Detection and Enumeration of Sulphate-Reducing Bacteria in Estuarine Sediments by Competitive PCR

The distribution of sulphate-reducing bacteria (SRB) in the sediments of the Colne River estuary, Essex, UK covering different saline concentrations of sediment porewater was investigated by the use of quantitative competitive PCR. Here, we show that a new PCR primer set and a new quantitative method using PCR are useful tools for the detection and the enumeration of SRB in natural environments. A PCR primer set selective for the dissimilatory sulphite reductase gene (dsr) of SRB was designed. PCR amplification using the single set of dsr-specific primers resulted in PCR products of the expected size from all 27 SRB strains tested, including Gram-negative and positive species. Sixty clones derived from sediment DNA using the primers were sequenced and all were closely related with the predicted dsr of SRB. These results indicate that PCR using the newly designed primer set are useful for the selective detection of SRB from a natural sample. This primer set was used to estimate cell numbers by dsr selective competitive PCR using a competitor, which was about 20% shorter than the targeted region of dsr. This procedure was applied to sediment samples from the River Colne estuary, Essex, UK together with simultaneous measurement of in situ rates of sulphate reduction. High densities of SRB ranging from 0.2 ? 5.7 × 10⁸ cells ml^{? 1} wet sediment were estimated by the competitive PCR assuming that all SRB have a single copy of dsr. Using these estimates cell specific sulphate reduction rates of 10^{? 17} to 10^{? 15} mol of SO₄ ^{2 ?} cell^{? 1} day^{? 1} were calculated, which is within the range of, or lower than, those previously reported for pure cultures of SRB. Our results show that the newly developed competitive PCR technique targeted to dsr is a powerful tool for rapid and reproducible estimation of SRB numbers in situ and is superior to the use of culture-dependent techniques.     

Relationships Between Environmental Factors, Bacterial Indicators, and the Occurrence of Enteric Viruses in Estuarine Sediments

Current standards for evaluation of the public health safety of recreational and shellfish-harvesting waters are based upon bacteriological analysis, but do not include an evaluation of the number of viruses. The objective of this study was to determine the occurrence of enteric viruses in estuarine sediments and to find a relationship, if any, between the presence of viruses in seawater or sediment or both and various biological and physicochemical characteristics of the environment. Viruses were found in greater numbers in sediment than in overlying seawater on a volume basis. Several types of enteroviruses were isolated: coxsackievirus types A16, B1, and B5, echovirus type 1, and poliovirus type 2. On several occasions, viruses were isolated from sediments when overlying seawaters met bacteriological water quality standards for recreational use. Statistical analysis of the relationship between viruses in seawater or in sediment and other variables measured yielded only one significant association: the number of viruses in sediment was found to be positively correlated with the number of fecal coliforms in sediment. No other physical, chemical, or biological characteristic of seawater or sediment that was measured showed statistically significant association with viral numbers. No correlation was found between bacterial indicators and virus in the overlying waters. The data indicated that evaluation of the presence of bacteria and viruses in sediment may provide additional insight into long-term water quality conditions and that indicator bacteria in water are not reflective of the concentration of enteric viruses in marine waters.     

Factors Influencing Bacterial Production in a Shallow Estuarine System

The bacterioplankton of the marine and brackish water zones of the complex system Ria de Aveiro was characterized as profiles of bacterial abundance and biomass productivity. During the warm season, total bacteria ranged from 0.2 to 8.5 x 109 cells L-1 and active bacteria number from 0.1 to 3.1 x 109 cells L-1. Total and active bacterial numbers were, on average, three times higher in brackish than in marine water. Bacterial productivity on different dates and different tides in the marine zone varied from 0.05 to 4.5 mg C L-1 h-1. Here the average productivity (1.1 mg C L-1 h-1) was 3.5 times less than in brackish water (average 3.8 mg C L-1 h-1; range 0.7-14.2 mg C L-1 h-1). Specific productivity varied from 0.05 to 2.61 fg C cell-1 h-1, a range that was similar throughout the ecosystem. However, specific productivity per active cell was 19% higher in brackish water. Bacterial production variation was best explained by the number of active bacteria, which, in turn, was highly associated with total bacterial number, temperature, and particulate organic carbon. In the marine zone, bacterial production was also influenced by depth and salinity. In the brackish zone, the set of independent variables explained a smaller percentage of bacterial production variation than in marine zone, suggesting greater importance of other variables. In the marine zone, and mainly near low tide, productivity was significantly higher (average 3.3 times) at the surface (down to 0.5 m) than in the deeper layers of the water column. This stratification of bacterial productivity was linked to the increased specific productivity per active cell, as no modification in the proportion of active cells in the population could be detected. The vertical profile of bacterial production in the deeper zone of this estuarine ecosystem, in which no clear salinity or thermal stratification occurs throughout the tidal cycle, seemed to reflect a biochemical stratification generated by increased phytoplankton exudation and/or by photochemical transformation of semilabile or recalcitrant organic compounds. Shallower water masses tend to blur this surface effect. The relative importance of photochemical transformation in the pattern of estuarine bacterial production will therefore tend to vary with the bathymetry of the system.     

