An improved procedure for shipboard enumeration of faecal indicator bacteria in marine sediments from sewage sludge disposal areas

An improved membrane filtration procedure for use on board ship to enumerate Escherichia coli and Group D faecal streptococci in marine sediments is described. Ultrasonication extraction combined with resuscitation of sublethally-injured cells yielded significantly higher counts of E. coli than sediments shaken by hand. Counts of E. coli were also higher on mFC agar (without rosalic acid) after a period of resuscitation on tryptone-soy agar supplemented with 0.1% yeast extract than on a 4% Teepol-lactose medium. Ultrasonication of sediments made no significant difference to counts of Group D faecal streptococci on KF-streptococcus agar. These improved isolation procedures allowed better discrimination of the area affected by sewage sludge at a disposal site off the northeast coast of England.     

An improved procedure for shipboard enumeration of faecal indicator bacteria in marine sediments from sewage sludge disposal areas

An improved membrane filtration procedure for use on board ship to enumerate Escherichia coli and Group D faecal streptococci in marine sediments is described. Ultrasonication extraction combined with resuscitation of sublethally-injured cells yielded significantly higher counts of E. coli than sediments shaken by hand. Counts of E. coli were also higher on mFC agar (without rosalic acid) after a period of resuscitation on tryptone-soy agar supplemented with 0.1% yeast extract than on a 4% Teepol-lactose medium. Ultrasonication of sediments made no significant difference to counts of Group D faecal streptococci on KF-streptococcus agar. These improved isolation procedures allowed better discrimination of the area affected by sewage sludge at a disposal site off the northeast coast of England.     

Rapid Method for the Radioisotopic Analysis of Gaseous End Products of Anaerobic Metabolism

A gas chromatographic procedure for the simultaneous analysis of (14)C-labeled and unlabeled metabolic gases from microbial methanogenic systems is described. H(2), CH(4), and CO(2) were separated within 2.5 min on a Carbosieve B column and were detected by thermal conductivity. Detector effluents were channeled into a gas proportional counter for measurement of radioactivity. This method was more rapid, sensitive, and convenient than gas chromatography-liquid scintillation techniques. The gas chromatography-gas proportional counting procedure was used to characterize the microbial decomposition of organic matter in anaerobic lake sediments and to monitor (14)CH(4) formation from H(2) and (14)CO(2) by Methanosarcina barkeri.     

A radioisotope assay for the quantification of hydrocarbon biodegradation potential in environmental samples

An enrichment culture method is described for quantifying the activity of hydrocarbon oxidizing bacteria in water and sediments. Application of the procedure indicated that the hydrocarbon oxidizing potential of environmental samples reflects the hydrocarbon burden of the area, the ability of the microflora to utilize hydrocarbons, and that lakes with large aquatic plant communities contain populations of hydrocarbon bacteria comparable to those found in oil-polluted harbors.     

Arsenic speciation in marine sediments: A comparison between two sequential extraction procedures

ABSTRACT

Arsenic distribution and mobility in marine sediments was investigated by means of total extraction and two sequential extraction procedures i.e. a modified sequential extraction procedure proposed by the European Standard, Measurementand Testing (SM&T) program, formerly the Community Bureau of Reference (BCR) procedure and a five steps sequential extraction based on the Wenzel extraction procedure, called modified Wenzel extraction. Sediments were collected from Cagliari’s harbour and Cagliari’s gulf, in Western Mediterranean Sea, Italy. The modified Wenzel extraction provided a more detailed As binding pattern and turned out to be much more appropriate than the modified SM&T procedure for gaining information regarding the mobilization of As within marine sediments. In the harbour, the largest part of As is contained in the residual fraction. At the contrary, As concentration, which is higher in the Cagliari’s gulf, is primarily associated with amorphous and crystalline hydrous Fe(Mn, Al) oxide. Moreover, this study suggests the possibility to segregate different types of marine sediments depending of human or industrial activities and to trace As contaminated marine sediments to determine the origin of contamination following the percentile of As contained in each extraction steps.     

