Autoclaved Sediment Research Articles

Potential Activities and Long Lifetimes of Organic Carbon-Degrading Extracellular Enzymes in Deep Subsurface Sediments of the Baltic Sea.

Heterotrophic microorganisms in marine sediments produce extracellular enzymes to hydrolyze organic macromolecules, so their products can be transported inside the cell and used for energy and growth. Therefore, extracellular enzymes may mediate the fate of organic carbon in sediments. The Baltic Sea Basin is a primarily depositional environment with high potential for organic matter preservation. The potential activities of multiple organic carbon-degrading enzymes were measured in samples obtained by the International Ocean Discovery Program Expedition 347 from the Little Belt Strait, Denmark, core M0059C. Potential maximum hydrolysis rates (Vmax) were measured at depths down to 77.9mbsf for the following enzymes: alkaline phosphatase, β-d-xylosidase, β-d-cellobiohydrolase, N-acetyl-β-d-glucosaminidase, β-glucosidase, α-glucosidase, leucyl aminopeptidase, arginyl aminopeptidase, prolyl aminopeptidase, gingipain, and clostripain. Extracellular peptidase activities were detectable at depths shallower than 54.95mbsf, and alkaline phosphatase activity was detectable throughout the core, albeit against a relatively high activity in autoclaved sediments. β-glucosidase activities were detected above 30mbsf; however, activities of other glycosyl hydrolases (β-xylosidase, β-cellobiohydrolase, N-acetyl-β-glucosaminidase, and α-glucosidase) were generally indistinguishable from zero at all depths. These extracellular enzymes appear to be extremely stable: Among all enzymes, a median of 51.3% of enzyme activity was retained after autoclaving for an hour. We show that enzyme turnover times scale with the inverse of community metabolic rates, such that enzyme lifetimes in subsurface sediments, in which metabolic rates are very slow, are likely to be extraordinarily long. A back-of-the-envelope calculation suggests enzyme lifetimes are, at minimum, on the order of 230days, and may be substantially longer. These results lend empirical support to the hypothesis that a population of subsurface microbes persist by using extracellular enzymes to slowly metabolize old, highly degraded organic carbon.

How to grow your cable bacteria: Establishment of a stable single-strain culture in sediment and proposal of Candidatus Electronema aureum GS

Cable bacteria are multicellular filamentous bacteria within the Desulfobulbaceae that couple the oxidation of sulfide to the reduction of oxygen over centimeter distances via long distance electron transport (LDET). So far, none of the freshwater or marine cable bacteria species have been isolated into pure culture. Here we describe a method for establishing a stable single-strain cable bacterium culture in partially sterilized sediment. By repeated transfers of a single cable bacterium filament from freshwater pond sediment into autoclaved sediment, we obtained strain GS, identified by its 16S rRNA gene as a member of Ca. Electronema. This strain was further propagated by transferring sediment clumps, and has now been stable within its semi-natural microbial community for several years. Its metagenome-assembled genome was 93% complete, had a size of 2.76 Mbp, and a DNA G + C content of 52%. Average Nucleotide Identity (ANI) and Average Amino Acid Identity (AAI) suggest the affiliation of strain GS to Ca. Electronema as a novel species. Cell size, number of outer ridges, and detection of LDET in the GS culture are likewise consistent with Ca. Electronema. Based on these combined features, we therefore describe strain GS as a new cable bacterium species of the candidate genus Electronema, for which we propose the name Candidatus Electronema aureum sp.nov. Although not a pure culture, this stable single-strain culture will be useful for physiological and omics-based studies; similar approaches with single-cell or single-filament transfers into natural medium may also aid the characterization of other difficult-to-culture microbes.

Potential of dissimilatory nitrate reduction pathways in polycyclic aromatic hydrocarbon degradation

