Assessment of factors influencing direct enumeration of viruses within estuarine sediments

Accurate enumeration of viruses within environmental samples is critical for investigations of the ecological role of viruses and viral infection within microbial communities. This report evaluates differences in viral and bacterial direct counts between estuarine sediment samples which were either immediately processed onboard ship or frozen at -20 degrees C and later processed. Viral and bacterial abundances were recorded at three stations spanning the length of the Chesapeake Bay in April and June 2003 within three sediment fractions: pore water (PW), whole sediment (WS), and sediment after pore water removal (AP). No significant difference in viral abundance was apparent between extracts from fresh or frozen sediments. In contrast, bacterial abundance was significantly lower in the samples subjected to freezing. Both bacterial and viral abundance showed significant differences between sediment fractions (PW, WS, or AP) regardless of the fresh or frozen status. Although pore water viral abundance has been used in the past as a measurement of viral abundance in sediments, this fraction accounted for only ca. 5% of the total sediment viral abundance across all samples. The effect of refrigerated storage of sediment viral extracts was also examined and showed that, within the first 2 h, viral abundance decreased ca. 30% in formalin-fixed extracts and 66% in unfixed extracts. Finally, the reliability of direct viral enumeration via epifluorescence microscopy was tested by using DNase treatment of WS extractions. These tests indicated that a large fraction (>86%) of the small SYBR gold fluorescing particles are likely viruses.     

High concentrations of viruses in the sediments of Lac Gilbert, Québec

Viruses were found to be very abundant in the top layer of the sediments of Lac Gilbert, Québec. Viruses were extracted from the sediments using pyrophosphate buffer, and viruses from the diluted extracts were pelleted onto grids and enumerated using transmission electron microscopy. Viral abundance in the sediments ranged from 6.5 × 10⁸ to 1.83 × 10¹⁰ ml^–1, which is 10- to 1,000-fold greater than the number observed in the water column. This increase corresponds well with the 100- to 1,000-fold increase in bacterial abundance in the sediments. Viral abundance differed significantly among the surface sediment samples taken at different bottom depths and among samples taken at different depths of the water column. Viral abundance also varied significantly between the oxic and anoxic zones of the water column and the sediments. The virus-to-bacteria ratio varied greatly among the different sediment sites but not among depths in the water column. Viral abundance in the water column was related to bacterial abundance and chlorophyll concentration, whereas viruses in the sediments were most abundant in sediments with high organic matter content. Elevated viral abundance and their erratic distribution in the sediments suggest that viruses might play an important role in sediment microbial dynamics. Correspondence to: Roxane Maranger     

Interannual dynamics of viriobenthos abundance and morphological diversity in Chesapeake Bay sediments

Despite significant implications of viral activity in sediment ecosystems, there are limited data describing how sediment viral assemblages respond to broader ecosystem changes. To document this, the spatial and temporal dynamics of viral and bacterial abundance (BA) and changes in the morphological distribution of viruses were examined within three salinity regions over 2 years. Viral abundances (VA) ranged from 0.2 to 17 × 10(10) viruses mL(-1) sediment while direct bacterial counts ranged from 3.8 to 37 × 10(8) cells mL(-1) sediment. Peaks and valleys in the abundance of extracted viruses and bacteria from surface sediments occurred simultaneously, with lows in February 2004 and highs in April 2003. Across all samples, viral and BA were positively correlated (P < 0.001). Vertical profiles showed a decrease in viral and BA with depth in sediments. Based on transmission electron microscopy results, viruses with diminutive capsids (20-50 nm) and from the Myoviridae and Podoviridae viral family types were dominant within surface sediments. The most morphologically diverse viral assemblages occurred in autumn samples from the sandy, polyhaline station and spring samples from the mesohaline station. Seasonal changes showed an average 72% decrease in VA from spring to winter. These observations support the view that viriobenthos assemblages are responsive to seasonal environmental changes and that viral processes have significant implications for the biogeochemical processes mediated by bacterial communities within Bay sediments.     

Distribution of viral abundance in the reef environment of Key Largo, Florida.

