The effect of substratum type,orientation and depth on the development of bacterial deep-sea biofilm communities grown on artificial substrata deployed in the Eastern Mediterranean

An increasing number of deep-sea studies have highlighted the importance of deep-sea biofouling, especially in relation to the protection of deep-sea instruments. In this study, the microbial communities developed on different substrata (titanium, aluminum, limestone, shale and neutrino telescope glass) exposed for 155 days at different depths (1500 m, 2500 m, 3500 m and 4500 m) and positions (vertical and horizontal) in the Eastern Mediterranean Deep Sea were compared. Replicated biofilm samples were analyzed using a Terminal Restriction Fragment Length Polymorphisms (T-RFLP) method. The restriction enzymes CfoI and RsaI produced similar total numbers (94, 93) of different T-RFLP peaks (T-RFs) along the vertical transect. In contrast, the mean total T-RF number between each sample according to substratum type and depth was higher in more samples when CfoI was used. The total species richness (S) of the bacterial communities differed significantly between the substrata, and depended on the orientation of each substratum within one depth and throughout the water column (ANOVA). T-RFLP analyses using the Jaccard similarity index showed that within one depth layer, the composition of microbial communities on different substrata was different and highly altered among communities developed on the same substratum but exposed to fouling at different depths. Based on Multidimensional Scaling Analyses (MDS), the study suggests that depth plays an important role in the composition of deep-sea biofouling communities, while substratum type and orientation of substrata throughout the water column are less important. To the authors’ knowledge, this is the first study of biofilm development in deep waters, in relation to the effects of substratum type, orientation and depth.     

The effect of surface colour on the formation of marine micro and macrofouling communities

The effect of substratum colour on the formation of micro- and macro fouling communities was investigated. Acrylic tiles, painted either black or white were covered with transparent sheets in order to ensure similar surface properties. All substrata were exposed to biofouling at 1?m depth for 40?d in the Marina Bandar al Rowdha (Muscat, Sea of Oman). Studies were conducted in 2010 over a time course of 5, 10 and 20?d, and in 2012 samples were collected at 7, 14 and 21?d. The densities of bacteria on the black and white substrata were similar with the exception of day 10, when the black substrata had a higher abundance than white ones. Pyrosequencing via 454 of 16S rRNA genes of bacteria from white and black substrata revealed that Alphaproteobacteria and Firmicutes were the dominant groups. SIMPER analysis demonstrated that bacterial phylotypes (uncultured Gammaproteobacteria, Actibacter, Gaetbulicola, Thalassobius and Silicibacter) and the diatoms (Navicula directa, Navicula sp. and Nitzschia sp.) contributed to the dissimilarities between communities developed on white and black substrata. At day 20, the highest amount of chlorophyll a was recorded in biofilms developed on black substrata. SIMPER analysis showed that Folliculina sp., Ulva sp. and Balanus amphitrite were the major macro fouling species that contributed to the dissimilarities between the communities formed on white and black substrata. Higher densities of these species were observed on black tiles. The results emphasise the effect of substratum colour on the formation of micro and macro fouling communities; substratum colour should to be taken into account in future studies.     

Mineralogy Influences Structure and Diversity of Bacterial Communities Associated with Geological Substrata in a Pristine Aquifer

Our understanding of mineralogical influences on subsurface microbial community structure and diversity has been difficult to assess due to difficulties in isolating this variable from others in the subsurface environment. In this study, biofilm coupons were used to isolate specific geological substrata from the surrounding geological matrix during colonization by microorganisms suspended in the surrounding groundwater for an 8-week period. Upon retrieval, the structure and diversity of the microbial community associated with each type of substratum was evaluated using 16S rDNA-based terminal-restriction fragment length polymorphism (T-RFLP). Phylogenetic affiliations of the populations associated with each type of substratum were established based on sequence analysis of near full-length 16S rDNA obtained through construction of a clone library. Hematite, quartz, and saprolite each harbored a community dominated by members of the division Proteobacteria (>67% of community). However, the different substrata selected for different subdivisions of bacteria within the Proteobacteria. After accounting for the influence exerted by substratum type on recovery of DNA from the attached populations, both phylogenetic data and Jaccard and Bray–Curtis similarity indices derived from terminal-restriction fragment (T-RF) profiles suggested a strong mineralogical influence on the structure and composition of the solid phase-associated community. The results suggest that mineralogical heterogeneity influences microbial community structure and diversity in pristine aquifers. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized authors.     

