The effect of sampling frequency and spatial and temporal variation in the density of fecal indicator bacteria on the assessment of coastal bathing water quality.

The time and location of sampling as well as the number of samples per season can influence a reliable assessment of bathing water quality. In this study, we investigated the spatio-temporal variation of fecal indicator bacteria (FIB) density and the effects of FIB variability and sampling frequency on the assessment of a single sample and the annual and final assessment of coastal bathing water quality. Increasing the number of samples from 10 to 20 per bathing season had a significant impact on bathing water quality assessment at sites where water quality fluctuations had previously been observed, resulting in a change in water quality to a lower category in 36 % of annual and 54 % of final assessments, suggesting that the minimum number of samples per season should be increased at such sites. Increasing the number of samples at sites assessed as excellent over a longer period had no impact on water quality assessment. Spatial and temporal variations in FIB density were significant at a considerable number of sites both in the single sample assessment and in the annual and final assessment. Bathing water quality was spatially unrepresentative at a quarter of the sites analyzed and temporally unrepresentative at a fifth, as there was at least one additional point with a lower bathing water quality than the official sampling point or the bathing water quality was lower in the afternoon than in the morning. When revising the current recreational water quality regulations, the impact of sampling frequency of and of spatio-temporal variation of FIB density on the relevance of bathing water quality assessment at sites subjected to pollution needs to be seriously considered.

Ecology of aerobic anoxygenic phototrophs on a fine-scale taxonomic resolution in Adriatic Sea unravelled by unsupervised neural network.

BACKGROUND: Aerobic anoxygenic phototrophs are metabolically highly active, diverse and widespread polyphyletic members of bacterioplankton whose photoheterotrophic capabilities shifted the paradigm about simplicity of the microbial food chain. Despite their considerable contribution to the transformation of organic matter in marine environments, relatively little is still known about their community structure and ecology at fine-scale taxonomic resolution. Up to date, there is no comprehensive (i.e. qualitative and quantitative) analysis of their community composition in the Adriatic Sea. RESULTS: Analysis was based on pufM gene metabarcoding and quantitative FISH-IR approach with the use of artificial neural network. Significant seasonality was observed with regards to absolute abundances (maximum average abundances in spring 2.136 ± 0.081 × 104 cells mL-1, minimum in summer 0.86 × 104 cells mL-1), FISH-IR groups (Roseobacter clade prevalent in autumn, other Alpha- and Gammaproteobacteria in summer) and pufM sequencing data agglomerated at genus-level. FISH-IR results revealed heterogeneity with the highest average relative contribution of AAPs assigned to Roseobacter clade (37.66%), followed by Gammaproteobacteria (35.25%) and general Alphaproteobacteria (31.15%). Community composition obtained via pufM sequencing was dominated by Gammaproteobacteria clade NOR5/OM60, specifically genus Luminiphilus, with numerous rare genera present in relative abundances below 1%. The use of artificial neural network connected this community to biotic (heterotrophic bacteria, HNA and LNA bacteria, Synechococcus, Prochlorococcus, picoeukaryotes, heterotrophic nanoflagellates, bacterial production) and abiotic environmental factors (temperature, salinity, chlorophyll a and nitrate, nitrite, ammonia, total nitrogen, silicate, and orthophosphate concentration). A type of neural network, neural gas analysis at order-, genus- and ASV-level, resulted in five distinct best matching units (representing particular environments) and revealed that high diversity was generally independent of temperature, salinity, and trophic status of the environment, indicating a potentially dissimilar behaviour of aerobic anoxygenic phototrophs compared to the general bacterioplankton. CONCLUSION: This research represents the first comprehensive analysis of aerobic anoxygenic phototrophs in the Adriatic Sea on a trophic gradient during a year-round period. This study is also one of the first reports of their genus-level ecology linked to biotic and abiotic environmental factors revealed by unsupervised neural network algorithm, paving the way for further research of substantial contribution of this important bacterial functional group to marine ecosystems.

Picoplankton diversity in an oligotrophic and high salinity environment in the central Adriatic Sea.

