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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The nitrogenase activity and phylogenetic diversity of nitrogen fixing microorganisms in several different cyanobacterial mat types from Guerrero Negro, Baja California, Mexico were investigated by acetylene reduction assay, and by amplification and sequencing of the nitrogenase nifH gene. Acetylene reduction assays performed on a Lyngbya sp. and two Microcoleus chthonoplastes dominated microbial mats showed a typical diel pattern of nitrogenase activity in these mats. The highest rates of activity were found at night, with 40 and 37 micromol C(2)H(4) m(-2) h(-1) measured in the Microcoleus mats, and 9 micromol C(2)H(4) m(-2) h(-1) in the Lyngbya mat. Nitrogenase sequences were obtained that clustered with sequences from cyanobacteria, gamma-Proteobacteria, and cluster 3 of nifH. In addition, novel and divergent sequences were also recovered. The composition of nifH sequence types recovered differed between the Lyngbya and Microcoleus mats. Interestingly, nifH sequences belonging to filamentous cyanobacteria were absent in most mat samples even though both mats were dominated by filamentous cyanobacteria. nifH sequences clustering with those of unicellular cyanobacteria were found, some of which were virtually identical to the nifH sequence from Halothece sp. MPI96P605, which had previously been isolated from the mat. In manipulation experiments, the Lyngbya and Microcoleus mats were allowed to re-colonize a cleared surface. In these developing mats, nifH sequences not previously observed in the mats were discovered. Our results showed that organisms capable of N(2) fixation were present in N(2) fixing mats, that the composition of the N(2) fixing communities differs between mats, and that filamentous cyanobacterial diazotrophs may not have a large role in the early stages of mat development.

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.

We applied nucleic acid-based molecular methods, combined with estimates of biomass (ATP), pigments, and microelectrode measurements of chemical gradients, to map microbial diversity vertically on a millimeter scale in a hypersaline microbial mat from Guerrero Negro, Baja California Sur, Mexico. To identify the constituents of the mat, small-subunit rRNA genes were amplified by PCR from community genomic DNA extracted from layers, cloned, and sequenced. Bacteria dominated the mat and displayed unexpected and unprecedented diversity. The majority (1,336) of the 1,586 bacterial 16S rRNA sequences generated were unique, representing 752 species (≥97% rRNA sequence identity) in 42 of the main bacterial phyla, including 15 novel candidate phyla. The diversity of the mat samples differentiated according to the chemical milieu defined by concentrations of O 2 and H 2 S. Bacteria of the phylum Chloroflexi formed the majority of the biomass by percentage of bulk rRNA and of clones in rRNA gene libraries. This result contradicts the general belief that cyanobacteria dominate these communities. Although cyanobacteria constituted a large fraction of the biomass in the upper few millimeters (>80% of the total rRNA and photosynthetic pigments), Chloroflexi sequences were conspicuous throughout the mat. Filamentous Chloroflexi bacteria were identified by fluorescence in situ hybridization within the polysaccharide sheaths of the prominent cyanobacterium Microcoleus chthonoplastes , in addition to free living in the mat. The biological complexity of the mat far exceeds that observed in other polysaccharide-rich microbial ecosystems, such as the human and mouse distal guts, and suggests that positive feedbacks exist between chemical complexity and biological diversity.

In many environments, biological nitrogen fixation can alleviate nitrogen limitation. The high rates of N 2 fixation often observed in cyanobacterial mats suggest that N 2 fixation may be an important source of N. In this study, organisms expressing nifH were identified in a Lyngbya sp.- and two Microcoleus chthonoplastes -dominated cyanobacterial mats. The pattern of nitrogenase activity was determined for the Lyngbya sp. mat and a Microcoleus chthonoplastes mat sampled directly in Guerrero Negro, Mexico. Their maximum rates were 23 and 15 μmol of C 2 H 4 m −2 h −1 , respectively. The second Microcoleus mat, which was maintained in a greenhouse facility, had a maximum rate of 40 μmol of C 2 H 4 m −2 h −1 . The overall diel pattern of nitrogenase activity in the three mats was similar, with the highest rates of activity occurring during the dark period. Analysis of nifH transcripts by reverse transcription-PCR revealed that several different organisms were expressing nifH during the dark period. nifH phylotypes recovered from these mats were similar to sequences from the unicellular cyanobacterial genera Halothece , Myxosarcina , and Synechocystis, the filamentous cyanobacterial genera Plectonema and Phormidium , and several bacterial nifH groups. The results of this study indicate that several different organisms, some of which were not previously known to fix nitrogen, are likely to be responsible for the observed dark-period nitrogenase activity in these cyanobacterial mats.