This study investigates the potential of an indigenous estuarine microbial consortium to degrade two polycyclic aromatic hydrocarbons (PAHs), naphthalene and fluoranthene, under nitrate-reducing conditions. Two physicochemically diverse sediment samples from the Lima Estuary (Portugal) were spiked individually with 25 mg L−1 of each PAH in laboratory designed microcosms. Sediments without PAHs and autoclaved sediments spiked with PAHs were run in parallel. Destructive sampling at the beginning and after 3, 6, 12, 30 and 63 weeks incubation was performed. Naphthalene and fluoranthene levels decreased over time with distinct degradation dynamics varying with sediment type. Next-generation sequencing (NGS) of 16 S rRNA gene amplicons revealed that the sediment type and incubation time were the main drivers influencing the microbial community structure rather than the impact of PAH amendments. Predicted microbial functional analyses revealed clear shifts and interrelationships between genes involved in anaerobic and aerobic degradation of PAHs and in the dissimilatory nitrate-reducing pathways (denitrification and dissimilatory nitrate reduction to ammonium - DNRA). These findings reinforced by clear biogeochemical denitrification signals (NO3− consumption, and NH4+ increased during the incubation period), suggest that naphthalene and fluoranthene degradation may be coupled with denitrification and DNRA metabolism. The results of this study contribute to the understanding of the dissimilatory nitrate-reducing pathways and help uncover their involvement in degradation of PAHs, which will be crucial for directing remediation strategies of PAH-contaminated anoxic sediments.

Mechanisms of the photochemical release of phosphate from resuspended sediments under solar irradiation

In previous studies, resuspended sediments that were exposed to simulated solar irradiation could release dissolved phosphate (PO43−). However, the mechanisms of phosphate release remain unclear. In this research, a battery of experiments was performed to reveal the mechanisms of the photochemical release of phosphate from resuspended sediments of a shallow eutrophic lake under solar irradiation. The results show that the PO43− released in resuspended sediments was significantly higher than that in the dark control or in water alone after treatment with solar irradiation for 6h. The results of sequential chemical extractions showed that the concentrations of labile organic, moderately labile organic and residual organic phosphorus decreased in the resuspended sediment after 6h of solar irradiation; of these, moderately labile organic phosphorus was the greatest contributor to the release of dissolved phosphate in resuspended sediment. Orthophosphate, phosphate monoesters, phosphate diesters and pyrophosphate were detected with 31P NMR. It is worth mentioning that the diester-P and pyro-P species disappeared after 6h of irradiation. In addition, enzyme activity and radical trapping experiments were applied to identify the roles of biomineralization and photochemical degradation during phosphate release from resuspended sediments under solar irradiation. The amount of PO43− released in fresh sediment was greater than that in the autoclaved sediment, which should be attributed to the higher alkaline phosphatase activity in the fresh sediment. However, the PO43− released from the photochemical degradation of organic phosphorus is the primary phosphate source during sediment resuspension under 6h of solar irradiation. The phosphate photorelease was inhibited when methanol was added to the suspension and decreased significantly when the concentration of methanol was increased from 0.5M to 2.0M. All of these results suggest that photochemical processes may lead to PO43− release from sediment in aquatic environments.

Bioleaching of heavy metals from contaminated sediments by the Aspergillus niger strain SY1

The objective of this study was to investigate the bioleaching of heavy metals from contaminated sediments by Aspergillus niger strain SY1. To achieve this, three targets were identified: (1) identify organic acids produced by the isolated A. niger strain SY1 from contaminated sediments, (2) compare the leaching ability and transformation of chemical speciation of heavy metals during the bioleaching processes, and (3) determine the toxic characteristic of sediment before and after bioleaching. The contaminated sediment was collected from the dredging of the Xihe River, China. The A. niger strain SY1 was isolated from this sediment. Bioleaching experiments were carried out in 250 ml autoclaved conical flasks with 10 g autoclaved sediment, 1 ml of spore suspension, and 99 ml culture medium; the flasks were placed in a shaking incubator (220 rpm) at 30 °C for 7 days. Toxicity characteristic leaching procedure (TCLP) tests were carried out according to USEPA-SW846 Method 1311, and the wheat and earthworm toxicity tests were carried out according to OECD “Guidelines for the Testing of Chemicals.” Fractionation of heavy metals was undertaken by the three-step sequential extraction procedure. The metabolites were determined with a HPLC system. There was 11.5 % leaching efficiency of Pb from the polluted sediment in the one-step bioleaching process; while in the two-step bioleaching process, the highest extraction efficiency of Pb was 65.4 %. In one-step bioleaching, 93.5 % Cd, 62.3 % Cu, and 68.2 % Zn were leached out; whereas, the highest metal extraction efficiencies of Cd, Cu, and Zn were 99.5, 56, 71.9, and 76.4 %, respectively, in two-step bioleaching. After the bioleaching, the metals remaining in the sediment were mainly found in the stable fractions. Cd, Pb, Cu, and Zn concentrations in extracted liquor of TCLP tests were reduced to far below the levels in two Chinese standards, and the sediment after bioleaching had a lower toxicity on wheat and earthworm. A. niger strain SY1 can effectively remove heavy metals in contaminated sediment. The bioleaching efficiencies of heavy metals in the two-step bioleaching were better than that in one-step bioleaching. After the bioleaching, metals remaining in the sediment were mainly found in the stable fractions, and the toxicity of it was reduced to a level for it to be used safely in landfill or used in land application. A. niger strain SY1 is a cost-effective, environmentally friendly, and efficient bioleacher of heavy metals.