The distribution of viral and microbial abundance in the Key Largo, Fla., reef environment was measured. Viral abundance was measured by transmission electron microscope direct counts and plaque titer on specific bacterial hosts in water and sediment samples from Florida Bay (Blackwater Sound) and along a transect from Key Largo to the outer edge of the reef tract in Key Largo Sanctuary. Water column viral direct counts were highest in Blackwater Sound of Florida Bay (1.2 x 10(7) viruses per ml), decreased to the shelf break (1.7 x 10(6) viruses per ml), and were inversely correlated with salinity (r = -0.97). Viral direct counts in sediment samples ranged from 1.35 x 10(8) to 5.3 x 10(8)/cm(3) of sediment and averaged nearly 2 orders of magnitude greater than counts in the water column. Viral direct counts (both sediment and water column measurements) exceeded plaque titers on marine bacterial hosts (Vibrio natriegens and others) by 7 to 8 orders of magnitude. Water column viral abundance did not correlate with bacterial direct counts or chlorophyll a measurements, and sediment viral parameters did not correlate with water column microbial, viral, or salinity data. Coliphage, which are indicators of fecal pollution, were detected in two water column samples and most sediment samples, yet their concentrations were relatively low (<2 to 15/liter for water column samples, and <2 to 108/cm(3) of sediment). Our findings indicate that viruses are abundant in the Key Largo environment, particularly on the Florida Bay side of Key Largo, and that processes governing their distribution in the water column (i.e., salinity and freshwater input) are independent of those governing their distribution in the sediment environment.     

Does Virus-Induced Lysis Contribute Significantly to Bacterial Mortality in the Oxygenated Sediment Layer of Shallow Oxbow Lakes?

Despite the recognition that viruses are ubiquitous components of aquatic ecosystems, the number of studies on viral abundance and the ecological role of viruses in sediments is scarce. In this investigation, the interactions between viruses and bacteria were studied in the oxygenated silty sediment layer of a mesotrophic oxbow lake. A long-term study (13 months) and a diel study revealed that viruses are a numerically important and dynamic component of the microbial community. The abundance and decay rates ranged from 4.3 × 10⁹ to 7.2 × 10⁹ particles ml of wet sediment⁻¹ and from undetectable to 22.2 × 10⁷ particles ml⁻¹ h⁻¹, respectively, and on average the values were 2 orders of magnitude higher than the values for the overlying water. In contrast to our expectations, viruses did not contribute significantly to the bacterial mortality in the sediment, since on average only 6% (range, 0 to 25%) of the bacterial secondary production was controlled by viruses. The low impact of viruses on the bacterial community may be associated with the quantitatively low viral burden that benthic bacteria have to cope with compared to the viral burden with which bacterial assemblages in the water column are confronted. The virus-to-bacterium ratio of the sediment varied between 0.9 and 3.2, compared to a range of 5.0 to 12.4 obtained for the water column. We speculate that despite high numbers of potential hosts, the possibility of encountering a host cell is limited by the physical conditions in the sediment, which is therefore not a favorable environment for viral proliferation. Our data suggest that viruses do not play an important role in the processing and transfer of bacterial carbon in the oxygenated sediment layer of the environment investigated.     

Bacterial Diversity in Shallow Oligotrophic Marine Benthos and Overlying Waters: Effects of Virus Infection,Containment, and Nutrient Enrichment