Intertidal stromatolites, covered by cyanobacterial mats, were recently discovered at Stocking Island, Exuma Cays, Bahamas. Ecophysiological responses (CO2 fixation, N2 fixation, and photoacclimation) of these cyanobacterial mats to experimental manipulations were examined to identify potential environmental variables controlling community structure and function. The mats exhibit horizontal zonation that shifts from soft to crusty to hard in a seaward direction. Cluster analysis of chemotaxonomic photopigments (chlorophylls and carotenoids) revealed that visually distinct mat types are composed of distinct phototrophic assemblages. Under reduced irradiance, diatoms within the mats photoacclimated by increasing accessory photopigments (diadinoxanthin, fucoxanthin, and chlorophyll c 1 c 2) and cyanobacteria reduced the photoprotective carotenoid echinenone. In a 4-day nutrient addition bioassay experiment, nitrate, phosphate, dissolved organic carbon, and trace metal enrichments did not enhance CO2 fixation, but phosphate enrichments tripled N2 fixation rates. The addition of DCMU increased N2 fixation rates relative to nonamended light and dark rates, indicating light (photosystem I) enhanced nitrogenase activity. Soft mats appear to represent the early stages of colonization and stabilization of mat communities. Active growth following stabilization results in the formation of partially-lithified crusty mats, which eventually become highly-lithified and form hard mats. Collectively, our results suggest that Stocking Island stromatolitic mats have low growth rates and consequently exhibit slow responses to increased nutrient availability and changes in ambient irradiance. In general, intertidal stromatolitic mats at Stocking Island appear to exhibit low rates of CO2 and N2 fixation relative to nonlithifying temperate cyanobacteral mats. Although production is low, respiration is likewise low, leading to the suggestion that high production to respiration ratios (P:R) may be necessary for lithification of intertidal stromatolitic mats.

Relative to biological demands, marine waters are often depleted in combined forms of nitrogen (Dugdale 1967). As such, the development of microbial mats in coastal marine environments is dependent on the fixation of atmospheric dinitrogen as the dominant source of biologically available N.

Intertidal marine microbial mats exhibited biologically mediated uptake of low molecular weight dissolved organic matter (DOM), including D-glucose, acetate, and an L-amino acid mixture at trace concentrations. Uptake of all compounds occurred in darkness, but was frequently enhanced under natural illumination. The photosystem 2 inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) generally failed to inhibit light-stimulated DOM uptake. Occasionally, light plus DCMU-amended treatments led to uptake rates higher than light-incubated samples, possibly due to phototrophic bacteria present in subsurface anoxic layers. Uptake was similar with either 3H- or 14C-labeled substrates, indicating that recycling of labeled CO2 via photosynthetic fixation was not interfering with measurements of light-stimulated DOM uptake. Microautoradiographs showed a variety of pigmented and nonpigmented bacteria and, to a lesser extent, cyanobacteria and eucaryotic microalgae involved in light-mediated DOM uptake. Light-stimulated DOM uptake was often observed in bacteria associated with sheaths and mucilage surrounding filamentous cyanobacteria, revealing a close association of organisms taking up DOM with photoautotrophic members of the mat community. The capacity for dark- and light-mediated heterotrophy, coupled to efficient retention of fixed carbon in the mat community, may help optimize net production and accretion of mats, even in oligotrophic waters.

