Short-term characterisation of climatic-environmental variables and microbial community diversity in a high-altitude Andean wetland (Salar de Huasco, Chile).

Microbial community structures are shaped by geochemical factors and their interactions with the lithosphere, hydrosphere, and atmosphere through the processes of chemical mobilisation and mineralisation. High-altitude wetlands and salt flats in the central Andes are characterised by pronounced physicochemical gradients and extreme climatic conditions, representing hotspots of microbial diversity. We here hypothesise about the existence of direct relationships between the local microbiology and the climate cyclicity variables based on meteorological and biogeochemical patterns that develop over a short time scale (five years). We have here analysed the interactions between hydrometeorological and biogeochemical variables and the microbial communities of the Salar de Huasco. These results were obtained by correlating 16S cDNA and DNA gene Illumina sequences with meteorological/satellite data collected both at monitoring stations and by remote sensing between the years 2015 and 2020. The precipitation levels and flooded areas (i.e., areas covered and/or saturated with permanent water) detected in the Salar de Huasco revealed a marked hydric cyclicity that correlated seasonally with intra-annual wet and dry seasons. Overall, at this site, wet periods occurred from December to April, and dry periods from May to November. Meteorological variables such as solar radiation, air temperature, relative humidity, wind speed, atmospheric pressure, and wind direction were well-defined, showing a potential association with the hydrogeology of the area, which is directly related to the wetlands' flooded areas. Finally, the microbial presence and potentially active microbial communities were determined through the sequencing of the 16S gene (DNA and cDNA, respectively), this were associated with climatic seasonality and spatially distributed physical and chemical heterogeneity. Other non-local inter-annual scale processes, such as El Niño-Southern Oscillation (ENSO) events, modify the physical and chemical context of the wetland, thus forming unique ecological niches in the Andean Mountains.

A Case Study for the Recovery of Authentic Microbial Ancient DNA from Soil Samples.

High Throughput DNA Sequencing (HTS) revolutionized the field of paleomicrobiology, leading to an explosive growth of microbial ancient DNA (aDNA) studies, especially from environmental samples. However, aDNA studies that examine environmental microbes routinely fail to authenticate aDNA, examine laboratory and environmental contamination, and control for biases introduced during sample processing. Here, we surveyed the available literature for environmental aDNA projects­from sample collection to data analysis­and assessed previous methodologies and approaches used in the published microbial aDNA studies. We then integrated these concepts into a case study, using shotgun metagenomics to examine methodological, technical, and analytical biases during an environmental aDNA study of soil microbes. Specifically, we compared the impact of five DNA extraction methods and eight bioinformatic pipelines on the recovery of microbial aDNA information in soil cores from extreme environments. Our results show that silica-based methods optimized for aDNA research recovered significantly more damaged and shorter reads (<100 bp) than a commercial kit or a phenol−chloroform method. Additionally, we described a stringent pipeline for data preprocessing, efficiently decreasing the representation of low-complexity and duplicated reads in our datasets and downstream analyses, reducing analytical biases in taxonomic classification.

A mesocosm study on bacteria-kelp interactions: Importance of nitrogen availability and kelp genetics.

Macroalgal holobiont studies involve understanding interactions between the host, its microbiota, and the environment. We analyzed the effect of bacteria-kelp interactions on phenotypic responses of two genetically distinct populations of giant kelp, Macrocystis pyrifera (north and south), exposed to different nitrogen (N) concentrations. In co-culture experiments with different N concentration treatments, we evaluated kelp growth responses and changes in three specific molecular markers associated with the N cycle, both in epiphytic bacteria (relative abundance of nrfA-gene: cytochrome c nitrite reductase) and macroalgae (expression of NR-gene: nitrate reductase; GluSyn-gene: glutamate synthase). Both kelp populations responded differently to N limitation, with M. pyrifera-south sporophytes having a lower specific growth rate (SGR) under N-limiting conditions than the northern population; M. pyrifera-north sporophytes showed no significant differences in SGR when exposed to low-N and high-N concentrations. This corresponded to a higher GluSyn-gene expression in the M. pyrifera-north sporophytes and the co-occurrence of specific nrfA bacterial taxa. These bacteria may increase ammonium availability under low-N concentrations, allowing M. pyrifera-north to optimize nutrient assimilation by increasing the expression of GluSyn. We conclude that bacteria-kelp interactions are important in enhancing kelp growth rates under low N availability, although this effect may be regulated by the genetic background of kelp populations.