Little is known of the factors shaping sediment bacterial communities, despite their high abundance and reports of high diversity. Two factors hypothesized to shape bacterial communities in the water column are nutrient (resource) availability and virus infection. The role these factors play in benthic bacterial diversity was assessed in oligotrophic carbonate–based sediments of Florida Bay (USA). Sediment–water mesocosm enclosures were made from 1-m diameter clear polycarbonate cylinders which were pushed into sediments to 201 cm sediment depth enclosing 80 L of water. Mesocosms were amended each day for 14 d with 10 µM NH ₄ ⁺ and 1 µM PO ₄ ^3– . In a second experiment, viruses from a benthic flocculent layer were concentrated and added back to flocculent layer samples which were collected near the mesocosm enclosures. Photosynthesis by microalgae in virus-amended incubations was monitored by pulse-amplitude modulated (PAM) fluorescence. In both experiments, bacterial diversity was estimated using automated rRNA intergenic spacer analysis (ARISA), a high-resolution fingerprinting approach. Initial sediment bacterial operational taxonomic unit (OTU) richness (236 ± 3) was higher than in the water column (148 ± 9), where an OTU was detectable when its amplified DNA represented >0.09% of the total amplified DNA. Effects on bacterial diversity and operational taxonomic unit (OTU) richness in nutrient-amended mesocosms may have been masked by the effects of containment, which stimulated OTU richness in the water column, but depressed OTU richness and diversity in sediments. Nutrient addition significantly elevated virus abundance and the ratio of viruses to bacteria (p < 0.05 for both) in the sediments, concomitant with elevated bacterial diversity. However, water column bacterial diversity (in unamended controls) was not affected by nutrient amendments, which may be due to rapid nutrient uptake by sediment organisms or adsorption of P to carbonate sediments. Addition of live viruses to benthic flocculent layer samples increased bacterial OTU diversity and richness compared with heat-killed controls; however, cluster analyses showed that the community structure in the virus-amended mesocosms varied greatly between replicates. Despite the strong effects upon eubacterial communities, photosynthesis of co-occurring protists and cyanobacteria was not significantly altered by the presence of virus concentrates. This study supports the hypothesis that nutrient availability plays a key role in shaping sediment bacterial communities, and also that viruses may regulate the abundance of the dominant competitors and allow less dominant organisms to maintain or increase their abundance in a community due to decreased competition for resources.     

Does virus-induced lysis contribute significantly to bacterial mortality in the oxygenated sediment layer of shallow oxbow lakes?

Despite the recognition that viruses are ubiquitous components of aquatic ecosystems, the number of studies on viral abundance and the ecological role of viruses in sediments is scarce. In this investigation, the interactions between viruses and bacteria were studied in the oxygenated silty sediment layer of a mesotrophic oxbow lake. A long-term study (13 months) and a diel study revealed that viruses are a numerically important and dynamic component of the microbial community. The abundance and decay rates ranged from 4.3 x 10(9) to 7.2 x 10(9) particles ml of wet sediment(-1) and from undetectable to 22.2 x 10(7) particles ml(-1) h(-1), respectively, and on average the values were 2 orders of magnitude higher than the values for the overlying water. In contrast to our expectations, viruses did not contribute significantly to the bacterial mortality in the sediment, since on average only 6% (range, 0 to 25%) of the bacterial secondary production was controlled by viruses. The low impact of viruses on the bacterial community may be associated with the quantitatively low viral burden that benthic bacteria have to cope with compared to the viral burden with which bacterial assemblages in the water column are confronted. The virus-to-bacterium ratio of the sediment varied between 0.9 and 3.2, compared to a range of 5.0 to 12.4 obtained for the water column. We speculate that despite high numbers of potential hosts, the possibility of encountering a host cell is limited by the physical conditions in the sediment, which is therefore not a favorable environment for viral proliferation. Our data suggest that viruses do not play an important role in the processing and transfer of bacterial carbon in the oxygenated sediment layer of the environment investigated.     

An Inexpensive,Accurate, and Precise Wet-Mount Method for Enumerating Aquatic Viruses

Viruses affect biogeochemical cycling, microbial mortality, gene flow, and metabolic functions in diverse environments through infection and lysis of microorganisms. Fundamental to quantitatively investigating these roles is the determination of viral abundance in both field and laboratory samples. One current, widely used method to accomplish this with aquatic samples is the “filter mount” method, in which samples are filtered onto costly 0.02-μm-pore-size ceramic filters for enumeration of viruses by epifluorescence microscopy. Here we describe a cost-effective (ca. 500-fold-lower materials cost) alternative virus enumeration method in which fluorescently stained samples are wet mounted directly onto slides, after optional chemical flocculation of viruses in samples with viral concentrations of <5 × 10⁷ viruses ml⁻¹. The concentration of viruses in the sample is then determined from the ratio of viruses to a known concentration of added microsphere beads via epifluorescence microscopy. Virus concentrations obtained by using this wet-mount method, with and without chemical flocculation, were significantly correlated with, and had precision equivalent to, those obtained by the filter mount method across concentrations ranging from 2.17 × 10⁶ to 1.37 × 10⁸ viruses ml⁻¹ when tested by using cultivated viral isolates and natural samples from marine and freshwater environments. In summary, the wet-mount method is significantly less expensive than the filter mount method and is appropriate for rapid, precise, and accurate enumeration of aquatic viruses over a wide range of viral concentrations (≥1 × 10⁶ viruses ml⁻¹) encountered in field and laboratory samples.     

