Evidence for bacterial urea production in marine sediments

Abstract The quantitative importance of bacteria in urea production and turnover in a defaunated sediment from a Danish estuary was studied. After collecting the sediment, benthic infauna was removed by sieving, followed by anoxic pre-incubation for a week. Yeast extract was added to half of the samples. The urea concentration, urea production/turnover rates and the net ammonium production rate, were followed during a 338 h incubation. The urea concentration was highest in the enriched sediment, although urea production and turnover rates were the same in the enriched and control sediment, indicating that the urea concentration was controlled by the urea turnover rate constant, k _urea. Net ammonium production, urea concetrations and teh urea turnover declined towards the end of the experiment. Comparison of the net ammonium production rate and the urea turnover rate in the enriched and control treatments, showed that urea hydrolysis accounted for up to 100% of the ammonium produced, from all sources. More than 98% of the urea which was hydrolyzed, originated from production during incubation. This suggests that urea was a major nitrogen excretion product from bacteria in this sediment, and that bacteria could also be an important source of urea in other marine sediments.     

Factors controlling anaerobic ammonium oxidation with nitrite in marine sediments

Factors controlling the anaerobic oxidation of ammonium with nitrate and nitrite were explored in a marine sediment from the Skagerrak in the Baltic-North Sea transition. In anoxic incubations with the addition of nitrite, approximately 65% of the nitrogen gas formation was due to anaerobic ammonium oxidation with nitrite, with the remainder being produced by denitrification. Anaerobic ammonium oxidation with nitrite exhibited a biological temperature response, with a rate optimum at 15 degrees C and a maximum temperature of 37 degrees C. The biological nature of the process and a 1:1 stoichiometry for the reaction between nitrite and ammonium indicated that the transformations might be attributed to the anammox process. Attempts to find other anaerobic ammonium-oxidizing processes in this sediment failed. The apparent K(m) of nitrite consumption was less than 3 microM, and the relative importance of ammonium oxidation with nitrite and denitrification for the production of nitrogen gas was independent of nitrite concentration. Thus, the quantitative importance of ammonium oxidation with nitrite in the jar incubations at elevated nitrite concentrations probably represents the in situ situation. With the addition of nitrate, the production of nitrite from nitrate was four times faster than its consumption and therefore did not limit the rate of ammonium oxidation. Accordingly, the rate of this process was the same whether nitrate or nitrite was added as electron acceptor. The addition of organic matter did not stimulate denitrification, possibly because it was outcompeted by manganese reduction or because transport limitation was removed due to homogenization of the sediment.     

Factors controlling eicosapentaenoic acid production in semicontinuous cultures of marine microalgae

Three marine microalgal species with a high content of eicosapentaenoic acid (EPA), Phaeodactylum tricornutum, Isochrysis galbana and Porphyridium cruentum, were cultured semicontinuously in order to study the effect of renewal rate on EPA productivity. The percentage of EPA in total fatty acids increased with increasing renewal rates in nitrogen limited cultures, but while for Phaeodactylum tricornutum and Isochrysis galbana a plateau around 20–25% of total fatty acids was reached with renewal rates that were not nitrogen-limiting, in Porphyridium cruentum EPA percentage increased continuously with increasing renewal rate even for those cultures that were nitrogen sufficient. Maximal EPA productivities of4.6 mg L^-1 day^-1 for Isochrysis galbana and 5.2 mg L^-1 day^-1 for Phaeodactylum tricornutum were achieved with renewal rates of 20% and 30% respectively. On the other hand for Porphyridium cruentum maximal EPA productivity, 5.3 mg L^-1 day^-1, was obtained with the maximal renewal rate tested. Results indicate that different culture strategies should be adopted for the production of a particular polyunsaturated fatty acid depending on the microalgal species being used. This revised version was published online in August 2006 with corrections to the Cover Date.     

Survival of fecal microorganisms in marine and freshwater sediments.