Greenhouse gases and biogeochemical diel fluctuations in a high-altitude wetland.

The landscapes of high-altitude wetland ecosystems are characterized by different kinds of aquatic sites, including ponds holding conspicuous microbial life. Here, we examined a representative pond of the wetland landscape for dynamics of greenhouse gases, and their association with other relevant biogeochemical conditions including diel shifts of microbial communities' structure and activity over two consecutive days. Satellite image analysis indicates that the area of ponds cover 238 of 381.3 Ha (i.e., 62.4%), representing a significant landscape in this wetland. Solar radiation, wind velocity and temperature varied daily and between the days sampled, influencing the biogeochemical dynamics in the pond, shifting the pond reservoir of inorganic versus dissolved organic nitrogen/phosphorus bioavailability, between day 1 and day 2. Day 2 was characterized by high dissolved organic nitrogen/phosphorus and N2O accumulation. CH4 presented a positive excess showing maxima at hours of high radiation during both days. The microbial community in the sediment was diverse and enriched in keystone active groups potentially related with GHG recycling including bacteria and archaea, such as Cyanobacteria, Verrucomicrobia, Rhodobacterales and Nanoarchaeaota (Woesearchaeia). Archaea account for the microbial community composition changes between both days and for the secondary productivity in the water measured during day 2. The results indicate that an intense recycling of organic matter occurs in the pond systems and that the activity of the microbial community is correlated with the availability of nutrients. Together, the above results indicate a net sink of CO2 and N2O, which has also been reported for other natural and artificial ponds. Overall, our two-day fluctuation study in a representative pond of a high-altitude wetland aquatic landscape indicates the need to explore in more detail the short-term besides the long-term biogeochemical variability in arid ecosystems of the Andes plateau, where wetlands are hotspots of life currently under high anthropogenic pressure.

Virus and Potential Host Microbes from Viral-Enriched Metagenomic Characterization in the High-Altitude Wetland, Salar de Huasco, Chile.

Salar de Huasco is a wetland in the Andes mountains, located 3800 m above sea level at the Chilean Altiplano. Here we present a study aimed at characterizing the viral fraction and the microbial communities through metagenomic analysis. Two ponds (H0 and H3) were examined in November 2015. Water samples were processed using tangential flow filtration to obtain metagenomes from which the DNA fraction of the sample was amplified and sequenced (HiSeq system, Illumina). The ponds were characterized by freshwater and the viral-like particles to picoplankton ratio was 12.1 and 2.3 for H0 and H3, respectively. A great number of unassigned viral sequences were found in H0 (55.8%) and H3 (32.8%), followed by the family Fuselloviridae 20.8% (H0) and other less relatively abundant groups such as Microviridae (H0, 11.7% and H3, 3.3%) and Inoviridae (H3, 2.7%). The dominant viral sequences in both metagenomes belong to the order Caudovirales, with Siphoviridae being the most important family, especially in H3 (32.7%). The most important bacteria phyla were Proteobacteria, Bacteroidetes and Firmicutes in both sites, followed by Cyanobacteria (H0). Genes encoding lysogenic and lytic enzymes (i.e., recombinases and integrases) were found in H0 and H3, indicating a potential for active viral replication at the time of sampling; this was supported by the presence of viral metabolic auxiliary genes at both sites (e.g., cysteine hydrolase). In total, our study indicates a great novelty of viral groups, differences in taxonomic diversity and replication pathways between sites, which contribute to a better understanding of how viruses balance the cycling of energy and matter in this extreme environment.

Aquatic Thermal Reservoirs of Microbial Life in a Remote and Extreme High Andean Hydrothermal System.

Hydrothermal systems are ideal to understand how microbial communities cope with challenging conditions. Lirima, our study site, is a polyextreme, high-altitude, hydrothermal ecosystem located in the Chilean Andean highlands. Herein, we analyze the benthic communities of three nearby springs in a gradient of temperature (42-72 °C represented by stations P42, P53, and P72) and pH, and we characterize their microbial diversity by using bacteria 16S rRNA (V4) gene metabarcoding and 16S rRNA gene clone libraries (bacteria and archaea). Bacterial clone libraries of P42 and P53 springs showed that the community composition was mainly represented by phototrophic bacteria (Chlorobia, 3%, Cyanobacteria 3%, at P42; Chlorobia 5%, and Chloroflexi 5% at P53), Firmicutes (32% at P42 and 43% at P53) and Gammaproteobacteria (13% at P42 and 29% at P53). Furthermore, bacterial communities that were analyzed by 16S rRNA gene metabarcoding were characterized by an overall predominance of Chloroflexi in springs with lower temperatures (33% at P42), followed by Firmicutes in hotter springs (50% at P72). The archaeal diversity of P42 and P53 were represented by taxa belonging to Crenarchaeota, Diapherotrites, Nanoarchaeota, Hadesarchaeota, Thaumarchaeota, and Euryarchaeota. The microbial diversity of the Lirima hydrothermal system is represented by groups from deep branches of the tree of life, suggesting this ecosystem as a reservoir of primitive life and a key system to study the processes that shaped the evolution of the biosphere.

