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Background: Guerrero Negro microbial mat is one of the best studied microbial mat ecosystems; however, the vertical functional organization has been less well studied. In this study, the microbial ecology, environmentally relevant adaptations and the potential evolutionary interlinking of the responsible genes between bacterial, archaeal and viral lineages in Guerrero Negro (GN) microbial mat were investigated using metagenomic sequencing across a vertical transect at millimeter scale. Through metagenomics, the current study provides a taxonomic description of the vertical taxonomic organization as well as a functional organization delineated between bacteria, archaea, eukarya and viruses in a GN microbial mat – revealing new insights into the ecology of these communities. Results: The community composition based on the number of unique genes recovered (n) and coverages (c) comprised bacteria (98.06 / 98.71 % n/c), archaea (1.82 / 1.19 % n/c), eukarya (0.07 / 0.07 % n/c) and viruses (0.05 / 0.03 % n/c). The greatest coverages of genes of bacteria and eukarya were detected in first layers, while the highest coverages of genes of archaea and viruses were found in deeper layers. Many genes potentially related to adaptation to the local environment (e.g., UV radiation, multidrug resistance, oxidative stress, and heavy metals) were detected. Those genes were found in bacterial, archaeal and viral lineages totalling 3255, 44, and 1 genes, respectively. We employed phylogenetics in order to investigate the evolutionary histories of genes with similar functional annotations that were taxonomically classified as coming from bacteria, archaea and viruses. Conclusions: A gene-focused analysis of bacteria, archaea, eukarya, and viruses showed a vertical partitioning of the community, both in taxonomic affinity and function. The presence of genes potentially involved in adaptation to environmental conditions may have implications and resilience to stress conditions. Phylogenetic analyses showed an interlinking between domains, where genes taxonomically classified as archaea and viruses formed clades suggesting they were phylogenetically relatively closely related to genes present in bacteria lineages – potentially indicating horizontal gene transfer in GN mat.

Some microscopic organisms grow together to build structures known as microbial mats. These mats are formed from several layers with different colors, and their structure depends on environmental conditions such as sunlight, humidity, and available food. Microbial mats are found in oceans, lakes, and coastal lagoons, as well as in extreme environments like deserts, polar regions, and hot springs. The study of fossils indicates that microbial mats were a common form of life on early Earth, and they have persisted on our planet ever since! Therefore, the study of modern mats helps us to understand microbial life in the past, and how they might help to regulate the Earth’s climate. Scientists believe that microbial mats can prosper on rocky planets like Earth, so they are studying mats in different terrestrial environments to help them to recognize evidence indicating the presence of mats on other worlds.

This study focuses on a Lyngbya cf. aestuarii dominated mat community from the intertidal zone of the Laguna Ojo de Liebre, Baja California Sur. In this environment, the mat is desiccated for several days between spring tides. While the mats were desiccated, photosynthetic activity was absent but recovered rapidly (~3 h) upon rehydration. It has been shown previously that the rate of photosynthetic recovery is dependent on both light intensity and salinity. In the current study, photosynthetic recovery was measured based on chlorophyll a fluorescence using pulse amplitude modulated (PAM) fluorometry. Upon the addition of water, photosystem II (PSII) complexes recovered the capacity for reaction centre excitation. However, these functional centres were initially closed. Respiratory activity early in recovery probably reduced the plastoquinone pool through the shared use of part of the photosynthetic transport chain, thus temporarily blocking electron transport downstream of PSII. The time that PSII complexes remained closed increased with light intensities above saturation. This condition is potentially damaging to the cyanobacteria since the exposure of closed PSII centres to high light intensities can lead to the production of singlet oxygen. After this initial lag period, PSII centres opened rapidly indicating an increase in the flow of electrons from PSII to PSI. The rate of photosynthetic recovery appeared to be limited primarily by the relatively slow return of functional PSII. Photosynthetic recovery rates were slower in salinities greater than those that naturally occur in the intertidal zone.

Metagenomic sequence data from defined mock communities is crucial for the assessment of sequencing platform performance and downstream analyses, including assembly, binning and taxonomic assignment. We report a comparison of shotgun metagenome sequencing and assembly metrics of a defined microbial mock community using the Oxford Nanopore Technologies (ONT) MinION, PacBio and Illumina sequencing platforms. Our synthetic microbial community BMock12 consists of 12 bacterial strains with genome sizes spanning 3.2–7.2 Mbp, 40–73% GC content, and 1.5–7.3% repeats. Size selection of both PacBio and ONT sequencing libraries prior to sequencing was essential to yield comparable relative abundances of organisms among all sequencing technologies. While the Illumina-based metagenome assembly yielded good coverage with few misassemblies, contiguity was greatly improved by both, Illumina + ONT and Illumina + PacBio hybrid assemblies but increased misassemblies, most notably in genomes with high sequence similarity to each other. Our resulting datasets allow evaluation and benchmarking of bioinformatics software on Illumina, PacBio and ONT platforms in parallel., Measurement(s)metagenomic data • sequence_assemblyTechnology Type(s)ONT MinION • Illumina sequencing • PacBio RS IIFactor Type(s)sequencing platformSample Characteristic - OrganismBacteriaSample Characteristic - Environmentmock community Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.10260740

The abundance and diversity of fungi were evaluated in a hypersaline microbial mat from Guerrero Negro, México, using a combination of quantitative polymerase chain reaction (qPCR) amplification of domain-specific primers, and metagenomic sequencing. Seven different layers were analyzed in the mat (Layers 1–7) at single millimeter resolution (from the surface to 7 mm in depth). The number of copies of the 18S rRNA gene of fungi ranged between 106 and 107 copies per g mat, being two logarithmic units lower than of the 16S rRNA gene of bacteria. The abundance of 18S rRNA genes of fungi varied significantly among the layers with layers 2–5 mm from surface contained the highest numbers of copies. Fifty-six fungal taxa were identified by metagenomic sequencing, classified into three different phyla: Ascomycota, Basidiomycota and Microsporidia. The prevalent genera of fungi were Thermothelomyces, Pyricularia, Fusarium, Colletotrichum, Aspergillus, Botrytis, Candida and Neurospora. Genera of fungi identified in the mat were closely related to genera known to have saprotrophic and parasitic lifestyles, as well as genera related to human and plant pathogens and fungi able to perform denitrification. This research suggests that fungi in the mat may participate in nutrient recycling, modification of community composition through parasitic activities, and denitrification.