Contrasting distributions of bacteriophages and eukaryotic viruses from contaminated coastal sediments

Viruses are ubiquitous, abundant and play an important role in all ecosystems. Here, we advance understanding of coastal sediment viruses by exploring links in the composition and abundance of sediment viromes to environmental stressors and sediment bacterial communities. We collected sediment from contaminated and reference sites in Sydney Harbour and used metagenomics to analyse viral community composition. The proportion of phages at contaminated sites was significantly greater than phages at reference sites, whereas eukaryotic viruses were relatively more abundant at reference sites. We observed shifts in viral and bacterial composition between contaminated and reference sites of a similar magnitude. Models based on sediment characteristics revealed that total organic carbon in the sediments explained most of the environmental stress-related variation in the viral dataset. Our results suggest that the presence of anthropogenic contaminants in coastal sediments could be influencing viral community composition with potential consequences for associated hosts and the environment.     

High virus-to-cell ratios indicate ongoing production of viruses in deep subsurface sediments

Marine sediments cover two-thirds of our planet and harbor huge numbers of living prokaryotes. Long-term survival of indigenous microorganisms within the deep subsurface is still enigmatic, as sources of organic carbon are vanishingly small. To better understand controlling factors of microbial life, we have analyzed viral abundance within a comprehensive set of globally distributed subsurface sediments. Phages were detected by electron microscopy in deep (320 m below seafloor), ancient (∼14 Ma old) and the most oligotrophic subsurface sediments of the world''s oceans (South Pacific Gyre (SPG)). The numbers of viruses (10⁴–10⁹ cm⁻³, counted by epifluorescence microscopy) generally decreased with sediment depth, but always exceeded the total cell counts. The enormous numbers of viruses indicate their impact as a controlling factor for prokaryotic mortality in the marine deep biosphere. The virus-to-cell ratios increased in deeper and more oligotrophic layers, exhibiting values of up to 225 in the deep subsurface of the SPG. High numbers of phages might be due to absorption onto the sediment matrix and a diminished degradation by exoenzymes. However, even in the oldest sediments, microbial communities are capable of maintaining viral populations, indicating an ongoing viral production and thus, viruses provide an independent indicator for microbial life in the marine deep biosphere.     

Accurate Estimation of Viral Abundance by Epifluorescence Microscopy

Virus enumeration by epifluorescence microscopy (EFM) is routinely done on preserved, refrigerated samples. Concerns about obtaining accurate and reproducible estimates led us to examine procedures for counting viruses by EFM. Our results indicate that aldehyde fixation results in rapid decreases in viral abundance. By 1 h postfixation, the abundance dropped by 16.4% ± 5.2% (n = 6), and by 4 h, the abundance was 20 to 35% lower. The average loss rates for glutaraldehyde- and formaldehyde-fixed samples over the first 2 h were 0.12 and 0.13 h⁻¹, respectively. By 16 days, viral abundance had decreased by 72% (standard deviation, 6%; n = 6). Aldehyde fixation of samples followed by storage at 4°C, for even a few hours, resulted in large underestimates of viral abundance. The viral loss rates were not constant, and in glutaraldehyde- and formaldehyde-fixed samples they decreased from 0.13 and 0.17 h⁻¹ during the first hour to 0.01 h⁻¹ between 24 and 48 h. Although decay rates changed over time, the abundance was predicted by using separate models to describe decay over the first 8 h and decay beyond 8 h. Accurate estimates of abundance were easily made with unfixed samples stained with Yo-Pro-1, SYBR Green I, or SYBR Gold, and slides could be stored at −20°C for at least 2 weeks or, for Yo-Pro-1, at least 1 year. If essential, samples can be fixed and flash frozen in liquid nitrogen upon collection and stored at −86°C. Determinations performed with fixed samples result in large underestimates of abundance unless slides are made immediately or samples are flash frozen. If protocols outlined in this paper are followed, EFM yields accurate estimates of viral abundance.     

