although they can cause epizootic events, the physiology of the histophagous ciliate-host interaction and krill (host) defenses remain unknown. From 1 oceanographic survey along the southwestern coast of the Baja California Peninsula near Bahía Magdalena and 2 in the Gulf of California, we investigated parasitoid-host physiological responses (fatty acid and oxidative stress indicators) of the subtropical krill Nyctiphanes simplex infected with the ciliate P. brintoni. All life stages of P. brintoni were associated with opportunistic bacterial assemblages that have not been explicitly investigated in other Pseudocollinia species (P. beringensis, P. oregonensis, and P. similis). Parasitoid ciliates exclusively infected adult females, which showed increased lipid content during gonad development. As the infection progressed, omega-3 eicosapentaenoic and docosahexaenoic fatty acids, which may act as energy sources to produce high numbers of ciliate transmission stages, were quickly depleted. Antioxidant enzymes, components of the crustacean defense system, varied throughout infection, but without inhibiting Pseudocollinia infection, i.e. higher levels of lipid oxidative damage were detected in late stages of infection. The ineffective response of the krill antioxidant defense system against histophagous ciliates and the bacteria associated with the ciliates suggests that Pseudocollinia ciliates are functionally analogous to krill predators and may have a strong influence on the population dynamics of krill.

Revisiting N2 fixation in Guerrero Negro intertidal microbial mats with a functional single-cell approach.

Photosynthetic microbial mats are complex, stratified ecosystems in which high rates of primary production create a demand for nitrogen, met partially by N2 fixation. Dinitrogenase reductase (nifH) genes and transcripts from Cyanobacteria and heterotrophic bacteria (for example, Deltaproteobacteria) were detected in these mats, yet their contribution to N2 fixation is poorly understood. We used a combined approach of manipulation experiments with inhibitors, nifH sequencing and single-cell isotope analysis to investigate the active diazotrophic community in intertidal microbial mats at Laguna Ojo de Liebre near Guerrero Negro, Mexico. Acetylene reduction assays with specific metabolic inhibitors suggested that both sulfate reducers and members of the Cyanobacteria contributed to N2 fixation, whereas (15)N2 tracer experiments at the bulk level only supported a contribution of Cyanobacteria. Cyanobacterial and nifH Cluster III (including deltaproteobacterial sulfate reducers) sequences dominated the nifH gene pool, whereas the nifH transcript pool was dominated by sequences related to Lyngbya spp. Single-cell isotope analysis of (15)N2-incubated mat samples via high-resolution secondary ion mass spectrometry (NanoSIMS) revealed that Cyanobacteria were enriched in (15)N, with the highest enrichment being detected in Lyngbya spp. filaments (on average 4.4 at% (15)N), whereas the Deltaproteobacteria (identified by CARD-FISH) were not significantly enriched. We investigated the potential dilution effect from CARD-FISH on the isotopic composition and concluded that the dilution bias was not substantial enough to influence our conclusions. Our combined data provide evidence that members of the Cyanobacteria, especially Lyngbya spp., actively contributed to N2 fixation in the intertidal mats, whereas support for significant N2 fixation activity of the targeted deltaproteobacterial sulfate reducers could not be found.

Evidence of novel phylogenetic lineages of methanogenic archaea from hypersaline microbial mats.

Methanogenesis in hypersaline and high-sulfate environments is typically dominated by methylotrophic methanogens because sulfate reduction is thermodynamically favored over hydrogenotrophic methanogenesis in these environments. We characterized the community composition of methanogenic archaea in both unmanipulated and incubated microbial mats from different hypersaline environments in Baja California Sur, Mexico. Clone libraries of methyl coenzyme-M reductase (mcrA) sequences and DGGE band patterns of 16S rRNA and mcrA sequences showed that the methanogen community in these microbial mats is dominated by methylotrophic methanogens of the genus Methanohalophilus. However, phylogenetic analyses of mcrA sequences from these mats also revealed two new lineages corresponding to putative hydrogenotrophic methanogens related with the strictly hydrogenotrophic order Methanomicrobiales. Stimulated methane production under decreased salinity and sulfate concentrations also suggested the presence of hydrogenotrophic methanogens in these samples. The relative abundance of mcrA gene and transcripts, estimated by SYBR green I qPCR assays, suggested the activity of different phylogenetic groups of methanogens, including the two novel clusters, in unmanipulated samples of hypersaline microbial mats. Using geochemical and molecular approaches, we show that substrate limitation and values of salinity and sulfate higher than 3 % and 25 mM (respectively) are potential environmental constraints for methanogenesis in these environments. Microcosm experiments with modifications of salinity and sulfate concentrations and TMA addition showed that upper salt and sulfate concentrations for occurrence of methylotrophic methanogenesis were 28 % and 263 mM, respectively. This study provides phylogenetic information about uncultivated and undescribed methanogenic archaea from hypersaline environments.