Microbial mats, due to stratification of the redox zones, have the potential to include a complete N cycle; however, an attempt to evaluate a complete N cycle in these ecosystems has not been yet made. In this study, the occurrence and rates of major N cycle processes were evaluated in intact microbial mats from Elkhorn Slough, Monterey Bay, CA, USA, and Baja California Sur, Mexico, under oxic and anoxic conditions using 15N-labeling techniques. All the major N transformation pathways, with the exception of anammox, were detected in both microbial mats. Nitrification rates were found to be low at both sites for both seasons investigated. The highest rates of ammonium assimilation were measured in Elkhorn Slough mats in April and corresponded to high in situ ammonium concentrations in the overlying water. Baja mats featured higher ammonification than ammonium assimilation rates, and this, along with their higher affinity for nitrate compared to ammonium and low dissimilatory nitrate reduction to ammonium rates, characterized their differences from Elkhorn Slough mats. Nitrogen fixation rates in Elkhorn Slough microbial mats were found to be low, implying that other processes, such as recycling and assimilation from water, are the main sources of N for these mats at the times sampled. Denitrification in all the mats was incomplete, with nitrous oxide as the end product and not dinitrogen. Our findings highlight N cycling features not previously quantified in microbial mats and indicate a need for further investigations of these microbial ecosystems. IMPORTANCE Nitrogen is essential for life. The nitrogen cycle on Earth is mediated by microbial activity and has had a profound impact on both the atmosphere and the biosphere throughout geologic time. Microbial mats, present in many modern environments, have been regarded as living records of the organisms, genes, and phylogenies of microbes, as they are one of the most ancient ecosystems on Earth. While rates of major nitrogen metabolic pathways have been evaluated in a number of ecosystems, they remain elusive in microbial mats. In particular, it is unclear what factors affect nitrogen cycling in these ecosystems and how morphological differences between mats impact nitrogen transformations. In this study, we investigate nitrogen cycling in two microbial mats having morphological differences. Our findings provide insight for further understanding of biogeochemistry and microbial ecology of microbial mats.

The landscapes of high-altitude wetland ecosystems are characterized by different kinds of aquatic sites, including ponds holding conspicuous microbial life. Here, we examined a representative pond of the wetland landscape for dynamics of greenhouse gases, and their association with other relevant biogeochemical conditions including diel shifts of microbial communities' structure and activity over two consecutive days. Satellite image analysis indicates that the area of ponds cover 238 of 381.3 Ha (i.e., 62.4%), representing a significant landscape in this wetland. Solar radiation, wind velocity and temperature varied daily and between the days sampled, influencing the biogeochemical dynamics in the pond, shifting the pond reservoir of inorganic versus dissolved organic nitrogen/phosphorus bioavailability, between day 1 and day 2. Day 2 was characterized by high dissolved organic nitrogen/phosphorus and N2O accumulation. CH4 presented a positive excess showing maxima at hours of high radiation during both days. The microbial community in the sediment was diverse and enriched in keystone active groups potentially related with GHG recycling including bacteria and archaea, such as Cyanobacteria, Verrucomicrobia, Rhodobacterales and Nanoarchaeaota (Woesearchaeia). Archaea account for the microbial community composition changes between both days and for the secondary productivity in the water measured during day 2. The results indicate that an intense recycling of organic matter occurs in the pond systems and that the activity of the microbial community is correlated with the availability of nutrients. Together, the above results indicate a net sink of CO2 and N2O, which has also been reported for other natural and artificial ponds. Overall, our two-day fluctuation study in a representative pond of a high-altitude wetland aquatic landscape indicates the need to explore in more detail the short-term besides the long-term biogeochemical variability in arid ecosystems of the Andes plateau, where wetlands are hotspots of life currently under high anthropogenic pressure.

The demand for food will outpace productivity of conventional agriculture due to projected growth of the human population, concomitant with shrinkage of arable land, increasing scarcity of freshwater, and a rapidly changing climate. While aquaponics has potential to sustainably supplement food production with minimal environmental impact, there is a need to better characterize the complex interplay between the various components (fish, plant, microbiome) of these systems to optimize scale up and productivity. Here, we investigated how the commonly-implemented practice of continued microbial community transfer from pre-existing systems might promote or impede productivity of aquaponics. Specifically, we monitored plant growth phenotypes, water chemistry, and microbiome composition of rhizospheres, biofilters, and fish feces over 61-days of lettuce (Lactuca sativa var. crispa) growth in nitrogen-limited aquaponic systems inoculated with bacteria that were either commercially sourced or originating from a pre-existing aquaponic system. Lettuce above- and below-ground growth were significantly reduced across replicates treated with a pre-existing aquaponic system inoculum when compared to replicates treated with a commercial inoculum. Reduced productivity was associated with enrichment in specific bacterial genera in plant roots, including Pseudomonas, following inoculum transfer from pre-existing systems. Increased productivity was associated with enrichment of nitrogen-fixing Rahnella in roots of plants treated with the commercial inoculum. Thus, we show that inoculation from a pre-existing system, rather than from a commercial inoculum, is associated with lower yields. Further work will be necessary to test the putative mechanisms involved.

