Physiological status and microbial diversity assessment of microbial mats: The signature lipid biomarker approach

Navarrete A., Urmeneta J., Cantu J.M., Vegas E., White D.C., Guerrero R.

Villanueva L., Navarrete A., Urmeneta J., White D.C., Guerrero R.

ABSTRACT A combined lipid biomarker-16S rRNA gene denaturing gradient gel electrophoresis analysis was used to monitor changes in the physiological status, biomass, and microbial composition of a microbial mat. In the morning hours, an increase in the biomass of layers containing a high density of phototrophs and a decrease in the growth rate in the deep layers were observed. The combined approach also revealed differences in major groups of microorganisms, including green nonsulfur, gram-positive, and heterotrophic bacteria.

Mills H.J., Martinez R.J., Story S., Sobecky P.A.

ABSTRACT In this study, the composition of the metabolically active fraction of the microbial community occurring in Gulf of Mexico marine sediments (water depth, 550 to 575 m) with overlying filamentous bacterial mats was determined. The mats were mainly composed of either orange- or white-pigmented Beggiatoa spp. Complementary 16S ribosomal DNA (crDNA) was obtained from rRNA extracted from three different sediment depths (0 to 2, 6 to 8, and 10 to 12 cm) that had been subjected to reverse transcription-PCR amplification. Domain-specific 16S PCR primers were used to construct 12 different 16S crDNA libraries containing 333 Archaea and 329 Bacteria clones. Analysis of the Archaea clones indicated that all sediment depths associated with overlying orange- and white-pigmented microbial mats were almost exclusively dominated by ANME-2 (95% of total Archaea clones), a lineage related to the methanogenic order Methanosarcinales . In contrast, bacterial diversity was considerably higher, with the dominant phylotype varying by sediment depth. An equivalent number of clones detected at 0 to 2 cm, representing a total of 93%, were related to the γ and δ classes of Proteobacteria , whereas clones related to δ- Proteobacteria dominated the metabolically active fraction of the bacterial community occurring at 6 to 8 cm (79%) and 10 to 12 cm (85%). This is the first phylogenetics-based evaluation of the presumptive metabolically active fraction of the Bacteria and Archaea community structure investigated along a sediment depth profile in the northern Gulf of Mexico, a hydrocarbon-rich cold-seep region.

BENDER J.

Microbial mats occur in nature as stratified communities of cyanobacteria and bacteria, but they can be cultured on large-scale and manipulated for a variety of functions. They are complex systems, but require few external inputs. The functional uses of mats broadly cover the areas of aquaculture and bioremediation. Preliminary research also points to promising uses in agriculture and energy production. Regarding aquaculture, mats were shown to produce protein, via nitrogen fixation , and were capable of supplying nutrition to tilapia ( Oreochromis niloticus ). Current research is examining the role of mats in the nitrification of nutrient-enriched effluents from aquaculture. Most research has addressed bioremediation, within which two majors categories of contaminants were examined: metals and radionuclides , and organic contaminants . Mats sequester or precipitate metals/radionuclides by surface absorption or by conditioning the surrounding chemical environment, thus bioconcentrating the metal/radionuclide in a small volume. Organic contaminants are degraded and may be completely mineralized. For agriculture mats hold promise as a soil amendment and nitrogen fertilizer . The use of mats in biohydrogen production has been verified, but is in a preliminary phase of development. We propose a comprehensive closed system based on microbial mats for aquaculture and waste management .

Caumette P., Guyoneaud R., Imhoff J.F., Süling J., Gorlenko V.

