Do restoration strategies in mangroves recover microbial diversity? A case study in the Yucatan peninsula.

Mangrove forests are fundamental coastal ecosystems for the variety of services they provide, including green-house gas regulation, coastal protection and home to a great biodiversity. Mexico is the fourth country with the largest extension of mangroves of which 60% occurs in the Yucatan Peninsula. Understanding the microbial component of mangrove forests is necessary for their critical roles in biogeochemical cycles, ecosystem health, function and restoration initiatives. Here we study the relation between the microbial community from sediments and the restoration process of mangrove forests, comparing conserved, degraded and restored mangroves along the northern coast of the Yucatan peninsula. Results showed that although each sampling site had a differentiated microbial composition, the taxa belonged predominantly to Proteobacteria (13.2-23.6%), Desulfobacterota (7.6-8.3%) and Chloroflexi (9-15.7%) phyla, and these were similar between rainy and dry seasons. Conserved mangroves showed significantly higher diversity than degraded ones, and restored mangroves recovered their microbial diversity from the degraded state (Dunn test p-value Benjamini-Hochberg adjusted = 0.0034 and 0.0071 respectively). The structure of sediment microbial ß-diversity responded significantly to the mangrove conservation status and physicochemical parameters (organic carbon content, redox potential, and salinity). Taxa within Chloroflexota, Desulfobacterota and Thermoplasmatota showed significantly higher abundance in degraded mangrove samples compared to conserved ones. This study can help set a baseline that includes the microbial component in health assessment and restoration strategies of mangrove forests.

Structure and composition of microbial communities in the water column from Southern Gulf of Mexico and detection of putative hydrocarbon-degrading microorganisms.

This study assessed the bacterioplankton community and its relationship with environmental variables, including total petroleum hydrocarbon (TPH) concentration, in the Yucatan shelf area of the Southern Gulf of Mexico. Beta diversity analyses based on 16S rRNA sequences indicated variations in the bacterioplankton community structure among sampling sites. PERMANOVA indicated that these variations could be mainly related to changes in depth (5 to 180 m), dissolved oxygen concentration (2.06 to 5.93 mg L-1), and chlorophyll-a concentration (0.184 to 7.65 mg m3). Moreover, SIMPER and one-way ANOVA analyses showed that the shifts in the relative abundances of Synechococcus and Prochlorococcus were related to changes in microbial community composition and chlorophyll-a values. Despite the low TPH content measured in the studied sites (0.01 to 0.86 µL L-1), putative hydrocarbon-degrading bacteria such as Alteromonas, Acinetobacter, Balneola, Erythrobacter, Oleibacter, Roseibacillus, and the MWH-UniP1 aquatic group were detected. The relatively high copy number of the alkB gene detected in the water column by qPCR and the enrichment of hydrocarbon-degrading bacteria obtained during lab crude oil tests exhibited the potential of bacterioplankton communities from the Yucatan shelf to respond to potential hydrocarbon impacts in this important area of the Gulf Mexico.

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.

Changes in the Rhizosphere Prokaryotic Community Structure of Halodule wrightii Monospecific Stands Associated to Submarine Groundwater Discharges in a Karstic Costal Area.