Diatom-Derived Carbohydrates as Factors Affecting Bacterial Community Composition in Estuarine Sediments

Microphytobenthic biofilms in estuaries, dominated by epipelic diatoms, are sites of high primary productivity. These diatoms exude large quantities of extracellular polymeric substances (EPS) comprising polysaccharides and glycoproteins, providing a substantial pool of organic carbon available to heterotrophs within the sediment. In this study, sediment slurry microcosms were enriched with either colloidal carbohydrates or colloidal EPS (cEPS) or left unamended. Over 10 days, the fate of these carbohydrates and changes in β-glucosidase activity were monitored. Terminal restriction fragment length polymorphism (T-RFLP), DNA sequencing, and quantitative PCR (Q-PCR) analysis of 16S rRNA sequences were used to determine whether sediment bacterial communities exhibited compositional shifts in response to the different available carbon sources. Initial heterotrophic activity led to reductions in carbohydrate concentrations in all three microcosms from day 0 to day 2, with some increases in β-glucosidase activity. During this period, treatment-specific shifts in bacterial community composition were not observed. However, by days 4 and 10, the bacterial community in the cEPS-enriched sediment diverged from those in colloid-enriched and unamended sediments, with Q-PCR analysis showing elevated bacterial numbers in the cEPS-enriched sediment at day 4. Community shifts were attributed to changes in cEPS concentrations and increased β-glucosidase activity. T-RFLP and sequencing analyses suggested that this shift was not due to a total community response but rather to large increases in the relative abundance of members of the γ-proteobacteria, particularly Acinetobacter-related bacteria. These experiments suggest that taxon- and substrate-specific responses within the bacterial community are involved in the degradation of diatom-derived extracellular carbohydrates.     

Microbial Formation of Ethane in Anoxic Estuarine Sediments

Estuarine sediment slurries produced methane and traces of ethane when incubated under hydrogen. Formation of methane occurred over a broad temperature range with an optimum above 65°C. Ethane formation had a temperature optimum at 40°C. Formation of these two gases was inhibited by air, autoclaving, incubation at 4 and 80°C, and by the methanogenic inhibitor, 2-bromoethanesulfonic acid. Ethane production was stimulated by addition of ethylthioethanesulfonic acid, and production from ethylthioethanesulfonic acid was blocked by 2-bromoethanesulfonic acid. A highly purified enrichment culture of a methanogenic bacterium obtained from sediments produced traces of ethane from ethylthioethanesulfonic acid. These results indicate that the small quantities of ethane found in anaerobic sediments can be formed by certain methanogenic bacteria.     

Ultraviolet Devitalization of Eight Selected Enteric Viruses in Estuarine Water

The effect of ultraviolet (UV) radiation on the devitalization of eight selected enteric viruses suspended in estuarine water was determined. The surviving fractions of each virus were calculated and then plotted against the UV exposure time for purposes of comparison. Analytical assessment of the survival data for each virus consisted of least squares regression analysis for determination of intercepts and slope functions. All data were examined for statistical significance. When the slope function of each virus was compared against the slope function of poliovirus type 1, the analytical findings indicated that poliovirus types 2 and 3, echovirus types 1 and 11, and coxsackievirus A-9 exhibited similar devitalization characteristics in that no statistically significant difference was found (P > 0.05). Conversely, the devitalization characteristics of coxsackievirus B-1 and reovirus type 1 were dissimilar from those of poliovirus type 1 in that a statistically significant difference was found between the slope functions (P < 0.05). This observed difference in devitalization of coxsackievirus B-1 and reovirus type 1 was attributed primarily to the frequency distribution of single and aggregate virions, the geometric configuration, the size of the aggregates, and the severity of aggregation. The devitalization curve of coxsackievirus B-1 was characteristic of a retardant die-away curve. The devitalization curve of reovirus type 1 was characteristic of a multihittype curve. The calculated devitalization half-life values for poliovirus types 1, 2, and 3; echovirus types 1 and 11; coxsackievirus types A-9 and B-1; and reovirus type 1 were 2.8, 3.1, 2.7, 2.8, 3.2, 3.1, 4.0, 4.0 sec, respectively. These basic data should facilitate an operative extrapolation of the findings to the applied situation. It was concluded that UV can be highly effective and provide a reliable safety factor in treating estuarine water.     