Four marine, phototrophic, purple sulfur bacteria (strains 5811T, 5812, BM-3 and BS-1) were isolated in pure culture from different brackish to marine sediments in the Mediterranean Sea, the White Sea and the Black Sea. Single cells of these strains were coccus-shaped, non-motile and did not contain gas vesicles. The colour of cell suspensions that were grown in the light was purple–red. Bacteriochlorophyll a and carotenoids of the okenone series were present as photosynthetic pigments. Photosynthetic membrane systems were of the vesicular type. Hydrogen sulfide, thiosulfate, elemental sulfur and molecular hydrogen were used as electron donors during photolithotrophic growth under anoxic conditions; carbon dioxide was utilized as the carbon source. During growth on sulfide, elemental sulfur globules were stored inside the cells. In the presence of hydrogen sulfide, several organic substances could be photoassimilated. Comparative 16S rDNA sequence analysis revealed an affiliation of these four strains to the genus Thiocapsa. Both phylogenetic analysis and the results of DNA–DNA hybridization studies revealed that these strains formed a separate cluster within the genus Thiocapsa. Thus, according to phenotypic characteristics and mainly the carotenoid composition, 16S rDNA sequence analysis and DNA–DNA hybridization data, it is proposed that these strains should be classified as a novel species, Thiocapsa marina sp. nov., with strain 5811T (=DSM 5653T=ATCC 43172T) as the type strain.

Fang J., Lovanh N., Alvarez P.J.

Phospholipid fatty acid (PLFA) analysis combined with (13)C-labeled tracers has been used recently as an environmental forensics tool to demonstrate microbial degradation of pollutants. This study investigated the effectiveness and limitations of this approach, applied to the biodegradation of toluene by five reference strains that express different aerobic toluene degradation pathways: Pseudomonas putida mt-2, P. putida F1, Burkholderia cepacia G4, B. pickettii PKO1, and P. mendocina KR1. The five strains were grown on mineral salts base medium amended with either 10 mM natural or [(13)C-ring]-labeled toluene. PLFA analysis showed that all five strains incorporated the toluene carbon into membrane fatty acids, as demonstrated by increases in the mass of fatty acids and their mass-spectrometry fragments for cells grown on (13)C-labeled toluene. Because of its ubiquitous presence and high abundance in bacteria, C16:0 fatty acid might be a useful biomarker for tracking contaminant degradation and (13)C flow. On the other hand, the (13)C-label (which was supplied at relatively high concentrations) generally exerted an inhibitory effect on fatty acid biosynthesis. Differences in fatty acid concentrations between cells grown on natural versus (13)C-labeled toluene would affect the interpretation of lipid profiles for microbial community analysis as indicated by principal component analysis of fatty acids. Therefore, caution should be exercised in linking lipid data with microbial population shifts in biodegradation experiments with (13)C-labeled tracers.

Vincent W.F., Mueller D.R., Bonilla S.

Microbial communities occur throughout the cryosphere in a diverse range of ice-dominated habitats including snow, sea ice, glaciers, permafrost, and ice clouds. In each of these environments, organisms must be capable of surviving freeze-thaw cycles, persistent low temperatures for growth, extremes of solar radiation, and prolonged dormancy. These constraints may have been especially important during global cooling events in the past, including the Precambrian glaciations. One analogue of these early Earth conditions is the thick, landfast sea ice that occurs today at certain locations in the Arctic and Antarctic. These ice shelves contain liquid water for a brief period each summer, and support luxuriant microbial mat communities. Our recent studies of these mats on the Markham Ice Shelf (Canadian high Arctic) by high performance liquid chromatography (HPLC) showed that they contain high concentrations of chlorophylls a and b, and several carotenoids notably lutein, echinenone and beta-carotene. The largest peaks in the HPLC chromatograms were two UV-screening compounds known to be produced by cyanobacteria, scytonemin, and its decomposition product scytonemin-red. Microscopic analyses of the mats showed that they were dominated by the chlorophyte genera cf. Chlorosarcinopsis, Pleurastrum, Palmellopsis, and Bracteococcus, and cyanobacteria of the genera Nostoc, Phormidium, Leptolyngbya, and Gloeocapsa. From point transects and localized sampling we estimated a total standing stock on this ice shelf of up to 11,200 tonnes of organic matter. These observations underscore the ability of microbial communities to flourish despite the severe constraints imposed by the cryo-ecosystem environment.