Bacterial community dynamics in the hyporheic zone of an intermittent stream

The dynamics of in situ bacterial communities in the hyporheic zone of an intermittent stream were described in high spatiotemporal detail. We assessed community dynamics in stream sediments and interstitial pore water over a two-year period using terminal-restriction fragment length polymorphism. Here, we show that sediments remained saturated despite months of drought and limited hydrologic connectivity. The intermittency of stream surface water affected interstitial pore water communities more than hyporheic sediment communities. Seasonal changes in bacterial community composition was significantly associated with water intermittency, phosphate concentrations, temperature, nitrate and dissolved organic carbon (DOC) concentrations. During periods of low- to no-surface water, communities changed from being rich in operational taxonomic units (OTUs) in isolated surface pools, to a few OTUs overall, including an overall decline in both common and rare taxa. Individual OTUs were compared between porewater and sediments. A total of 19% of identified OTUs existed in both porewater and sediment samples, suggesting that bacteria use hyporheic sediments as a type of refuge from dessication, transported through hydrologically connected pore spaces. Stream intermittency impacted bacterial diversity on rapid timescales (that is, within days), below-ground and in the hyporheic zone. Owing to the coupling of intermittent streams to the surrounding watershed, we stress the importance of understanding connectivity at the pore scale, consequences for below-ground and above-ground biodiversity and nutrient processing, and across both short- and long-time periods (that is, days to months to years).     

Metagenomic Analysis of Viral Communities in (Hado)Pelagic Sediments

In this study, we analyzed viral metagenomes (viromes) in the sedimentary habitats of three geographically and geologically distinct (hado)pelagic environments in the northwest Pacific; the Izu-Ogasawara Trench (water depth = 9,760 m) (OG), the Challenger Deep in the Mariana Trench (10,325 m) (MA), and the forearc basin off the Shimokita Peninsula (1,181 m) (SH). Virus abundance ranged from 10⁶ to 10¹¹ viruses/cm³ of sediments (down to 30 cm below the seafloor [cmbsf]). We recovered viral DNA assemblages (viromes) from the (hado)pelagic sediment samples and obtained a total of 37,458, 39,882, and 70,882 sequence reads by 454 GS FLX Titanium pyrosequencing from the virome libraries of the OG, MA, and SH (hado)pelagic sediments, respectively. Only 24−30% of the sequence reads from each virome library exhibited significant similarities to the sequences deposited in the public nr protein database (E-value <10⁻³ in BLAST). Among the sequences identified as potential viral genes based on the BLAST search, 95−99% of the sequence reads in each library were related to genes from single-stranded DNA (ssDNA) viral families, including Microviridae, Circoviridae, and Geminiviridae. A relatively high abundance of sequences related to the genetic markers (major capsid protein [VP1] and replication protein [Rep]) of two ssDNA viral groups were also detected in these libraries, thereby revealing a high genotypic diversity of their viruses (833 genotypes for VP1 and 2,551 genotypes for Rep). A majority of the viral genes predicted from each library were classified into three ssDNA viral protein categories: Rep, VP1, and minor capsid protein. The deep-sea sedimentary viromes were distinct from the viromes obtained from the oceanic and fresh waters and marine eukaryotes, and thus, deep-sea sediments harbor novel viromes, including previously unidentified ssDNA viruses.     

