Viability and Biomass of <Emphasis Type="Italic">Micrococcus luteus</Emphasis> DE2008 at Different Salinity Concentrations Determined by Specific Fluorochromes and CLSM-Image Analysis

In previous studies, our group developed a method based on Confocal Laser Scanning Microscopy and Image Analysis (CLSM-IA) to analyze the diversity and biomass of cyanobacteria in microbial mats. However, this method cannot be applied to heterotrophic microorganisms, as these do not have autofluorescence. In this article, we present a method that combines CLSM-IA and Hoechst 33342 and SYTOX Green fluorochromes (FLU-CLSM-IA) to determine the viability and biomass of Micrococcus luteus DE2008, isolated from a saline microbial mat (Ebro Delta, Tarragona, Spain). The method has been applied to assess the effect of salinity on this microorganism. A reduction in viability and biomass (live cells) was observed as the salt concentration increases. The largest effect was at 100‰ NaCl with a cell death of 27.25% and a decrease in total and individual biomass of 39.75 and 0.009 mgC/cm³, respectively, both with respect to optimal growth (10 ‰ NaCl). On the other hand, another important contribution of this article was that combining the FLU-CLSM-IA results with those achieved by plate counts enabled us to determine, for first time, the viability and the total biomass of the “dormant cells” (66.75% of viability and 40.59 mgC/cm³ of total biomass at 100‰ NaCl). FLU-CLSM-IA is an efficient, fast, and reliable method for making a total count of cells at pixel level, including the dormant cells, to evaluate the viability and the biomass of a hetetrophic microorganism, M. luteus DE2008.     

4-Chlorophenol degradation by a bacterial consortium: development of a granular activated carbon biofilm reactor

A bacterial consortium that can degrade chloro- and nitrophenols has been isolated from the rhizosphere of Phragmitis communis. Degradation of 4-chlorophenol (4-CP) by a consortium attached to granular activated carbon (GAC) in a biofilm reactor was evaluated during both open and closed modes of operation. During the operation of the biofilm reactor, 4-CP was not detected in the column effluent, being either adsorbed to the GAC or biodegraded by the consortium. When 4-CP at 100 mg l⁻¹ was fed to the column in open mode operation (20 mg g⁻¹ GAC total supply), up to 27% was immediately available for biodegradation, the rest being adsorbed to the GAC. Biodegradation continued after the system was returned to closed mode operation, indicating that GAC bound 4-CP became available to the consortium. Biofilm batch cultures supplied with 10–216 mg 4-CP g⁻¹ GAC suggested that a residual fraction of GAC-bound 4-CP was biologically unavailable. The consortium was able to metabolise 4-CP after perturbations by the addition of chromium (Cr VI) at 1–5 mg l⁻¹ and nitrate at concentrations up to 400 mg l⁻¹. The development of the biofilm structure was analysed by scanning electron microscopy and confocal laser scanning microscopy (CLSM) techniques. CLSM revealed a heterogeneous structure with a network of channels throughout the biofilm, partially occupied by microbial exopolymer structures. Received: 17 March 1999 / Received revision: 27 May 1999 / Accepted: 28 May 1999     

In Situ Determination of the Effects of Lead and Copper on Cyanobacterial Populations in Microcosms

Background

Biomass has been studied as biomarker to evaluate the effect of heavy metals on microbial communities. Nevertheless, the most important methodological problem when working with natural and artificial microbial mats is the difficulty to evaluate changes produced on microorganism populations that are found in thicknesses of just a few mm depth.

Methodology/Principal Findings

Here, we applied for first time a recently published new method based on confocal laser scanning microscopy and image-program analysis to determine in situ the effect of Pb and Cu stress in cyanobacterial populations.

Conclusions/Significance

The results showed that both in the microcosm polluted by Cu and by Pb, a drastic reduction in total biomass for cyanobacterial and Microcoleus sp. (the dominant filamentous cyanobacterium in microbial mats) was detected within a week. According to the data presented in this report, this biomass inspection has a main advantage: besides total biomass, diversity, individual biomass of each population and their position can be analysed at microscale level. CLSM-IA could be a good method for analyzing changes in microbial biomass as a response to the addition of heavy metals and also to other kind of pollutants.     