Active microbiome structure and its association with environmental factors and viruses at different aquatic sites of a high-altitude wetland.

Salar de Huasco is a high-altitude wetland characterized by a highly diverse microbial life adapted to extreme climatic and environmental conditions. Our study aims to determine active microbial community structure changes within different aquatic sites and its relationship with environmental factors and viruses as potential drivers of diversification in different aquatic areas of this ecosystem. In this study, bacteria and archaea composition (16S rRNA subunit pyrolibraries) and picoplankton and viral abundance were determined at ponds, springs and lagoon sites of the wetland during wet and dry seasons (February and July 2012, respectively). In general, mixosaline waters (1,400-51,000 µS/cm) usually found in ponds and lagoon presented higher picoplanktonic abundances compared to freshwater (<800 µS/cm) spring sites, ranging from 1.07 × 105 to 1.83 × 107  cells/ml. Viral abundance and viral to picoplankton ratio (VPR) also presented greater values at ponds compared to spring sites, reaching up to 4.78 × 108 viruses-like particles and up to 351 for VPR. In general, ponds hold a higher microbial diversity and complexity associated also with the presence of microbial mats compared with water sources or lagoon (Shannon index H' 2.6-3.9 vs. <2.0). A greater richness of archaea was also detected in ponds characterized by functional groups such as known methanogens and ammonia oxidizers, and uncultured groups. In total, our results indicate that among the different aquatic sites of the wetland, ponds presented a great microbial community diversification associated to a higher top-down control by viruses which may influence nutrient and greenhouse gases cycling.

Microbial community composition and trophic role along a marked salinity gradient in Laguna Puilar, Salar de Atacama, Chile.

The geological, hydrological and microbiological features of the Salar de Atacama, the most extensive evaporitic sedimentary basin in the Atacama Desert of northern Chile, have been extensively studied. In contrast, relatively little attention has been paid to the composition and roles of microbial communities in hypersaline lakes which are a unique feature in the Salar. In the present study biochemical, chemical and molecular biological tools were used to determine the composition and roles of microbial communities in water, microbial mats and sediments along a marked salinity gradient in Laguna Puilar which is located in the "Los Flamencos" National Reserve. The bacterial communities at the sampling sites were dominated by members of the phyla Bacteroidetes, Chloroflexi, Cyanobacteria and Proteobacteria. Stable isotope and fatty acid analyses revealed marked variability in the composition of microbial mats at different sampling sites both horizontally (at different sites) and vertically (in the different layers). The Laguna Puilar was shown to be a microbially dominated ecosystem in which more than 60% of the fatty acids at particular sites are of bacterial origin. Our pioneering studies also suggest that the energy budgets of avian consumers (three flamingo species) and dominant invertebrates (amphipods and gastropods) use minerals as a source of energy and nutrients. Overall, the results of this study support the view that the Salar de Atacama is a heterogeneous and fragile ecosystem where small changes in environmental conditions may alter the balance of microbial communities with possible consequences at different trophic levels.

Distribution of greenhouse gases in hyper-arid and arid areas of northern Chile and the contribution of the high altitude wetland microbiome (Salar de Huasco, Chile).