Earth's atmospheric composition has changed significantly over geologic time. Many redox active atmospheric constituents have left evidence of their presence, while inert constituents such as dinitrogen gas (N2 ) are more elusive. In this study, we examine two potential biological indicators of atmospheric N2 : the morphological and isotopic signatures of heterocystous cyanobacteria. Biological nitrogen fixation constitutes the primary source of fixed nitrogen to the global biosphere and is catalyzed by the oxygen-sensitive enzyme nitrogenase. To protect this enzyme, some filamentous cyanobacteria restrict nitrogen fixation to microoxic cells (heterocysts) while carrying out oxygenic photosynthesis in vegetative cells. Heterocysts terminally differentiate in a pattern that is maintained as the filaments grow, and nitrogen fixation imparts a measurable isotope effect, creating two biosignatures that have previously been interrogated under modern N2 partial pressure (pN2 ) conditions. Here, we examine the effect of variable pN2 on these biosignatures for two species of the filamentous cyanobacterium Anabaena. We provide the first in vivo estimate of the intrinsic isotope fractionation factor of Mo-nitrogenase (efix = -2.71 ± 0.09‰) and show that, with decreasing pN2 , the net nitrogen isotope fractionation decreases for both species, while the heterocyst spacing decreases for Anabaena cylindrica and remains unchanged for Anabaena variabilis. These results are consistent with the nitrogen fixation mechanisms available in the two species. Application of these quantifiable effects to the geologic record may lead to new paleobarometric measurements for pN2 , ultimately contributing to a better understanding of Earth's atmospheric evolution.

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.

The demand for food will outpace productivity of conventional agriculture due to projected growth of the human population, concomitant with shrinkage of arable land, increasing scarcity of freshwater, and a rapidly changing climate. Efforts to increase conventional agricultural output come with significant environmental impacts stemming from deforestation and excessive use of chemicals, including soil salinization, erosion, and nutrient runoffs. While aquaponics has potential to sustainably supplement food production with minimal environmental impact, there is a need to better characterize the complex interplay between the various components (fish, plant, microbiome) of these systems to optimize scale up and productivity. For instance, much of our knowledge of beneficial and detrimental microbial communities vis-à-vis crop productivity comes from studies on plant-microbiome interactions in soil. Here, we investigated how the practice of continued transfer of microbial communities from pre-existing systems might promote or impede productivity of aquaponics. Specifically, we monitored plant growth phenotypes, water chemistry, and microbiome composition of rhizospheres, biofilters, and fish feces over 61-days of lettuce (Lactuca sativa) growth in aquaponic systems inoculated with bacteria that were either commercially sourced or originating from a pre-existing aquaponic system. Strikingly, L. sativa plant and root growth was significantly reduced across all replicates inoculated with the established microbiome. Further analyses revealed the reduced productivity was potentially a consequence of plant-specific pathogen enrichment, including Pseudomonas, through transfer of microbiomes from pre-existing systems – a phenomenon consistent with negative feedbacks in soil ecology. These findings underscore the need for diagnostic tools to monitor microbiome composition, detect negative feedbacks early, and minimize pathogen accumulation in aquaponic systems.

Summary Monitoring microbial communities aboard the International Space Station (ISS) is essential to maintaining astronaut health and the integrity of life-support systems. Using assembled genomes of ISS-derived microbial isolates as references, recruiting metagenomic reads from an astronaut's nasal microbiome revealed no recruitment to a Staphylococcus aureus isolate from samples before launch, yet systematic recruitment across the genome when sampled after 3 months aboard the ISS, with a median percent identity of 100%. This suggests that either a highly similar S. aureus population colonized the astronaut's nasal microbiome while the astronaut was aboard the ISS or that it may have been below detection before spaceflight, instead supporting a shift in community composition. This work highlights the value in generating genomic libraries of microbes from built-environments such as the ISS and demonstrates one way such data can be integrated with metagenomics to facilitate the tracking and monitoring of astronaut microbiomes and health., Graphical Abstract, Highlights • Understanding built-environment microbiomes is critical for human space exploration • Reference-guided metagenomics is a powerful approach for monitoring microbiomes • We show potential microbial colonization of an astronaut microbiome while aboard the ISS, Microbiology; Space Science

The demonstration of a mutualistic interaction requires evidence of benefits for both partners as well as stability of the association over multiple generations. A synthetic mutualism between the freshwater microalga Chlorella sorokiniana and the soil-derived plant growth-promoting bacterium (PGPB) Azospirillum brasilense was created when both microorganisms were co-immobilized in alginate beads. Using stable isotope enrichment experiments followed by high-resolution secondary ion mass spectrometry (SIMS) imaging of single cells, we demonstrated transfer of carbon and nitrogen compounds between the two partners. Further, using fluorescent in situ hybridization (FISH), mechanical disruption and scanning electron microscopy, we demonstrated the stability of their physical association for a period of 10 days after the aggregated cells were released from the beads. The bacteria significantly enhanced the growth of the microalgae while the microalgae supported growth of the bacteria in a medium where it could not otherwise grow. In conclusion, we propose that this microalga-bacterium association is a true synthetic mutualism independent of co-evolution. (155 words).

Microbial mats, due to their high microbial diversity, have the potential to express most biogeochemical cycling processes, highlighting their prospective use in bioremediation of various environmental contaminants. In this study the mechanisms of nitrogen attenuation were investigated in naturally occurring microbial mats from Elkhorn Slough, Monterey Bay, CA, USA, and Baja California Sur, Mexico. Key processes responsible for this removal were evaluated using quantification of functional genes related to nitrification, denitrification, and nitrogen fixation. Both microbial mats were capable of removing high (up to 2 mM) concentrations of ammonium and nitrate. Ammonium assimilation rates measured for Elkhorn Slough mats showed that this process was responsible for most of the ammonium uptake in these mats. While Elkhorn Slough mats did not show any evidence of nitrogen removal pathways other than microbial assimilation, Baja mats exhibited the potential for nitrification, denitrification, and DNRA as well as assimilation. The results of this study demonstrate the potential of microbial mats for bioremediation of nitrogenous pollutants independent of the mechanisms responsible for their removal.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Hypersaline photosynthetic microbial mats are stratified microbial communities known for their taxonomic and metabolic diversity and strong light-driven day-night environmental gradients. In this study of the upper photosynthetic zone of hypersaline microbial mats of Elkhorn Slough, California (USA), we show how metagenome sequencing can be used to meaningfully assess microbial ecology and genetic partitioning in these complex microbial systems. Mapping of metagenome reads to the dominant Cyanobacteria observed in the system, Coleofasciculus (Microcoleus) chthonoplastes, was used to examine strain variants within these metagenomes. Highly conserved gene subsystems indicated a core genome for the species, and a number of variant genes and subsystems suggested strain level differentiation, especially for nutrient utilization and stress response. Metagenome sequence coverage binning was used to assess ecosystem partitioning of remaining microbes to both reconstruct the model organisms in silico and identify their ecosystem functions as well as to identify novel clades and propose their role in the biogeochemical cycling of mats. Functional gene annotation of these bins (primarily of Proteobacteria, Bacteroidetes, and Cyanobacteria) recapitulated the known biogeochemical functions in microbial mats using a genetic basis, and revealed significant diversity in the Bacteroidetes, presumably in heterotrophic cycling. This analysis also revealed evidence of putative phototrophs within the Gemmatimonadetes and Gammaproteobacteria residing in microbial mats. This study shows that metagenomic analysis can produce insights into the systems biology of microbial ecosystems from a genetic perspective and to suggest further studies of novel microbes.

