Variation in Microbial Biomass and Community Structure in Sediments of Eutrophic Bays as Determined by Phospholipid Ester-Linked Fatty Acids

The distribution of phospholipid ester-linked fatty acids (PLFA) in sediments of eutrophic bays (Hiroshima Bay and Aki Nada) was studied to quantify the microbial biomass, community structure, and nutritional status. A total of 63 fatty acids in the range of C₁₀ to C₂₄ were determined. They consist of saturated fatty acids, branched fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids, and variation was revealed in the relative proportions of these fatty acids in sediments. On the basis of the PLFA concentration in sediments, the calculated microbial biomass showed variation (mean ± standard deviation = 0.70 × 10⁸ ± 0.53 × 10⁸ cells per g [dry weight] of sediment) in the eutrophic bays. In sediments, a higher amount of biomass was observed in the coastal area of Hiroshima Bay than that observed in the rest of the bay and adjacent Aki Nada. The microbial community structure of the present study area, as characterized by the PLFA profiles, showed very low percentages of polyunsaturated fatty acids and long-chain fatty acids characteristic of microeukary-otes and terrestrial input, respectively, and high percentages of fatty acids characteristic of bacteria. The distribution of PLFA profiles also showed the relative contribution of both aerobic and anaerobic bacteria, especially sulfate-reducing bacteria, in the study area. The relative proportions of PLFA revealed distinctive differences among the stations of the study area, as is evidenced from six clusters obtained for the PLFA profiles. The results of Tukey's honestly significant difference test further confirmed that the sediments in the coastal area of Hiroshima Bay were significantly enriched by a number of fatty acids when compared with other areas investigated where relatively few fatty acids were present in significant quantities. No marked variation in environmental parameters in the surface- and bottom-water samples was observed, indicating the absence of any water movement in the study area. Furthermore, low redox potential and the levels of sulfide in the sediment revealed the reduced condition of the sediment. The existing environmental conditions and pollution of the study area were attributed to the observed microbial community structure in the sediments.     

Relations Between Bacterial Biomass and Carbon Cycle in Marine Sediments: An Early Diagenetic Model

A new model for early diagenetic processes has been developed through a new formula explicitly accounting for microbial population dynamics. Following a mechanistic approach based on enzymatic reactions, a new model has been proposed for oxic mineralisation and denitrification. It incorporates the dynamics of bacterial metabolism. We find a general formula for inhibition processes of which some other mathematical expressions are particular cases. Moreover a fast numerical algorithm has been developed. It allows us to perform simulations of different diagenetic models in non-steady states. We use this algorithm to compare our model to a classical one (Soetaert et al., 1996). Dynamical evolutions of a perturbation of particulate organic carbon (POC) input are studied for both models. The results are very similar for stationary cases. But with variable inputs, the bacterial biomass dynamics brings about noticeable differences, and these are discussed.     

Screening of Marine Bacterial Producers of Polyunsaturated Fatty Acids and Optimisation of Production

Water samples from three different environments including Mid Atlantic Ridge, Red Sea and Mediterranean Sea were screened in order to isolate new polyunsaturated fatty acids (PUFAs) bacterial producers especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Two hundred and fifty-one isolates were screened for PUFA production and among them the highest number of producers was isolated from the Mid-Atlantic Ridge followed by the Red Sea while no producers were found in the Mediterranean Sea samples. The screening strategy included a simple colourimetric method followed by a confirmation via GC/MS. Among the tested producers, an isolate named 66 was found to be a potentially high PUFA producer producing relatively high levels of EPA in particular. A Plackett–Burman statistical design of experiments was applied to screen a wide number of media components identifying glycerol and whey as components of a production medium. The potential low-cost production medium was optimised by applying a response surface methodology to obtain the highest productivity converting industrial by-products into value-added products. The maximum achieved productivity of EPA was 20 mg/g, 45 mg/l, representing 11 % of the total fatty acids, which is approximately five times more than the amount produced prior to optimisation. The production medium composition was 10.79 g/l whey and 6.87 g/l glycerol. To our knowledge, this is the first investigation of potential bacteria PUFA producers from Mediterranean and Red Seas providing an evaluation of a colourimetric screening method as means of rapid screening of a large number of isolates.     