Estimates of bacterial productivity in marine sediments and water from a temperate saltmarsh lagoon

Tritiated thymidine incorporation (TTI) into DNA was used to estimate bacterial productivity in sediment and water samples from two sites in Langebaan Lagoon, South Africa. Routine analysis of isotope dilution showed seasonal variations of approximately threefold in the thymidine precursor pool sizes for bacterial assemblages from each site. Dual label incorporation of [³H]-thymidine and ¹⁴C-leucine into DNA and protein, respectively, showed that pelagic but not sediment assemblages were in a balanced state of growth during TTI. This is the first report of dual label measurements of bacterial production in sediments. Sediments supported bacterial productivities that exceeded those in the water column by factors from five- to 950-fold, whereas bacterial abundance supported by sediments exceeded that in the water column by more than 3 orders of magnitude. Estimates of bacterial productivities in sediments were coincident with levels of organic content in sediments, but not with bacterial abundance. Measurements of TTI activity for 5 different benthic microhabitats at one lagoon site showed highest activity associated with seagrass beds (2.11 ± 0.84 nmol thymidine hours^–1 g-1 dry weight), whereas activities decreased with depth (0.46 ± 0.21 nmol thymidine hours^–1 g^–I dry weight) below sediment surface.Offprint requests to: B. J. Tibbles.     

Effects of fish farming on microbial enzyme activities and densities: comparison between three Mediterranean sites

AIM: The effects of fish farming on microbial enzyme activities and heterotrophic bacterial density were investigated in three Mediterranean sites before and after the start of mariculture. METHODS AND RESULTS: Microbial activities were measured on water and sediment samples by using fluorogenic substrates specific for leucine aminopeptidase, beta-glucosidase and alkaline phosphatase (AP); bacterial counts were determined by Marine agar plates. SIGNIFICANCE AND IMPACT OF THE STUDY: Comparison of activity and abundance values obtained before and after the experiment showed that fish farming mainly affected the levels of microbial activities; they were significantly enhanced both in water and sediments, reaching an increase of 183.66 times for AP in Castellammare Gulf. After mariculture, no significant variations were recorded in heterotrophic bacterial density in the waters, while significant changes were observed in the sediments. Effects induced appeared to be extended not only to stations in which cages were located, but also to control sites far from the direct influence of fish farming.     

Accurate estimation of viral abundance by epifluorescence microscopy

Virus enumeration by epifluorescence microscopy (EFM) is routinely done on preserved, refrigerated samples. Concerns about obtaining accurate and reproducible estimates led us to examine procedures for counting viruses by EFM. Our results indicate that aldehyde fixation results in rapid decreases in viral abundance. By 1 h postfixation, the abundance dropped by 16.4% +/- 5.2% (n = 6), and by 4 h, the abundance was 20 to 35% lower. The average loss rates for glutaraldehyde- and formaldehyde-fixed samples over the first 2 h were 0.12 and 0.13 h(-1), respectively. By 16 days, viral abundance had decreased by 72% (standard deviation, 6%; n = 6). Aldehyde fixation of samples followed by storage at 4 degrees C, for even a few hours, resulted in large underestimates of viral abundance. The viral loss rates were not constant, and in glutaraldehyde- and formaldehyde-fixed samples they decreased from 0.13 and 0.17 h(-1) during the first hour to 0.01 h(-1) between 24 and 48 h. Although decay rates changed over time, the abundance was predicted by using separate models to describe decay over the first 8 h and decay beyond 8 h. Accurate estimates of abundance were easily made with unfixed samples stained with Yo-Pro-1, SYBR Green I, or SYBR Gold, and slides could be stored at -20 degrees C for at least 2 weeks or, for Yo-Pro-1, at least 1 year. If essential, samples can be fixed and flash frozen in liquid nitrogen upon collection and stored at -86 degrees C. Determinations performed with fixed samples result in large underestimates of abundance unless slides are made immediately or samples are flash frozen. If protocols outlined in this paper are followed, EFM yields accurate estimates of viral abundance.     

Importance of Micromonospora spp. as colonizers of cellulose in freshwater lakes as demonstrated by quantitative reverse transcriptase PCR of 16S rRNA

The relative abundance of micromonosporas in the bacterial communities inhabiting cellulose baits, water columns, and sediments of two freshwater lakes was determined by quantitative PCR (qPCR) of reverse-transcribed 16S rRNA. Micromonospora spp. were shown to be significant members of the active bacterial population colonizing cellulosic substrates in the lake sediment, and their increased prevalence with greater depth was confirmed by enumeration of CFU.     