By combining qualitative 16S metabarcoding and quantitative CARD-FISH methods with neural gas analysis, different patterns of the picoplankton community were revealed at finer taxonomic levels in response to changing environmental conditions in the Adriatic Sea. We present the results of a one-year study carried out in an oligotrophic environment where increased salinity was recently observed. We have shown that the initial state of community structure changes according to environmental conditions and is expressed as qualitative and quantitative changes. A general pattern of increasing diversity under harsh environmental conditions, particularly under the influence of increasing salinity at the expense of community abundance was observed. Considering the trend of changing seawater characteristics due to climate change, this study helps in understanding a possible structural change in the microbial community of the Adriatic Sea that could affect higher levels of the marine food web.

How do open coastal fire episodes' impact sea surface microlayer neuston communities?

Human-induced climate change is expected to increase the frequency and severity of vegetation fires. The Mediterranean region is considered particularly prone to fire episodes in summer. It is well known that pyrogenic particles are an important source of external nutrients for the marine environment, especially in oligotrophic areas. In this study, the plankton components of the sea surface layers were integrated to evaluate, for the first time, their dynamics over six months and their response to fire events in a typical coastal area of the Adriatic Sea. Concentrations of nutrients and organic compounds, together with plankton communities were significantly higher in the sea surface microlayer (SML, < 1 mm thick), than in the underlying water from 1 m depth. The piconeuston community and chlorophyll a responded with extreme abundance and concentration to the most intense fire event that enriched the SML with NH4+. Phytoneuston abundance increased with a delay of 2 weeks, while diversity indices decreased slightly after the fire events. The large abundances of the studied piconeuston parameters could be explained by the high availability of organic compounds and the immediate availability of NH4+, while the phytoneuston community responded to an increased NO3- concentration, triggered by the fire events. We confirmed that fast-acting marine heterotrophs are important members of biogeochemical cycles associated with fire events and that, together with phytoplankton, they are unavoidable parameters to detect environmental changes.

Enhanced mercury reduction in the South Atlantic Ocean during carbon remineralization.

Mercury (Hg) in seawater is subject to interconversions via (photo)chemical and (micro)biological processes that determine the extent of dissolved gaseous mercury (DGM) (re)emission and the production of monomethylmercury. We investigated Hg speciation in the South Atlantic Ocean on a GEOTRACES cruise along a 40°S section between December 2011 and January 2012 (354 samples collected at 24 stations from surface to 5250 m maximum depth). Using statistical analysis, concentrations of methylated mercury (MeHg, geometric mean 35.4 fmol L-1) were related to seawater temperature, salinity, and fluorescence. DGM concentrations (geometric mean 0.17 pmol L-1) were related to water column depth, concentrations of macronutrients and dissolved inorganic carbon (DIC). The first-ever observed linear correlation between DGM and DIC obtained from high-resolution data indicates possible DGM production by organic matter remineralization via biological or dark abiotic reactions. DGM concentrations projected from literature DIC data using the newly discovered DGM-DIC relationship agreed with published DGM observations.

Lineage-Specific Growth Curves Document Large Differences in Response of Individual Groups of Marine Bacteria to the Top-Down and Bottom-Up Controls.