Development of Sulfidogenic Sludge from Marine Sediments and Trichloroethylene Reduction in an Upflow Anaerobic Sludge Blanket Reactor

The importance of microbial sulfate reduction relies on the various applications that it offers in environmental biotechnology. Engineered sulfate reduction is used in industrial wastewater treatment to remove large concentrations of sulfate along with the chemical oxygen demand (COD) and heavy metals. The most common approach to the process is with anaerobic bioreactors in which sulfidogenic sludge is obtained through adaptation of predominantly methanogenic granular sludge to sulfidogenesis. This process may take a long time and does not always eliminate the competition for substrate due to the presence of methanogens in the sludge. In this work, we propose a novel approach to obtain sulfidogenic sludge in which hydrothermal vents sediments are the original source of microorganisms. The microbial community developed in the presence of sulfate and volatile fatty acids is wide enough to sustain sulfate reduction over a long period of time without exhibiting inhibition due to sulfide. This protocol describes the procedure to generate the sludge from the sediments in an upflow anaerobic sludge blanket (UASB) type of reactor. Furthermore, the protocol presents the procedure to demonstrate the capability of the sludge to remove by reductive dechlorination a model of a highly toxic organic pollutant such as trichloroethylene (TCE). The protocol is divided in three stages: (1) the formation of the sludge and the determination of its sulfate reducing activity in the UASB, (2) the experiment to remove the TCE by the sludge, and (3) the identification of microorganisms in the sludge after the TCE reduction. Although in this case the sediments were taken from a site located in Mexico, the generation of a sulfidogenic sludge by using this procedure may work if a different source of sediments is taken since marine sediments are a natural pool of microorganisms that may be enriched in sulfate reducing bacteria.     

Synoptic determination of living/dead and active/dormant bacterial fractions in marine sediments

The most widely used methods for the estimation of the living/dead fractions of bacterial cells involve specific stains that are able to reveal membrane integrity. Here, we have compared two different probes (propidium iodide and ethidium homodimer-2) that have different molecular weights and steric hindrance effects. We have also combined this method with the staining/destaining procedure that is currently used in the identification of potentially active cells. The procedure for marine sediments described here allows the synoptic (i.e. from the same filter) identification of: (i) the number of living bacteria; (ii) the number of active vs. dormant cells within this living fraction; (iii) the bacterial fraction with an intact nucleoid region without membrane integrity; and (iv) dead cells (devoid of the nucleoid region and without membrane integrity). Our results demonstrate that the concentration of propidium is crucial for the correct estimation of the dead bacterial fraction, ethidium homodimer-2 allows efficient and accurate estimates that are independent of the concentrations used and the sample storage. The active bacterial fraction represented c. 40% of the total bacterial abundance, the inactive/dormant fraction c. 30%, and the dead fraction was, on average, c. 30%. This method allows the processing of a large number of samples with high precision and at relatively low cost, and thus it provides additional synoptic insights into the metabolic state of bacteria in marine sediments.     

Contrasting two methods for determining trace metal partitioning in oxidized lake sediments

A simultaneous (SIM) sediment extraction procedure for low carbonate sediments, which partitions sediment-bound trace metals (Fe, Mn, Zn, Cu, and Cd) into easily reducible (associated with Mn oxides), reducible (associated with Fe oxides) and alkaline extracted (bound to organic) metal is presented. The SIM method was compared to the sequential (SEQ) extraction procedure of Tessier et al. (1979). Both methods showed good agreement for the partitioning of Zn and Cd among the easily reducible, reducible and organic components of sediment. Both methods also showed the same general distribution of Mn, Fe and Cu among the three sediment components, however concentrations of metals recovered by the two methods differed; less Mn and Fe and more Cu was recovered from sediments by the SEQ vs. the SIM procedure. Less recovery of Mn is in part attributed to the loss of this metal in the `in between' reagent rinses required in the SEQ procedure. Greater recovery of Cu by the SEQ vs. the SIM method may be due to the pretreatment of sediment with strong reducing agents prior to the step used for liberating organically bound metals. Advantages of a SIM over the SEQ include rapid sample processing time (i.e. the treatment of 40 samples per day vs. 40 samples in three days), plus minimal sample manipulation. Hence, for partitioning metals into easily reducible, reducible and organic sediment components in sediments low in carbonate, we recommend the use of a SIM extraction over that of a SEQ procedure.     

A Rapid and Simple Method for Estimating Sulfate Reduction Activity and Quantifying Inorganic Sulfides

A simplified passive extraction procedure for quantifying reduced inorganic sulfur compounds from sediments and water is presented. This method may also be used for the estimation of sulfate reduction rates. Efficient extraction of FeS, FeS(inf2), and S(sup2-) was obtained with this procedure; however, the efficiency for S(sup0) depended on the form that was tested. Passive extraction can be used with samples containing up to 20 mg of reduced sulfur. We demonstrated the utility of this technique in a determination of both sulfate reduction rates and reduced inorganic sulfur pools in marine and freshwater sediments. A side-by-side comparison of the passive extraction method with the established single-step distillation technique yielded comparable results with a fraction of the effort.     