Preservation of Estuarine Sediments for Lipid Analysis of Biomass and Community Structure of Microbiota

Various methods were tested for preserving estuarine sediments in the field before biochemical analysis of the microbiota. Total microbial biomass was determined as lipid phosphate (LP), and the fatty acids of the microbial lipids were used as indicators of community structure. Control samples were sieved to remove macroinvertebrates and plant materials and were extracted immediately in the field. Other samples were preserved both before and after sieving and stored for 5 days before analysis. Freezing resulted in a 50% decline in LP and significant decreases in many fatty acids. Refrigeration resulted in a 19% decrease in LP but no change in the fatty acids. Samples preserved with Formalin before sieving exhibited no significant change in LP but substantial increases in many fatty acids, which were probably derived from the macroinvertebrates. Sieved samples preserved with Formalin showed a 17 to 18% decline in LP but no change in the fatty acids. Ideally, samples should be sieved and extracted immediately in the field. However, short-term refrigeration and longer-term preservation of sieved samples with Formalin may be acceptable compromises.     

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.     

trans-Monounsaturated Acids in a Marine Bacterial Isolate

[This corrects the article on p. 849 in vol. 41.].     

Abiotic Racemization Kinetics of Amino Acids in Marine Sediments

The ratios of d- versus l-amino acids can be used to infer the sources and composition of sedimentary organic matter. Such inferences, however, rely on knowing the rates at which amino acids in sedimentary organic matter racemize abiotically between the d- and the l-forms. Based on a heating experiment, we report kinetic parameters for racemization of aspartic acid, glutamic acid, serine, and alanine in bulk sediment from Aarhus Bay, Denmark, taken from the surface, 30 cm, and 340 cm depth below seafloor. Extrapolation to a typical cold deep sea sediment temperature of 3°C suggests racemization rate constants of 0.50×10⁻⁵–11×10⁻⁵ yr⁻¹. These results can be used in conjunction with measurements of sediment age to predict the ratio of d:l amino acids due solely to abiotic racemization of the source material, deviations from which can indicate the abundance and turnover of active microbial populations.     

Comparison of Rapid Methods for Analysis of Bacterial Fatty Acids

When rapid gas-liquid chromatography methods for determination of bacterial fatty acids were compared, results showed that saponification was required for total fatty acid analysis. Transesterification with boron-trihalide reagents (BF(3)-CH(3)OH, BCl(3)-CH(3)OH) caused extensive degradation of cyclopropane acids and was less effective than saponification in releasing cellular hydroxy fatty acids. Digestion of cells with tetramethylammonium hydroxide was unsatisfactory because of extraneous gas-liquid chromatography peaks and because of lower recovery of branched-chain and hydroxy fatty acids. A simple, rapid saponification procedure which can be used for total cellular fatty acid analysis of freshly grown cells is described.     

Community Structure, Cellular rRNA Content, and Activity of Sulfate-Reducing Bacteria in Marine Arctic Sediments

The community structure of sulfate-reducing bacteria (SRB) of a marine Arctic sediment (Smeerenburgfjorden, Svalbard) was characterized by both fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization by using group- and genus-specific 16S rRNA-targeted oligonucleotide probes. The SRB community was dominated by members of the Desulfosarcina-Desulfococcus group. This group accounted for up to 73% of the SRB detected and up to 70% of the SRB rRNA detected. The predominance was shown to be a common feature for different stations along the coast of Svalbard. In a top-to-bottom approach we aimed to further resolve the composition of this large group of SRB by using probes for cultivated genera. While this approach failed, directed cloning of probe-targeted genes encoding 16S rRNA was successful and resulted in sequences which were all affiliated with the Desulfosarcina-Desulfococcus group. A group of clone sequences (group SVAL1) most closely related to Desulfosarcina variabilis (91.2% sequence similarity) was dominant and was shown to be most abundant in situ, accounting for up to 54.8% of the total SRB detected. A comparison of the two methods used for quantification showed that FISH and rRNA slot blot hybridization gave comparable results. Furthermore, a combination of the two methods allowed us to calculate specific cellular rRNA contents with respect to localization in the sediment profile. The rRNA contents of Desulfosarcina-Desulfococcus cells were highest in the first 5 mm of the sediment (0.9 and 1.4 fg, respectively) and decreased steeply with depth, indicating that maximal metabolic activity occurred close to the surface. Based on SRB cell numbers, cellular sulfate reduction rates were calculated. The rates were highest in the surface layer (0.14 fmol cell⁻¹ day⁻¹), decreased by a factor of 3 within the first 2 cm, and were relatively constant in deeper layers.     