Anaerobic Oxidation of Acetylene by Estuarine Sediments and Enrichment Cultures

Acetylene disappeared from the gas phase of anaerobically incubated estuarine sediment slurries, and loss was accompanied by increased levels of carbon dioxide. Acetylene loss was inhibited by chloramphenicol, air, and autoclaving. Addition of ¹⁴C₂H₂ to slurries resulted in the formation of ¹⁴CO₂ and the transient appearance of ¹⁴C-soluble intermediates, of which acetate was a major component. Acetylene oxidation stimulated sulfate reduction; however, sulfate reduction was not required for the loss of C₂H₂ to occur. Enrichment cultures were obtained which grew anaerobically at the expense of C₂H₂.     

Temporal Occurrence of Vibrio Species and Aeromonas hydrophila in Estuarine Sediments

Marine sediments were assayed for their concentration of Vibrio spp. and Aeromonas hydrophila over 1 year. A temporal variation was observed in which A. hydrophila, and to a lesser degree V. fluvialis, were found in the winter months, V. parahaemolyticus and V. vulnificus predominated in the spring and summer, with non-O-1 V. cholerae and V. alginolyticus detected in the late summer and fall. These organisms were found in greatest numbers in the top 5 cm of sediment, but were detectable down to 15 cm. Epidemiological data revealed a predominance of non-O-1 V. cholerae infections at the time the organisms were observed to flourish in the sediments.     

Detection of Animal Viruses in Coastal Seawater and Sediments

Animal viruses, predominantly enteroviruses, were detected in shallow water at bottom depths and in clastic marine sediments. Viruses accumulated in sandy and slimy deposits of the sea bottom near the shore and could be easily released into water by means of simple mechanical shaking.     

Enumeration, Isolation, and Characterization of Beggiatoa from Freshwater Sediments

An accurate most-probable-number enumeration method was developed for counting the number of Beggiatoa trichomes from various freshwater sediments. The medium consisted of extracted hay, diluted soil extract, 0.05% acetate, and 15 to 35 U of catalase per ml. The same enrichment medium, but without the acetate, was the best enrichment medium from which to obtain pure cultures because it supported good growth of the beggiatoas without allowing them to be overgrown by other bacteria. A total of 32 strains of Beggiatoa were isolated from seven different freshwater habitats and partially characterized. The strains were separated into five groups based on several preliminary characteristics. Four of the groups contained cells with trichomes of approximately the same diameter (1.5 to 2.7 μm) and may be Beggiatoa leptomitiformis or an unnamed species. The fifth group appeared to be Beggiatoa alba. With the exception of three strains, all of the strains deposited sulfur in the presence of hydrogen sulfide, and all strains grew heterotrophically and deposited poly-β-hydroxybutyrate and volutin when grown on acetate supplemented with low concentrations of other organic nutrients. Thin sections of sulfur-bearing trichomes indicated that the sulfur granules were external to the cytoplasmic membrane and that they were surrounded by an additional membrane.     

Anaerobic ortho Dechlorination of Polychlorinated Biphenyls by Estuarine Sediments from Baltimore Harbor

Reductive dechlorination of the ortho moiety of polychlorinated biphenyls (PCBs) as well as of meta and para moieties is shown to occur in anaerobic enrichments of Baltimore Harbor sediments. These estuarine sediments ortho dechlorinated 2,3,5,6-chlorinated biphenyl (CB), 2,3,5-CB, and 2,3,6-CB in freshwater or estuarine media within a relatively short period of 25 to 44 days. ortho dechlorination developed within 77 days in marine medium. High levels of ortho dechlorination (>90%) occurred when harbor sediments were supplied with only 2,3,5-CB. Incubation with 2,3,4,5,6-CB or 2,3,4,5-CB resulted in the formation of the ortho dechlorination product 3,5-CB; however, para dechlorination of these congeners always preceded ortho chlorine removal. ortho dechlorination of PCBs is an exceedingly rare event that has not been reported previously for marine or estuarine conditions. The activity was reproducible and could be sustained through sequential transfers. In contrast, freshwater sediments incubated under the same conditions exhibited only meta and para dechlorinations. The results indicate that unique anaerobic dechlorinating activity is catalyzed by microorganisms in the estuarine sediments from Baltimore Harbor.     