Rates and pathways of methane production were determined from photosynthetic soft microbial mats and gypsum-encrusted endoevaporites collected in hypersaline environments from California, Mexico and Chile, as well as an organic-rich mud from a pond in the El Tatio volcanic fields, Chile. Samples (mud, soft mats and endoevaporites) were incubated anaerobically with deoxygenated site water, and the increase in methane concentration through time in the headspaces of the incubation vials was used to determine methane production rates. To ascertain the substrates used by the methanogens, 13C-labeled methylamines, methanol, dimethylsulfide, acetate or bicarbonate were added to the incubations (one substrate per vial) and the stable isotopic composition of the resulting methane was measured. The vials amended with 13C-labeled methylamines produced the most 13C-enriched methane, generally followed by the 13C-labeled methanol-amended vials. The stable isotope data and the methane production rates were used to determine first order rate constants for each of the substrates at each of the sites. Estimates of individual substrate use revealed that the methylamines produced 55–92 % of the methane generated, while methanol was responsible for another 8–40 %.

Cyanobacterial organic matter excretion is crucial to carbon cycling in many microbial communities, but the nature and bioavailability of this C depend on unknown physiological functions. Cyanobacteria-dominated hypersaline laminated mats are a useful model ecosystem for the study of C flow in complex communities, as they use photosynthesis to sustain a more or less closed system. Although such mats have a large C reservoir in the extracellular polymeric substances (EPSs), the production and degradation of organic carbon is not well defined. To identify extracellular processes in cyanobacterial mats, we examined mats collected from Elkhorn Slough (ES) at Monterey Bay, California, for glycosyl and protein composition of the EPS. We found a prevalence of simple glucose polysaccharides containing either α or β (1,4) linkages, indicating distinct sources of glucose with differing enzymatic accessibility. Using proteomics, we identified cyanobacterial extracellular enzymes, and also detected activities that indicate a capacity for EPS degradation. In a less complex system, we characterized the EPS of a cyanobacterial isolate from ES, ESFC-1, and found the extracellular composition of biofilms produced by this unicyanobacterial culture were similar to that of natural mats. By tracing isotopically labeled EPS into single cells of ESFC-1, we demonstrated rapid incorporation of extracellular-derived carbon. Taken together, these results indicate cyanobacteria reuse excess organic carbon, constituting a dynamic pool of extracellular resources in these mats.

In this paper we report a method for experimental measurement of photosynthetic productivity as a function of simulated depth in open pond raceways for algae cultivation. Knowledge of the depth dependence of photosynthetic productivity aids in designing ponds with optimal depth with respect to biomass productivity and capital and operating costs. To simulate depth, we (i) measured irradiance attenuation coefficients of liquid algal cultures as a function of wavelength in the range of 400 to 700 nm, (ii) reproduced the magnitude and spectral content of the irradiance that would exist at various depths within open ponds using a programmable LED array, and (iii) measured photosynthetic rate as oxygen evolution under irradiances corresponding to various depths. We report the depth distribution of photosynthetic rate in simulated 20 cm deep ponds of the green alga Chlorella vulgaris and the cyanobacterium Spirulina platensis at a biomass concentration of 0.19 g dry biomass per liter (g/l). Under an incident irradiance corresponding to full sunlight, the compensation depth for Chlorella was 12 cm. Below this depth, net oxygen consumption due to respiration had a magnitude equal to 15% that of the total oxygen production above the compensation depth. For Spirulina, negative net oxygen production was not observed at any depth, but the top 13 cm of the pond accounted for 90% of its total oxygen production. These productivity cross-sections, in addition to knowledge of the dependence of capital and operating costs on pond depth, enable the design of open ponds for optimal depth for maximum return on investment.

Large-scale cultivation of microalgal biomass in open systems can benefit from the low cost of using natural sunlight, as opposed to artificial light, but may encounter problems with photoinhibition, high evaporation rates, potential contamination and high energy demand. Wavelength selective luminescent solar concentrator (LSC) panels can solve some of these problems when incorporated into low-cost sheltered structures for algal biomass production that concurrently produce their own electricity by harnessing select portions of solar energy, not used for algal growth. The LSC panels in this study contained a fluorescent dye, Lumogen Red 305, which transmits blue and red wavelengths used for photosynthesis with high efficiency, while absorbing the green wavelengths and re-emitting them as red wavelengths. The fluorescently generated red wavelengths are either transmitted to boost algal growth, or waveguided and captured by photovoltaic cells to be converted into electricity. We found that different strains of microalgae (currently used commercially) grew equally well under the altered spectral conditions created by the luminescent panels, compared to growth under the full solar spectrum. Thus this technology presents a new approach wherein algae can be grown under protected, controlled conditions, while the cost of operations is offset by the structure's internal electrical production, without any loss to algal growth rate or achievable biomass density.