Bacterial communities in the initial stage of marine biofilm formation on artificial surfaces

Succession of bacterial communities during the first 36 h of biofilm formation in coastal water was investigated at 3 approximately 15 h intervals. Three kinds of surfaces (i.e., acryl, glass, and steel substratum) were submerged in situ at Sacheon harbor, Korea. Biofilms were harvested by scraping the surfaces, and the compositions of bacterial communities were analyzed by terminal restriction fragment length polymorphism (T-RFLP), and cloning and sequencing of 16S rRNA genes. While community structure based on T-RFLP analysis showed slight differences by substratum, dramatic changes were commonly observed for all substrata between 9 and 24 h. Identification of major populations by 16S rRNA gene sequences indicated that gamma-Proteobacteria (Pseudomonas, Acinetobacter, Alteromonas, and uncultured gamma-Proteobacteria) were predominant in the community during 0 approximately 9 h, while the ratio of alpha-Proteobacteria (Loktanella, Methylobacterium, Pelagibacter, and uncultured alpha-Proteobacteria) increased 2.6 approximately 4.8 folds during 24 approximately 36 h of the biofilm formation, emerging as the most predominant group. Previously, alpha-Proteobacteria were recognized as the pioneering organisms in marine biofilm formation. However, results of this study, which revealed the bacterial succession with finer temporal resolution, indicated some species of gamma-Proteobacteria were more important as the pioneering population. Measures to control pioneering activities of these species can be useful in prevention of marine biofilm formation.     

Effects of environment on electric field detection by small spotted catshark Scyliorhinus canicula (L.)

This study evaluated the influence of environment (substratum type and depth) on the electroreception capabilities of small spotted catsharks Scyliorhinus canicula in response to prey-simulating electric fields. In experiments where electric fields (applied current 15 μA) were presented beneath different substrata (sand, pebbles, rocks and control) it was found that search effort was not different between substrata or S. canicula sexes, however, both rates of turning and biting towards active electrodes were decreased over pebbles and rocks compared with sand and the control (no substratum). There was no significant effect of sex on turn and bite rates over any substrata. Electric fields were then presented beneath different depths of sand to examine the depth-limits of fish electroreception. Turn and bite rates were significantly lower at depths below 10 mm, with no bites towards electrodes made when they were >30 mm depth. Search effort was not found to be different between different burial depth treatments or between sexes. These results indicate substratum type and depth influences the ability of S. canicula to detect prey-simulating electric fields. This variation in electroreceptive performance may influence space use of sharks.     

Impact of Substratum Surface on Microbial Community Structure and Treatment Performance in Biological Aerated Filters

The impact of substratum surface property change on biofilm community structure was investigated using laboratory biological aerated filter (BAF) reactors and molecular microbial community analysis. Two substratum surfaces that differed in surface properties were created via surface coating and used to develop biofilms in test (modified surface) and control (original surface) BAF reactors. Microbial community analysis by 16S rRNA gene-based PCR-denaturing gradient gel electrophoresis (DGGE) showed that the surface property change consistently resulted in distinct profiles of microbial populations during replicate reactor start-ups. Pyrosequencing of the bar-coded 16S rRNA gene amplicons surveyed more than 90% of the microbial diversity in the microbial communities and identified 72 unique bacterial species within 19 bacterial orders. Among the 19 orders of bacteria detected, Burkholderiales and Rhodocyclales of the Betaproteobacteria class were numerically dominant and accounted for 90.5 to 97.4% of the sequence reads, and their relative abundances in the test and control BAF reactors were different in consistent patterns during the two reactor start-ups. Three of the five dominant bacterial species also showed consistent relative abundance changes between the test and control BAF reactors. The different biofilm microbial communities led to different treatment efficiencies, with consistently higher total organic carbon (TOC) removal in the test reactor than in the control reactor. Further understanding of how surface properties affect biofilm microbial communities and functional performance would enable the rational design of new generations of substrata for the improvement of biofilm-based biological treatment processes.     