Bacterial Community Structure Along Moisture Gradients in the Parafluvial Sediments of Two Ephemeral Desert Streams

Microorganisms inhabiting stream sediments mediate biogeochemical processes of importance to both aquatic and terrestrial ecosystems. In deserts, the lateral margins of ephemeral stream channels (parafluvial sediments) are dried and rewetted, creating periodically wet conditions that typically enhance microbial activity. However, the influence of water content on microbial community composition and diversity in desert stream sediments is unclear. We sampled stream margins along gradients of wet to dry sediments, measuring geochemistry and bacterial 16S rRNA gene composition, at streams in both a cold (McMurdo Dry Valleys, Antarctica) and hot (Chihuahuan Desert, New Mexico, USA) desert. Across the gradients, sediment water content spanned a wide range (1.6–37.9% w/w), and conductivity was highly variable (12.3–1,380 μS cm⁻²). Bacterial diversity (at 97% sequence similarity) was high and variable, but did not differ significantly between the hot and cold desert and was not correlated with sediment water content. Instead, conductivity was most strongly related to diversity. Water content was strongly related to bacterial 16S rRNA gene community composition, though samples were distributed in wet and dry clusters rather than as assemblages shifting along a gradient. Phylogenetic analyses showed that many taxa from wet sediments at the hot and cold desert site were related to, respectively, halotolerant Gammaproteobacteria, and one family within the Sphingobacteriales (Bacteroidetes), while dry sediments at both sites contained a high proportion of taxa related to the Acidobacteria. These results suggest that bacterial diversity and composition in desert stream sediments is more strongly affected by hydrology and conductivity than temperature.     

Fatty Acids in the Lipids of Marine and Terrestrial Nitrifying Bacteria

Fatty acids in the lipids of 19 marine and terrestrial nitrifying bacteria have been analyzed. Ammonia-oxidizing bacteria have a very simple acid composition; palmitic and palmitoleic acid account for 96 to 100% of the total acids. The fatty acids of nitrite-oxidizing bacteria cover a wider range, from C(14) to C(19), but from two to four acids still account for more than 80% of the total acids. Branched iso- and anteiso-acids are present in traces only in 2 of the 19 bacteria. The chemical and morphological similarity between blue-green algae and these bacteria is discussed.     

Influence of Particle Size on Bacterial Community Structure in Aquatic Sediments as Revealed by 16S rRNA Gene Sequence Analysis

Bacterial communities associated with sediment particles were examined using PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing. Particle size influenced community structure, with attached bacterial assemblages separating into 63- to 125-, 125- to 1,000-, and 1,000- to 2,000-μm fractions. Differences were particularly pronounced for the Verrucomicrobia-Planctomycetes, whose numbers were significantly reduced on coarser particles.     

Characterization of Specific Membrane Fatty Acids as Chemotaxonomic Markers for Sulfate-Reducing Bacteria Involved in Anaerobic Oxidation of Methane

Membrane fatty acids were extracted from a sediment core above marine gas hydrates at Hydrate Ridge, NE Pacific. Anaerobic sediments from this environment are characterized by high sulfate reduction rates driven by the anaerobic oxidation of methane (AOM). The assimilation of methane carbon into bacterial biomass is indicated by carbon isotope values of specific fatty acids as low as m 103. Specific fatty acids released from bacterial membranes include C 16:1 y 5c , C 17:1 y 6c , and cyC 17:0 y 5,6 , all of which have been fully characterized by mass spectrometry. These unusual fatty acids continuously display the lowest i 13 C values in all sediment horizons and two of them are detected in high abundance (i.e., C 16:1 y 5c and cyC 17:0 y 5,6 ). Combined with microscopic examination by fluorescence in situ hybridization specifically targeting sulfate-reducing bacteria (SRB) of the Desulfosarcina/Desulfococcus group, which are present in the aggregates of AOM consortia in extremely high numbers, these specific fatty acids appear to provide a phenotypic fingerprint indicative for SRB of this group. Correlating depth profiles of specific fatty acid content and aggregate number in combination with pore water sulfate data provide further evidence of this finding. Using mass balance calculations we present a cell-specific fatty acid pattern most likely displaying a very close resemblance to the still uncultured Desulfosarcina/Desulfococcus species involved in AOM.     