Belowground seagrass associated microbial communities regulate biogeochemical dynamics in the surrounding sediments and influence seagrass physiology and health. However, little is known about the impact of environmental stressors upon interactions between seagrasses and their prokaryotic community in coastal ecosystems. Submerged groundwater discharges (SGD) at Dzilam de Bravo, Yucatán, Mexico, causes lower temperatures and salinities with higher nutrient loads in seawater, resulting in Halodule wrightii monospecific stands. In this study, the rhizospheric archaeal and bacterial communities were characterized by 16S rRNA Illumina sequencing along with physicochemical determinations of water, porewater and sediment in a 400 m northwise transect from SGD occurring at 300 m away from coastline. Core bacterial community included Deltaproteobacteria, Bacteroidia and Planctomycetia, possibly involved in sulfur metabolism and organic matter degradation while highly versatile Bathyarchaeia was the most abundantly represented class within the archaeal core community. Beta diversity analyses revealed two significantly different clusters as result of the environmental conditions caused by SGD. Sites near to SGD presented sediments with higher redox potentials and sand contents as well as lower organic matter contents and porewater ammonium concentrations compared with the furthest sites. Functional profiling suggested that denitrification, aerobic chemoheterotrophy and environmental adaptation processes could be better represented in these sites, while sulfur metabolism and genetic information processing related profiles could be related to SGD uninfluenced sites. This study showed that the rhizospheric prokaryotic community structure of H. wrightii and their predicted functions are shaped by environmental stressors associated with the SGD. Moreover, insights into the archaeal community composition in seagrasses rhizosphere are presented.

Sediment microbial community structure associated to different ecological types of mangroves in Celestún, a coastal lagoon in the Yucatan Peninsula, Mexico.

Mangroves are unique coastal ecosystems, which have many important ecological functions, as they are a reservoir of many marine species well adapted to saline conditions and are fundamental as sites of carbon storage. Although the microbial contribution to nutrient cycling in these ecosystems has been well recognized, there is a lack of information regarding the microbial composition and structure of different ecological types of mangrove forests. In this study, we characterized the microbial community (Bacteria and Archaea) in sediments associated with five ecological types of mangrove forests in a coastal lagoon dominated by Avicennia germinans and Rhizophora mangle, through 16S rRNA-V4 gene sequencing. Overall, Proteobacteria (51%), Chloroflexi (12%), Gemmatimonadetes (5%) and Planctomycetes (6%) were the most abundant bacterial phyla, while Thaumarchaeota (30%), Bathyarchaeota (21%) and Nanoarchaeaeota (18%) were the dominant archaeal phyla. The microbial composition associated with basin mangroves dominated by Avicennia germinans was significantly different from the other ecological types, which becomes relevant for restoration strategies.

Changes in the sediment microbial community structure of coastal and inland sinkholes of a karst ecosystem from the Yucatan peninsula.

The karst underground river ecosystem of Yucatan peninsula is composed of cave systems and sinkholes. The microbial diversity of water from this underground river has been studied, but, structure of the microbial community in its cave sediments remained largely unknown. Here we describe how the microbial community structure of these sediments changes due to different environmental conditions found in sediment zones along the caves of a coastal and an inland sinkhole. We found that dominant microbial groups varied according to the type of sinkhole (Coastal: Chloroflexi and Crenarchaeota; inland: Methylomirabilota and Acidobacteriota) and that the community structures differed both among sinkhole types, and within the sediment zones that were studied. These microorganisms are associated with different types of metabolism, and differed from a microbial community dominated by sulfate reducers at the coastal sinkhole, to one dominated by methylotrophs at the inland sinkhole, suggesting there are biogeochemical processes in the coastal and inland sinkholes that lead to changes in the microbial composition of the underground river ecosystem's sediments. Our results suggest sediments from unexplored sinkhole caves are unique environmental niches with distinct microbial assemblages that putatively play an important role in the biogeochemical cycles of these ecosystems.

Assessing the Effect of Chemical Dispersant Nokomis 3-F4 on the Degradation of a Heavy Crude Oil in Water by a Marine Microbial Consortium.

Degradation efficiency of a heavy crude oil by a marine microbial consortium was evaluated in this study, with and without the addition of a chemical dispersant (Nokomis 3-F4). 15.50% of total petroleum hydrocarbons (TPH) were removed after 15 days of incubation without dispersant, with a degradation rate of 2.39 ± 0.22 mg L-1 day-1. In contrast, the addition of Nokomis 3-F4 increased TPH degradation up to 30.81% with a degradation rate of 5.07 ± 0.37 mg L-1 day-1. 16S rRNA gene sequencing indicated a dominance of the consortium by Achromobacter and Alcanivorax. Nonetheless, significant increases in the relative abundance of Martelella and Ochrobactrum were observed with the addition of Nokomis 3-F4. These results will contribute to further environmental studies of the Gulf of Mexico, where Nokomis 3-F4 can be used as chemical dispersant.