Klappenbach J.A., Pierson B.K.

We report the phylogenetic and physiological characterization of a mesophilic and halophilic member of the filamentous anoxygenic phototrophic (FAP) bacteria, provisionally named ‘Candidatus Chorothrix halophila’ gen. nov. sp. nov., that has been maintained in a highly enriched culture in our laboratory for over a decade. Phylogenetic analysis of small-subunit RNA-encoding sequences places ‘Candidatus Chlorothrix halophila’ in a clade that includes cultivated members of the genera Chloroflexus and Oscillochloris. Physiological studies demonstrated sulfide-dependent photosynthetic uptake of 14C-labeled bicarbonate. Enzymatic assays for the activity of propionyl-coenzyme A synthase indicated that ‘Candidatus Chlorothrix halophila’ does not use the 3-hydroxypropionate cycle of Chloroflexus aurantiacus OK-70-fl for autotrophic carbon assimilation. New concepts regarding the taxonomy and phylogeny of FAP bacteria have emerged from this work.

Spring S., Merkhoffer B., Weiss N., Kroppenstedt R.M., Hippe H., Stackebrandt E.

Taxonomic studies were performed on four strains (D-1/D-an/IIT, C/C-an/B1T, A-1/C-an/C1T and A-1/C-an/IT) of anaerobic, gram-positive, spore-forming bacteria originally isolated from a mat sample retrieved from a shallow, moated area around Lake Fryxell, an Antarctic freshwater lake. Phylogenetic analyses based on 16S rRNA gene sequence data indicated that these strains are affiliated with cluster I clostridia and form a coherent group with Clostridium estertheticum and Clostridium laramiense. Similarity values among 16S rRNA gene sequences within this assemblage ranged between 96.7 and 99.8%. Despite the close phylogenetic relationship, several distinguishing phenotypic traits were found among the novel strains using a polyphasic approach. All strains were psychrophilic, but the temperature optimum for growth differed markedly, ranging from 4 to 16 degrees C. In addition, substrate utilization patterns, fermentation end products, cellular fatty acid profiles and morphological traits enabled a clear differentiation between the strains. DNA-DNA hybridization experiments revealed that each of the four novel strains represents a distinct species, with DNA-DNA similarity values to related strains in the range 16-62%. In contrast, the type strains of C. estertheticum and C. laramiense shared 79% DNA-DNA similarity, indicating a close relationship at the species level. On the basis of genetic and phenotypic properties, it is proposed to designate four novel species of the genus Clostridium to harbour the newly isolated strains: Clostridium frigoris sp. nov. (type strain D-1/D-an/IIT=DSM 14204T=ATCC BMAA-579T), Clostridium lacusfryxellense sp. nov. (type strain C/C-an/B1T=DSM 14205T=ATCC BAA-580T), Clostridium bowmaniisp. nov. (type strain A-1/C-an/C1T=DSM 14206T=ATCC BAA-581T) and Clostridium psychrophilum sp. nov. (type strain A-1/C-an/IT=DSM 14207T=ATCC BAA-582T). It is also proposed to unite C. laramiense and C. estertheticum under C. estertheticum. The subspecies C. estertheticum subsp. laramiense subsp. nov. is established, represented by strain ATCC 51254T (=DSM 14884T). The type strain of C. estertheticum subsp. estertheticum remains NCIMB 12511T (=DSM 8809T).

Urmeneta J., Navarrete A., Huete J., Guerrero R.