Arctic cyanobacteria and limnological properties of their environment: Bylot Island, Northwest Territories, Canada (73°N, 80°W)

Cyanobacteria were a major constituent of phototrophic communities in the lakes, ponds and streams of Bylot Island, in the Canadian high Arctic. The waters spanned a range of temperatures (1.8–16.8°C in late July), pH regimes (6.2–9.2) and conductivities (1.5–1700 μS cm⁻¹) but nutrient concentrations were consistently low (< 1 μg dissolved reactive P l⁻¹ at all sites; < 10 μg NO₃-N l⁻¹ at most sites). Picoplanktonic species (Synechococcus spp.) were often the numerical dominants in the plankton, and periphytic filamentous species (Oscillatoriaceae) commonly formed thick (5–50 mm) benthic mats. Bloom-forming species of cyanobacteria were either absent or poorly represented even in Chla-rich ponds. The total community biomass ranged from 0.1 to 29.8 μg Chla l⁻¹ in the plankton and from 1.1 to 34.8 μg Chla cm⁻² in the benthos. The in vivo absorbance characteristics of isolates from these environments indicated a genetically diverse range of species in each group of Arctic cyanobacteria. Growth versus irradiance relationships were determined for each of the isolates and similarly revealed large genetic differences (maximum growth rates from 0.17 to 0.61 day⁻¹), even between morphologically identical taxa. A comparison of nutrients, pigment concentrations and species composition underscores the strong similarities between freshwater ecosystems in the north and south polar zones. Received: 3 June 1996 / Accepted: 3 November 1996     

Phosphate Addition and Plant Species Alters Microbial Community Structure in Acidic Upland Grassland Soil

Agricultural improvement (addition of fertilizers, liming) of seminatural acidic grasslands across Ireland and the UK has resulted in significant shifts in floristic composition, soil chemistry, and microbial community structure. Although several factors have been proposed as responsible for driving shifts in microbial communities, the exact causes of such changes are not well defined. Phosphate was added to grassland microcosms to investigate the effect on fungal and bacterial communities. Plant species typical of unimproved grasslands (Agrostis capillaris, Festuca ovina) and agriculturally improved grasslands (Lolium perenne) were grown, and phosphate was added 25 days after seed germination, with harvesting after a further 50 days. Phosphate addition significantly increased root biomass (p < 0.001) and shoot biomass (p < 0.05), soil pH (by 0.1 U), and microbial activity (by 5.33 mg triphenylformazan [TPF] g⁻¹ soil; p < 0.001). A slight decrease (by 0.257 mg biomass-C g⁻¹ soil; p < 0.05) in microbial biomass after phosphate addition was found. The presence of plant species significantly decreased soil pH (p < 0.05; by up to 0.2 U) and increased microbial activity (by up to 6.02 mg TPF g⁻¹ soil) but had no significant effect on microbial biomass. Microbial communities were profiled using automated ribosomal intergenic spacer analysis. Multidimensional scaling plots and canonical correspondence analysis revealed that phosphate addition and its interactions with upland grassland plant species resulted in considerable changes in the fungal and bacterial communities of upland soil. The fungal community structure was significantly affected by both phosphate (R = 0.948) and plant species (R = 0.857), and the bacterial community structure was also significantly affected by phosphate (R = 0.758) and plant species (R = 0.753). Differences in microbial community structure following P addition were also revealed by similarity percentage analysis. These data suggest that phosphate application may be an important contributor to microbial community structural change during agricultural management of upland grasslands.     

Benthic moss pillars in Antarctic lakes

Unique pillar-like colonies of aquatic mosses, rising from cyanobacterial and algal mats, have been discovered in some freshwater lakes in the vicinity of Syowa Station (69°00′S, 39°35′E), continental Antarctica. These moss pillars are about 40 cm in diameter and up to 60 cm high and occur at the lake bottoms mainly between 3 and 5 m depth. The primary component is a species of Leptobryum, a genus unknown in the continental Antarctic terrestrial bryoflora and as an aquatic genus elsewhere in the world. Bryum pseudotriquetrum is often an associated species. In longitudinal section the pillars reveal several whitish layers formed by mineral sediment and dead cyanobacteria. It is speculated that the biomass of aquatic mosses at the bottom of many Antarctic lakes is considerably greater than that previously estimated. Accepted: 11 April 1999     