Effects of arbuscular mycorrhizal inoculation and phosphorus supply on the growth and nutrient uptake of Kandelia obovata (Sheue, Liu & Yong) seedlings in autoclaved soil

This study evaluated the interactive effect of arbuscular mycorrhizal fungi (AMF) inoculation and exogenous phosphorus supply on soil phosphotases, plant growth, and nutrient uptake of Kandelia obovata (Sheue, Liu & Yong). We aimed to explore the ecophysiological function of AMF in mangrove wetland ecosystems, and to clarify the possible survival mechanism of mangrove species against nutrient deficiency. K. obovata seedlings with or without AMF inoculation (mixed mangrove AMF), were cultivated for six months in autoclaved sediment medium which was supplemented with KH2PO4 (0, 15, 30, 60, 120mgkg−1). Then the plant growth, nitrogen and phosphorus content, root vitality, AMF colonization and soil phosphatase activity were analyzed. The inoculated AMF successfully infected K. obovata roots, developed intercellular hyphae, arbuscular (Arum-type), and vesicle structures. Arbuscular mycorrhizal fungi colonization ranged from 9.04 to 24.48%, with the highest value observed under 30 and 60mgkg−1 P treatments. Soil P supply, in the form of KH2PO4, significantly promoted the height and biomass of K. obovata, enhanced root vitality and P uptake, while partially inhibiting soil acid (ACP) and alkaline phosphotase (ALP) activities. Without enhancing plant height, the biomass, root vitality and P uptake were further increased when inoculated with AMF, and the reduction on ACP and ALP activities were alleviated. Phosphorus supply resulted in the decrease of leaf N–P ratio in K. obovata, and AMF inoculation strengthened the reduction, thus alleviating P limitation in plant growth. Arbuscular mycorrhizal fungi inoculation and adequate P supply (30mgkg−1 KH2PO4) enhanced root vitality, maintained soil ACP and ALP activities, increased plant N and P uptake, and resulted in greater biomass of K. obovata. Mutualistic symbiosis with AMF could explain the survival strategies of mangrove plants under a stressed environment (waterlogging and nutrient limitation) from a new perspective.

Transformation of 17α-Estradiol, 17β-Estradiol, and Estrone in Sediments Under Nitrate- and Sulfate-Reducing Conditions

The natural manure-borne hormones, 17α-estradiol (17α-E2), 17β-estradiol (17β-E2), and estrone (E1), are routinely detected in surface water near agricultural land and wastewater treatment facilities. Once in the stream network, hormones may enter the sediment bed where they are subject to anaerobic conditions. This study focuses on the difference in anaerobic transformation rates and formation of metabolites from 17α-E2, 17β-E2, and E1 (applied at ∼3.66 μmol kg(-1) of sediment on a dry weight basis) under nitrate- and sulfate-reducing conditions. Sediment extracts were analyzed using negative electrospray ionization tandem mass spectrometry. Under both redox conditions, degradation was stereospecific and followed similar trends in half-lives, 17β-E2 < 17α-E2 < E1, with degradation considerably slower under sulfate-reducing conditions. Both E2 isomers were predominantly converted to E1; however, isomeric conversion also occurred with peak concentrations of ∼1.7 mol % of 17β-E2 formed in 17α-E2 amended sediments and peak concentrations of ∼2.4 mol % of 17α-E2 formed from 17β-E2. In E1-amended systems, E1 transformed to E2 with preferential formation of the more potent 17β isomer up to ∼30 mol % suggesting that isomer interconversion is through E1. Sediments, therefore, may serve as both a sink and a source of the more estrogenic compound E2. Transformation of amended hormones in autoclaved sediments was markedly slower than in nonautoclaved sediments. Results support the inclusion of isomer-specific behavior and the potential for reversible transformation and interconversion in anaerobic sediments in modeling fate in stream networks and developing risk management strategies.