Nematicidal Activity of the Endophyte Serratia ureilytica against Nacobbus aberrans in Chili Plants (Capsicum annuum L.) and Identification of Genes Related to Biological Control.

The genus Serratia is widely distributed in soil, water, plants, animals, invertebrates, and humans. Some species of this genus have antifungal, antibacterial, and nematicidal activity. In this work, the nematicidal activity of the endophytic strain of Serratia sp. in chili, Capsicum annuum L., is reported, where at a bacterial concentration of 4 × 109 cel/mL, the penetration of nematodes into the roots significantly decreased by 91 and 55% at 7 and 21 days after inoculation. This bacterial concentration also significantly decreased the number of galls, eggs, egg masses and reproduction factor produced by Nacobbus aberrans in Chili plants, with respect to the control where this bacterial strain was not applied. In the analysis of the genome of the strain, based on average nucleotide identity (ANI), the isolate could be affiliated to the species Serratia ureilytica. The size of the genome is 5.4 Mb, with a 59.3% content of GC. Genes related to the synthesis of chitinases, siderophores, proteases C, serralisins, hemolysin, and serrawettin W2 that have been reported for biocontrol of nematodes were identified in the genome. It is the first report of Serratia ureilytica with nematicidal activity. Based on these results of nematicidal activity, this strain can be evaluated in the field as an alternative in the biocontrol of Nacobbus aberrans in chili cultivation.

First record of entomopathogenic nematodes from Yucatán State, México and their infectivity capacity against Aedes aegypti.

BACKGROUND: Biological control using entomopathogenic nematodes (EPN) has demonstrated good potential to contribute to the integral control of mosquito larvae, which as adults are vectors of diseases such as Dengue fever, Zika and Chikungunya. However, until now there are no records of the presence of EPN or their killing capacity in Yucatán state, southern México. The objectives of the current study were: (1) to report the entomopathogenic nematodes present in Yucatán soils and (2) to determine the killing capacity of the most frequent and abundant EPN against Aedes aegypti mosquito larvae and the microbial community developed by Ae. Aegypti exposed to this EPN. METHODS: The nematodes were collected by the insect trap technique using the great wax moth Galleria mellonella. Internal transcribed spacer (ITS), 28S gene of ribosomal DNA and phylogenetic analyses were performed to identify the EPN. For the bioassay, four concentrations of the most frequent and abundant EPN were tested: 1,260:1 infective juveniles (IJs) per mosquito larvae, 2,520 IJs:1, 3,780 IJs:1 and 5,040 IJs:1. High-throughput sequencing of the 16S rRNA gene was used to identify bacterial amplicon sequences in the mosquito larvae infected with EPN. RESULTS: Six isolates of Heterorhabditis were recovered from 144 soil samples. Heterorhabditis indica (four isolates) was the most frequent and abundant EPN, followed by Heterorhabditis n. sp. (two isolates). Both nematodes are reported for the first time for Yucatán state, Mexico. The concentration of 2,520 IJs:1 produced 80% of mosquito larvae mortality in 48 h. Representative members of Photorhabdus genus were numerically dominant (74%) in mosquito larvae infected by H. indica. It is most likely that these bacteria produce secondary toxic metabolites that enhance the mortality of these mosquito larvae.

Local dynamics of a white syndrome outbreak and changes in the microbial community associated with colonies of the scleractinian brain coral Pseudodiploria strigosa.