The survival of culturable fecal coliforms, fecal streptococci, and Clostridium perfringens spores in freshwater and marine sediments from sites near sewage outfalls was studied. In laboratory studies, the inhibition of protozoan predators with cycloheximide allowed the fecal coliforms to grow in the sediment whereas the presence of predators resulted in a net die-off. C. perfringens spores did not appear either to be affected by predators or to die off throughout the duration of the experiments (28 days). Studies using in situ membrane diffusion chambers showed that, with the exception of C. perfringens, die-off of the test organisms to 10% of their initial numbers occurred in both marine and freshwater sediments within 85 days. The usual exponential decay model could not be applied to the sediment survival data, with the exception of the data for fecal streptococci. It was concluded that application of the usual decay model to the fecal coliform data was confounded by the complex relationship between growth and predation. The survival of seeded Escherichia coli in marine sediment was studied by using an enumeration method which detected viable but nonculturable bacteria. Throughout the duration of the experiment (68 days), the same proportion of E. coli organisms remained culturable, suggesting that sediment provides a favorable, nonstarvation environment for the bacteria.     

Bacterial activity and production in near-surface estuarine and freshwater sediments

Abstract: Two indices of bacterial production, thymidine incorporation and the frequency of divided and dividing cells were measured, along with a suite of measurements of aerobic and anaerobic bacterial activity, to investigate the relationship between bacterial cell production and organic carbon mineralisation at three different sediment sites: a sheltered intertidal estuarine mudflat (Kingoodie Bay), a riverside mudbank (Ashleworth Quay) and an intertidal mudflat in a hydraulically dynamic estuary (Aust Warth). Organic carbon mineralisation was dominated by anaerobic processes at all three sites: sulfate reduction at the two estuarine sites (equivalent to 76% and 61% of oxygen uptake) and methanogenesis at the freshwater site (56%). Although all three sites had similar bacterial population sizes, activities in Kingoodie Bay were 2–3 times higher than at Aust Warth or Ashleworth Quay. Thymidine incorporation rates and Numbers of Dividing and Divided Cells correlated strongly at all three sites. Thymidine incorporation rates were spatially uncoupled from zones of principal anaerobic activity, providing in situ evidence that sulfate-reducing bacteria and methanogens do not incorporate radiolabelled thymidine into DNA during growth. Cell yield was lower in the anaerobic zone, as subsurface peaks in anaerobic mineralisation were not matched by increases in bacterial productivity. However, as anaerobic degradation processes were so dominant, anaerobic productivity still accounted for the majority of cell production.     

High bacterial diversity in permanently cold marine sediments.

A 16S ribosomal DNA (rDNA) clone library from permanently cold marine sediments was established. Screening 353 clones by dot blot hybridization with group-specific oligonucleotide probes suggested a predominance of sequences related to bacteria of the sulfur cycle (43.4% potential sulfate reducers). Within this fraction, the major cluster (19.0%) was affiliated with Desulfotalea sp. and other closely related psychrophilic sulfate reducers isolated from the same habitat. The cloned sequences showed between 93 and 100% similarity to these bacteria. Two additional groups were frequently encountered: 13% of the clones were related to Desulfuromonas palmitatis, and a second group was affiliated with Myxobacteria spp. and Bdellovibrio spp. Many clones (18.1%) belonged to the gamma subclass of the class Proteobacteria and were closest to symbiotic or free-living sulfur oxidizers. Probe target groups were further characterized by amplified rDNA restriction analysis to determine diversity within the groups and within the clone library. Rarefaction analysis suggested that the total diversity assessed by 16S rDNA analysis was very high in these permanently cold sediments and was only partially revealed by screening of 353 clones.     

The effect of temperature and algal biomass on bacterial production and specific growth rate in freshwater and marine habitats

We analyzed heterotrophic, pelagic bacterial production and specific growth rate data from 57 studies conducted in fresh, marine and estuarine/coastal waters. Strong positive relationships were identified between 1) bacterial production and bacterial abundance and 2) bacterial production and algal biomass. The relationship between bacterial production and bacterial abundance was improved by also considering water temperature. The analysis of covariance model revealed consistent differences between fresh, marine and estuarine/coastal waters, with production consistently high in estuarine/coastal environments. The log-linear regression coefficient of abundance was not significantly different from 1.00, and this linear relationship permitted the use of specific growth rate (SGR in day⁻¹) as a dependent variable. A strong relationship was identified between specific growth rate and temperature. This relationship differed slightly across the three habitats. A substantial portion of the residual variation from this relationship was accounted for by algal biomass, including the difference between marine and estuarine/coastal habitats. A small but significant difference between the fresh- and saltwater habitats remained. No significant difference between the chlorophyll effect in different habitats was identified. The model of SGR against temperature and chlorophyll was much weaker for freshwater than for marine environments. For a small subset of the data set, mean cell volume accounted for some of the residual variation in SGR. Pronounced seasonality, fluctuations in nutrient quality, and variation of the grazing environment may contribute to the unexplained variation in specific growth.     

