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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.

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).

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.

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.

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 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.

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.

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.

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.

The microboring activity of endolithic cyanobacteria plays a major role in the formation of lithified laminae in modern marine stromatolites in the Exuma Cays, Bahamas. These stromatolites are composed primarily of fine grained carbonate sand that is trapped and bound by the filamentous cyanobacteria Schizothrix sp. Periodic introduction of coccoid endolithic cyanobacteria, Solentia sp., results in formation of lithified horizons, 200 to 1000 micron thick. We used SEM and petrographic analyses to examine both naturally occurring lithified layers dominated by endoliths and fused oolitic crusts generated in the laboratory by activity of endolithic cyanobacteria (Solentia sp.). Fused grain crusts consist of micritized grains that are welded together at point contacts. Micritization results from extensive microboring and rapid (days to weeks) carbonate precipitation within the bore holes. This precipitation appears to occur concurrently with further endolithic activity within the grain, Infilling of bore holes that cross from one grain to another at point contacts results in grain welding, Thus, while microboring destroys original grain textures, at the same time the endolith activity plays a constructional role in stromatolite growth by forming lithified layers of welded grains. These framework structures help to stabilize and preserve the stromatolite deposits.

The nonheterocystous filamentous cyanobacterium strain ESFC-1 has recently been isolated from a marine microbial mat system, where it was identified as belonging to a recently discovered lineage of active nitrogen-fixing microorganisms. Here, we report the draft genome sequence of this isolate. The assembly consists of 3 scaffolds and contains 5,632,035 bp with a GC content of 46.5%.

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.

[1] The Bolivian Altiplano is a harsh environment for life with high solar irradiation (visible and UVR), below freezing temperatures, and some of the lowest precipitation rates on the planet. However, microbial life is visibly abundant in small isolated refugia of spring or snowmelt-fed lakes. In this study, we characterized the cyanobacterial composition of a variety of microbial mats present in three lake systems: Laguna Blanca, Laguna Verde (elevation 4300 m), and a summit lake in the Licancabur Volcano cone (elevation 5970 m). These lakes and their adjacent geothermal springs present an interesting diversity of environments within a geographically small region (5 km2). From these sites, 78 cyanobacterial cultures were isolated in addition to ∼400 cyanobacterial 16S rRNA gene sequences from environmental genomic DNA. Based on microscopy, cultivation, and molecular analyses, these communities contained many heterocytous, nitrogen-fixing cyanobacteria (e.g., Calothrix, Nostoc, Nodularia) as well as a large number of cyanobacteria belonging to the form-genus Leptolyngbya. More than a third (37%) of all taxa in this study were new species (≤96% 16S rRNA gene sequence identity), and 11% represented new and novel taxa distantly related (≤93% identity) to any known cyanobacteria. This is one of the few studies to characterize cyanobacterial communities based on both cultivation-dependent and cultivation-independent analyses.

Summary Methanogenesis was characterized in hypersaline microbial mats from Guerrero Negro, Baja California Sur, Mexico both in situ and after long-term manipulation in a greenhouse environment. Substrate addition experiments indicate methanogenesis to occur primarily through the catabolic demethylation of non-competitive substrates, under field conditions. However, evidence for the coexistence of other metabolic guilds of methanogens was obtained during a previous manipulation of sulfate concentrations. To fully characterize methanogenesis in these mats, in the absence of competition for reducing equivalents with sulfate-reducing microorganisms, we maintained microbial mats for longer than 1 year under conditions of lowered sulfate and salinity levels. The goal of this study was to assess whether observed differences in methane production during sulfate and salinity manipulation were accompanied by shifts in the composition of methanogen communities. Culture-independent techniques targeting methyl coenzyme M reductase genes (mcrA) were used to assess the dynamics of methanogen assemblages. Clone libraries from mats sampled in situ or maintained at field-like conditions in the greenhouse were exclusively composed of sequences related to methylotrophic members of the Methanosarcinales. Increases in pore water methane concentrations under conditions of low sulfate correlated with an observed increase in the abundance of putatively hydrogenotrophic mcrA, related to Methanomicrobiales. Geochemical and molecular data provide evidence of a significant shift in the metabolic pathway of methanogenesis from a methylotroph-dominated system in high-sulfate environments to a mixed community of methylotrophic and hydrogenotrophic methanogens under low sulfate conditions.