Microbial Reductive Dechlorination of Aroclor 1260 in Anaerobic Slurries of Estuarine Sediments

Reductive dechlorination of Aroclor 1260 was investigated in anaerobic slurries of estuarine sediments from Baltimore Harbor (Baltimore, Md.). The sediment slurries were amended with 800 ppm Aroclor 1260 with and without the addition of 350 μM 2,3,4,5-tetrachlorobiphenyl (2,3,4,5-CB) or 2,3,5,6-tetrachlorobiphenyl (2,3,5,6-CB) and incubated in triplicate at 30°C under methanogenic conditions in an artificial estuarine medium. After 6 months, extensive meta dechlorination and moderate ortho dechlorination of Aroclor 1260 occurred in all incubated cultures except for sterilized controls. Overall, total chlorines per biphenyl decreased by up to 34%. meta chlorines per biphenyl decreased by 65, 55, and 45% and ortho chlorines declined by 18, 12, and 9%, respectively, when 2,3,4,5-CB, 2,3,5,6-CB, or no additional congener was supplied. This is the first confirmed report of microbial ortho dechlorination of a commercial polychlorinated biphenyl mixture. In addition, compared with incubated cultures supplied with Aroclor 1260 alone, the dechlorination of Aroclor 1260 plus 2,3,4,5-CB or 2,3,5,6-CB occurred with shorter lag times (31 to 60 days versus 90 days) and was more extensive, indicating that the addition of a single congener stimulated the dechlorination of Aroclor 1260.     

Effect of Salinity on Mercury-Methylating Activity of Sulfate-Reducing Bacteria in Estuarine Sediments

The biomethylation of mercury was measured in anoxic estuarine sediments that ranged in salinity from 0.03 to 2.4% with or without added molybdate, an inhibitor of sulfate reducers. Mercury methylation was inhibited by molybdate by more than 95%, regardless of sediment salinity. In the absence of inhibitor, high-salinity sediments methylated mercury at only 40% of the level observed in low-salinity sediments. In response to molybdate inhibition of sulfate reducers, methanogenesis increased up to 258% in high-salinity sediments but only up to 25% in low-salinity sediments. In contrast to an earlier low-salinity isolate, a Desulfovibrio desulfuricans strain from high-salinity sediment required 0.5 M sodium for optimal growth and mercury methylation activity. The formation of negatively charged mercuric chloride complexes at high salinity did not noticeably interfere with the methylation process. Results of these studies demonstrate that sulfate reducers are responsible for mercury methylation in anoxic estuarine sediments, regardless of the prevailing salinity.     

Habitat Segregation of a Functional Gene Encoding Nitrate Ammonification in Estuarine Sediments

Distribution and diversity of nrfA gene encoding dissimilatory nitrite reduction to ammonium (DNRA) in the sediments of the Colne River, North Essex, UK, were investigated. Sequencing cloned nrfA fragments amplified from environmental DNA enabled structure analysis of the bacterial community responsible for this pathway. The DNA was extracted from the sediment samples at different depths from the estuary ranging from freshwater to seawater regions, and amplified using specific PCR primer pairs targeting for the nrfA gene. Analysis of the nrfA clones showed two distinct clusters corresponding to their origins, namely, divided into the stable sites (marine and freshwater regions) and the unstable sites (brackish water region), where the tidal rise and fall constantly disturbs the environmental conditions. In addition, the nrfA clones from the deeper layer of the sediment formed a more homogenous community than those from the surface layer of the sediment. This may be due to more isolated and anaerobic conditions kept in the deeper sediment less influenced by the overlying water and other environmental factors. Most of the nrfA clones from the Colne estuarine sediments formed several distinct clusters including known nitrate ammonifiers such as Aeromonas, Shewanella, Desulfovibrio and Sulfurospillum. One of which was, however, related to Bacteroides but still quite divergent (～70% identity) and the rest forming unknown clusters of supposedly uncultured members of bacteria. This is the first trial to describe the nrfA partial sequences derived from a natural environment, with reference to their habitat-specific community structure.     