Marine bacterioplankton represent a diverse assembly of species differing largely in their abundance, physiology, metabolic activity, and role in microbial food webs. To analyze their sensitivity to bottom-up and top-down controls, we performed a manipulation experiment where grazers were removed, with or without the addition of phosphate. Using amplicon-reads normalization by internal standard (ARNIS), we reconstructed growth curves for almost 300 individual phylotypes. Grazer removal caused a rapid growth of most bacterial groups, which grew at rates of 0.6 to 3.5 day-1, with the highest rates (>4 day-1) recorded among Rhodobacteraceae, Oceanospirillales, Alteromonadaceae, and Arcobacteraceae. Based on their growth response, the phylotypes were divided into three basic groups. Most of the phylotypes responded positively to both grazer removal as well as phosphate addition. The second group (containing, e.g., Rhodobacterales and Rhizobiales) responded to the grazer removal but not to the phosphate addition. Finally, some clades, such as SAR11 and Flavobacteriaceae, responded only to phosphate amendment but not to grazer removal. Our results show large differences in bacterial responses to experimental manipulations at the phylotype level and document different life strategies of marine bacterioplankton. In addition, growth curves of 130 phylogroups of aerobic anoxygenic phototrophs were reconstructed based on changes of the functional pufM gene. The use of functional genes together with rRNA genes may significantly expand the scientific potential of the ARNIS technique. IMPORTANCE Growth is one of the main manifestations of life. It is assumed generally that bacterial growth is constrained mostly by nutrient availability (bottom-up control) and grazing (top-down control). Since marine bacteria represent a very diverse assembly of species with different metabolic properties, their growth characteristics also largely differ accordingly. Currently, the growth of marine microorganisms is typically evaluated using microscopy in combination with fluorescence in situ hybridization (FISH). However, these laborious techniques are limited in their throughput and taxonomical resolution. Therefore, we combined a classical manipulation experiment with next-generation sequencing to resolve the growth dynamics of almost 300 bacterial phylogroups in the coastal Adriatic Sea. The analysis documented that most of the phylogroups responded positively to both grazer removal and phosphate addition. We observed significant differences in growth kinetics among closely related species, which could not be distinguished by the classical FISH technique.

Artificial neural network analysis of microbial diversity in the central and southern Adriatic Sea.

Bacteria are an active and diverse component of pelagic communities. The identification of main factors governing microbial diversity and spatial distribution requires advanced mathematical analyses. Here, the bacterial community composition was analysed, along with a depth profile, in the open Adriatic Sea using amplicon sequencing of bacterial 16S rRNA and the Neural gas algorithm. The performed analysis classified the sample into four best matching units representing heterogenic patterns of the bacterial community composition. The observed parameters were more differentiated by depth than by area, with temperature and identified salinity as important environmental variables. The highest diversity was observed at the deep chlorophyll maximum, while bacterial abundance and production peaked in the upper layers. The most of the identified genera belonged to Proteobacteria, with uncultured AEGEAN-169 and SAR116 lineages being dominant Alphaproteobacteria, and OM60 (NOR5) and SAR86 being dominant Gammaproteobacteria. Marine Synechococcus and Cyanobium-related species were predominant in the shallow layer, while Prochlorococcus MIT 9313 formed a higher portion below 50 m depth. Bacteroidota were represented mostly by uncultured lineages (NS4, NS5 and NS9 marine lineages). In contrast, Actinobacteriota were dominated by a candidatus genus Ca. Actinomarina. A large contribution of Nitrospinae was evident at the deepest investigated layer. Our results document that neural network analysis of environmental data may provide a novel insight into factors affecting picoplankton in the open sea environment.

Changes in the Trophic Pathways within the Microbial Food Web in the Global Warming Scenario: An Experimental Study in the Adriatic Sea.

A recent analysis of the Mediterranean Sea surface temperature showed significant annual warming. Since small picoplankton microorganisms play an important role in all major biogeochemical cycles, fluxes and processes occurring in marine systems (the changes at the base of the food web) as a response to human-induced temperature increase, could be amplified through the trophic chains and could also significantly affect different aspects of the structure and functioning of marine ecosystems. In this study, manipulative laboratory growth/grazing experiments were performed under in situ simulated conditions to study the structural and functional changes within the microbial food web after a 3 °C increase in temperature. The results show that a rise in temperature affects the changes in: (1) the growth and grazing rates of picoplankton, (2) their growth efficiency, (3) carrying capacities, (4) sensitivity of their production and grazing mortality to temperature, (5) satisfying protistan grazer carbon demands, (6) their preference in the selection of prey, (7) predator niche breadth and their overlap, (8) apparent uptake rates of nutrients, and (9) carbon biomass flow through the microbial food web. Furthermore, temperature affects the autotrophic and heterotrophic components of picoplankton in different ways.

Is TBX agar a suitable medium for monitoring Escherichia coli in bathing water using the membrane filtration method?