Dinitrogen fixation by exposed communities on the rim of Tikehau atoll (Tuamotu Archipelago, French Polynesia)

Various types of sub-aerially exposed microbial mats, including emergent mats, beach sand, beach rock and Kopara mats, are widespread on the 78 km (25 km²) of rim surrounding the Tikehau atoll lagoon. These mats form laminated accretions or diffuse microbial communities growing under high insolation and temperatures, and are therefore subject to desiccation. Both heterocystous and non-heterocystous cyanobacteria occur in these mats. Using acetylene reduction techniques, nitrogenase activity was observed at all sites over a period of 5 years and was 3–17 times higher during daylight than at night in all communities except for beach rock. ¹⁵N₂ measurements indicated a molar ratio of acetylene reduction to N₂ fixed of 1.6 for all exposed communities. Estimated N₂ fixation ranged from 1.44 to 8.0 mg N m⁻² day⁻¹ in these exposed communities (mean of 4.66 mg N m⁻² day⁻¹) with beachrock showing the highest rates. For the whole reef rim, daily N₂ fixation amounted to 98.42 kg N day⁻¹ which represents 28% of the rate of fixation in the entire lagoon (area 400 km²).     

Dinitrogen-Fixing Cyanobacteria in Microbial Mats of Two Shallow Coral Reef Ecosystems

Dinitrogen-fixing organisms in cyanobacterial mats were studied in two shallow coral reef ecosystems: La Reunion Island, southwestern Indian Ocean, Sesoko (Okinawa) Island, and northwestern Pacific Ocean. Rapidly expanding benthic miniblooms, frequently dominated by a single cyanobacterial taxon, were identified by microscopy and molecular tools. In addition, nitrogenase activity by these blooms was measured in situ. Dinitrogen fixation and its contribution to mat primary production were calculated using ¹⁵N₂ and ¹³C methods. Dinitrogen-fixing cyanobacteria from mats in La Reunion and Sesoko showed few differences in taxonomic composition. Anabaena sp. among heterocystous and Hydrocoleum majus and Symploca hydnoides among nonheterocystous cyanobacteria occurred in microbial mats of both sites. Oscillatoria bonnemaisonii and Leptolyngbya spp. occurred only in La Reunion, whereas Hydrocoleum coccineum dominated in Sesoko. Other mats dominated by Hydrocoleum lyngbyaceum, Phormidium laysanense, and Trichocoleus tenerrimus occurred at lower frequencies. The 24-h nitrogenase activity, as measured by acetylene reduction, varied between 11 and 324 nmoles C₂H₂ reduced μg⁻¹ Chl a. The highest values were achieved by heterocystous Anabaena sp. performed mostly during the day. Highest values for nonheterocystous cyanobacteria were achieved by H. coccineum mostly during the night. Daily nitrogen fixation varied from nine (Leptolyngbya) to 238 nmoles N₂ μg⁻¹ Chl day⁻¹ (H. coccineum). Primary production rates ranged from 1,321 (S. hydnoides) to 9,933 nmoles C μg⁻¹ Chl day⁻¹ (H. coccineum). Dinitrogen fixation satisfied between 5% and 21% of the nitrogen required for primary production.     

Artificial Cyanobacterial Mats: Growth, Structure, and Vertical Zonation Patterns

Abstract The formation of cyanobacterial mats (originally induced by incubation of sediment cores in which metazoans and most other eukaryotes had been removed) was followed over approximately 2.6 years. The thickness of the mats increased at a rate of 2–3 mm per year because of accumulation of empty cyanobacterial sheaths and as a result of carbonate deposition; the fraction of living biomass remained relatively constant over at least 2 years, but there was a slow accumulation of nonliving organic C (≈ 1 mmol yr⁻¹). Biota composition (dominated by five types of filamentous cyanobacteria, unicellular cyanobacteria, diatoms, anoxygenic phototrophs, and heterotrophic bacteria) and vertical zonation patterns in the upper 2–3 mm of the mats were also almost constant over time. Using transmission electron microscopy and stereological analysis it was possible to quantify the vertical distribution of major groups of organisms. Received: 20 December 1999; Accepted: 10 June 2000; Online Publication: 28 August 2000     

Quantification of water and biomass in small colony variant PAO1 biofilms by confocal Raman microspectroscopy