Baseline characterization of aerobic hydrocarbon degrading microbial communities in deep-sea sediments of the Great Australian Bight,Australia

Exploratory drilling for deep-sea oil and gas resources is planned for the Great Australian Bight (GAB). There is scant knowledge of the region's benthic ecosystems and no baseline information of the region's indigenous oil degrading bacteria. To address this knowledge gap, we used next generation sequencing (NGS) of three marker genes (alkB, c23o and pmoA) to detect and characterize the microbial communities capable of aerobic hydrocarbon degradation. Unique, highly novel microbial communities capable of degrading hydrocarbons occur in surface sediments at depths between 200 and 2800 m. Clustering at 97% demonstrated differences in community structure with depth, changing most markedly between 400 and 1000 m depth on the continental slope, and identified putative functional ‘ecotypes’ related to depth. Observed differences in community structure showed strong correlations with temperature, other physicochemical properties of the overlying water column and are further modulated by differences in sediment grain size. This study provides important baseline data on hydrocarbon degrading microbial communities prior to the start of petroleum resource extraction. Our data will inform future ecological monitoring of the GAB deep-sea ecosystem.     

Biofilm diatom community structure: influence of temporal and substratum variability

Diatoms, which are early autotrophic colonisers, are an important constituent of the biofouling community in the marine environment. The effects of substratum and temporal variations on the fouling diatom community structure in a monsoon-influenced tropical estuary were studied. Fibreglass and glass coupons were exposed every month for a period of 4 days and the diatom population sampled at 24 h intervals, over a period of 14 months. The planktonic diatom community structure differed from the biofilm community. Pennate diatoms dominated the biofilms whilst centric diatoms were dominant in the water column. Among the biofilm diatoms, species belonging to the genera Navicula, Amphora, Nitzschia, Pleurosigma and Thalassionema were dominant. On certain occasions, the influence of planktonic blooms was also seen on the biofilm community. A comparative study of biofilms formed on the two substrata revealed significant differences in density and diversity. However species composition was almost constant. In addition to substratum variations, the biofilm diatom community structure also showed significant seasonal variations, which were attributed to physico-chemical and biological changes in both the water and substratum. Temporal variations in the tychopelagic diatoms of the water were also observed to exert an influence on the biofilm diatom community. Variations in diatom communities may determine the functional ecosystem of the benthic environment.     

Profile of Microbial Community Structure and Presence of Endolithic Microorganisms Inside a Deep-Sea Rock

Microbial community structure in the depth profile of a deep-sea sedimentary rock collected from the Sanriku Escarpment in the Japan Trench at a depth of 6337 m were analyzed using enrichment culture methods and culture-independent molecular phylo-genetic techniques. The rock was subsampled at four depths (S1 to S4; from the surface to the inside), and carbon concentrations and colony-forming units (CFU) were determined under several culture conditions. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified 16S rRNA gene (rDNA) sequences indicated that a shift in bacterial and archaeal ribotype structures occurred in the sections at different depths from the surface. rDNA clone analysis revealed a significant change in microbial rDNA community structure. Bacterial community rDNA in sections S1 to S3 consisted of typical marine bacteria mainly members of the f and n -subclass of Proteobacteria, while the inner most section, S4, contained rDNA signatures for the g -subclass of Proteobacteria and the High G + C Gram-Positive Group. Major archaeal rDNA clones shifted from Marine Group I (S1) to Thermococcales (S2-S4). The changes in bacterial and archaeal rDNA community structure indicated the possible infiltration of seawater and microorganisms into the rock and strongly suggested the isolation of endolithic microbial communities over the geological history of the rock.     