Acceleration of Nonylphenol and 4-tert-Octylphenol Degradation in Sediment by Phragmites australis and Associated Rhizosphere Bacteria

We investigated biodegradation of technical nonylphenol (tNP) in Phragmites australis rhizosphere sediment by conducting degradation experiments using sediments spiked with tNP. Accelerated tNP removal was observed in P. australis rhizosphere sediment, whereas tNP persisted in unvegetated sediment without plants and in autoclaved sediment with sterile plants, suggesting that the accelerated tNP removal resulted largely from tNP biodegradation by rhizosphere bacteria. Three bacterial strains, Stenotrophomonas sp. strain IT-1 and Sphingobium spp. strains IT-4 and IT-5, isolated from the rhizosphere were capable of utilizing tNP and 4-tert-octylphenol as a sole carbon source via type II ipso-substitution. Oxygen from P. australis roots, by creating highly oxygenated conditions in the sediment, stimulated cell growth and the tNP-degrading activity of the three strains. Moreover, organic compounds from P. australis roots functioned as carbon and energy sources for two strains, IT-4 and IT-5, supporting cell growth and tNP-degrading activity. Thus, P. australis roots elevated the cell growth and tNP-degrading activity of the three bacterial strains, leading to accelerated tNP removal. These results demonstrate that rhizoremediation of tNP-contaminated sediments using P. australis can be an effective strategy.

Short-term indicators of seagrass transplant stress in response to sediment bacterial community disruption

Abstract Bacterial communities in sediments engage in activities that determine the pathways and rates of organic matter remineralization. Changes in bacterial community composition might result in greater restoration success if interactions among seagrasses, sediment, and bacterial communities were elucidated. A manipulative experiment that disrupted the sediment bacterial community examined the response of Thalassia testudinum transplants. Planting units were transported to a suitable location and replanted with either autoclaved, transplant, or donor site sediments. Sediments from the rhizosphere were sub-sampled for analysis of the 16S rDNA gene. The autoclave treatment group had significantly lower bacterial diversity than the other treatments, as measured with terminal restriction fragment length polymorphism. Significantly higher mortality of the transplants was observed in the autoclave treatment group, while mortality in the donor and transplant treatment groups was low relative to the control group. Rhizosphere disruption through the removal of surrounding sediments did not increase mortality, while manipulating the composition of the bacterial community increased mortality in transplants. A native bacterial community was a critical component for minimizing short-term stress associated with a seagrass transplant, which suggests a tighter coupling between seagrasses and sediment bacterial communities than previously thought.

Importance of the Sedimentary Matrix for Anaerobic Oil Degradation by Marine Bacterial Assemblages

ABSTRACT The microbial conversion of petroleum hydrocarbons is increasingly employed in bioremediation efforts. In marine sediments, oxygen levels are characteristically depleted, so anaerobic degradation coupled to sulfate reduction dominates. Prior studies have noted that anaerobic degradation is much reduced in the absence of sediments. In this study, a simple centrifugation protocol was used to extract sediment porewaters to obtain a sediment-free bacterial assemblage capable of anaerobic hydrocarbon degradation. In addition, the factors in sediment that were important to degradation rates were determined. Experiments were designed to differentiate among the effects of increased surface area associated with individual grains in sediments, differing levels of organic constituents in sediments and water, and disparate microbiota within the sedimentary matrix and those free living. Anaerobic alkane degradation, sulfate levels and bacterial community structure were monitored over 90 days in five treatments consisting of Bonny Light crude oil added to (1) intact sediment, (2) sediment-free supernate from centrifuged sediment, (3) supernate plus autoclaved sediment, (4) supernate plus organic-free (combusted) sediment, and (5) a control, with autoclaved supernate plus autoclaved sediment. Lack of surface area associated with sediment grains had little effect on degradation. Separation of porewaters from the sedimentary matrix resulted in loss of bacterial biomass, although this had only a temporary negative effect on degradation rates. Reduction of organic matter due to sediment removal had the largest effect, resulting initially in lower degradation rates. However, sulfate depletion in low organic treatments was also reduced so that long-term loss of alkanes was enhanced.

