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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.

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.

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 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.

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 (N

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 %.

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.

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.

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.

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.

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

Past studies of hydrogen cycling in hypersaline microbial mats have shown an active nighttime cycle, with production largely from Cyanobacteria and consumption from sulfate-reducing bacteria (SRB). However, the mechanisms and magnitude of hydrogen cycling have not been extensively studied. Two mats types near Guerrero Negro, Mexico -- permanently submerged Microcoleus microbial mats (GN-S), and intertidal Lyngbya microbial mats (GN-I) -- were used in microcosm diel manipulation experiments with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), molybdate, ammonium addition, and physical disruption to understand the processes responsible for hydrogen cycling between mat microbes. Across microcosms, H2 production occurred under dark anoxic conditions with simultaneous production of a suite of organic acids. H2 production was not significantly affected by inhibition of nitrogen fixation, but rather appears to result from constitutive fermentation of photosynthetic storage products by oxygenic phototrophs. Comparison to accumulated glycogen and to CO2 flux indicated that, in the GN-I mat, fermentation released almost all of the carbon fixed via photosynthesis during the preceding day, primarily as organic acids. Across mats, although oxygenic and anoxygenic phototrophs were detected, cyanobacterial [NiFe]-hydrogenase transcripts predominated. Molybdate inhibition experiments indicated that SRBs from a wide distribution of dsrA phylotypes were responsible for H2 consumption. Incubation with 13C-acetate and nanoSIMS (secondary ion mass-spectrometry) indicated higher uptake in both Chloroflexi and SRBs relative to other filamentous bacteria. These manipulations and diel incubations confirm that Cyanobacteria were the main fermenters in Guerrero Negro mats and that the net flux of nighttime fermentation byproducts (not only hydrogen) was largely regulated by the interplay between Cyanobacteria, SRBs, and Chloroflexi.

The advent of oxygenic photosynthesis on Earth may have increased global biological productivity by a factor of 100-1,000 (ref. 1), profoundly affecting both geochemical and biological evolution. Much of this new productivity probably occurred in microbial mats, which incorporate a range of photosynthetic and anaerobic microorganisms in extremely close physical proximity. The potential contribution of these systems to global biogeochemical change would have depended on the nature of the interactions among these mat microorganisms. Here we report that in modern, cyanobacteria-dominated mats from hypersaline environments in Guerrero Negro, Mexico, photosynthetic microorganisms generate H2 and CO-gases that provide a basis for direct chemical interactions with neighbouring chemotrophic and heterotrophic microbes. We also observe an unexpected flux of CH4, which is probably related to H2-based alteration of the redox potential within the mats. These fluxes would have been most important during the nearly 2-billion-year period during which photosynthetic mats contributed substantially to biological productivity-and hence, to biogeochemistry-on Earth. In particular, the large fluxes of H2 that we observe could, with subsequent escape to space, represent a potentially important mechanism for oxidation of the primitive oceans and atmosphere.

Microbial mats on the surfaces of modern, marine stromatolites at Highborne Cay, Bahamas, were investigated to assess the role of microbial processes in stromatolite formation. The Highborne Cay stromatolitic mats contain Schizothix as the dominant cyanobacterium and show millimeter-scale lamination: Some layers in the mat are soft (unlithified), whereas other layers are crusty (lithified). Lithified layers within the mats correspond to micritic horizons composed of thin (20‐50 mm) micritic crusts, which commonly overlie truncated, micritized carbonate sand grains. These features are identical to lithified laminae in the underlying stromatolite; the micritic crusts are similar in thickness to micritic laminae in many ancient stromatolites. Biogeochemical parameters in a representative stromatolitic mat from Highborne Cay were measured to identify the role of bacteria in precipitation and dissolution of CaCO3. Depth distributions of O2 ,H S 2 , and pH were determined with microelectrode measurements in the field. Oxygen profiles were used to calculate photosynthesis and aerobic respiration. Sulfate reduction was determined using 35 SO4 22 and sulfide oxidation potential was measured in homogenized samples. Our results indicate that cyanobacterial photosynthesis, sulfate reduction, and anaerobic sulfide oxidation in stromatolitic mats at Highborne Cay are responsible for CaCO3 precipitation, whereas aerobic respiration and aerobic sulfide oxidation cause CaCO 3 dissolution. A close coupling of photosynthesis and aerobic respiration in the uppermost few millimeters of the mats results in no, or very little, net lithification. Sulfur cycling, on the other hand, shows a close correlation with the formation of lithified micritic layers. Photosynthesis, combined with sulfate reduction and sulfide oxidation results in net lithification. Sulfate reduction rates are high in the uppermost lithified layer and, on a diel basis, consume 33‐38% of the CO2 fixed by the cyanobacteria. In addition, this lithified layer contains a significant population of sulfide-oxidizing bacteria and shows a high sulfide oxidation potential. These findings argue that photosynthesis coupled to sulfate reduction and sulfide oxidation is more important than photosynthesis coupled to aerobic respiration in the formation of lithified micritic laminae in Highborne Cay stromatolites.