Northern Chile harbors different bioclimatic zones including hyper-arid and arid ecosystems and hotspots of microbial life, such as high altitude wetlands, which may contribute differentially to greenhouse gases (GHG) such as carbon dioxide (CO2), methane (CH4) and nitrous oxide (N2O). In this study, we explored ground level GHG distribution and the potential role of a wetland situated at 3800 m.a.s.l, and characterized by high solar radiation < 1600 W m-2, extreme temperature ranges (-12 to 24 °C) and wind stress (< 17 m s-1). The water source of the wetland is mainly groundwater springs, which generates streams and ponds surrounded by peatlands. These sites support a rich microbial aquatic life including diverse bacteria and archaea communities, which transiently form more complex structures, such as microbial mats. In this study, GHG were measured in the water and above ground level air at the wetland site and along an elevation gradient in different bioclimatic areas from arid to hyper-arid zones. The microbiome from the water and sediments was described by high-throughput sequencing 16S rRNA and rDNA genes. The results indicate that GHG at ground level were variable along the elevation gradient potentially associated with different bioclimatic zones, reaching high values at the high Andean steppe and variable but lower values in the Atacama Desert and at the wetland. The water areas of the wetland presented high concentrations of CH4 and CO2, particularly at the spring areas and in air bubbles below microbial mats. The microbial community was rich (> 40 phyla), including archaea and bacteria potentially active in the different matrices studied (water, sediments and mats). Functional microbial groups associated with GHG recycling were detected at low frequency, i.e., < 2.5% of total sequences. Our results indicate that hyper-arid and arid areas of northern Chile are sites of GHG exchange associated with various bioclimatic zones and particularly in aquatic areas of the wetland where this ecosystem could represent a net sink of N2O and a source for CH4 and CO2.

The activity of nitrifying microorganisms in a high-altitude Andean wetland.

High-altitude wetland holds freshwater springs, evaporitic ponds and lagoon with variable salinity and nutrients, potentially influencing the ecology of nitrifying communities. In this study, nitrifying microorganisms in Salar de Huasco (Chile) were surveyed to determine bacterial and archaeal contribution to ammonium (AO), nitrite oxidation (NO), ammonium uptake (AU) during wet and dry seasons. The activity signals from these groups were assessed by specific amoA-qPCR transcription, 15N tracer studies and addition of group specific inhibitor experiments for nitrifying microorganisms (N1-guanyl-1, 7-diaminoheptane [GC7]-archaeal specific and allylthiourea [ATU]-bacterial specific). Nitrifying communities, i.e. Nitrosopumilus, Nitrosospira, Nitrosomonas, Kuenenia and Nitrospira, were more frequent (∼0.25% of 16S rRNA sequences) at low salinity sites. Bacterial amoA-qPCR transcripts also increased at low salinity and along in situ ammonium increase observed between wet/dry seasons. Nutrient changes through time and 15N tracer experiments results showed that AO and NO were detected and peaked mainly at low salinity-high ammonium sites (<37 000 µS cm-1 and >0.3 µM), whereas AU was predominant at evaporitic sites. Our results indicate that salinity and ammonium affect the nitrifying communities that are potentially more active at low-salinity sites but persistent at saltier evaporitic areas of the wetland when ammonium is available.

Rhodobacter sp. Rb3, an aerobic anoxygenic phototroph which thrives in the polyextreme ecosystem of the Salar de Huasco, in the Chilean Altiplano.

The Salar de Huasco is an evaporitic basin located in the Chilean Altiplano, which presents extreme environmental conditions for life, i.e. high altitude (3800 m.a.s.l.), negative water balance, a wide salinity range, high daily temperature changes and the occurrence of the highest registered solar radiation on the planet (> 1200 W m-2). This ecosystem is considered as a natural laboratory to understand different adaptations of microorganisms to extreme conditions. Rhodobacter, an anoxygenic aerobic phototrophic bacterial genus, represents one of the most abundant groups reported based on taxonomic diversity surveys in this ecosystem. The bacterial mat isolate Rhodobacter sp. strain Rb3 was used to study adaptation mechanisms to stress-inducing factors potentially explaining its success in a polyextreme ecosystem. We found that the Rhodobacter sp. Rb3 genome was characterized by a high abundance of genes involved in stress tolerance and adaptation strategies, among which DNA repair and oxidative stress were the most conspicuous. Moreover, many other molecular mechanisms associated with oxidative stress, photooxidation and antioxidants; DNA repair and protection; motility, chemotaxis and biofilm synthesis; osmotic stress, metal, metalloid and toxic anions resistance; antimicrobial resistance and multidrug pumps; sporulation; cold shock and heat shock stress; mobile genetic elements and toxin-antitoxin system were detected and identified as potential survival mechanism features in Rhodobacter sp. Rb3. In total, these results reveal a wide set of strategies used by the isolate to adapt and thrive under environmental stress conditions as a model of polyextreme environmental resistome.

Bacterial Survival under Extreme UV Radiation: A Comparative Proteomics Study of Rhodobacter sp., Isolated from High Altitude Wetlands in Chile.