Quantitative Centrifugation To Extract Benthic Protozoa from Freshwater Sediments

Two methods for extracting protists from freshwater sediment are described: (i) an adapted isopycnic centrifugation technique for sandy and gyttja-like sediments and (ii) a rate zonal centrifugation technique for sediments rich in particulate organic material (litter-like sediments). The recoveries of protists during isopycnic centrifugation in media of several densities were compared. No significant losses in sodium diatrizoate and Percoll were recorded. After known amounts of nanoflagellates were added to azoic sediments, the protists were extracted and counted. For sandy sediments, we found 100% recovery, and for the gyttja-like sediments we found a maximum recovery of 94%. The recovery of protozoa extracted from litter-like sediments, characteristic of littoral systems, depends on a given centrifugal force, on time, and on the dimensions of the flagellates. A recovery model which takes into account cell dimensions and centrifugation characteristics gives the minimum expected recovery.     

Alkane Biodegradation Genes from Chronically Polluted Subantarctic Coastal Sediments and Their Shifts in Response to Oil Exposure

Although sediments are the natural hydrocarbon sink in the marine environment, the ecology of hydrocarbon-degrading bacteria in sediments is poorly understood, especially in cold regions. We studied the diversity of alkane-degrading bacterial populations and their response to oil exposure in sediments of a chronically polluted Subantarctic coastal environment, by analyzing alkane monooxygenase (alkB) gene libraries. Sequences from the sediment clone libraries were affiliated with genes described in Proteobacteria and Actinobacteria, with 67?% amino acid identity in average to sequences from isolated microorganisms. The majority of the sequences were most closely related to uncultured microorganisms from cold marine sediments or soils from high latitude regions, highlighting the role of temperature in the structuring of this bacterial guild. The distribution of alkB sequences among samples of different sites and years, and selection after experimental oil exposure allowed us to identify ecologically relevant alkB genes in Subantarctic sediments, which could be used as biomarkers for alkane biodegradation in this environment. 16?S rRNA amplicon pyrosequencing indicated the abundance of several genera for which no alkB genes have yet been described (Oleispira, Thalassospira) or that have not been previously associated with oil biodegradation (Spongiibacter-formerly Melitea-, Maribius, Robiginitomaculum, Bizionia and Gillisia). These genera constitute candidates for future work involving identification of hydrocarbon biodegradation pathway genes.     

Rapid method for direct extraction of DNA from soil and sediments.

A rapid method for the direct extraction of DNA from soil and sediments was developed. The indigenous microorganisms in the soil and sediments were lysed by using lysozyme and a freeze-thaw procedure. The lysate was extracted with sodium dodecyl sulfate and phenol-chloroform. In addition to a high recovery efficiency (greater than 90%), the yields of DNA were high (38 and 12 micrograms/g [wet weight] from sediments and soil, respectively). This method generated minimal shearing of the extracted DNA. The crude DNA could be further purified with an Elutip-d column if necessary. An additional advantage of this method is that only 1 g of sample is required, which allows for the analysis of small samples and the processing of many samples in a relatively short (7 h) period.     

DNA extractions from deep subseafloor sediments: novel cryogenic-mill-based procedure and comparison to existing protocols

Extracting DNA from deep subsurface sediments is challenging given the complexity of sediments types, low biomasses, resting structures (spores, cysts) frequently encountered in deep sediments, and the potential presence of enzymatic inhibitors. Promising results for cell lysis efficiency were recently obtained by use of a cryogenic mill (Lipp et al., 2008). These findings encouraged us to devise a DNA extraction protocol using this tool. Thirteen procedures involving a combination of grinding in liquid nitrogen (for various durations and beating rates) with different chemical solutions (phenol, chloroform, SDS, sarkosyl, proteinase, GTC), or with use of DNA recovery kits (MagExtractor®) were compared. Effective DNA extraction was evaluated in terms of cell lysis efficiency, DNA extraction efficiency, DNA yield and determination of prokaryotic diversity. Results were compared to those obtained by standard protocols: the FastDNA®SPIN kit for soil and the Zhou protocol. For most sediment types grinding in a cryogenic mill at a low beating rate in combination with direct phenol-chloroform extraction resulted in much higher DNA yields than those obtained using classical procedures. In general (except for clay-rich sediments), this procedure provided high-quality crude extracts for direct downstream nested-PCR, from cell numbers as low as 1.1 × 10⁶ cells/cm³. This procedure is simple, rapid, low-cost, and could be used with minor modifications for large-scale DNA extractions for a variety of experimental goals.     