Continental shelf sediments of the central Chile upwelling area are dominated by the presence of dense mats of the filamentous, sulfur-depositing bacterium Thioploca spp. We examined rates and pathways of S and methane cycling in these sediments along a transect from the Bay of Concepcion to the continental slope. Sulfate reduction rates (170–4670 nmol cm−3 d−1) were equal to or exceeded rates reported for other subtidal marine sediments. Elemental S and pyrite were the dominant end-products of sulfate reduction in Thioploca mats on the continental shelf, whereas, in the highly-reducing, Beggiatoa-dominated sediments of the nearby Bay of Concepcion, acid-volatile S was the principal end-product. Dissolved organic C values were lowest at the stations with the highest sulfate reduction rates and increased offshore. Sediment porewater methane concentrations in all surface sediments were low (

These case studies focus on the CH4 emissions from wastewater treatment in photosynthetic oxidation ponds and facultative sludge lagoons. These area-intensive processes are for cryophilic-to-mesophilic treatment and storage and are uncovered, except for a layer of aerobic liquid. Pond/lagoon treatment technologies represent some of the lowest energy processes available and as such are attractive options from an indirect GHG perspective. While it is intuitive that sludge treatment lagoons have significant anaerobic zones, oxidation ponds have also been shown to develop anaerobic layers, particularly in the deeper portions. Literature also suggests that CH4 evolution is mitigated by methanotrophic bacteria in the water column that can aerobically oxidize CH4, as occurs naturally in lakes and other environments. Because each system has potentially significant anaerobic volumes, quantifying the levels of CH4 emissions would provide insight into the significance of direct CH4 emissions and whether improvements are warranted to address fugitive emissions in these otherwise very sustainable technologies. These tasks provide a better understanding of the wastewater treatment industry's true impacts from a GHG standpoint. With that understanding better solutions may be developed to remediate their impact as warranted by their significance. This title belongs to WERF Research Report Series . ISBN: 9781780404875 (eBook)