The Raman spectra, water content, and biomass density of wild-type (WT) Pseudomonas aeruginosa PAO1, small colony variant (SCV) PAO1, and Pseudoalteromonas sp. NCIMB 2021 biofilms were compared in order to determine their variation with strain and species. Living, fully submerged biofilms were analyzed in situ by confocal Raman microspectroscopy for up to 2 weeks. Water to biomass ratios (W/BRs), which are the ratios of the O–H stretching vibration of water at 3,450 cm⁻¹ to the C–H stretching band characteristic of biomass at 2,950 cm⁻¹, were used to estimate the biomass density and cell density by comparison with W/BRs of protein solutions and bacterial suspensions, respectively, on calibration curves. The hydration within SCV biofilm colonies was extremely heterogeneous whereas W/BRs were generally constant in young WT biofilm colonies. The mean biomass in biofilm colonies of WT or colony cores of SCV was typically equivalent to 16% to 27% protein (w/v), but was 10% or less for NCIMB 2021. The corresponding cell densities were 7.5 to >10 × 10¹⁰ cfu mL⁻¹ for SCV, while the maximum cell density for NCIMB biofilms was 2.8 × 10¹⁰ cfu mL⁻¹.     

Microbial mats on the Orkney Islands revisited: microenvironment and microbial community composition

The microenvironment and community composition of microbial mats developing on beaches in Scapa Flow (Orkney Islands) were investigated. Analysis of characteristic biomarkers (major fatty acids, hydrocarbons, alcohols, and alkenones) revealed the presence of different groups of bacteria and microalgae in mats from Waulkmill and Swanbister beach, including diatoms, Haptophyceae, cyanobacteria, and sulfate-reducing bacteria. These analyses also indicated the presence of methanogens, especially in Swanbister beach mats, and therefore a possible role of methanogenesis for the carbon cycle of these sediments. High amounts of algal lipids and slightly higher numbers (genera, abundances) of cyanobacteria were found in Waulkmill Bay mats. However, overall only a few genera and low numbers of unicellular and filamentous cyanobacteria were present in mats from Waulkmill and Swanbister beach, as deduced from CLSM (confocal laser scanning microscopy) analysis. Spectral scalar irradiance measurements with fiber-optic microprobes indicated a pronounced heterogeneity concerning zonation and density of mainly anoxygenic phototrophs in Swanbister Bay mats. By microsensor and T-RFLP (terminal restriction fragment length polymorphism) analysis in Swanbister beach mats, the depth distribution of different populations of purple and sulfate-reducing bacteria could be related to the microenvironmental conditions. Oxygen, but also sulfide and other (inorganic and organic) sulfur compounds, seems to play an important role in the stratification and diversity of these two major bacterial groups involved in sulfur cycling in Swanbister beach mats.     

Nutrients and light effects on stream biofilms: a combined assessment with CLSM,structural and functional parameters

Nutrients and light are the most determinant factors for microbial benthic assemblages in oligotrophic forested streams. We investigated the importance of nutrients and light availability on the structure and the function of epilithic biofilms in a Mediterranean forested stream (Fuirosos, Spain). Biofilms grew on artificial substrata in both enriched and unenriched reaches where shade conditions were simulated. Four different treatments were generated: higher light unenriched, lower light unenriched, higher light enriched (HL-E) and lower light enriched. Chlorophyll a, bacterial density, extracellular polymeric substances (EPS), extracellular leucine aminopeptidase (LAmP) and alkaline phosphatase (APase) activities were analysed during the colonisation at days 4, 9, 16, 22 and 52. At day 52, confocal laser scanning microscopy (CLSM) was used to determine differences in biofilm architecture. CLSM evidenced differences in thickness and structural complexity of biofilms grown in different conditions. Biofilms in HL-E were the thickest and had the most complex structure. The CLSM highlighted that the EPS was agglomerated in the upper layer of enriched-grown biofilms, but evenly distributed through the biofilm in unenriched biofilms. CLSM 3D images suggested that cyanobacteria increased under higher nutrient conditions. Nutrient enrichment caused the decrease of APase activity. Interaction between the two factors affected LAmP activity. HL-E had the highest LAmP and the lowest APase activities, an indication that biofilm responses to nutrients mostly occurred with high-light availability. Our results revealed that the conjoint availability of light and nutrients caused the highest changes in biofilm spatial organisation, microbial structure and functioning in oligotrophic forested streams.