Nonbiological removal of cis-dichloroethylene and 1,1-dichloroethylene in aquifer sediment containing magnetite.

The U.S. EPA Technical Protocol for Evaluating Natural Attenuation of Chlorinated Solvents in Groundwater emphasizes biological reductive dechlorination as the primary mechanism for destruction of chlorinated solvents. However, biological reductive dechlorination could not explain the removal of cis-dichloroethylene (cis-DCE) and 1,1-DCE from a plume of contaminated groundwater in Minnesota. Several recent laboratory studies have demonstrated that common iron minerals such as magnetite can also transform chlorinated alkenes. Laboratory microcosms were constructed with sediment from three depth intervals in the aquifer near the source of the plume. The microcosms were autoclaved to prevent biological transformations. In these autoclaved sediments, the rates of removal of cis-DCE in samples from the shallow, intermediate, and deeper depth intervals in the aquifer were 0.58 +/- 0.09, 2.29 +/- 0.26, and 0.31 +/- 0.08 per year at 95% confidence. The rate of removal of 1,1-DCE in sediment from the shallow interval was 1.37 +/- 0.50 per year. The rates of removal in the microcosms are similar to the rates of attenuation observed in the field. Magnetite was identified in the sediment by X-ray diffraction and optical microscopy. Published rates of transformation of cis-DCE by magnetite are consistent with the rates of removal in the microcosm study.

Different methods for extracting bacteria from freshwater sediment and a simple method to measure bacterial production in sediment samples

The efficiency of different treatments was tested to extract bacterial cells from freshwater sediment samples. The influence of sonication, density gradient centrifugation, fixation by formalin and centrifugation speed on bacterial recovery was investigated. The method developed by Smith and Azam [Mar. Microb. Food Webs 6 (1992) 107] to measure microbial activity on bacterioplankton ( 3H-leucine incorporation), was also evaluated in sediment samples. After 1 min of sonication bacterial abundance was reduced by about 47% in diluted sediments with tetrasodium pyrophosphate. With the addition of Percoll after sonication, bacterial counts were not significantly different ( P<0.05). Fixation by formalin increased bacterial counts using sonication. However, higher bacterial abundance was estimated in non-sonicated samples. Bacterial abundance in samples centrifuged at 7000× g with and without Percoll was not significantly different ( P<0.05). Highest bacterial abundance was obtained after centrifugation at low speed (750× g). Bacterial abundance decreased with higher centrifugation speed (750, 1500 and 3000× g), the difference, however, was not significant. Bacterial production ranged from 0.10 μg C cm −3 d −1 in autoclaved sediment to 0.27 μg C cm −3 d −1 in untreated sediment. The radioactivity measured in controls of both untreated and autoclaved sediment was high (70 and 91%, respectively), indicating a high level of leucine adsorption in sediment particles. In contrast, radioactivity in control samples previously centrifuged was markedly lower (6%). Despite the high values of radioactivity in the controls, bacterial production in untreated sediment was significantly higher than in centrifuged sediment ( P<0.05).

Survival of Inoculants in Polluted Sediments: Effect of Strain Origin and Carbon Source Competition

Inoculation of polluted environments with specialized strains is an important strategy in bioremediation. Especially the efficacy and safety of genetically engineered biodegradative microorganisms could be improved further, if host organisms with predictable survival in specific target ecosystems were identified and could be engineered to carry catabolic gene cassettes. Here we empirically tested whether strains originally isolated from the target ecosystem (indigenous inoculants) had superior survival rates compared to strains derived from geographically and ecologically different locations (nonindigenous inoculants). We experimentally tested this approach by isolating various strains of Pseudomonas and related genera from a highly polluted river sediment (Spittelwasser River) and following their survival in sediment slurries from the same river in comparison with that of nonindigenous strains. Both indigenous and nonindigenous inoculants grew and maintained cell densities of between 107 and 109 cfu ml−1 in autoclaved sediment slurries for up to 70 days, indicating that they were not negatively affected by abiotic factors, especially potentially toxic pollutants. In live sediment slurries, however, all inoculants died out at varying rates. If live sediment slurries were amended with a carbon source that the inoculant could degrade, most strains were able to grow, regardless of their original source. In some cases, high cell densities of the inoculant were maintained for up to 70 days, even after exhaustion of the added carbon source. Reisolating these strains and monitoring their survival in sediment slurries from a different sampling date did not reveal increased survival rates compared to the original strains. It is concluded that the use of indigenous isolates for bioremediation and as hosts for constructing genetically engineered organisms does not provide any advantage in dynamic, highly competitive environments. For safety reasons, therefore, a limited number of well-characterized strains whose pathogenic potential is known and for which ecological information can be obtained, should be used. To ensure survival of the inoculant, either the site must be engineered to provide a temporary advantage for the introduced strain, or the inoculant must be able to exploit a specific niche better than the indigenous microbial community.