A picture on the wall: innovative mapping reveals cold-water coral refuge in submarine canyon

Cold-water corals are azooxanthellate species found throughout the ocean at water depths down to 5000 m. They occur in patches, reefs or large mound structures up to 380 m high, and as ecosystem engineers create important habitats for a diverse fauna. However, the majority of these habitats are now within reach of deep-sea bottom trawling. Many have been severely damaged or are under threat, despite recent protection initiatives. Here we present a cold-water coral habitat type that so far has been overlooked--quite literally--and that has received minimal impact from human activities. Vertical and overhanging cliffs in deep-sea canyons, revealed using an innovative approach to marine habitat mapping, are shown to provide the perfect substratum for extensive cold-water coral-based communities. Typical canyon-related processes, including locally enhanced internal tides and focussed downslope organic carbon transport, provide favourable environmental conditions (current regime, food input) to sustain the communities, even outside the optimal depth and density envelopes reported elsewhere in the NE Atlantic. Our findings show that deep-sea canyons can form natural refuges for faunal communities sensitive to anthropogenic disturbance, and have the potential to fulfil the crucial role of larval sources for the recolonisation of damaged sites elsewhere on the margin.     

Biofilm diatom community structure: Influence of temporal and substratum variability

Abstract

Diatoms, which are early autotrophic colonisers, are an important constituent of the biofouling community in the marine environment. The effects of substratum and temporal variations on the fouling diatom community structure in a monsoon-influenced tropical estuary were studied. Fibreglass and glass coupons were exposed every month for a period of 4 days and the diatom population sampled at 24 h intervals, over a period of 14 months. The planktonic diatom community structure differed from the biofilm community. Pennate diatoms dominated the biofilms whilst centric diatoms were dominant in the water column. Among the biofilm diatoms, species belonging to the genera Navicula, Amphora, Nitzschia, Pleurosigma and Thalassionema were dominant. On certain occasions, the influence of planktonic blooms was also seen on the biofilm community. A comparative study of biofilms formed on the two substrata revealed significant differences in density and diversity. However species composition was almost constant. In addition to substratum variations, the biofilm diatom community structure also showed significant seasonal variations, which were attributed to physico-chemical and biological changes in both the water and substratum. Temporal variations in the tychopelagic diatoms of the water were also observed to exert an influence on the biofilm diatom community. Variations in diatom communities may determine the functional ecosystem of the benthic environment.     

Using Species-Area Relationships to Inform Baseline Conservation Targets for the Deep North East Atlantic

Demands on the resources of the deep-sea have increased in recent years. Consequently, the need to create and implement a comprehensive network of Marine Protected Areas (MPAs) to help manage and protect these resources has become a global political priority. Efforts are currently underway to implement MPA networks in the deep North East Atlantic. To ensure these networks are effective, it is essential that baseline information be available to inform the conservation planning process. Using empirical data, we calculated conservation targets for sessile benthic invertebrates in the deep North East Atlantic for consideration during the planning process. We assessed Species-Area Relationships across two depth bands (200–1100 m and 1100–1800 m) and nine substrata. Conservation targets were predicted for each substratum within each depth band using z-values obtained from fitting a power model to the Species-Area Relationships of observed and estimated species richness (Chao1). Results suggest an MPA network incorporating 10% of the North East Atlantic’s deep-sea area would protect approximately 58% and 49% of sessile benthic species for the depth bands 200–1100 m and 1100–1800 m, respectively. Species richness was shown to vary with substratum type indicating that, along with depth, substratum information needs to be incorporated into the conservation planning process to ensure the most effective MPA network is implemented in the deep North East Atlantic.     