© 2017 The Author(s). Cyanobacterial mats are laminated microbial ecosystems which occur in highly diverse environments and which may provide a possible model for early life on Earth. Their ability to produce hydrogen also makes them of interest from a biotechnological and bioenergy perspective. Samples of an intertidal microbial mat from the Elkhorn Slough estuary in Monterey Bay, California, were transplanted to a greenhouse at NASA Ames Research Center to study a 24-h diel cycle, in the presence or absence of molybdate (which inhibits biohydrogen consumption by sulfate reducers). Here, we present metagenomic analyses of four samples that will be used as references for future metatranscriptomic analyses of this diel time series.

Remote effects (occurring without physical contact) of two plant growth-promoting bacteria (PGPB) Azospirillum brasilense Cd and Bacilus pumilus ES4 on growth of the green microalga Chlorella sorokiniana UTEX 2714 were studied. The two PGPB remotely enhanced the growth of the microalga, up to six-fold, and its cell volume by about three-fold. In addition to phenotypic changes, both bacteria remotely induced increases in the amounts of total lipids, total carbohydrates, and chlorophyll a in the cells of the microalga, indicating an alteration of the microalga’s physiology. The two bacteria produced large amounts of volatile compounds, including CO2, and the known plant growth-promoting volatile 2,3-butanediol and acetoin. Several other volatiles having biological functions in other organisms, as well as numerous volatile compounds with undefined biological roles, were detected. Together, these bacteria-derived volatiles can positively affect growth and metabolic parameters in green microalgae without physical attachment of the bacteria to the microalgae. This is a new paradigm on how PGPB promote growth of microalgae which may serve to improve performance of Chlorella spp. for biotechnological applications.

Microalgae have great potential to be used as part of a regenerative life support system and to facilitate in-situ resource utilization (ISRU) on long-duration human space missions. Little is currently known, however, about microalgal responses to the space environment over long (months) or even short (hours to days) time scales. We describe here the development of biological support subsystems for a prototype “3U” (i.e., three conjoined 10-cm cubes) nanosatellite, called GraviSat, designed to experimentally elucidate the effects of space microgravity and the radiation environment on microalgae and other microorganisms. The GraviSat project comprises the co-development of biological handling-and-support technologies with implementation of integrated measurement hardware for photosynthetic efficiency and physiological activity in support of long-duration (3–12 months) space missions. It supports sample replication in a fully autonomous system that will grow and analyze microalgal cultures in 120 μL wells around the circumference of a microfluidic polymer disc; the cultures will be launched while in stasis, then grown in orbit. The disc spins at different rotational velocities to generate a range of artificial gravity levels in space, from microgravity to multiples of Earth gravity. Development of the biological support technologies for GraviSat comprised the screening of more than twenty microalgal strains for various physical, metabolic and biochemical attributes that support prolonged growth in a microfluidic disc, as well as the capacity for reversible metabolic stasis. Hardware development included that necessary to facilitate accurate and precise measurements of physical parameters by optical methods (pulse amplitude modulated fluorometry) and electrochemical sensors (ion-sensitive microelectrodes). Nearly all microalgal strains were biocompatible with nanosatellite materials; however, microalgal growth was rapidly inhibited (∼1 week) within sealed microwells that did not include dissolved bicarbonate due to CO 2 starvation. Additionally, oxygen production by some microalgae resulted in bubble formation within the wells, which interfered with sensor measurements. Our research achieved prolonged growth periods (>10 months) without excess oxygen production using two microalgal strains, Chlorella vulgaris UTEX 29 and Dunaliella bardawil 30 861, by lowering light intensities (2–10 μmol photons m −2 s −1 ) and temperature (4–12 °C). Although the experiments described here were performed to develop the GraviSat platform, the results of this study should be useful for the incorporation of microalgae in other satellite payloads with low-volume microfluidic systems.

Photosynthetic oxidation ponds are a low-cost method for secondary treatment of wastewater using natural and more energy-efficient aeration strategies. Methane (CH(4)) is produced during the anaerobic digestion of organic matter, but only some of it is oxidized in the water column, with the remaining CH(4) escaping into the atmosphere. In order to characterize the CH(4) flux in two photosynthetic oxidation ponds in a wastewater treatment plant in northern California, the isotopic compositions and concentrations of CH(4) were measured in the water column, in bubbles and in flux chambers, over a period of 12 to 21 months to account for seasonal trends in CH(4) emissions. Methane flux varied seasonally throughout the year, with an annual average flux of 5.5 g CH(4) m⁻² d⁻¹ Over half of the CH(4) flux, 56.1-74.4% v/v, was attributed to ebullition. The oxidation efficiency of this system was estimated at 69.1%, based on stable carbon isotopes and a calculated fractionation factor of 1.028. This is the first time, to our knowledge, that a fractionation factor for CH(4) oxidation has been empirically determined for oxidation ponds. Quantifying CH(4) emissions from these systems is essential to properly identify their contribution and to mitigate their impact on global warming.

The recent reports of methane in the atmosphere of Mars, as well as the findings of hypersaline paleoenvironments on that planet, have underscored the need to evaluate the importance of biological (as opposed to geological) trace gas production and consumption, particularly in hypersaline environments. Methane in the atmosphere of Mars may be an indication of extant life, but it may also be a consequence of geologic activity and/or the thermal alteration of ancient organic matter. On Earth these methane sources can be distinguished using stable isotopic analyses and the ratio of methane (C1) to C2 and C3 alkanes present in the gas source (C1/(C2 + C3)). We report here that methane produced in hypersaline environments on Earth has an isotopic composition and alkane content outside the values presently considered to indicate a biogenic origin. Methane-rich bubbles released from sub-aqueous substrates contained δ13CCH4 and δ2HCH4 values ranging from −65‰ to −35‰ and −350‰ to −140‰ respectively. Higher salinity endoevaporites yielded what would be considered non-biogenic methane based upon stable isotopic and alkane content, however incubation of crustal and algal mat samples resulted in methane production with similar isotopic values. Radiocarbon analysis indicated that the production of the methane was from recently fixed carbon. An extension of the isotopic boundaries of biogenic methane is necessary in order to avoid the possibility of false negatives returned from measurements of methane on Mars and other planetary bodies.