Dechlorination of chlorobenzenes in anaerobic estuarine sediment

The reductive dechlorination of chlorobenzenes (CBs) in an anaerobic estuarine sediment was examined. The sediment was preexposed to various anthropogenic chemicals from the surrounding industries. The sulfate content (20 mmoles/l) in the interstitial waters indicated that the sediment was sulfidogenic. All the CBs were transformed following a first-order reaction kinetics with rate constants ranging from 0.0016 to 0.0389 day−1 or half-lives between 17 and 433 days. Disappearance of CBs in autoclaved sediment were observed but to a much less extent than in the test sediment. Detected intermediates indicated that the removal of chlorine atom occurred at all possible positions on the aromatic ring. The contribution of different dechlorination pathways were estimated using branched chain first-order irreversible reaction kinetics. The results showed that the preferential dechlorination was in the following order: two chlorine atoms on both sides &amp;gt; one of the adjacent chlorine atoms &amp;gt; chlorine with no adjacent chlorine atoms. The major pathway observed for CBs followed a thermodynamically most favorable step that gave the highest energy for dechlorination.

Dechlorination of chlorobenzenes in anaerobic estuarine sediment

The reductive dechlorination of chlorobenzenes (CBs) in an anaerobic estuarine sediment was examined. The sediment was preexposed to various anthropogenic chemicals from the surrounding industries. The sulfate content (20 mmoles/l) in the interstitial waters indicated that the sediment was sulfidogenic. All the CBs were transformed following a first-order reaction kinetics with rate constants ranging from 0.0016 to 0.0389 day-1 or half-lives between 17 and 433 days. Disappearance of CBs in autoclaved sediment were observed but to a much less extent than in the test sediment. Detected intermediates indicated that the removal of chlorine atom occurred at all possible positions on the aromatic ring. The contribution of different dechlorination pathways were estimated using branched chain first-order irreversible reaction kinetics. The results showed that the preferential dechlorination was in the following order: two chlorine atoms on both sides > one of the adjacent chlorine atoms > chlorine with no adjacent chlorine atoms. The major pathway observed for CBs followed a thermodynamically most favorable step that gave the highest energy for dechlorination.

Bioavailability and Transformation of Highly Chlorinated Dibenzo-p-Dioxins and Dibenzofurans in Anaerobic Soils and Sediments

The long-term fate of environmentally relevant concentrations (ppb) of penta- to heptachlorinated dibenzo-p-dioxin (PCDD) and dibenzofuran (PCDF) congeners was evaluated in both high and low organic carbon anaerobic sediments. The inocula were derived from historically contaminated environments and were amended with a mixture of aliphatic and aromatic acids to stimulate anaerobic microbial activity. The long-term (>2 years) removal patterns of sediment-sorbed PCDD/PCDF in both sediments could be explained by labile and resistant PCDD/PCDF desorption components, presumably due to intraparticle diffusion-controlled mass transfer limitations. Mass transfer limitations were based on incubation time-dependent decreased extraction efficiencies of PCDD/PCDF from autoclaved controls. Lesser chlorinated congeners were observed in both active and autoclaved sediments, accounting for up to 30% of the PCDD/F spiked. Whereas tetraCDD accumulated from hepta-, hexa-, and pentaCDD/F in active sediments, the extent of dechlorination in autoclaved sediments was limited to removal of one to two chlorines. These observations provide the first evidence for the contribution of biologically mediated reductive dechlorination of PCDD/PCDF to the overall fate of these contaminants in the environment and may lead the way to designing in situ transformation/biodegradation systems.