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) that can withstand prolonged storage in complex biological media (1-4). An all-solid-state ion-selective-electrode (ASSISE) is especially attractive for the aforementioned applications. The electrode should have the following favorable characteristics: easy construction, low maintenance, and (potential for) miniaturization, allowing for batch processing. A microfabricated ASSISE intended for quantifying H(+), Ca(2+), and CO3(2-) ions was constructed. It consists of a noble-metal electrode layer (i.e. Pt), a transduction layer, and an ion-selective membrane (ISM) layer. The transduction layer functions to transduce the concentration-dependent chemical potential of the ion-selective membrane into a measurable electrical signal. The lifetime of an ASSISE is found to depend on maintaining the potential at the conductive layer/membrane interface (5-7). To extend the ASSISE working lifetime and thereby maintain stable potentials at the interfacial layers, we utilized the conductive polymer (CP) poly(3,4-ethylenedioxythiophene) (PEDOT) (7-9) in place of silver/silver chloride (Ag/AgCl) as the transducer layer. We constructed the ASSISE in a lab-on-a-chip format, which we called the multi-analyte biochip (MAB) (Figure 1). Calibrations in test solutions demonstrated that the MAB can monitor pH (operational range pH 4-9), CO3(2-) (measured range 0.01 mM - 1 mM), and Ca(2+) (log-linear range 0.01 mM to 1 mM). The MAB for pH provides a near-Nernstian slope response after almost one month storage in algal medium. The carbonate biochips show a potentiometric profile similar to that of a conventional ion-selective electrode. Physiological measurements were employed to monitor biological activity of the model system, the microalga Chlorella vulgaris. The MAB conveys an advantage in size, versatility, and multiplexed analyte sensing capability, making it applicable to many confined monitoring situations, on Earth or in space. Biochip Design and Experimental Methods The biochip is 10 x 11 mm in dimension and has 9 ASSISEs designated as working electrodes (WEs) and 5 Ag/AgCl reference electrodes (REs). Each working electrode (WE) is 240 μm in diameter and is equally spaced at 1.4 mm from the REs, which are 480 μm in diameter. These electrodes are connected to electrical contact pads with a dimension of 0.5 mm x 0.5 mm. The schematic is shown in Figure 2. Cyclic voltammetry (CV) and galvanostatic deposition methods are used to electropolymerize the PEDOT films using a Bioanalytical Systems Inc. (BASI) C3 cell stand (Figure 3). The counter-ion for the PEDOT film is tailored to suit the analyte ion of interest. A PEDOT with poly(styrenesulfonate) counter ion (PEDOT/PSS) is utilized for H(+) and CO3(2-), while one with sulphate (added to the solution as CaSO4) is utilized for Ca(2+). The electrochemical properties of the PEDOT-coated WE is analyzed using CVs in redox-active solution (i.e. 2 mM potassium ferricyanide (K3Fe(CN)6)). Based on the CV profile, Randles-Sevcik analysis was used to determine the effective surface area (10). Spin-coating at 1,500 rpm is used to cast ~2 μm thick ion-selective membranes (ISMs) on the MAB working electrodes (WEs). The MAB is contained in a microfluidic flow-cell chamber filled with a 150 μl volume of algal medium; the contact pads are electrically connected to the BASI system (Figure 4). The photosynthetic activity of Chlorella vulgaris is monitored in ambient light and dark conditions.

Diel rates of nitrogen (N,) fixation (acetylene reduction) and primary production (14C0, fixation) were examined seasonally on a North Carolina Atlantic coastal, intertidal, benthic microbial mat community dominated by the filamentous, nonheterocystous cyanobacterial genera Microcoleus and Lyngbya. Highest hourly and daily rates of Nz and CO2 fixation were observed during spring through fall. During this period, an inverse temporal relationship was noted between these processes, with CO, fixation closely tracking irradiance and Nz fixation rates remaining low during daylight and becoming maximal at night. Under the influence of the photosynthetic (PS 2) inhibitor 3-(3,4 dichlorophenyl)- 1,l dimethylurea (DCMU), daytime N, fixation was enhanced, indicating in situ 0, inhibition of Nz fixation. The most pronounced DCMU stimulation of daytime N2 fixation was in spring-fall. Both N2 and CO, fixation rates were lower in winter. Winter patterns of diel N, fixation were the reverse of those in summer, with maximum rates at midday. The reversal was related to seasonal changes in daily and hourly photosynthetic rates, leading to differential 0, suppression of N, fixation. Seasonal changes in cyanobacterial community composition and bacterial diazotrophy may have played additional roles in determining diel rates and patterns of N2 fixation and mat production. Results indicate a more important role for bacteria in the dynamics of mat Nz fixation than has been previously recognized.

Benthic photosynthetic microorganisms are widespread in shallow-water sediments, microenvironments that are commonly assumed to be virtually opaque to ultraviolet radiation (UVR). We used a newly developed optical microprobe to measure the submillimeter penetration of solar UVR into a variety of these microenvironments. UVR trapping due to strong scattering occurred at the surface of some sedirnents, resulting in a surface maximum of scalar irradiance (E,) that could be significantly larger that the Incident radiation. In the subsurface, E,, was typically extingu~shed in a quasiexponential manner, with attenuation coefficients (310 nm) ranging from 4 to 21 mm-', depending on sediment type. Ultrav~olet B (at 310 nm) was extingu~shed to 1 "h of the incident between 1.25 and 0 23 mm from the surface W~thin the euphotic zones of these sediments, however, the space-averaged UVB scalar irradiance was very h ~ y h , between 15 and 33'!,, of the incident In natural waters, for example, the same parameter varies between 3 and g':(, of the incident. Thus, in fact, photosynthesis in these environments must develop under strong UV stress, and it must be regarded as potentially labile to the effects of ozone depletion.