     

The use of microscopy and three-dimensional visualization to evaluate the structure of microbial biofilms cultivated in the calgary biofilm device

Microbes frequently live within multicellular, solid surface-attached assemblages termed biofilms. These microbial communities have architectural features that contribute to population heterogeneity and consequently to emergent cell functions. Therefore, three-dimensional (3D) features of biofilm structure are important for understanding the physiology and ecology of these microbial systems. This paper details several protocols for scanning electron microscopy and confocal laser scanning microscopy (CLSM) of biofilms grown on polystyrene pegs in the Calgary Biofilm Device (CBD). Furthermore, a procedure is described for image processing of CLSM data stacks using amira™, a virtual reality tool, to create surface and/or volume rendered 3D visualizations of biofilm microorganisms. The combination of microscopy with microbial cultivation in the CBD — an apparatus that was designed for highthroughput susceptibility testing — allows for structure-function analysis of biofilms under multivariate growth and exposure conditions.     

Effects on plant production after addition of labile carbon to arctic/alpine soils

Biomass production was analysed in Festuca vivipara, grown for 3 months in pots with non-sterilized or sterilized soil after factorial addition of three levels of labile carbon combined with high and low levels of N and P. The soil was a nutrient-poor subarctic heath soil. In the non-sterilized soil plant biomass production increased strongly only in the treatment with high levels of both N and P, which suggests that both nutrients limited plant growth. In the sterilized soil addition of a high level of N without P addition gave almost the same growth response as in the combined NP treatment. This was because of a more than 30-fold increase of inorganic phosphorus in the soil as P was released from the killed microbial biomass after sterilization. Sugar addition reduced plant growth in all treatments. The reduction in plant growth was dose dependent within the range of 0–450 μg C g⁻¹ soil added to the non-sterilized soil, but the response levelled off at 233 μg C g⁻¹ soil in the soil that had been sterilized at the start of the experiment. The plant response, together with observed depletion of soil inorganic N and P, indicated that the microbial biomass immobilized nutrients efficiently and reduced plant growth when extra labile carbon was added. The inhibition of growth was lower, however, in the soil which had been sterilized, probably because of a slow recovery of the microbial populations in it. Two of the nutrient-carbon solutions closely matched the N, P and C concentrations in a solution containing leaf extracts of Cassiope tetragona and Betula tortuosa that had been used previously to test for possible allelopathic effects of compounds in the leaf extracts. These extracts also reduced plant growth. The growth reduction was equally large or larger after nutrient-sugar addition than after addition of leaf extracts in three out of the four possible combinations of species and sterilized or non-sterilized soil. In the fourth case (Betula extract added to sterilized soil), the effect was larger when leaf extract was added than after addition of the nutrient-carbon solution. This could be due to a low rate of microbial degradation of phytotoxic substances in this soil because of a slow recovery of the microbial populations after sterilization. The generally stronger or equal effect of the nutrient-sugar addition compared to the leaf extract addition leads to the conclusion that microbial nutrient immobilization and microbial competition for nutrients increased as a function of labile carbon addition with the extract. Hence, it appears that enhanced microbial activity and microbial nutrient immobilization rather than phytotoxic effects was the primary reasons for the reduced biomass production in F. vivipara even after addition of the leaf extracts. Received: 25 October 1996 / Accepted: 11 June 1997     

Survival of Akinetes (Resting-State Cells of Cyanobacteria) in Low Earth Orbit and Simulated Extraterrestrial Conditions