A comparison of phosphorus immobilization in sediments of freshwater and coastal marine systems

The extent to which sediments of aquatic systems immobilize or release phosphorus can affect dramatically the P content of overlying waters. Data from 48 different aquatic systems suggests that there may be a major difference between fresh- and salt-water systems in this immobilization. Under oxic conditions (water overlying sediments had dissolved oxygen > 0.5 mg/L) P is strongly immobilized in sediments of most fresh-water systems. In sediments of most salt-water systems P is released from sediments and behaves, essentially, as a conservative tracer of benthic decomposition. This difference in P cycling is large enough to have an influence on the often cited difference in phytoplankton nutrient limitation between fresh- and salt-water systems.     

Phylogenetic analysis of the bacterial communities in marine sediments.

For the phylogenetic analysis of microbial communities present in environmental samples microbial DNA can be extracted from the sample, 16S rDNA can be amplified with suitable primers and the PCR, and clonal libraries can be constructed. We report a protocol that can be used for efficient cell lysis and recovery of DNA from marine sediments. Key steps in this procedure include the use of a bead mill homogenizer for matrix disruption and uniform cell lysis and then purification of the released DNA by agarose gel electrophoresis. For sediments collected from two sites in Puget Sound, over 96% of the cells present were lysed. Our method yields high-molecular-weight DNA that is suitable for molecular studies, including amplification of 16S rRNA genes. The DNA yield was 47 micrograms per g (dry weight) for sediments collected from creosote-contaminated Eagle Harbor, Wash. Primers were selected for the PCR amplification of (eu)bacterial 16S rDNA that contained linkers with unique 8-base restriction sites for directional cloning. Examination of 22 16S rDNA clones showed that the surficial sediments in Eagle Harbor contained a phylogenetically diverse population of organisms from the Bacteria domain (G. J. Olsen, C. R. Woese, and R. Overbeek, J. Bacteriol. 176:1-6, 1994) with members of six major lineages represented: alpha, delta, and gamma Proteobacteria; the gram-positive high G+C content subdivision; clostridia and related organisms; and planctomyces and related organisms. None of the clones were identical to any representatives in the Ribosomal Database Project small subunit RNA database. The analysis of clonal representives in the first report using molecular techniques to determine the phylogenetic composition of the (eu)bacterial community present in coastal marine sediments.     

Oxidation of short-chain fatty acids by sulfate-reducing bacteria in freshwater and in marine sediments

Colony counts of acetate-, propionate- and l-lactate-oxidizing sulfate-reducing bacteria in marine sediments were made. The vertical distribution of these organisms were equal for the three types considered. The highest numbers were found just beneath the border of aerobic and anaerobic layers.Anaerobic mineralization of acetate, propionate and l-lactate was studied in the presence and in the absence of sulfate. In freshwater and in marine sediments, acetate and propionate were oxidized completely with concomitant reduction of sulfate. l-Lactate was always fermented. Lactate-oxidizing, sulfate-reducing bacteria, belonging to the species Desulfovibrio desulfuricans, and lactate-fermenting bacteria were found in approximately equal amounts in the sediments. Acetate-oxidizing, sulfate-reducing bacteria could only be isolated from marine sediments, they belonged to the genus Desulfobacter and oxidized only acetate and ethanol by sulfate reduction. Propionate-oxidizing, sulfate-reducing bacteria belonged to the genus Desulfobulbus. They were isolated from freshwater as well as from marine sediments and showed a relatively large range of usable substrates: hydrogen, formate, propionate, l-lactate and ethanol were oxidized with concomitant sulfate reduction. l-Lactate and pyruvate could be fermented by most of the isolated strains.     