Salar de Huasco, defined as a polyextreme environment, is a high altitude saline wetland in the Chilean Altiplano (3800 m.a.s.l.), permanently exposed to the highest solar radiation doses registered in the world. We present here the first comparative proteomics study of a photoheterotrophic bacterium, Rhodobacter sp., isolated from this remote and hostile habitat. We developed an innovative experimental approach using different sources of radiation (in situ sunlight and UVB lamps), cut-off filters (Mylar, Lee filters) and a high-throughput, label-free quantitative proteomics method to comprehensively analyze the effect of seven spectral bands on protein regulation. A hierarchical cluster analysis of 40 common proteins revealed that all conditions containing the most damaging UVB radiation induced similar pattern of protein regulation compared with UVA and visible light spectral bands. Moreover, it appeared that the cellular adaptation of Rhodobacter sp. to osmotic stress encountered in the hypersaline environment from which it was originally isolated, might further a higher resistance to damaging UV radiation. Indeed, proteins involved in the synthesis and transport of key osmoprotectants, such as glycine betaine and inositol, were found in very high abundance under UV radiation compared to the dark control, suggesting the function of osmolytes as efficient reactive oxygen scavengers. Our study also revealed a RecA-independent response and a tightly regulated network of protein quality control involving proteases and chaperones to selectively degrade misfolded and/or damaged proteins.

A Functional Perspective Analysis of Macroalgae and Epiphytic Bacterial Community Interaction.

Macroalgae are photosynthetic, multicellular, sessile eukaryotic organisms that offer diverse habitats for the colonization of epiphytic bacteria, therefore establishing biological interactions of diverse complexity. This review focusses on the interactions between macroalgae and their Epiphytic Bacterial Community (EBC); the main aims are to ascertain whether (1) the epiphytic bacterial groups differ at the phylum and genus levels of the macroalgae; (2) the methodologies used so far to study these microorganisms are related in any way to eventual variations of the EBCs on macroalgae; and (3) the EBC of macroalgae has a functional means rather a simple taxonomic grouping. Results showed firstly the taxonomic grouping of macroalgae does not explain the composition and structure of the EBCs. Secondly, the methodology used is important for describing EBCs; and thirdly, multiple bacteria can have the same function and thus to describe the functionality of EBCs it is important to recognize host-specific and generalist bacteria. We recommend the incorporation of a complementary approach between the taxonomic composition and the functional composition analyzes of EBCs, as well as the use of methodological tools that allow analysis of interactions between the EBCs and their hosts, based on the "holobiont" concept.

Bacterial Active Community Cycling in Response to Solar Radiation and Their Influence on Nutrient Changes in a High-Altitude Wetland.

Microbial communities inhabiting high-altitude spring ecosystems are subjected to extreme changes in solar irradiance and temperature throughout the diel cycle. Here, using 16S rRNA gene tag pyrosequencing (cDNA) we determined the composition of actively transcribing bacteria from spring waters experimentally exposed through the day (morning, noon, and afternoon) to variable levels of solar radiation and light quality, and evaluated their influence on nutrient recycling. Solar irradiance, temperature, and changes in nutrient dynamics were associated with changes in the active bacterial community structure, predominantly by Cyanobacteria, Verrucomicrobia, Proteobacteria, and 35 other Phyla, including the recently described Candidate Phyla Radiation (e.g., Parcubacteria, Gracilibacteria, OP3, TM6, SR1). Diversity increased at noon, when the highest irradiances were measured (3.3-3.9 H', 1125 W m-2) compared to morning and afternoon (0.6-2.8 H'). This shift was associated with a decrease in the contribution to pyrolibraries by Cyanobacteria and an increase of Proteobacteria and other initially low frequently and rare bacteria phyla (< 0.5%) in the pyrolibraries. A potential increase in the activity of Cyanobacteria and other phototrophic groups, e.g., Rhodobacterales, was observed and associated with UVR, suggesting the presence of photo-activated repair mechanisms to resist high levels of solar radiation. In addition, the percentage contribution of cyanobacterial sequences in the afternoon was similar to those recorded in the morning. The shifts in the contribution by Cyanobacteria also influenced the rate of change in nitrate, nitrite, and phosphate, highlighted by a high level of nitrate accumulation during hours of high radiation and temperature associated with nitrifying bacteria activity. We did not detect ammonia or nitrite oxidizing bacteria in situ, but both functional groups (Nitrosomona and Nitrospira) appeared mainly in pyrolibraries generated from dark incubations. In total, our results reveal that both the structure and the diversity of the active bacteria community was extremely dynamic through the day, and showed marked shifts in composition that influenced nutrient recycling, highlighting how abiotic variation affects potential ecosystem functioning.