Cyanobacteria are photosynthetic organisms that have been considered for space applications, such as oxygen production in bioregenerative life support systems, and can be used as a model organism for understanding microbial survival in space. Akinetes are resting-state cells of cyanobacteria that are produced by certain genera of heterocystous cyanobacteria to survive extreme environmental conditions. Although they are similar in nature to endospores, there have been no investigations into the survival of akinetes in extraterrestrial environments. The aim of this work was to examine the survival of akinetes from Anabaena cylindrica in simulated extraterrestrial conditions and in Low Earth Orbit (LEO). Akinetes were dried onto limestone rocks and sent into LEO for 10 days on the ESA Biopan VI. In ground-based experiments, the rocks were exposed to periods of desiccation, vacuum (0.7 × 10⁻³ kPa), temperature extremes (−80 to 80°C), Mars conditions (−27°C, 0.8 kPa, CO₂) and UV radiation (325–400 nm). A proportion of the akinete population was able to survive a period of 10 days in LEO and 28 days in Mars simulated conditions, when the rocks were not subjected to UV radiation. Furthermore, the akinetes were able to survive 28 days of exposure to desiccation and low temperature with high viability remaining. Yet long periods of vacuum and high temperature were lethal to the akinetes. This work shows that akinetes are extreme-tolerating states of cyanobacteria that have a practical use in space applications and yield new insight into the survival of microbial resting-state cells in space conditions.     

Cyanobacterial mats from hot springs produce antimicrobial compounds and quorum-sensing inhibitors under natural conditions

Polar (water) and non-polar (ethyl acetate) extracts from the cyanobacterial layer (top 1–3 mm) of four hot spring microbial mats in the Sultanate of Oman were tested for their antibacterial, antidiatom and quorum-sensing inhibitory activities under natural conditions. The chemical composition of the active extracts was analysed using gas chromatography–mass spectrometry (GC-MS). Cyanobacteria within these mats were identified by direct microscopy while the total bacterial community composition was compared using automated ribosomal intergenic spacer analysis (ARISA). Only the extracts from Bowshar and Nakhl mats showed antibacterial properties against Bacillus sp., Micrococcus luteus, Shigella sonnei, Salmonella enterica and Klebsiella pneumoniae. All tested extracts inhibited the growth of the benthic diatom Amphora coffeaeformis. Extracts from Bowshar, Rustaq and Nakhl inhibited quorum-sensing of the reporter strains Chromobacterium violaceum CV017 and Agrobacterium tumefaciens NTL4. The highest bioactivity was recorded for ethyl acetate extracts from Nakhl mats, which had the lowest number of operational taxonomic units (OTUs). Using GC-MS, 74 chemical compounds were obtained, however with different distribution among the four mat extracts (similarity < 43%). Various cyanobacteria, belonging mainly to Chroococcus, Phormidium, Leptolyngbya, Spirulina and Lyngbya were detected in the different mats, and each mat had its unique bacterial community, as confirmed by ARISA profiles. We conclude that antimicrobial and quorum-sensing inhibitory compounds can be produced by hot spring mat microorganisms under natural conditions and the differences in these compounds could be attributed to the differences in the mats’ bacterial composition as well as the physical–chemical conditions of the springs.     

Bioremediation of pentachlorophenol-contaminated soil by bioaugmentation using activated soil

The use of an indigenous microbial consortium, pollutant-acclimated and attached to soil particles (activated soil), was studied as a bioaugmentation method for the aerobic biodegradation of pentachlorophenol (PCP) in a contaminated soil. A 125-l completely mixed soil slurry (10% soil) bioreactor was used to produce the activated soil biomass. Results showed that the bioreactor was very effective in producing a PCP-acclimated biomass. Within 30 days, PCP-degrading bacteria increased from 10⁵ cfu/g to 10⁸ cfu/g soil. Mineralization of the PCP added to the reactor was demonstrated by chloride accumulation in solution. The soil-attached consortium produced in the reactor was inhibited by PCP concentrations exceeding 250 mg/l. This high level of tolerance was attributed to the beneficial effect of the soil particles. Once produced, the activated soil biomass remained active for 5 weeks at 20 °C and for up to 3 months when kept at 4 °C. The activated attached soil biomass produced in the completely mixed soil slurry bioreactor, as well as a PCP-acclimated flocculent biomass obtained from an air-lift immobilized-soil bioreactor, were used to stimulate the bioremediation of a PCP-impacted sandy soil, which had no indigenous PCP-degrading microorganisms. Bioaugmentation of this soil by the acclimated biomass resulted in a 99% reduction (from 400 mg/kg to 5 mg/kg in 130 days) in PCP concentration. The PCP degradation rates obtained with the activated soil biomass, produced either as a biomass attached to soil particles or as a flocculent biomass, were similar. Received: 31 March 1997 / Received revision: 22 July 1997 / Accepted: 25 August 1997