The effect of manipulations of freshwater sediments on responses of benthic invertebrates in whole‐sediment toxicity tests

AbstractManipulations of freshwater sediment were performed to remove indigenous organisms prior to conducting toxicity tests with three species of benthic invertebrates. The effects of these treatments on end points in bioassays were compared within and between two sediments, i.e., a “clean” sediment and a “contaminated” sediment. In addition, the effects of manipulations on the physicochemical structure of the two sediments and the presence of metals, PAHs, and PCBs in the contaminated sediment were examined. The amphipod Hyalella azteca was most sensitive to the manipulations and had low survival in sediment that was sterilized. Growth (milligrams dry weight per individual) was affected by the presence of contaminants. Survival of Chironomus riparius was not affected by any manipulation but was reduced by contaminants as well as indigenous organisms. Growth of C. riparius was higher in autoclaved sediment but lower in sediment containing endemic tubificid worms. Production of young by Tubifex tubifex increased in sediment that was irradiated, possibly due to increased detrital material. Particle size distribution, metals, nutrients, and PAHs varied little as a function of manipulation; however, sieving of sediment through 250‐μm mesh did reduce percent total organic carbon (TOC), percent loss on ignition (LOI), and concentrations of some PCBs in either clean or contaminated sediment. Manipulation of sediments to remove endemic species should be determined on a case‐by‐case basis and is specific to the organisms used in toxicity tests.

Cadmium sorption by bacteria and freshwater sediment

Sorption of cadmium by sediment bacteria and freshwater sediment was investigated using diffusion chambers to simulate the water-sediment interface. Diffusion chambers were constructed to provide two compartments separated by a dialysis membrane. Diffusion of cadmium across the membrane was monitored after pure cultures of sediment bacteria or lake sediments were added to the sediment side of a diffusion chamber. Cellular accumulation of cadmium by cadmium-sensitive and cadmium-resistant bacteria removed between 20% and 80% of the dissolved cadmium from the simulated water column and pore water. Cellular accumulation of cadmium was greatest for cadmium-sensitive isolates that were tested. Sediment with an intact microbial community sequestered 80% of the cadmium added to sediment, whereas autoclaved sediment retained 97% of the metal that was added. Addition of glucose to cadmium-amended sediment decreased retention of cadmium by untreated and autoclaved sediments, resulting in elevated concentrations of dissolved cadmium in the simulated water column.

Survival ofEscherichia coliK12 in seawater

The fate of an auxotrophic Escherichia coli K12 strain (NF1830) in coastal water was investigated. The E. coli K12 were enumerated after incubation for varying times in seawater. Incubated in raw seawater at 15 and 20°C, the NF1830 decreased from 106 cfu/ml to below detection within six days of incubation, but when incubated at 7°C it persisted longer. The NF1830 was capable of cell division in sterile seawater. Growth was also shown to occur in raw seawater in the presence of autoclaved sediment. The E. coli K12 decreased in number at a much lower rate when incubated in seawater treated with eukaryotic inhibitors. These findings suggest that the die-off of the auxotrophic E. coli K12 strain seen in the raw seawater was caused by grazing of bacterial predators in the seawater.

Binding of exogenous DNA to marine sediments and the effect of DNA/sediment binding on natural transformation ofPseudomonas stutzeristrain ZoBell in sediment columns

The capacity of marine sediments to facilitate natural transformation of Pseudomonas stutzeri strain ZoBell was investigated. The role of DNA/ sediment binding on transformation frequency was also explored. While transformation frequencies increased as a function of DNA concentrations from 0 to 1.0 μg of DNA from rifampin-resistant strains for filter transformations, transformation in autoclaved sediment columns displayed a reduction in transformation frequency in response to low DNA concentrations (0–1.0 μg/cm3 sediment). Maximum transformation frequencies were obtained on filters at 1.0 μg exogenous DNA, however, maximum frequencies were not reached in sediments until a DNA concentration of 3.0 μg/cm3 sediment was added. When autoclaved sediments were pre-loaded with excess calf thymus DNA, this reduction in response of transformation frequency (cf. filter transformation) was eliminated, i.e., transformation frequencies reached saturation in the preloaded sediments at 1.0 μg DNA/cm3. Autoclaved sediments were shown to bind DNA at a concentration of about 3.6 μg/cm3 sediment, and maximum transformation frequencies were only obtained when these sediments were saturated with DNA. These data indicate that autoclaved marine sediments have the capacity to bind DNA in a form that prevents its availability for natural transformation and only after sediments are saturated with DNA does exogenous DNA become biologically active for natural transformation.