Isolation and identification of several strains of cyanobacteria from microbial mats of the Ebro Delta, Spain, are described. A series of tenfold dilutions was the first step of isolation. Self-isolation techniques, which use one or several physiological characteristics of a cyanobacterium, were applied in some cases to obtain enrichment cultures. Twelve filamentous strains were isolated and stored in axenic culture. As only a few cyanobacterial species can be frozen and revived without any cryoprotective agent, preservation of isolated strains was assayed with several cryoprotective solutions. Methanol and glycerol were not suitable as cryoprotective agents for most of the isolates. Dimethyl sulfoxide (DMSO) was apparently the best cryoprotector. A new method, which used a filter paper as a growing substratum that later could be directly stored at −80°C, was successfully used. A morphological study of each strain under light and electron microscopy was made to classify them. All isolated strains belong to phylum BX, Class 1, subsection III of the Bergey's manual of systematic bacteriology, 2nd ed., vol. 1. Most genera are included in the LPP group as Lyngbya aestuarii and Microcoleus chthonoplastes.

Zaar A., Fuchs G., Golecki J.R., Overmann J.

A new strain of purple sulfur bacterium was isolated from a marine microbial mat sampled in Great Sippewissett Salt Marsh at the Atlantic coast (Woods Hole, Mass., USA). Single cells of strain AZ1 were coccus-shaped, highly motile by means of a single flagellum, and did not contain gas vesicles. Intracellular membranes were of the vesicular type. However, additional concentric membrane structures were present. The photosynthetic pigments were bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series, with rhodopin as the dominant carotenoid. Hydrogen sulfide (up to 11 mM), sulfur, thiosulfate, and molecular hydrogen were used as electron donors during anaerobic phototrophic growth. During growth on sulfide, elemental sulfur globules were transiently stored inside the cells. Strain AZ1 is much more versatile than most other Chromatiaceae with respect to electron donor and organic substrates. In the presence of CO2, it is capable of assimilating C1–C5 fatty acids, alcohols, and intermediates of the tricarboxylic acid cycle. Strain AZ1 could also grow photoorganotrophically with acetate as the sole photosynthetic electron donor. Chemotrophic growth in the dark under microoxic conditions was not detected. Optimum growth occurred at pH 6.5–6.7, 30–35 °C, ≥50 µmol quanta m−2 s−1, and 2.4–2.6% NaCl. The DNA base composition was 64.5 mol% G+C. Comparative sequence analysis of the 16S rRNA gene confirmed that the isolate is a member of the family Chromatiaceae. Sequence similarity to the most closely related species, Thiorhodococcus minor DSMZ 11518T, was 97.8%; however, the value for DNA-DNA hybridization between both strains was only 20%. Because of the low genetic similarity and since strain AZ1 physiologically differs considerably from all other members of the Chromatiaceae, including Trc. minor, the new isolate is described as a new species of the genus Thiorhodococcus, Thiorhodococcus drewsii sp. nov.

Hirschler-Réa A., Matheron R., Riffaud C., Mouné S., Eatock C., Herbert R.A., Willison J.C., Caumette P.

Microbial mats developing in the hypersaline lagoons of a commercial saltern in the Salin-de-Giraud (Rhône delta) were found to contain a red layer fully dominated by spirilloid phototrophic purple bacteria underlying a cyanobacterial layer. From this layer four strains of spirilloid purple bacteria were isolated, all of which were extremely halophilic. All strains were isolated by using the same medium under halophilic photolithoheterotrophic conditions. One of them, strain SG 3105 was a purple non-sulfur bacterial strain closely related to Rhodovibrio sodomensis with a 16S rDNA sequence similarity of 98.8%. The three other isolated strains, SG 3301T, SG 3302 and SG 3304, were purple sulfur bacteria and were found to be very similar. The cells were motile by a polar tuft of flagella. Photosynthetic intracytoplasmic membranes of the lamellar stack type contained BChl a and spirilloxanthin as the major carotenoid. Phototrophic growth with sulfide as electron donor was poor; globules of elemental sulfur were present outside the cells. In the presence of sulfide and CO2 good growth occurred with organic substrates. Optimum growth occurred in the presence of 9-12% (w/v) NaCl at neutral pH (optimal pH 6.8-7) and at 30-35 degrees C. The DNA base composition of strains SG 3301T and SG 3304 were 74.5 and 74.1 mol% G + C, respectively. According to the 16S rDNA sequences, strains SG 3301T and SG 3304 belonged to the genus Halorhodospira, but they were sufficiently separated morphologically, physiologically and genetically from other recognized Halorhodospira species to be described as a new species of the genus. They are, therefore, described as Halorhodospira neutriphila sp. nov. with strain SG 3301T as the type strain (=DSM 15116T).