Exposure to moderate doses of UV B (0.35 to 0.79 W m(sup-2) s(sup-1) or 0.98 to 2.2 (mu)mol of photons m(sup-2) s(sup-1) at 310 nm) caused the surface layers of microbial mats from Solar Lake, Sinai, Egypt, to become visibly lighter green. Concurrent with the color change were rapid and dramatic reductions in gross photosynthesis and in the resultant high porewater oxygen concentrations in the surface layers of the mats. The depths at which both maximum gross photosynthesis and maximum oxygen concentrations occurred were displaced downward. In contrast, gross photosynthesis in the deeper layers of the mats increased in response to UV B incident upon the surface. The cessation of exposure to UV B partially reversed all of these changes. Taken together, these responses suggest that photoautotrophic members of the mat community, most likely the dominant cyanobacterium Microcoleus chthonoplastes, were migrating in response to the added UV B. The migration phenomenon was also observed in response to increases in visible radiation and UV A, but UV B was ca. 100-fold more effective than visible radiation and ca. 20-fold more effective than UV A in provoking the response. Migrating microorganisms within this mat are apparently able to sense UV B directly and respond behaviorally to limit their exposure to UV. Because of strong vertical gradients of light and dissolved substances in microbial mats, the migration and the resultant vertical redistribution of photosynthetic activity have important consequences for both the photobiology of the cyanobacteria and the net primary productivity of the mat ecosystem.

The purpose of this study was to determine the production and N2 fixation responses of a hypersaline mat community following a reduction in salinity and nutrient enrichment. Cyanobateria-dominated microbial mat samples were collected from hypersaline Storr's Lake and normal seawater salinity Pigeon Creek and preincubated at ambient (90%.) and reduced (45%.) salinities following no nutrient as well as inorganic nutrient (NO3−, PO4−, trace metals) and dissolved organic carbon (DOC, as mannitol) enrichment. CO2 and N2 fixation rates were determined 2 and 4 days later. In addition, DOC (trace concentrations of 3H-labeled glucose and amino acids) uptake was measured in mats under normal and hypersaline conditions. A reduction in salinity from 90 to 45%. significantly enhanced CO2 and N2 fixation rates, but inorganic nutrient and DOC additions did not significantly enhance rates compared with the controls. Dissolved organic carbon/dissolved organic nitrogen (DON) uptake was not influenced over the entire range of salinities (45–90%.) used in this study. When salinity-induced osmotic stress was relieved, mats underwent enhanced primary production and nitrogen fixation. Abiotic stress, induced by hypersaline conditions in Bahamian lagoons, results in lower productivity of the microbial mat communities and this stress may outweigh the typical limiting factors regulating phototrophic community primary production.

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.

N(2) fixation is a key process in photosynthetic microbial mats to support the nitrogen demands associated with primary production. Despite its importance, groups that actively fix N(2) and contribute to the input of organic N in these ecosystems still remain largely unclear. To investigate the active diazotrophic community in microbial mats from the Elkhorn Slough estuary, Monterey Bay, CA, USA, we conducted an extensive combined approach, including biogeochemical, molecular and high-resolution secondary ion mass spectrometry (NanoSIMS) analyses. Detailed analysis of dinitrogenase reductase (nifH) transcript clone libraries from mat samples that fixed N(2) at night indicated that cyanobacterial nifH transcripts were abundant and formed a novel monophyletic lineage. Independent NanoSIMS analysis of (15)N(2)-incubated samples revealed significant incorporation of (15)N into small, non-heterocystous cyanobacterial filaments. Mat-derived enrichment cultures yielded a unicyanobacterial culture with similar filaments (named Elkhorn Slough Filamentous Cyanobacterium-1 (ESFC-1)) that contained nifH gene sequences grouping with the novel cyanobacterial lineage identified in the transcript clone libraries, displaying up to 100% amino-acid sequence identity. The 16S rRNA gene sequence recovered from this enrichment allowed for the identification of related sequences from Elkhorn Slough mats and revealed great sequence diversity in this cluster. Furthermore, by combining (15)N(2) tracer experiments, fluorescence in situ hybridization and NanoSIMS, in situ N(2) fixation activity by the novel ESFC-1 group was demonstrated, suggesting that this group may be the most active cyanobacterial diazotroph in the Elkhorn Slough mat. Pyrotag sequences affiliated with ESFC-1 were recovered from mat samples throughout 2009, demonstrating the prevalence of this group. This work illustrates that combining standard and single-cell analyses can link phylogeny and function to identify previously unknown key functional groups in complex ecosystems.