The nonheterocystous filamentous cyanobacterium, strain ESFC-1, is a recently described member of the order Oscillatoriales within the Cyanobacteria. ESFC-1 has been shown to be a major diazotroph in the intertidal microbial mat system at Elkhorn Slough, CA, USA. Based on phylogenetic analyses of the 16S RNA gene, ESFC-1 appears to belong to a unique, genus-level divergence; the draft genome sequence of this strain has now been determined. Here we report features of this genome as they relate to the ecological functions and capabilities of strain ESFC-1. The 5,632,035 bp genome sequence encodes 4914 protein-coding genes and 92 RNA genes. One striking feature of this cyanobacterium is the apparent lack of either uptake or bi-directional hydrogenases typically expected within a diazotroph. Additionally, a large genomic island is found that contains numerous low GC-content genes and genes related to extracellular polysaccharide production and cell wall synthesis and maintenance.

The results of a numerical study on the simulation of pulse amplitude modulated (PAM) fluorometry within dense suspensions of photosynthetic microorganisms are presented. The Monte Carlo method was used to solve the radiative transfer equation in an algae-filled cuvette, taking into account absorption, anisotropic scattering, and fluorescence, as well as Fresnel reflections at interfaces. This method was used to simulate the transport of excitation and fluorescence light in a common laboratory fluorometer. In this fluorometer, detected fluorescence originates from a multitude of locations within the algal suspension, which can be exposed to very different fluence rates. The fluorescence-weighted fluence rate is reported, which is the local fluence rate of actinic light, averaged over all locations from which detected fluorescence originated. A methodology is reported for recovering the fluorescence-weighted fluence rate as a function of the transmittance of measuring light and actinic light through the sample, which are easily measured with common laboratory fluorometers. The fluorescence-weighted fluence rate can in turn be used as a correction factor for recovering intrinsic physiological parameters, such as the functional cross section of Photosystem II, from apparent (experimental) values. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1601-1615, 2016.

Salt flats (sabkha) are a recognized habitat for microbial life in desert environments and as analogs of habitats for possible life on Mars. Here we report on the physical setting and microbiology of interdune sabkhas among the large dunes in the Rub' al Khali (the Empty Quarter) in Liwa Oasis, United Arab Emirates. The salt flats, composed of gypsum and halite, are moistened by relatively fresh ground water. The result is a salinity gradient that is inverted compared to most salt flat communities with the hypersaline layer at the top and freshwater layers below. We describe and characterize a rich photosynthetically-based microbial ecosystem that is protected from the arid outside environment by a translucent salt crust. Gases collected from sediments under shallow ponds in the sabkha contain methane in concentrations as high as 3400 ppm. The salt crust could preserve biomarkers and other evidence for life in the salt after it dries out. Chloride-filled depressions have been identified on Mars and although surface flow of water is unlikely on Mars today, ground water is possible. Such a near surface system with modern groundwater flowing under ancient salt deposits could be present on Mars and could be accessed by surface rovers.

Alkaline soils occupy approximately 5 % of the Earth’s land surface (7 million km), and this may increase due to the global trend towards increasing desertification, yet the extent to which these soils modulate carbon dynamics on regional and global scales is inadequately studied and poorly understood. Railroad Valley (RRV) playa (Nevada, USA) is a semi-arid playa with highly alkaline soils (pH > 10) and no vegetation. The extreme, alkaline environment and absence of vascular vegetation make RRV an ideal site to investigate the role of physiochemical processes of soil-atmosphere CO2 exchange. Both field and laboratory investigations were conducted. This work shows how the atmospheric CO2 mixing ratio decreases at nighttime at RRV playa to a value well below the average global background CO2 concentration. Laboratory investigations using soil samples collected at RRV playa confirmed that CO2 uptake by RRV playa soils occurs when temperatures are decreased. Both field and laboratory studies suggest that the alkaline RRV soil acts as a CO2 reservoir during colder periods, such as at nighttime. These results highlight the importance of investigating carbon dynamics in previously understudied environments. Given how little information is available on the CO2 flux in desert and semi-arid alkaline ecosystems lacking vegetation, our findings draw attention to these environments as becoming increasingly important for carbon fluxes on regional and global scales.

Motivated by the increasingly abundant evidence for hypersaline environments on Mars and reports of methane in its atmosphere, we examined methanogenesis in hypersaline ponds in Baja California Sur, Mexico, and in northern California, USA. Methane-rich bubbles trapped within or below gypsum/halite crusts have δ¹³C values near -40‰. Methane with these relatively high isotopic values would typically be considered thermogenic; however, incubations of crust samples resulted in the biological production of methane with similar isotopic composition. A series of measurements aimed at understanding the isotopic composition of methane in hypersaline systems was therefore undertaken. Methane production rates, as well as the concentrations and isotopic composition of the particulate organic carbon (POC), were measured. Methane production was highest from microbial communities living within gypsum crusts, whereas POC content at gypsum/halite sites was low, generally less than 1% of the total mass. The isotopic composition of the POC ranged from -26‰ to -10‰. To determine the substrates used by the methanogens, ¹³C-labeled methylamines, methanol, acetate, and bicarbonate were added to individual incubation vials, and the methane produced was monitored for ¹³C content. The main substrates used by the methanogens were the noncompetitive substrates, the methylamines, and methanol. When unlabeled trimethylamine (TMA) was added to incubating gypsum/halite crusts in increasing concentrations, the isotopic composition of the methane produced became progressively lower; the lowest methane δ¹³C values occurred when the most TMA was added (1000 μM final concentration). This decrease in the isotopic composition of the methane produced with increasing TMA concentrations, along with the high in situ methane δ¹³C values, suggests that the methanogens within the crusts are operating at low substrate concentrations. It appears that substrate limitation is decreasing isotopic fractionation during methanogenesis, which results in these abnormally high biogenic methane δ¹³C values.