The significance of viruses to mortality in aquatic microbial communities

A variety of approaches including enumeration of visibly infected microbes, removal of viral particles, decay of viral infectivity, and measurements of viral production rates have been used to infer the impact of viruses on microbial mortality. The results are surprisingly consistent and suggest that, on average, about 20% of marine heterotrophic bacteria are infected by viruses and 10–20% of the bacterial community is lysed daily by viruses. The effect of viruses on phytoplankton is less certain, but ca. 3% of Synechococcus biomass may be lysed daily. The fraction of primary productivity this represents depends upon the relative biomass and growth rate of Synechococcus. Virus enrichment experiments suggest that the productivity of eukaryotic phytoplankton would be ca. 2% higher in the absence of viruses. Overall, probably about 2–3% of primary productivity is lost to viral lysis. There is considerable variation about these estimates; however, they represent a starting point for incorporating viral-mediated processes into aquatic ecosystem models.     

Binding of phosphate in sediment accumulation areas of the eastern Gulf of Finland,Baltic Sea

The relationships between P and components binding P were studied by analysing the concentrations of N, P, Fe, Mn, Ca and Al in sediments and pore water along the estuarine transect of the River Neva in August 1995. The high sediment organic matter concentration resulted in low surface redox potential and high pore-water o-P concentration, whereas the abundance of amphipods resulted in high surface redox potentials and low pore-water o-P concentration. However, despite the variation in sediment organic matter and the abundance of amphipods, very reduced conditions and slightly variable concentrations of Tot-P (0.7–1.1 mg g^–1 DW) were observed in the 10–15 cm sediment depth along the estuarine gradient, indicating that the pools of mobile P were largely depleted within the depth of 0–15 cm. Multiple regression analysis demonstrated that organic matter and Tot-Fe concentration of the sediment were closely related to the variation in Tot-P concentration of the sediments (r ² = 0.817, n=32). In addition, the high total Fe:P ratio suggested that there is enough Fe to bind P in sediments along the estuarine gradient. However, low Fe_diss concentrations in the pore water of reduced sediment (redox-potential <–50 mV) indicated efficient precipitation of FeS (FeS and FeS₂), incapable to efficiently bind P. Consequently, the low Fe_diss:o-P ratio (< 1) recorded in pore water in late summer implied that Fe³⁺ oxides formed by diffusing Fe_diss in the oxic zone of the sediments were insufficient to bind the diffusing o-P completely. The measured high o-P concentrations in the near-bottom water are consistent with this conclusion. However, there was enough Fe_diss in pore water to form Fe³⁺ oxides to bind upwards diffusing P in the oxic sediment layer of the innermost Neva estuary and the areas bioturbated by abundant amphipods.     

Proportion of prokaryotes enumerated as viruses by epifluorescence microscopy

It is well known that there are prokaryotes small in size (e.g. ultra-microprokaryotes) that pass through a 0.2-μm filter. As bacterial and viral abundances are determined by epifluorescence microscopy and the differentiation between them is based on particle size, some bacteria can be erroneously enumerated as viruses, namely in marine waters where bacteria are small. However, there is no information on the proportion of prokaryotes that could be misidentified as viruses by epifluorescence microscopy. In this work, we assessed, in water samples collected in the estuarine system Ria de Aveiro (Portugal), the proportion of prokaryotes that could be counted as viruses by the current widespread epifluorescence microscopy and, for the first time, by fluorescence in situ hybridization (FISH). The total number of particles was determined on membranes of 0.2 and 0.02 μm after staining with 4′,6-diamidino-2-phenylindole (DAPI), and the number of prokaryotes (Bacteria and Archaea) was determined by FISH for both pore size membranes. The results show that, in the marine zone of the estuarine system, 28 % of particles enumerated as virus-like particles were prokaryotes, but, in the brackish water zone, only 13 % of the particles counted as viruses were actually prokaryotic cells. Epifluorescence microscopy overestimates viral abundance, and also the ratio viruses:prokaryotes, and this error must be taken into consideration because it can vary significantly within a system. In fact, in the marine zone of an estuarine system, the overestimation of viral abundance can be twice as high as in the brackish water zone.