Reef corals in the Mexican Reef System have been severely affected by the emergence of a white syndrome that resembles both White Plague II and SCTLD descriptions. Meandroid scleractinian coral species are among the most severely affected. To gain insight into this affliction we conducted a broad study in the brain coral Pseudodiploria strigosa at a rear reef site in the NE Mexican Caribbean. We describe macro and microscopical signals of the disease, characterize the outbreak dynamics, the tissue histopathology, explore immunological responses in the individuals, and compare microbial assemblages associated with the surface mucus layer of healthy and unhealthy colonies. At the study site, the white syndrome outbreak on P. strigosa showed a high incidence rate in summer-fall and a low one in winter, as well as low survival expectation of diseased colonies at the end of the study. After 306 days of observation, out of 96 tracked colonies, eight remained apparently healthy and seven were diseased. No effective resistance to colony disease progression was observed once white syndrome signs developed. Tissue loss rate during the study varied among colonies (mean = 10.8 cm2, s.d. = 7.8 cm2) suggesting a complex relation between causal agents and colony resistance. The deterioration of tissues was evidenced from the basal to the surface body wall of polyps (up to 66% hypertrophy and liquefactive necrosis in unhealthy colonies), implying that microscopic alterations begin before macroscopic signals develop, suggesting this may be a systemic disease. We measured high levels of phenoloxidase (two orders of magnitude higher PO activity than P. strigosa affected by BBD) and antibacterial activity without significant reduction in unhealthy samples from the mucus layer, indicative of an enhanced immunological response. Results showed that opportunistic bacteria dominated damaged colonies, where six genera of the Bacteroidia class were found with significant changes in unhealthy colonies after DeSeq2 analysis. Nevertheless, histological observations did not support infection of the tissues. The opportunistic overload seems to be contained within the mucus layer but may be associated with the mortality of tissues in a yet unclear way. Future research should focus on experimental infections, the tracking of natural infections, and the immunocompetence of corals in the face of environmental pressures due to local, regional, and global impacts. If environmental deterioration is the primary cause of the continuing emergence and re-emergence of lethal coral diseases, as has been proposed by many authors, the only true option to effectively help preserve the coral reef biodiversity and services, is to restore the environmental quality of reef waters at the local scale and reduce greenhouse gases at the global scale.

Assessing the Diversity of Benthic Sulfate-Reducing Microorganisms in Northwestern Gulf of Mexico by Illumina Sequencing of dsrB Gene.

This study investigates the community composition, structure, and abundance of sulfate-reducing microorganisms (SRM) in surficial sediments of the Northwestern Gulf of Mexico (NWGoM) along a bathymetric gradient. For these purposes, Illumina sequencing and quantitative PCR (qPCR) of the dissimilatory sulfite reductase gene beta subunit (dsrB gene) were performed. Bioinformatic analyses indicated that SRM community was predominantly composed by members of Proteobacteria and Firmicutes across all the samples. However, Actinobacteria, Thermodesulfobacteria, and Chlorobi were also detected. Phylogenetic analysis indicated that unassigned dsrB sequences were related to Deltaproteobacteria and Nitrospirota superclusters, Euryarchaeota, and to environmental clusters. PCoA ordination revealed that samples clustered in three different groups. PERMANOVA indicated that water depth, temperature, redox, and nickel and cadmium content were the main environmental drivers for the SRM communities in the studied sites. Alpha diversity and abundance of SRM were lower for deeper sites, suggesting decreasing sulfate reduction activity with respect to water depth. This study contributes with the understanding of distribution and composition of dsrAB-containing microorganisms involved in sulfur transformations that may contribute to the resilience and stability of the benthic microbial communities facing metal and hydrocarbon pollution in the NWGoM, a region of recent development for oil and gas drilling.

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.

A succession of marine bacterial communities in batch reactor experiments during the degradation of five different petroleum types.