Jonkers H., Abed R.

Thar R., Kühl M.

ABSTRACT We describe the morphology and behavior of a hitherto unknown bacterial species that forms conspicuous veils (typical dimensions, 30 by 30 mm) on sulfidic marine sediment. The new bacteria were enriched on complex sulfidic medium within a benthic gradient chamber in oxygen-sulfide countergradients, but the bacteria have so far not been isolated in pure culture, and a detailed characterization of their metabolism is still lacking. The bacteria are colorless, gram-negative, and vibrioid-shaped (1.3- to 2.5- by 4- to 10-μm) cells that multiply by binary division and contain several spherical inclusions of poly-β-hydroxybutyric acid. The cells have bipolar polytrichous flagella and exhibit a unique swimming pattern, rotating and translating along their short axis. Free-swimming cells showed aerotaxis and aggregated at ca. 2 μM oxygen within opposing oxygen-sulfide gradients, where they were able to attach via a mucous stalk, forming a cohesive whitish veil at the oxic-anoxic interface. Bacteria attached to the veil kept rotating and adapted their stalk lengths dynamically to changing oxygen concentrations. The joint action of rotating bacteria on the veil induced a homogeneous water flow from the oxic water region toward the veil, whereby the oxygen uptake rate could be enhanced up to six times, as shown by model calculations. The veils showed a pronounced succession pattern. New veils were generated de novo within 24 h and had a homogeneous whitish translucent appearance. Bacterial competitors or eukaryotic predators were apparently kept away by the low oxygen concentration prevailing at the veil surface. Frequently, within 2 days the veil developed a honeycomb pattern of regularly spaced holes. After 4 days, most veils were colonized by grazing ciliates, leading to the fast disappearance of the new bacteria. Several-week-old veils finally developed into microbial mats consisting of green, purple, and colorless sulfur bacteria.

Guyoneaud R., Mouné S., Eatock C., Bothorel V., Hirschler-Réa A., Willison J., Duran R., Liesack W., Herbert R., Matheron R., Caumette P.

Three new spirilloid phototrophic purple nonsulfur bacteria were isolated in pure culture from three different environments: strain CE2105 from a brackish lagoon in the Arcachon Bay (Atlantic coast, France), strain SE3104 from a saline sulfur spring in the Pyrenees (Navarra, Spain) , and strain AT2115 a microbial mat (Tetiaroa Atoll, Society Islands). Single cells of the three strains were spiral-shaped and highly motile. Their intracellular photosynthetic membranes were of the vesicular type. Bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series were present as photosynthetic pigments. Optimal growth occurred under photoheterotrophic conditions and in the presence of 0.5–4% w/v NaCl. These features are similar to those described for Roseospira mediosalina. Comparative sequence analysis of their 16S rRNA genes placed these strains within the α-subclass of Proteobacteria, in a cluster together with Roseospira mediosalina and Rhodospira trueperi. They form a closely related group of slightly to moderately halophilic spiral-shaped purple nonsulfur bacteria. However, the three new isolates exhibited some differences in their physiology and genetic characteristics. Consequently, we propose that they are members of three new species within the genus Roseospira, Roseospira marina sp. nov., Roseospira navarrensis sp. nov., and Roseospira thiosulfatophila sp. nov., with strains CE2105, SE3104, and AT2115 as the type strains, respectively. As a consequence, an emended description of the genus Roseospira is also given.

Boadella J., Butturini A., Doménech-Pascual A., Freixinos Z., Perujo N., Urmeneta J., Vidal A., Romaní A.M.