The use of a suitable method for the enumeration of indicator microorganisms is of crucial importance for reliable monitoring and assessment of the quality of bathing waters. Among other characteristics, the method should be selective enough and ensure acceptable relative recovery of target microorganisms. This study presents the basic parameters, relative recovery and categorical performance characteristics of Tryptone Bile X-glucuronide (TBX) agar for Escherichia coli (E. coli) enumeration in bathing water samples using the membrane filtration method.The results of the relative recovery study, in which TBX agar was compared against temperature-modified ISO 9308-1:2014, showed that in order to achieve a satisfactory relative recovery of E. coli with TBX agar at 44 ± 0.5 °C, the resuscitation period on a non-selective medium (Minerals Modified Glutamate Agar, MMGA) at 36 ± 2 °C is crucial. Incubation on a double-layer MMGA/TBX medium with a 6-h resuscitation period and alternating incubation on single-layer MMGA and TBX agar with a 4-h resuscitation period resulted in acceptable and very similar relative recovery. The achieved performance characteristics of the tested medium, double-layer MMGA/TBX agar, are acceptable. The selectivity was matrix-dependent and was 60.6% for inland and 69.9% for coastal waters. No significant effect of the resuscitation period on selectivity was recorded. Finally, the results showed that when the resuscitation period on a non-selective medium is included, TBX agar is a suitable medium for E. coli enumeration in bathing water samples using the membrane filtration method and that its use, theoretically, would not have negative effects on the assessment of bathing water quality.

Temperature and phosphorus interacts in controlling the picoplankton carbon flux in the Adriatic Sea: an experimental versus field study.

Temperature and phosphorus positively interacted in controlling picoplankton biomass production and its transfer towards higher trophic levels. Two complementary approaches (experimental and field study) indicated several coherent patterns: (1) the impact of temperature on heterotrophic bacteria was high at temperatures lower than 16°C and levelled off at higher temperatures, whereas this impact on autotrophic picoplankton was linear along the entire range of the investigated temperatures; (2) the addition of phosphorus increased the values of picoplankton production and grazing, but did not change the nature of their relationships with temperature substantially; (3) the picoplankton carbon flux towards higher trophic levels was larger during the warmer months (grazing by HNF dominated during the warmer period and by ciliates during the colder period) and also strengthened in conditions without phosphorus limitation; (4) the hypothesis that the available phosphorus can be better utilized at higher temperatures was confirmed for both autotrophic and heterotrophic picoplankton; (5) the hypothesis that the rise in temperature stimulates growth only in conditions of sufficient phosphorus was confirmed only for heterotrophic bacteria. Therefore, in the global warming scenario, an increase of the picoplankton carbon flux towards higher trophic levels can be expected in the Adriatic Sea, particularly under unlimited phosphorus conditions.

Relations between mercury fractions and microbial community components in seawater under the presence and absence of probable phosphorus limitation conditions.

Microbial transformations of toxic monomethylmercury (MMHg) and dissolved gaseous mercury (DGM) at the lower levels of the marine food web are not well understood, especially in oligotrophic and phosphorus-limited seas. To examine the effects of probable phosphorus limitation (PP-limitation) on relations between mercury (Hg) fractions and microorganisms, we determined the total mercury (THg), total methylated mercury (MeHg), DGM, and microbiological and chemical parameters in the Central Adriatic Sea. Using statistical analysis, we assessed the potential microbial effects on Hg transformations and bioaccumulation. Only in the absence of PP-limitation conditions (NO-PP-limitation) is MeHg significantly related to most chemical and microbial parameters, indicating metabolism-dependent Hg transformations. The heterotrophic activity of low nucleic acid bacteria (abundant in oligotrophic regions) seems responsible for most of Hg methylation under NO-PP-limitation. Under these conditions, DGM is strongly related to microbial fractions and chlorophyll a, indicating biological DGM production, which is probably not metabolically induced, as most of these relations are also observed under PP-limitation. MMHg biomagnification was observed through an increased bioaccumulation factor from microseston to mesozooplankton. Our results indicate that Hg transformations and uptake might be enhanced under NO-PP-limitation conditions, emphasizing their impact on the transfer of Hg to higher trophic levels.

Status of faecal pollution in ports: A basin-wide investigation in the Adriatic Sea.