Marine microbial communities might be subjected to accidental petroleum spills; however, some bacteria can degrade it, making these specific bacteria valuable for bioremediation from petroleum contamination. Thus, characterizing the microbial communities exposed to varying types of petroleum is essential. We evaluated five enriched microbial communities from the northwest Gulf of Mexico (four from the water column and one from sediments). Enrichments were performed using five types of petroleum (extra light, light, medium, heavy and extra heavy), to reveal the microbial succession using a 16S rDNA amplicon approach. Four communities were capable of degrading from extra light to heavy petroleum. However, only the community from sediment was able to degrade the extra heavy petroleum. Successional changes in the microbial communities' structures were specific for each type of petroleum where genus Dietzia, Gordonia, Microvirga, Rhizobium, Paracoccus, Thalassobaculum, Sphingomonas, Moheibacter, Acinetobacter, Pseudohongiella, Porticoccus, Pseudoalteromonas, Pseudomonas, Shewanella, and Planctomyces presented differential abundance between the treatments.

Changes in the Bacterioplankton Community Structure from Southern Gulf of Mexico During a Simulated Crude Oil Spill at Mesocosm Scale.

The southern Gulf of Mexico (sGoM) is highly susceptible to receiving environmental impacts due to the recent increase in oil-related activities. In this study, we assessed the changes in the bacterioplankton community structure caused by a simulated oil spill at mesocosms scale. The 16S rRNA gene sequencing analysis indicated that the initial bacterial community was mainly represented by Gamma-proteobacteria, Alpha-proteobacteria, Flavobacteriia, and Cyanobacteria. The hydrocarbon degradation activity, measured as the number of culturable hydrocarbonoclastic bacteria (CHB) and by the copy number of the alkB gene, was relatively low at the beginning of the experiment. However, after four days, the hydrocarbonoclastic activity reached its maximum values and was accompanied by increases in the relative abundance of the well-known hydrocarbonoclastic Alteromonas. At the end of the experiment, the diversity was restored to similar values as those observed in the initial time, although the community structure and composition were clearly different, where Marivita, Pseudohongiella, and Oleibacter were detected to have differential abundances on days eight-14. These changes were related with total nitrogen (p value = 0.030 and r2 = 0.22) and polycyclic aromatic hydrocarbons (p value = 0.048 and r2 = 0.25), according to PERMANOVA. The results of this study contribute to the understanding of the potential response of the bacterioplankton from sGoM to crude oil spills.

Assessment of the bacterial community structure in shallow and deep sediments of the Perdido Fold Belt region in the Gulf of Mexico.

The Mexican region of the Perdido Fold Belt (PFB), in northwestern Gulf of Mexico (GoM), is a geological province with important oil reservoirs that will be subjected to forthcoming oil exploration and extraction activities. To date, little is known about the native microbial communities of this region, and how these change relative to water depth. In this study we assessed the bacterial community structure of surficial sediments by high-throughput sequencing of the 16S rRNA gene at 11 sites in the PFB, along a water column depth gradient from 20 to 3,700 m, including five shallow (20-600 m) and six deep (2,800-3,700 m) samples. The results indicated that OTUs richness and diversity were higher for shallow sites (OTUs = 2,888.2 ± 567.88; H' = 9.6 ± 0.85) than for deep sites (OTUs = 1,884.7 ± 464.2; H' = 7.74 ± 1.02). Nonmetric multidimensional scaling (NMDS) ordination revealed that shallow microbial communities grouped separately from deep samples. Additionally, the shallow sites plotted further from each other on the NMDS whereas samples from the deeper sites (abyssal plains) plotted much more closely to each other. These differences were related to depth, redox potential, sulfur concentration, and grain size (lime and clay), based on the environmental variables fitted with the axis of the NMDS ordination. In addition, differential abundance analysis identified 147 OTUs with significant fold changes among the zones (107 from shallow and 40 from deep sites), which constituted 10 to 40% of the total relative abundances of the microbial communities. The most abundant OTUs with significant fold changes in shallow samples corresponded to Kordiimonadales, Rhodospirillales, Desulfobacterales (Desulfococcus), Syntrophobacterales and Nitrospirales (GOUTA 19, BD2-6, LCP-6), whilst Chromatiales, Oceanospirillales (Amphritea, Alcanivorax), Methylococcales, Flavobacteriales, Alteromonadales (Shewanella, ZD0117) and Rhodobacterales were the better represented taxa in deep samples. Several of the OTUs detected in both deep and shallow sites have been previously related to hydrocarbons consumption. Thus, this metabolism seems to be well represented in the studied sites, and it could abate future hydrocarbon contamination in this ecosystem. The results presented herein, along with biological and physicochemical data, constitute an available reference for further monitoring of the bacterial communities in this economically important region in the GoM.