LINKING BENTHIC ALGAL BIOMASS TO STREAM SUBSTRATUM TOPOGRAPHY1

The physical properties of substrata significantly influence benthic algal development. We explored the relationships among substratum surface texture and orientation with epilithic microphytobenthic biomass accumulation at the whole‐substratum and micrometer scales. Unglazed clay tiles set at three orientations (horizontal, vertical, and 45°), and six substrata of varying surface roughness were deployed in a prairie stream for 3 weeks. Substrata were analyzed for loosely attached, adnate, and total benthic algal biomass as chl a, and confocal laser scanning microscopy was used to measure substrata microtopography (i.e., roughness, microscale slope angles, and three‐dimensional surface area). At the whole‐substratum level, vertical substrata collected significantly (P < 0.05) less algal biomass, averaging 34% and 36% less than horizontal and 45° substrata, respectively. Benthic algal biomass was also significantly less on smoother surfaces; glass averaged 29% less biomass than stream rocks. At the microscale level, benthic algal biomass was the greatest at intermediate values, peaking at a mean roughness of approximately 17 μm, a mean microscale slope of 50°, and a projected/areal surface area ratio of 2:1. The proportion of adnate algae increased with surface roughness (26% and 67% for glass and brick, respectively), suggesting that substratum type changes the efficiency of algal removal by brushing. Individual substrata and microsubstrata characteristics can have a strong effect on benthic algae development and potentially affect reach scale algal variability as mediated by geomorphology.     

Shifts in archaeal communities associated with lithological and geochemical variations in subsurface Cretaceous rock

Subsurface microbial community structure in relation to geochemical gradients and lithology was investigated using a combination of molecular phylogenetic and geochemical analyses. Discreet groundwater and substratum samples were obtained from depths ranging from 182 to 190 m beneath the surface at approximately 10-cm intervals using a multilevel sampler (MLS) that straddled Cretaceous shale and sandstone formations at a site in the southern San Juan Basin in New Mexico. DNA and RNA were extracted directly from quartzite sand substratum loaded into individual cells of the MLS and colonized in situ. Polymerase chain reaction (PCR)-mediated T-RFLP analysis of archaeal rRNA genes (rDNA) in conjunction with partial sequencing analysis of archaeal rDNA libraries and quantitative RNA hybridization with oligonucleotide probes were used to probe community structure and function. Although total microbial populations remained relatively constant over the entire depth interval sampled, significant shifts in archaeal populations, predominantly methanogens, were observed. These shifts coincided with the geochemical transition from relatively high methane (26 mM), low sulphate (< 3 mg l(-1)) conditions in the region adjacent to the organic matter-rich shale to relatively low-methane (< 0.5 mM), high-sulphate (48 mg l(-1)) conditions in the organic-poor sandstone beneath the shale. These results indicated that active, phylogenetically diverse archaeal communities were present in the subsurface Cretaceous rock environment at this site and that major archaeal clades shifted dramatically over scales of tens of centimetres, corresponding to changes in the lithology and geochemical gradients.     

Preliminary characterization of thin biofilms by optical microscopy

A simple non‐invasive technique has been used that employs conventional optical microscopy and a glass flow cell to observe biofilms formed on opaque thin substrata. The technique allows the roughness of the biofilm and the substratum to be evaluated, and the biofilm thickness to be easily measured. The biofilm density may be quantified through colour gradients. In addition, some details of biofilm growth processes like the formation of water channels and pores, and interactions between planktonic and sessile cells can be visualized. Results related to the development of thin biofilms and their response to the environment under different conditions are reported. Pure and mixed microbial cultures and different solid substrata were assessed.     

Depth distributions of coral reef fishes: the influence of microhabitat structure,settlement, and post-settlement processes

Many coral reef fishes have restricted depth ranges that are established at settlement or soon after, but the factors limiting these distributions are largely unknown. This study examines whether the availability of microhabitats (reef substrata) explains depth limits, and evaluates whether juvenile growth and survival are lower beyond these limits. Depth-stratified surveys of reef fishes at Kimbe Bay (Papua New Guinea) showed that the abundance of new settlers and the cover of coral substrata differed significantly among depths. A field experiment investigated whether settling coral reef fishes preferred particular depths, and whether these depth preferences were dependent on microhabitat. Small patch reefs composed of identical coral substrata were set up at five depths (3, 6, 10, 15 and 20 m), and settlement patterns were compared to those on unmanipulated reef habitat at the same five depths. For all species, settlement on patch reefs differed significantly among depths despite uniform substratum composition. For four of the six species tested, depth-related settlement patterns on unmanipulated habitat and on patch reefs did not differ, while for the other two, depth ranges were greater on the patch reefs than on unmanipulated habitat. A second experiment examined whether depth preferences reflected variation in growth and survival when microhabitat was similar. Newly settled individuals of Chrysiptera parasema and Dascyllus melanurus were placed, separately, on patch reefs at five depths (as above) and their survival and growth monitored. D. melanurus, which is restricted to shallow depths, had highest survival and growth at the shallowest depth. Depth did not affect either survival or growth of C. parasema, which has a broader depth range than D. melanurus (between 6 and 15 m). This suggests that the fitness costs potentially incurred by settling outside a preferred depth range may depend on the strength of the depth preference.     