Ports are subject to a variety of anthropogenic impacts, and there is mounting evidence of faecal contamination through several routes. Yet, little is known about pollution in ports by faecal indicator bacteria (FIB). FIB spatio-temporal dynamics were assessed in 12 ports of the Adriatic Sea, a semi-enclosed basin under strong anthropogenic pressure, and their relationships with environmental variables were explored to gain insight into pollution sources. FIB were abundant in ports, often more so than in adjacent areas; their abundance patterns were related to salinity, oxygen, and nutrient levels. In addition, a molecular method, quantitative (q)PCR, was used to quantify FIB. qPCR enabled faster FIB determination and water quality monitoring that culture-based methods. These data provide robust baseline evidence of faecal contamination in ports and can be used to improve the management of routine port activities (dredging and ballast water exchange), having potential to spread pathogens in the sea.

Performance characteristics of the temperature-modified ISO 9308-1 method for the enumeration of Escherichia coli in marine and inland bathing waters.

This study defines performance characteristics of the temperature-modified ISO 9308-1 method for E. coli enumeration in bathing water. After a 4-hour resuscitation period at 36 ±â€¯2 °C, the incubation temperature was changed to 44 ±â€¯0.5 °C. Elevated incubation temperature significantly suppressed the growth of thermo-intolerant bacteria, and enhanced the selectivity of Chromogenic Coliform Agar (CCA) up to 49.5% for inland and up to 66.0% for coastal water. Consequently, most of the selectivity-related performance characteristics are improved. Relative recovery was determined by comparing an alternative method against the reference, ISO 9308-1:2014 method, following the criteria set out in ISO 17994:2014. Temperature modification did not significantly alter the results and the methods were evaluated as "not different" for both, coastal and inland waters. Chromogenic Coliform Agar was assessed as a suitable medium for reliable E. coli enumeration in bathing water when incubated for 17-19 h at 44 ±â€¯0.5 °C after the 4-5 h resuscitation period at 36 ±â€¯2 °C.

The effect of temperature increase on microbial carbon fluxes in the Adriatic Sea: an experimental approach.

An assessment of the temperature increase effect on processes within the microbial food web provides a better insight into the carbon transfer and energy flow processes in marine environments in the global warming perspective. Modified laboratory dilution experiments that allow simultaneous estimates of protozoan grazing and viral lysis on picoplankton groups (bacteria, Prochlorococcus, Synechococcus and pico-eukaryotic algae) under in situ and 3°C above in situ temperatures were performed at seasonal scale. Picoplankton mortality due to grazing was generally higher than that caused by viral lysis, especially in the cold months. The largest part of HNF carbon demand was satisfied by grazing on bacteria throughout the year. Although ciliates satisfied their carbon demand predominantly through grazing on HNF and bacteria, the role of autotrophic picoplankton (APP) as their prey increased significantly in the cold months. Bacteria constituted the most important host for viruses throughout the year. However, during the warm months, APP groups were also significant hosts for viral infection. Under the warming condition the amount of picoplankton biomass transferred to protozoan grazers exceeded the lysed biomass, suggesting that global warming could further increase picoplankton carbon flow toward higher trophic levels in the Adriatic Sea.

Distribution of aerobic anoxygenic phototrophs in the Eastern Adriatic Sea.

The spatial patterns of aerobic anoxygenic phototrophs abundances were investigated, for the first time, in the Adriatic Sea. Also, the spatial patterns of the whole picoplankton community as well as the environmental factors that potentially influence these patterns were highlighted. AAP abundances was in average 66.9 ± 66.8 × 103 cell mL-1, and their proportion in total bacteria was 7.3 ± 4.3%. These values are in the upper range of AAP abundances observed in marine environments. Multivariate analyses proved that environmental factors influenced the picoplankton community interdependently. Chl a was the main driving factor for the picoplankton community, accounting for 33.3% of picoplankton community variance, followed by NO2- (17.9% of variance explained) and temperature (14.2% of variance explained). Chl a showed stronger correlation with AAPs, non-pigmented bacteria and Picoeucaryotes than with cyanobacteria. Abundance of cyanobacteria was stronger correlated to salinity and the N:P ratio than to nutrient concentrations.