[1] Modern microbial mats have been used as analogs for early life because of the longevity of microbial life on Earth. Mats collected from hypersaline salterns in Baja California were maintained for over a year and a half under both normal (85 ppt salinity, 50 mM SO42−) and reduced salinity (35 ppt salinity; near modern seawater) and sulfate concentrations (20 mM, ≤1 mM SO42−) to assess carbon processing under sulfate conditions more similar to the Archean oceans. As sulfate was removed from the mats by diffusion out into the overlying water, methane concentrations within the mats increased. Highest methane concentrations occurred in mats with reduced salinity and little sulfate. The δ13C values of bulk particulate organic matter in all of the mats averaged −11‰, similar to what had been observed previously for these Microcoleus mats. In mats with sulfate, pore water concentrations of dissolved inorganic carbon (DIC) δ13C values averaged about −3‰. However, in the mats with ≤1 mM sulfate concentrations, the DIC δ13C values increased substantially with depth, from about −1‰ in the overlying water to +10‰ by 20 mm depth. Rates of methanogenesis, calculated from pore water dissolved methane concentration profiles, were too low to account for the total increase in DIC δ13C values. These positive isotopic signatures, however, are also consistent with the occurrence of acetogenesis, as are the higher acetate concentrations in the low-sulfate mats. Acetogens may be poised to become successful competitors for substrates in these mats, given the right environmental conditions.

The salinity and sulfate concentrations of microbial mats collected from hypersaline salterns were mainipulated to examine the effects of low sulfate concentrations and lowered salinity on carbon metabolism. As sulfate was slowly removed from the mats, methane production and flux increased, with highest fluxes and concentration in mats at lowered salinity (35 ppt) and low sulfate concentrations. The δ 13 C values of bulk particulate organic carbon (POC) ranged from about -10 to -12 p, similar to what had been observed previously for these cyanobacterial mats. In mats with higher sulfate concentrations, pore water profiles of dissolved inorganic carbon (DIC) δ 13 C values decreased with depth. However, in the mats with lowered sulfate, the DIC δ 13 C values increased substantially, from about -1 p in the overlying water to +12 p by 20 mm depth. Although the increase in DIC δ 13 C values is consistent with biogenic methanogenesis, the measured methane concentrations in these mats were not great enough to be the sole cause of the increase. These positive isotopic values, as well as the higher acetate concentrations observed in the low-sulfate mats, are also consistent with the occurrence of acetogenesis.

For three billion years, before the Cambrian diversification of life, laminated carbonate build-ups called stromatolites were widespread in shallow marine seas. These ancient structures are generally thought to be microbial in origin and potentially preserve evidence of the Earth's earliest biosphere. Despite their evolutionary significance, little is known about stromatolite formation, especially the relative roles of microbial and environmental factors in stromatolite accretion. Here we show that growth of modern marine stromatolites represents a dynamic balance between sedimentation and intermittent lithification of cyanobacterial mats. Periods of rapid sediment accretion, during which stromatolite surfaces are dominated by pioneer communities of gliding filamentous cyanobacteria, alternate with hiatal intervals. These discontinuities in sedimentation are characterized by development of surface films of exopolymer and subsequent heterotrophic bacterial decomposition, forming thin crusts of microcrystalline carbonate. During prolonged hiatal periods, climax communities develop, which include endolithic coccoid cyanobacteria. These coccoids modify the sediment, forming thicker lithified laminae. Preservation of lithified layers at depth creates millimetre-scale lamination. This simple model of modern marine stromatolite growth may be applicable to ancient stromatolites.