AbstractSaline shallow lakes in arid and semi-arid regions frequently undergo drying episodes, leading to significant variations in salinity and water availability. Research on the impacts of salinity and drought on the structure and function of biofilms in hypersaline shallow lakes is limited. This study aimed to understand the potential changes of biofilms in playa-lake sediments during the drying process. Sediments were sampled at different depths (surface, subsurface) and hydrological periods (wet, retraction, and dry), which included a decrease in water activity (aw, the availability of water for microbial use) from 0.99 to 0.72. aw reduction caused a greater effect on functional variables compared to structural variables, indicating the high resistance of the studied biofilms to changes in salinity and water availability. Respiration and hydrolytic extracellular enzyme activities exhibited higher values under high aw, while phenol oxidase activity and prokaryote biomass increased at lower aw. This shift occurred at both depths but was more pronounced at the surface, possibly due to the more extreme conditions (up to 0.7 aw). The increased levels of extracellular polymeric substances and carotenoids developed at low aw may help protect microorganisms in high salinity and drought environments. However, these harsh conditions may interfere with the activity of hydrolytic enzymes and their producers, while promoting the growth of resistant prokaryotes and their capacity to obtain C and N sources from recalcitrant compounds. The resilience of biofilms in hypersaline lakes under extreme conditions is given by their resistant biochemichal structure and the adaptability of their microbial functioning.

Sherysheva N.G., Osipov G.A., Khalko V.V.

The structure and function of bacterial communities in sediments of aquatic ecosystems have been considered using the molecular method of gas chromatography—mass spectrometry (GC–MS). Specific substances of microbial origin have been discovered in the samples and assigned to most probable genera or species. The local bank of microbial markers has been designed for lake silts. The composition of bacteriobenthic community is determined quantitatively using the algebra algorithm of decomposition of the total fatty acids profile into individual taxonomic components.

Paissé S., Goñi-Urriza M., Stadler T., Budzinski H., Duran R.

Kharlamenko V.I., Imbs A.B., Tarasov V.G.

The physiological state of littoral and sublittoral microbial communities in a marine shallow-water hydrothermal ecosystem (Kraternaya Bight) was studied using lipid biomarkers. The ratio trans/cis (n-7) isomers of monoenic fatty acids (FAs) of polar lipids in intertidal and subtidal algobacterial and bacterial mats of the bight exceeded 0.1 significantly; this indicated a stress state in bacteria. No concomitant increase was found in the ratio of cyclopropane fatty acids to 16: 1 and 18: 1 (n-7) cis monoenic fatty acids. In bottom sediments, the ratio trans/cis (n-7) isomers of monoenic fatty acids was below 0.1. A positive correlation (r = 0.71) was revealed between the ratio trans/cis isomers of (n-7) monoenic fatty acids and the content of saturated fatty acids.

Bordenave S., Goñi-urriza M., Vilette C., Blanchard S., Caumette P., Duran R.

The aim of this work was to characterize bacterial ring-hydroxylating dioxygenase (RHD) diversity in a pristine microbial mat and follow their diversity changes in response to heavy fuel oil contamination. In order to describe the RHDs diversity, new degenerate primers were designed and a nested-PCR approach was developed to gain sensitivity and wider diversity. RHD diversity in artificially contaminated mats maintained in microcosms and in chronically contaminated mats was analysed by clone libraries and terminal restriction fragment length polymorphism (T-RFLP) at genomic and transcriptomic levels. The RHD diversity in the pristine microbial mat was represented by Pseudomonas putida nahAc-like genes and no increase of diversity was detected after 1 year of oil contamination. The diversity observed in a 30 year chronically polluted microbial mat was represented by four main RHD clusters and two new genes revealing higher polyaromatic hydrocarbon (PAH) degradation capacity. This study illustrates that a single petroleum contamination (such as oil spill) is not enough to involve a detectable modification of RHD diversity. The new degenerate primers described here allowed RHD gene amplification from pristine and contaminated samples thereby showing their diversity. The proposed approach solves one of the main problems of functional gene analysis providing effective amplification of the environmental diversity of the targeted genes.