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.

Methanogenic and Sulfate-Reducing Activities in a Hypersaline Microbial Mat and Associated Microbial Diversity.

Methanogenesis and sulfate reduction are important microbial processes in hypersaline environments. However, key aspects determining substrate competition between these microbial processes have not been well documented. We evaluated competitive and non-competitive substrates for stimulation of both processes through microcosm experiments of hypersaline microbial mat samples from Guerrero Negro, Baja California Sur, Mexico, and we assessed the effect of these substrates on the microbial community composition. Methylotrophic methanogenesis evidenced by sequences belonging to methanogens of the family Methanosarcinaceae was found as the dominant methanogenic pathway in the studied hypersaline microbial mat. Nevertheless, our results showed that incubations supplemented with acetate and lactate, performed in absence of sulfate, also produced methane after 40 days of incubation, apparently driven by hydrogenotrophic methanogens affiliated to the family Methanomicrobiaceae. Sulfate reduction was mainly stimulated by addition of acetate and lactate; however, after 40 days of incubation, an increase of the H2S concentrations in microcosms amended with trimethylamine and methanol was also observed, suggesting that these substrates are putatively used for sulfate reduction. Moreover, 16S rRNA gene sequencing analysis showed remarkable differences in the microbial community composition among experimental treatments. In the analyzed sample amended with acetate, sulfate-reducing bacteria (SRB) belonging to the family Desulfobacteraceae were dominant, while members of Desulfohalobiaceae, Desulfomicrobiaceae, and Desulfovibrionaceae were found in the incubation with lactate. Additionally, we detected an unexpected high abundance of unclassified Hydrogenedentes (near 25%) in almost all the experimental treatments. This study contributes to better understand methanogenic and sulfate-reducing activities, which play an important role in the functioning of hypersaline environments.

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.

Phylogenetic diversity of methyl-coenzyme M reductase (mcrA) gene and methanogenesis from trimethylamine in hypersaline environments.

Methanogens have been reported in complex microbial communities from hypersaline environments, but little is known about their phylogenetic diversity. In this work, methane concentrations in environmental gas samples were determined while methane production rates were measured in microcosm experiments with competitive and non-competitive substrates. In addition, the phylogenetic diversity of methanogens in microbial mats from two geographical locations was analyzed: the well studied Guerrero Negro hypersaline ecosystem, and a site not previously investigated, namely Laguna San Ignacio, Baja California Sur, Mexico. Methanogenesis in these microbial mats was suspected based on the detection of methane (in the range of 0.00086 to 3.204 %) in environmental gas samples. Microcosm experiments confirmed methane production by the mats and demonstrated that it was promoted only by non-competitive substrates (trimethylamine and methanol), suggesting that methylotrophy is the main characteristic process by which these hypersaline microbial mats produce methane. Phylogenetic analysis of amino acid sequences of the methyl coenzyme-M reductase (mcrA) gene from natural and manipulated samples revealed various methylotrophic methanogens belonging exclusively to the family Methanosarcinaceae. Moderately halophilic microorganisms of the genus Methanohalophilus were predominant (>60 % of mcrA sequences retrieved). Slightly halophilic and marine microorganisms of the genera Methanococcoides and Methanolobus, respectively, were also identified, but in lower abundances.