In situ stimulation of bacterial sulfate reduction in sulfate-limited freshwater lake sediments

Abstract By adding sulfate in the form of solid gypsum, it was possible to transform in situ a predominantly methanogenic sediment ecosystem into a sulfate-reducing one. The concentrations of sulfate, sulfide, methane, acetate, propionate, soluble iron, and manganese were determined in the porewater before and after the transition. Although sulfate was no longer limiting, acetate and propionate continued to accumulate and reached much higher concentrations than under sulfate-limited conditions. Metabolic activities of fermenting bacteria and of sulfate reducers, which belong to the group that incompletely oxidizes organic material, might be responsible for the increased production of volatile fatty acids. The elevated concentrations of soluble Fe(II)²⁺ and Mn(II)²⁺ observed in the porewater stem from iron and manganese compounds which may be reduced chemically by hydrogen sulfide and other microbially produced reducing agents or directly through increased activities of the iron and manganese reducing bacteria. In the horizon with high sulfate-reducing activities the methane concentrations in the porewater were lower than in non-stimulated sediment regions. The shape of the concentration depth profile indicates methane consumption through sulfate reducing processes. The in situ experiment demonstrates the response of a natural microbial ecosystem to fluctuations in the environmental conditions.     

Proteinase production by halophilic isolates from marine sediments

Twenty-one strains of spore-forming bacteria were isolated from marine sediments of the Tyrrhenian seaboard (Livorno, Italy) and identified asBacillus licheniformis by their morphological, physiological and biochemical characteristics. All strains were able to grow at 15% NaCl and on media prepared with 100% seawater. In these conditions 75% of the strains were able to produce a bacitracin-like antibiotic and 100% of the strains showed proteolytic activity. Particularly, all strains showed proteinase production with an activity optimum at pH 8.5 and 60°C. Three strains produced high levels of proteolytic activity only when cultured in the presence of seawater.     

Bacterial production in freshwater sediments: Cell specific versus system measures

Estimates of bacterial production based on total trichloroacetic acid (TCA)-precipitable [methyl-³H]thymidine incorporation and frequency of dividing cell (FDC) techniques were compared to sediment respiration rates in Lake George, New York. Bacterial growth rates based on thymidine incorporation ranged from 0.024 to 0.41 day^–1, while rates based on FDC ranged from 1.78 to 2.48 day^–1. Respiration rates ranged from 0.11 to 1.8mol O₂·hour^–1·g dry weight sediment^–1. Thymidine incorporation yielded production estimates which were in reasonable agreement with respiration rates. Production estimates based on FDC were 4- to 190-fold higher than those predicted from respiration rates.     

Ectoenzyme activity and bacterial secondary production in nutrient-impoverished and nutrient-enriched freshwater mesocosms

This report presents results on relationships between the kinetics (V_max and K_m) of -glucosidase (GLCase) and aminopeptidase (AMPase) activity, and dissolved organic carbon (DOC) and bacterial secondary production in freshwater mesocosms of differing degrees of eutrophication. These relationships varied in different mesocosms and depended on the trophic status of water and the exudation rates of organic carbon (EOC) by phytoplankton. Close coupling of bacterial production to V_max of GLCase activity was observed only in nutrient-enriched mesocosms. The relationship between GLCase and DOC content was also significant in enriched water. There was no correlation between the V_max, of GLCase and DOC and bacterial production in nutrient-impoverished and control (mesotrophic) enclosures. However, the V_max of AMPase correlated well to DOC and bacterial production in these mesocosms. AMPase activity did not correlate with DOC and bacterial production in nutrient-impoverished mesocosms. There was no relationship between bacterial biomass and enzyme activity in all studied mesocosms. Comparison of the rates of phytoplankton production of EOC and rates of the bacterial organic carbon demand (BOCD) in nutrient-impoverished mesocosms showed that EOC flux constituted, on average, 90% of BOCD. However, in nutrient-enriched mesocosms EOC contributed only, on average, 27% to the BOCD; thus, in these mesocosms, bacteria were probably organic-carbon limited. It is hypothesized that to bypass substrate limitation, bacteria produced GLCase and AMPase. These enzymes had a high specific activity and high affinity to their substrates and efficiently hydrolyzed polysaccharides and proteins, thereby supplying microorganisms with readily utilizable products of enzyme catalysis. Offprint requests to: R.J. Chróst.     