Rapid method for direct extraction of DNA from soil and sediments.

A rapid method for the direct extraction of DNA from soil and sediments was developed. The indigenous microorganisms in the soil and sediments were lysed by using lysozyme and a freeze-thaw procedure. The lysate was extracted with sodium dodecyl sulfate and phenol-chloroform. In addition to a high recovery efficiency (greater than 90%), the yields of DNA were high (38 and 12 micrograms/g [wet weight] from sediments and soil, respectively). This method generated minimal shearing of the extracted DNA. The crude DNA could be further purified with an Elutip-d column if necessary. An additional advantage of this method is that only 1 g of sample is required, which allows for the analysis of small samples and the processing of many samples in a relatively short (7 h) period.     

A new standardized sediment bioassay protocol using the amphipod Hyalella azteca (Saussure)

A new standardized bioassay procedure for testing the chronic toxicity of sediments to Hyalella azteca was developed. Tests were initiated with 0–1 wk old amphipods exposed to sediments from Hamilton Harbour, Toronto Harbour, and Lake Ontario for 4 to 8 weeks. Both survival and growth were significantly reduced in the Hamilton Harbour sediments relative to those from the lake after 4 weeks. Exposures of 8 weeks resulted in greater variability; survival of amphipods in sediments from one of the harbour stations, and growth in sediments from both harbour stations with surviving young were not statistically different from survival and growth in lake sediments. Growth and survival in lake sediments were comparable to cultures grown with cotton gauze and no sediment after 4 weeks, but survival was poorer by week 8. Replication was good in 12 out of 13 tests done in duplicate; the difference in survival between replicates averaged 2.2 animals (20 amphipods/replicate, 4 week exposure). We propose that 4 week exposures of young (0–1 wk old) Hyalella would provide a suitable standardized chronic toxicity test for sediments. A detailed protocol on the methodology is presented.     

Simultaneous Recovery of Extracellular and Intracellular DNA Suitable for Molecular Studies from Marine Sediments

The occurrence of high extracellular DNA concentrations in aquatic sediments (concentrations that are 3 to 4 orders of magnitude greater than those in the water column) might play an important role in biogeochemical cycling, as well as in horizontal gene transfer through natural transformation. Since isolation of extracellular DNA from sediments is a difficult and unsolved task, in this study we developed an efficient procedure to recover simultaneously DNA associated with microbial cells and extracellular DNA from the same sediment sample. This procedure is specifically suitable for studying extracellular DNA because it avoids any contamination with DNA released by cell lysis during handling and extraction. Applying this procedure to different sediment types, we obtained extracellular DNA concentrations that were about 10 to 70 times higher than the intracellular DNA concentrations. Using specific targeted prokaryotic primers, we obtained evidence that extracellular DNA recovered from different sediments did not contain amplifiable 16S rRNA genes. By contrast, using DNA extracted from microbial cells as the template, we always amplified 16S rRNA genes. Although 16S rRNA genes were not detected in extracellular DNA, analyses of the sizes of extracellular DNA indicated the presence of high-molecular-weight fragments that might have contained other gene sequences. This protocol allows investigation of extracellular DNA and its possible participation in natural transformation processes.     

Simultaneous recovery of extracellular and intracellular DNA suitable for molecular studies from marine sediments

The occurrence of high extracellular DNA concentrations in aquatic sediments (concentrations that are 3 to 4 orders of magnitude greater than those in the water column) might play an important role in biogeochemical cycling, as well as in horizontal gene transfer through natural transformation. Since isolation of extracellular DNA from sediments is a difficult and unsolved task, in this study we developed an efficient procedure to recover simultaneously DNA associated with microbial cells and extracellular DNA from the same sediment sample. This procedure is specifically suitable for studying extracellular DNA because it avoids any contamination with DNA released by cell lysis during handling and extraction. Applying this procedure to different sediment types, we obtained extracellular DNA concentrations that were about 10 to 70 times higher than the intracellular DNA concentrations. Using specific targeted prokaryotic primers, we obtained evidence that extracellular DNA recovered from different sediments did not contain amplifiable 16S rRNA genes. By contrast, using DNA extracted from microbial cells as the template, we always amplified 16S rRNA genes. Although 16S rRNA genes were not detected in extracellular DNA, analyses of the sizes of extracellular DNA indicated the presence of high-molecular-weight fragments that might have contained other gene sequences. This protocol allows investigation of extracellular DNA and its possible participation in natural transformation processes.     