Hydrogen (H(2)) release from photosynthetic microbial mats has contributed to the chemical evolution of Earth and could potentially be a source of renewable H(2) in the future. However, the taxonomy of H(2)-producing microorganisms (hydrogenogens) in these mats has not been previously determined. With combined biogeochemical and molecular studies of microbial mats collected from Elkhorn Slough, Monterey Bay, California, we characterized the mechanisms of H(2) production and identified a dominant hydrogenogen. Net production of H(2) was observed within the upper photosynthetic layer (0-2 mm) of the mats under dark and anoxic conditions. Pyrosequencing of rRNA gene libraries generated from this layer demonstrated the presence of 64 phyla, with Bacteriodetes, Cyanobacteria and Proteobacteria dominating the sequences. Sequencing of rRNA transcripts obtained from this layer demonstrated that Cyanobacteria dominated rRNA transcript pyrotag libraries. An OTU affiliated to Microcoleus spp. was the most abundant OTU in both rRNA gene and transcript libraries. Depriving mats of sunlight resulted in an order of magnitude decrease in subsequent nighttime H(2) production, suggesting that newly fixed carbon is critical to H(2) production. Suppression of nitrogen (N(2))-fixation in the mats did not suppress H(2) production, which indicates that co-metabolic production of H(2) during N(2)-fixation is not an important contributor to H(2) production. Concomitant production of organic acids is consistent with fermentation of recently produced photosynthate as the dominant mode of H(2) production. Analysis of rRNA % transcript:% gene ratios and H(2)-evolving bidirectional [NiFe] hydrogenase % transcript:% gene ratios indicated that Microcoelus spp. are dominant hydrogenogens in the Elkhorn Slough mats.

Hypersaline microbial mat communities have recently been shown to be more diverse than once thought. The variability in community composition of hypersaline mats, both in terms of spatial and temporal dimensions, is still poorly understood. Because this information is essential to understanding the complex biotic and abiotic interactions within these communities, terminal restriction fragment analysis and 16S rRNA gene sequencing were used to characterize the near-surface community of a hypersaline microbial mat in Guerrero Negro, Mexico. Core samples were analyzed to assay community variability over large regional scales (centimeter to kilometer) and to track depth-related changes in population distribution at 250-μm intervals over a diel period. Significant changes in total species diversity were observed at increasing distances across the mat surface; however, key species (e.g. Microcoleus sp.) were identified throughout the mat. The vertical position and abundance of >50% of the 60 peaks detected varied dramatically over a diel cycle, including Beggiatoa sp., cyanobacteria, Chloroflexus sp., Halochromatium sp., Bacteroidetes sp. and several as-yet-identified bacteria. Many of these migrations correlated strongly with diel changes in redox conditions within the mat, contributing to strong day–night community structure differences.

Lyngbya mats in the intertidal of the Laguna Ojo de Liebre are metabolically active for only a few days every 2 weeks during spring tides, with environmental conditions varying greatly during these periods of hydration. Pulse amplitude modulated fluorometry (PAM) and oxygen measurements were used to measure photosynthetic activity during the first few hours after rehydration under various light intensities and salinities. Upon rehydration, a transitory maximum in respiratory activity (10–30 min) occurred before the resumption of photosynthesis, which could recover in about 2 h. Salinities outside the mats' natural range (35–50 psu) were detrimental to photosynthetic recovery. Both high (100 psu) and low (0–10 psu) salinities slowed recovery as well as lowered the overall photosynthetic yield. Photosynthesis was initiated earlier and recovered more rapidly with increasing light intensity. In addition, the positive effect of light on rates of recovery was disproportionately greater at lower salinities (10–25 psu) where high light (500 W·m−2) counteracted the negative effects of low-salt stress early in recovery. However, higher light intensities became photoinhibitory later in recovery (>2 h). Photosynthesis did not recover uniformly within the mat. Filaments deeper in the mat most likely recovered later than those near the surface due to high light attenuation. The ability of the mats to tolerate desiccation and take advantage of hydration periods even when conditions are suboptimal enables these mats to predominate in the intertidal environment.

This article presents the design, construction and characterization of a novel type of light probe for measuring the angular radiance distribution of light fields. The differential acceptance angle (DAA) probe can resolve the directionality of a light field in environments with steep light gradients, such as microbial mats, without the need to remove, reorient, and reinsert the probe, a clear advantage over prior techniques. The probe consists of an inner irradiance sensor inside a concentric, moveable light-absorbing sheath. The radiative intensity in a specific zenith direction can be calculated by comparing the irradiance onto the sensor at different acceptance angles. We used this probe to measure the angular radiance distribution of two sample light fields, and observed good agreement with a conventional radiance probe. The DAA probe will aid researchers in understanding light transfer physics in dense microbial communities and expedite validation of numerical radiative transfer models for these environments.

Summary Diazotrophic community structure in microbial mats from Guerrero Negro (GN), Baja California, Mexico, was studied using polymerase chain reaction amplification of the nifH gene and a newly developed nifH oligonucleotide microarray. Ninety-six oligonucleotide probes designed for nifH sequences from cultivated isolates and the environment were printed on glass microarrays. Phylogenetic analysis showed that the probes represented all of the main nifH clusters. Specificity was tested by (i) evaluation of cross hybridization using individual targets, and (ii) comparison of the observed hybridization signals and those predicted from the sequences cloned from microbial mats. Signal intensity had a positive relationship with target concentration and the percentage identity between probe and target. Under moderate stringency and high target concentration, specificity of the probes varied from 77% to 100% with the individual targets tested. At the end of a 7-month long nutrient manipulation experiment in GN microbial mats, no expression of nitrogen fixation under nitrogen loading was detected, although a diverse community of diazotrophs was detected. The diversity in diazotrophic population present was higher than in the population expressing the nifH gene, and there were taxa specific differences in response to nutrients. The nifH microarray is a powerful tool for diazotroph community analysis in the marine environment.