Molecular evidence for the broad distribution of anaerobic ammonium-oxidizing bacteria in freshwater and marine sediments

Previously available primer sets for detecting anaerobic ammonium-oxidizing (anammox) bacteria are inefficient, resulting in a very limited database of such sequences, which limits knowledge of their ecology. To overcome this limitation, we designed a new primer set that was 100% specific in the recovery of approximately 700-bp 16S rRNA gene sequences with >96% homology to the "Candidatus Scalindua" group of anammox bacteria, and we detected this group at all sites studied, including a variety of freshwater and marine sediments and permafrost soil. A second primer set was designed that exhibited greater efficiency than previous primers in recovering full-length (1,380-bp) sequences related to "Ca. Scalindua," "Candidatus Brocadia," and "Candidatus Kuenenia." This study provides evidence for the widespread distribution of anammox bacteria in that it detected closely related anammox 16S rRNA gene sequences in 11 geographically and biogeochemically diverse freshwater and marine sediments.     

Monitoring freshwater sediments

The objectives of the SENSPOL Expert Meeting on 'Monitoring Freshwater Sediments' held in Antwerp, Belgium, 12-13 September, 2001, were firstly to identify and define problems and secondly to develop a realistic strategy to solve these problems. Both of the stakeholder groups (governmental authorities and the dredging industry) present at the workshop participated in detailed discussions to elucidate the role of sensors in the field of sediments and sediment/water interfaces. The 19 invited experts were agreed that in situ monitoring systems are needed to monitor freshwater sediments. New recognised tools for sediment monitoring would help industry to meet the governmental sediment quality criteria and to handle the data concerning historic river contamination and geological background data. The need to monitor by effect-related studies together with chemical monitoring was stressed. The main focus for development of new sensor tools should be for on site determination of certain priority pollutants where there would be advantage over existing methods or where no suitable method exists, and to monitor biological effects (alarm systems and effect-related on site tests). Sensing technologies would also be useful to monitor bioavailability in sediments in situ to provide information for risk assessment. In addition, they could be of use to monitor bioremediation in situ. A useful role was forseen in dredging sediments, for in situ sediment screening and to guide treatment of dredged material. The new sensing tools presented, included determination of metal concentrations in sediments using the diffuse gradients in thin films (DGT) technique (Lancaster University, UK), an analytical protocol for determination of metal speciation in sediments (Universitat Autonoma de Barcelona, Spain), microbiotests for determination of sediment toxicity (University of Ghent, Belgium), a portable whole cell sensors device for heavy metal bioavailability (VITO, Belgium) and a microfabricated sensor array system for Pb concentration profile measurement in the microM range at the liquid-solid interface (University of Geneva, Switzerland).     

Factors relevant in bacterial pyrroloquinoline quinone production

Quinoprotein content and levels of external pyrroloquinoline quinone (PQQ) were determined for several bacteria under a variety of growth conditions. From these data and those from the literature, a number of factors can be indicated which are relevant for PQQ production. Synthesis of PQQ is only started if synthesis of a quinoprotein occurs, but quinoprotein synthesis does not depend on PQQ synthesis. The presence of quinoprotein substrates is not necessary for quinoprotein and PQQ syntheses. Although the extent of PQQ production was determined by the type of organism and quinoprotein produced, coordination between quinoprotein and PQQ syntheses is loose, since underproduction and overproduction of PQQ with respect to quinoprotein were observed. The results can be interpreted to indicate that quinoprotein synthesis depends on the growth rate whereas PQQ synthesis does not. In that view, the highest PQQ production can be achieved under limiting growth conditions, as was shown indeed by the much higher levels of PQQ produced in fed-batch cultures compared with those produced in batch experiments. The presence of nucleophiles, especially amino acids, in culture media may cause losses of PQQ due to transformation into biologically inactive compounds. Some organisms continued to synthesize PQQ de novo when this cofactor was administered exogenously. Most probably PQQ cannot be taken up by either passive diffusion or active transport mechanisms and is therefore not able to exert feedback regulation on its biosynthesis in these organisms.