Estimation of methanogen biomass by quantitation of coenzyme M

Determination of the role of methanogenic bacteria in an anaerobic ecosystem often requires quantitation of the organisms. Because of the extreme oxygen sensitivity of these organisms and the inherent limitations of cultural techniques, an accurate biomass value is very difficult to obtain. We standardized a simple method for estimating methanogen biomass in a variety of environmental matrices. In this procedure we used the thiol biomarker coenzyme M (CoM) (2-mercaptoethanesulfonic acid), which is known to be present in all methanogenic bacteria. A high-performance liquid chromatography-based method for detecting thiols in pore water (A. Vairavamurthy and M. Mopper, Anal. Chim. Acta 78:363-370, 1990) was modified in order to quantify CoM in pure cultures, sediments, and sewage water samples. The identity of the CoM derivative was verified by using liquid chromatography-mass spectroscopy. The assay was linear for CoM amounts ranging from 2 to 2,000 pmol, and the detection limit was 2 pmol of CoM/ml of sample. CoM was not adsorbed to sediments. The methanogens tested contained an average of 19.5 nmol of CoM/mg of protein and 0.39 +/- 0.07 fmol of CoM/cell. Environmental samples contained an average of 0.41 +/- 0.17 fmol/cell based on most-probable-number estimates. CoM was extracted by using 1% tri-(N)-butylphosphine in isopropanol. More than 90% of the CoM was recovered from pure cultures and environmental samples. We observed no interference from sediments in the CoM recovery process, and the method could be completed aerobically within 3 h. Freezing sediment samples resulted in 46 to 83% decreases in the amounts of detectable CoM, whereas freezing had no effect on the amounts of CoM determined in pure cultures. The method described here provides a quick and relatively simple way to estimate methanogenic biomass.     

A versatile and sensitive tritium-based radioassay for measuring hydrogenase activity in aquatic sediments

We present a method for the measurement of hydrogenase (H(2)ase) activity in aquatic sediments. The assay is based on the H(2)ase-mediated isotopic exchange between dissolved molecular hydrogen (H(2)) and water. A slurry of sediment material is incubated with a tritiated hydrogen (HT) headspace in a glass syringe on a rotary shaker. The method includes a procedure for preparing HT from radiolabeled sodium borohydride, which is a useful alternative to purchasing HT directly. A method for measuring HT specific activity based on liquid scintillation counting is also presented. Validation tests were run using live and frozen cultures of Clostridium pasteurianum and Desulfovibrio vulgaris, and freshly collected marine sediments. Adherence to Michaelis-Menten kinetics was demonstrated. An interassay coefficient of variation of 15% was determined using frozen C. pasteurianum cultures as reference material. Serial dilutions of cultures and sediments showed that measured H(2)ase activity scales with cell concentration, and indicate that the method can detect C. pasteurianum cell concentrations of between 300 and 3000 cells/ml. This technique allows measurement of H(2)ase activity in a variety of environmental samples, and will be particularly useful in the study of deep marine sediments with low microbial activity.     

Ammonium adsorption in sediments of a tropical mangrove forest (Thailand) and a temperate Wadden Sea area (Denmark)

Dimensionless ammonium adsorption coefficients (K) were determined for tropical mangrove forest sediments and temperate Wadden Sea sediments. The K values were obtained from ammonium adsorption isotherms of KCl (2 M) extractable ammonium versus dissolved ammonium in the porewater; relationships that can be described by a linear model. Linearity was evident even at low porewater concentrations for mangrove sediment, according to isotherms based on KCl extractions on untreated sediment profiles. K-values were low in mangrove forest sediments (0.04–0.10), and higher in Wadden Sea sediments (0.17–1.12). The vertical range in K-values was larger at the vegetated sites, with highest values in subsurface sediments, which suggests differences in quantity and quality of the fine organic and inorganic fractions. The low ammonium adsorption in the mangrove sediments can be explained by a higher concentration of competitive cations, especially iron, in this iron-rich environment. The low adsorption of ammonium in mangrove sediments and vegetated surface sediment of the Wadden Sea was negatively related to the organic content of the sediments, which is in contrast to findings for other marine sediments. We suggest that organic material may have a diluting effect on the exchange capacity in fine-grained sediments, and that organic coatings may block ion exchange sites on clay surfaces. Thus, there may be a organic-rich ‘wetland’ versus organic-poor ‘sediment’ effect. This revised version was published online in July 2006 with corrections to the Cover Date.