On three separate occasions we investigated morphological and physiological aspects of bacterial associations with planktonic aggregates of the ubiquitous marine N2 fixing cyanobacterium Trichodesmium sp. Close associations generally characterized Trichodesmium blooms; associations were present during day- and night-time. Colonization by both rod-shaped and filamentous heterotrophic bacteria occurred on Trichodesmiun aggregates actively fixing N2 (acetylene reduction). Scanning electron and optical microscopy showed bacteria located both around and within aggregates. Microautoradiography demonstrated that associated bacteria largely mediated utilization of trace additions of 3H-labeled carbohydrates (fructose, glucose, mannitol) and amino acids, whereas Trichodesmium utilized amino acids only. Oxygen measurements using microelectrodes revealed high localized oxygen consumption among aggregates, with rapid (within a minute) changes from supersaturated to subsaturated oxygen following the transition from photosynthetic illuminated to dark periods. Stab culturing techniques confirmed the presence of heterotrophic N2 fixers among aggregate-associated bacteria. Parallel deployment of oxygen microelectrodes, the tetrazolium salt 2,3,5 triphenyl tetrazolium chloride (TTC) and acetylene reduction assays demonstrated microaerophilic requirements for expression of nitrogenase activity among cultured bacteria. Trichodesmium aggregates are characterized by dynamic nutrient and oxygen regimes, which promote and maintain simultaneous and contiguous oxygenic photosynthesis and N2 fixation. In part, the above-mentioned consortial interactions with a variety of heterotrophic bacteria facilitate Trichodesmium biomass production and bloom formation in nitrogen depleted, oligotrophic tropical/subtropical waters.

The growth rates of five strains of the marine ascomycete Corollospora maritima Werderm., isolated from different geographical regions, show significant differences in their responses to temperature. At 30 °C, strains isolated from warm waters are able to grow at higher rates than cold-water isolates. At 10 °C, strains isolated from cold waters grow at higher rates than warm-water isolates (P < 0.05). A strain isolated from temperate waters shows growth rates intermediate between those of the cold- and warm-water isolates. Strains of C. maritima thus appear to be adapted to grow best near the temperatures at which they are found in nature. It is suggested that the cosmopolitan classification of C. maritima be reexamined, and that the species consists of distinct physiological races which maintain characteristic temperature responses in the laboratory.

Diel variations in N 2 fixation (acetylene reduction), CO 2 fixation, and oxygen concentrations were measured, on three separate occasions, in a marine microbial mat located on Shackleford Banks, North Carolina. Nitrogenase activity (NA) was found to be inversely correlated with CO 2 fixation and, in two of the three diel periods studied, was higher at night than during the day. Oxygen concentrations within the top 3 mm of the mat ranged from 0 to 400 μM on a diel cycle; anaerobic conditions generally persisted below 4 mm. NA in the mat was profoundly affected by naturally occurring oxygen concentrations. Experimentally elevated oxygen concentrations resulted in a significant depression of NA, whereas the addition of the Photosystem II inhibitor 3(3,4-dichlorophenyl)-1,1-dimethylurea decreased oxygen concentrations within the mat and resulted in a significant short-term enhancement of NA. Mat N 2 -fixing microorganisms include cyanobacteria and heterotrophic, photoautotrophic, and chemolithotrophic eubacteria. Measured (whole-mat) NA is probably due to a combination of the NA of each of these groups of organisms. The relative contributions of each group to whole-mat NA probably varied during diel and seasonal (successional) cycles. Reduced compounds derived from photosynthetic CO 2 fixation appeared to be an important source of energy for NA during the day, whereas heterotrophic or chemolithotrophic utilization of reduced compounds appeared to be an important source of energy for NA at night, under reduced ambient oxygen concentrations. Previous estimates of N 2 fixation calculated on the basis of daytime measurements may have seriously underestimated diel and seasonal nitrogen inputs in mat systems.

Among the nitrogen (N(2))-fixing Cyanobacteria, the filamentous, nonheterocystous marine Oscillatoria spp. (Trichodesmium) appears enigmatic; it exhibits N(2) fixation in the presence of oxygenic photosynthesis without structural protection of the N(2-)fixing apparatus (nitrogenase) from potential inhibition by molecular oxygen (O(2)). Characteristically, N(2) fixation is largely confined to aggregates (bundles) of filaments. Previous work has suggested that spatial partitioning of photosynthesis and of N(2) fixation occurs in the bundles as a means of allowing both processes to occur contemporaneously. The probing of freshly sampled bundles with O(2) microelectrodes directly confirmed such partitioning by showing the presence of O(2-)depleted (reduced) microzones in photosynthetically active, N(2-)fixing bundles. Bundle size was directly related to both the development of internal reduced microzones and cellular N(2) fixation rates. By enhancing microzone formation, bundles optimize N(2) fixation as a means of supporting Oscillatoria spp. blooms in surficial, nitrogen-depleted tropical and subtropical waters.