Microbial Activity Response to Solar Radiation across Contrasting Environmental Conditions in Salar de Huasco, Northern Chilean Altiplano.

In high altitude environments, extreme levels of solar radiation and important differences of ionic concentrations over narrow spatial scales may modulate microbial activity. In Salar de Huasco, a high-altitude wetland in the Andean mountains, the high diversity of microbial communities has been characterized and associated with strong environmental variability. Communities that differed in light history and environmental conditions, such as nutrient concentrations and salinity from different spatial locations, were assessed for bacterial secondary production (BSP, 3H-leucine incorporation) response from short-term exposures to solar radiation. We sampled during austral spring seven stations categorized as: (a) source stations, with recently emerged groundwater (no-previous solar exposure); (b) stream running water stations; (c) stations connected to source waters but far downstream from source points; and (d) isolated ponds disconnected from ground sources or streams with a longer isolation and solar exposure history. Very high values of 0.25 µE m-2 s-1, 72 W m-2 and 12 W m-2 were measured for PAR, UVA, and UVB incident solar radiation, respectively. The environmental factors measured formed two groups of stations reflected by principal component analyses (near to groundwater sources and isolated systems) where isolated ponds had the highest BSP and microbial abundance (35 microalgae taxa, picoeukaryotes, nanoflagellates, and bacteria) plus higher salinities and PO43- concentrations. BSP short-term response (4 h) to solar radiation was measured by 3H-leucine incorporation under four different solar conditions: full sun, no UVB, PAR, and dark. Microbial communities established in waters with the longest surface exposure (e.g., isolated ponds) had the lowest BSP response to solar radiation treatments, and thus were likely best adapted to solar radiation exposure contrary to ground source waters. These results support our light history (solar exposure) hypothesis where the more isolated the community is from ground water sources, the better adapted it is to solar radiation. We suggest that factors other than solar radiation (e.g., salinity, PO43-, NO3-) are also important in determining microbial productivity in heterogeneous environments such as the Salar de Huasco.

Changes in epiphytic bacterial communities of intertidal seaweeds modulated by host, temporality, and copper enrichment.

This study reports on the factors involved in regulating the composition and structure of bacterial communities epiphytic on intertidal macroalgae, exploring their temporal variability and the role of copper pollution. Culture-independent, molecular approaches were chosen for this purpose and three host species were used as models: the ephemeral Ulva spp. (Chlorophyceae) and Scytosiphon lomentaria (Phaeophyceae) and the long-living Lessonia nigrescens (Phaeophyceae). The algae were collected from two coastal areas in Northern Chile, where the main contrast was the concentration of copper in the seawater column resulting from copper-mine waste disposals. We found a clear and strong effect in the structure of the bacterial communities associated with the algal species serving as host. The structure of the bacterial communities also varied through time. The effect of copper on the structure of the epiphytic bacterial communities was significant in Ulva spp., but not on L. nigrescens. The use of 16S rRNA gene library analysis to compare bacterial communities in Ulva revealed that they were composed of five phyla and six classes, with approximately 35 bacterial species, dominated by members of Bacteroidetes (Cytophaga-Flavobacteria-Bacteroides) and α-Proteobacteria, in both non-polluted and polluted sites. Less common groups, such as the Verrucomicrobiae, were exclusively found in polluted sites. This work shows that the structure of bacterial communities epiphytic on macroalgae is hierarchically determined by algal species > temporal changes > copper levels.

Changes in bacterial community structure associated with coastal copper enrichment.

Marine bacterial communities isolated from the water column, sediment, the rock surface, and the green seaweed Ulva compressa were studied in an intertidal ecosystem. The study area included a coastal zone chronically affected by copper mine waste disposals. Bacterial community composition was analyzed by terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes, and multivariate analyses of T-RFLP data sets were used for comparisons. Results showed that diversity and richness indexes were not able to detect differences among compartments. However, comparisons within the same compartment clearly showed that copper enrichment was associated with changes in the composition of the bacterial communities and revealed that the magnitude of the effect depends on the compartment being considered. In this context, communities from sediments appeared as the most affected by copper enrichment. The present study also demonstrated that intertidal bacterial communities were dominated by Gammaproteobacteria, Firmicutes, and Actinobacteria and the changes in these communities were mainly due to changes in their relative abundances.