Reliability of CARD-FISH Procedure for Enumeration of Archaea in Deep-Sea Surficial Sediments

The enumeration of Archaea in deep-sea sediment samples is still limited, although different methodological procedures have been applied. Among these, catalysed reporter deposition-fluorescence in situ hybridisation (CARD-FISH) technique is a promising tool for estimation of archaeal abundance in deep-sea sediment samples. Comparing different permeabilisation treatments, the best results obtained both on archaeal pure cultures and on natural assemblages were with hydrochloric acid (0.1 M) and proteinase K (0.004 U/ml) treatments. The application of CARD-FISH on deep-sea sediments revealed that Archaea reach up to 41% of total prokaryotic cells. Specific probes for planktonic Archaea showed that marine Crenarchaea dominated archaeal seafloor communities. No clear bathymetric trends were observed for archaeal abundances and the morphology of continental margin (slope vs. canyon) seems not to have a direct influence on archaeal relative abundances. The site-specific sediment habitat—both abiotic environmental setting and sedimentary organic matter quality—explain up to 65% of variance of archaeal, crenarchaeal and euryarchaeal relative abundance, suggesting a wide ecophysiological adaptation to deep-sea benthic ecosystems. The findings demonstrate that Archaea are an important component of benthic microbial assemblages so far neglected, and hence they lay the groundwork for more focused research on their ecological importance in the functioning of deep-sea benthic ecosystems.     

Application of Most-Probable-Number Statistics to Direct Enumeration of Microorganisms

A novel method for rapid determination of total microbial cell numbers was investigated. The method involves the application of most-probable-number estimation statistics to direct microscopic counting of microbial cells by using a particle sizing graticule. Its accuracy and reliability were tested with computer simulations of bacterial cell distributions encountered in ecological studies. Good estimates of cell numbers were obtained when the cell density varied from 3 to 6,000 cells per field, i.e., over 3 orders of magnitude. Low levels of contagion did not markedly influence cell estimates, although high levels, corresponding to discrete scattered microcolonies, did. However, these could be recognized visually. Estimates of cell numbers in Breed smears confirmed its speed and good correlation with the standard quadrat counting technique under real experimental conditions.     

Ecological Risk Assessment of Arsenic and Metals in Surface Sediments from Estuarine and Coastal Areas of the Southern Bohai Sea,China

Distribution and magnitude of arsenic and metals in surface sediments collected from the coastal and estuarine areas of the southern Bohai Sea, China, were investigated. Sediments from the estuarine and coastal areas of the Jie and Xiaoqing Rivers contained highest concentrations of arsenic, cadmium, copper, mercury, and zinc. Mean concentrations of Cu, Zn, Cr, Pb, and Cd were higher than background concentrations determined for the areas. The magnitude of both enrichment factors (EF) and geoaccumulation indices (I_geo) suggested that pollution with As and metals was occurring along estuarine and coastal areas of the southern Bohai Sea. Risk analysis also suggested that concentrations of As and metals were sufficiently elevated as to cause adverse biological effects in the study area. According to the ecological risk index (RI) values, the upstream of the Jie River has a very high ecological risk for the waterbody. The data provided in this study are considered crucial for controlling and remediation of As and metals’ pollution of the southern Bohai Sea.     

Higher Abundance of Bacteria than of Viruses in Deep Mediterranean Sediments

The interactions between viral abundance and bacterial density, biomass, and production were investigated along a longitudinal transect consisting of nine deep-sea stations encompassing the entire Mediterranean basin. The numbers of viruses were very low (range, 3.6 × 10⁷ to 12.0 × 10⁷ viruses g⁻¹) and decreased eastward. The virus-to-bacterium ratio was always < 1.0, indicating that the deep-sea sediments of the Mediterranean Sea are the first example of a marine ecosystem not numerically dominated by viruses. The lowest virus numbers were found where the lowest bacterial metabolism and turnover rates and the largest cell size were observed, suggesting that bacterial doubling time might play an important role in benthic virus development.     

Rapid Assessment of Intertidal Wetland Sediments

Urbanization of coastal areas poses a severe threat to ecologically valuable intertidal wetlands. This paper presents a pragmatic approach called Rapid Assessment for Intertidal Wetland Sediments (RAITWS) for evaluating the sediment quality of intertidal wetlands. RAITWS involves construction of reference groups, selection of a subset of environmental variables, matching of test sites to reference groups, prediction of the benthic fauna community structure (e.g. of macroinvertebrates) at test sites, evaluation of the Observation to Expectation ratio (O/E ratio), quantification of environmental variables with series of dynamic numerical models, and interpretation of the O/E findings. The proposed method extends the existing rapid biological assessment approach from static to dynamic applications. In particular, RAITWS provides a fast method of assessing intertidal wetland sites which are undergoing ecological change due to nearby coastal development.