Cyanobacterial mats collected in hypersaline salterns were incubated in a greenhouse under low sulfate concentrations ([SO4]) and examined for their primary productivity and emissions of methane and other major carbon species. Atmospheric greenhouse warming by gases such as carbon dioxide and methane must have been greater during the Archean than today in order to account for a record of moderate to warm paleoclemates, despite a less luminous early sun. It has been suggested that decreased levels of oxygen and sulfate in Archean oceans could have significantly stimulated microbial methanogenesis relative to present marine rates, with a resultant increase in the relative importance of methane in maintaining the early greenhouse. We maintained modern microbial mats, models of ancient coastal marine communities, in artificial brine mixtures containing both modern [SO4=] (ca. 70 mM) and "Archean" [SO4] (less than 0.2 mM). At low [SO4], primary production in the mats was essentially unaffected, while rates of sulfate reduction decreased by a factor of three, and methane fluxes increased by up to ten-fold. However, remineralization by methanogenesis still amounted to less than 0.4 % of the total carbon released by the mats. The relatively low efficiency of conversion of photosynthate to methane is suggested to reflect the particular geometry and chemical microenvironment of hypersaline cyanobacterial mats. Therefore, such mats w-ere probably relatively weak net sources of methane throughout their 3.5 Ga history, even during periods of low- environmental levels oxygen and sulfate.

Physiological and molecular phylogenetic approaches were used to investigate variation among 12 cyanobacterial strains in their tolerance of sulfide, an inhibitor of oxygenic photosynthesis. Cyanobacteria from sulfidic habitats were found to be phylogenetically diverse and exhibited an approximately 50-fold variation in photosystem II performance in the presence of sulfide. Whereas the degree of tolerance was positively correlated with sulfide levels in the environment, a strain's phenotype could not be predicted from the tolerance of its closest relatives. These observations suggest that sulfide tolerance is a dynamic trait primarily shaped by environmental variation. Despite differences in absolute tolerance, similarities among strains in the effects of sulfide on chlorophyll fluorescence induction indicated a common mode of toxicity. Based on similarities with treatments known to disrupt the oxygen-evolving complex, it was concluded that sulfide toxicity resulted from inhibition of the donor side of photosystem II.

Photosynthetic microbial mat communities were obtained from marine hypersaline saltern ponds, maintained in a greenhouse facility, and examined for the effects of salinity variations. Because these microbial mats are considered to be useful analogs of ancient marine communities, they offer insights about evolutionary events during the3 billion year time interval wherein mats co-evolved with Earth's lithosphere and atmosphere. Although photosynthetic mats can be highly dynamic and exhibit extremely high activity, the mats in the present study have been maintained for1 year with relatively minor changes. The major groups of microorganisms, as assayed using microscopic, genetic, and biomarker methodologies, are essentially the same as those in the original field samples. Field and greenhouse mats were similar with respect to rates of exchange of oxygen and dissolved inorganic carbon across the mat-water interface, both during the day and at night. Field and greenhouse mats exhibited similar rates of efflux of methane and hydrogen. Manipulations of salinity in the water overlying the mats produced changes in the community that strongly resemble those observed in the field. A collaboratory testbed and an array of automated features are being developed to support remote scientific experimentation with the assistance of intelligent software agents. This facility will permit teams of investigators the opportunity to explore ancient environmental conditions that are rare or absent today but that might have influenced the early evolution of these photosynthetic ecosystems.

The simple biochemistry of H2 is critical to a large number of microbial processes, affecting the interaction of organisms with each other and with the environment. The sensitivity of each of these processes to H2 can be described collectively, through the quantitative language of thermodynamics. A necessary prerequisite is to understand the factors that, in turn, control H2 partial pressures. These factors are assessed for two distinctly different ecosystems. In anoxic sediments from Cape Lookout Bight (North Carolina, USA), H2 partial pressures are strictly maintained at low, steady-state levels by H2-consuming organisms, in a fashion that can be quantitatively predicted by simple thermodynamic calculations. In phototrophic microbial mats from Baja California (Mexico), H2 partial pressures are controlled by the activity of light-sensitive H2-producing organisms, and consequently fluctuate over orders of magnitude on a daily basis. The differences in H2 cycling can subsequently impact any of the H2-sensitive microbial processes in these systems. In one example, methanogenesis in Cape Lookout Bight sediments is completely suppressed through the efficient consumption of H2 by sulfate-reducing bacteria; in contrast, elevated levels of H2 prevail in the producer-controlled phototrophic system, and methanogenesis occurs readily in the presence of 40 mM sulfate.

Methane production has been observed in a number of hypersaline environments, and it is generally thought that this methane is produced through the use of noncompetitive substrates, such as the methylamines, dimethylsulfide and methanol. Stable isotope measurements of the produced methane have also suggested that the methanogens are operating under conditions of substrate limitation. Here, substrate limitation in gypsum-hosted endoevaporite and soft-mat hypersaline environments was investigated by the addition of trimethylamine, a noncompetitive substrate for methanogenesis, and dried microbial mat, a source of natural organic matter. The δ 13 C values of the methane produced after amendments were compared to those in unamended control vials. At all hypersaline sites investigated, the δ 13 C values of the methane produced in the amended vials were statistically lower (by 10 to 71‰) than the unamended controls, supporting the hypothesis of substrate limitation at these sites. When substrates were added to the incubation vials, the methanogens within the vials fractionated carbon isotopes to a greater degree, resulting in the production of more 13 C-depleted methane. Trimethylamine-amended samples produced lower methane δ 13 C values than the mat-amended samples. This difference in the δ 13 C values between the two types of amendments could be due to differences in isotope fractionation associated with the dominant methane production pathway (or substrate used) within the vials, with trimethylamine being the main substrate used in the trimethylamine-amended vials. It is hypothesized that increased natural organic matter in the mat-amended vials would increase fermentation rates, leading to higher H 2 concentrations and increased CO 2 /H 2 methanogenesis.

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.