Influence of substratum on the variability of benthic biofilm stable isotope signatures: implications for energy flow to a primary consumer

Benthic biofilms have been identified using stable isotope analysis (SIA) as an important resource supporting many freshwater food webs. However, biofilm δ¹³C signatures are highly variable in freshwaters, which may hamper our understanding of energy flow through food webs in these systems. There has been little consideration of the influence that substratum may have on biofilm δ¹³C signature variability and energy flows to primary consumers. We investigated the effect of organic and inorganic substrata on biofilm dynamics by examining: (1) temporal variability of biofilm stable isotope (δ¹³C, δ¹⁵N) signatures on allochthonous leaf-litter (Eucalyptus camaldulensis) and cobble substrata over 12 months in a lowland river in south-eastern Australia; and (2) the effect of substrata on biofilm energy flows to a grazer snail, Physa acuta (Gastropoda: Physidae), using SIA and ecological stoichiometry in a laboratory experiment. The temporal study indicated that cobble biofilm varied significantly in δ¹³C signature during the 12 months (up to 11‰), whereas the δ¹³C signature of leaf biofilm was less variable (less than 2‰). In contrast, biofilm δ¹⁵N signatures varied temporally on both cobble (2.6‰) and leaf (1‰) substrata. This suggests that leaf biofilm was more reliant on leaf tissue for carbon and therefore less limited by carbon supply than cobble biofilm whereas for nitrogen biofilm on both substrata was reliant on external sources. In the laboratory experiment, snails fed leaf biofilm reflected more of an allochthonous δ¹³C signature than cobble biofilm fed snails, suggesting assimilation of leaf carbon via the heterotrophic microbial community within the biofilm. Snails grew largest on cobble biofilm, which had lower C:N ratios than leaf biofilm. Our results demonstrate that the type of substratum can influence the temporal variability of biofilm δ¹³C signatures and energy flow to primary consumers.     

Cross-depth analysis of marine bacterial networks suggests downward propagation of temporal changes

Interactions among microbes and stratification across depths are both believed to be important drivers of microbial communities, though little is known about how microbial associations differ between and across depths. We have monitored the free-living microbial community at the San Pedro Ocean Time-series station, monthly, for a decade, at five different depths: 5 m, the deep chlorophyll maximum layer, 150 m, 500 m and 890 m (just above the sea floor). Here, we introduce microbial association networks that combine data from multiple ocean depths to investigate both within- and between-depth relationships, sometimes time-lagged, among microbes and environmental parameters. The euphotic zone, deep chlorophyll maximum and 890 m depth each contain two negatively correlated ‘modules'' (groups of many inter-correlated bacteria and environmental conditions) suggesting regular transitions between two contrasting environmental states. Two-thirds of pairwise correlations of bacterial taxa between depths lagged such that changes in the abundance of deeper organisms followed changes in shallower organisms. Taken in conjunction with previous observations of seasonality at 890 m, these trends suggest that planktonic microbial communities throughout the water column are linked to environmental conditions and/or microbial communities in overlying waters. Poorly understood groups including Marine Group A, Nitrospina and AEGEAN-169 clades contained taxa that showed diverse association patterns, suggesting these groups contain multiple ecological species, each shaped by different factors, which we have started to delineate. These observations build upon previous work at this location, lending further credence to the hypothesis that sinking particles and vertically migrating animals transport materials that significantly shape the time-varying patterns of microbial community composition.