Chemoautotrophic Carbon Fixation Rates and Active Bacterial Communities in Intertidal Marine Sediments

Chemoautotrophy has been little studied in typical coastal marine sediments, but may be an important component of carbon recycling as intense anaerobic mineralization processes in these sediments lead to accumulation of high amounts of reduced compounds, such as sulfides and ammonium. We studied chemoautotrophy by measuring dark-fixation of ¹³C-bicarbonate into phospholipid derived fatty acid (PLFA) biomarkers at two coastal sediment sites with contrasting sulfur chemistry in the Eastern Scheldt estuary, the Netherlands. At one site where free sulfide accumulated in the pore water right to the top of the sediment, PLFA labeling was restricted to compounds typically found in sulfur and ammonium oxidizing bacteria. At the other site, with no detectable free sulfide in the pore water, a very different PLFA labeling pattern was found with high amounts of label in branched i- and a-PLFA besides the typical compounds for sulfur and ammonium oxidizing bacteria. This suggests that other types of chemoautotrophic bacteria were also active, most likely Deltaproteobacteria related to sulfate reducers. Maximum rates of chemoautotrophy were detected in first 1 to 2 centimeters of both sediments and chemosynthetic biomass production was high ranging from 3 to 36 mmol C m⁻² d⁻¹. Average dark carbon fixation to sediment oxygen uptake ratios were 0.22±0.07 mol C (mol O₂)⁻¹, which is in the range of the maximum growth yields reported for sulfur oxidizing bacteria indicating highly efficient growth. Chemoautotrophic biomass production was similar to carbon mineralization rates in the top of the free sulfide site, suggesting that chemoautotrophic bacteria could play a crucial role in the microbial food web and labeling in eukaryotic poly-unsaturated PLFA was indeed detectable. Our study shows that dark carbon fixation by chemoautotrophic bacteria is a major process in the carbon cycle of coastal sediments, and should therefore receive more attention in future studies on sediment biogeochemistry and microbial ecology.     

Impacts of Bioremediation Schemes on Bacterial Population in Naphthalene-Contaminated Marine Sediments

Microcosm experiments were conduced in which the surface of marine sediment was contaminated with naphthalene and subjected to either of three different bioremediation schemes, i.e., biostimulation (BS) by supplementing with slow-release nitrogen and phosphorus fertilizers, bioaugmentation (BA) by inoculating with Cycloclasticus sp. E2, an aromatics-degrading bacterium identified to play an important role for aromatic-hydrocarbon degradation in marine environments and combination (CB) of BS and BA. These three schemes were found to be similarly effective for removing naphthalene, while naphthalene disappearance in sediment without any treatment (WT) was slower than those in the treated sediments. Shifts in bacterial populations during and after bioremediation were analyzed by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments. It was found that the Cycloclasticus rRNA type occurred as the strongest bands in the course of naphthalene degradation. Clustering analysis of DGGE profiles showed that bacterial populations in the WT, BS and CB sediments differed consistently from those in the uncontaminated control, while the profile for the BA sediment was finally included in the cluster for uncontaminated control sediments after a 150-day treatment. The results suggest that bioaugmentation with ecologically competent pollutant-degrading bacteria is an ecologically promising bioremediation scheme.     

Aliphatic Hydrocarbons and Fatty Acids of Some Marine and Freshwater Microorganisms

Gas chromatography and combined gas chromatography-mass spectrometry have been used to study the fatty acids and hydrocarbons of a bacterium from the Pacific Ocean, Vibrio marinus, a freshwater blue-green alga, Anacystis nidulans, and algal mat communities from the Gulf of Mexico. Both types of microorganisms (bacteria and algae) showed relatively simple hydrocarbon and fatty acid patterns, the hydrocarbons predominating in the region of C-17 and the fatty acids in the range of C-14 to C-18. The patterns of V. marinus were more comparable to those of the algal populations than to patterns reported for other bacteria. An incomplete correlation between fatty acids and hydrocarbons in both types of organisms was observed, making it difficult to accept the concept that the biosynthesis of hydrocarbons follows a simple fatty acid decarboxylation process.     

Effect of Silicate Grain Shape, Structure, and Location on the Biomass and Community Structure of Colonizing Marine Microbiota

Microbiota colonizing silica grains of the same size and water pore space, but with a different microtopography, showed differences in biomass and community structure after 8 weeks of exposure to running seawater. The absence of surface cracks and crevices resulted in a marked diminution of the total microbial biomass measured as lipid phosphate and total extractable palmitic acid. With increasing smoothness of the sand grain surface, examination of the community structure showed a marked decrease in procaryotes and algal microeucaryotes, with a relative increase in microeucaryotic grazers. A comparison of the colonizing sediment incubated in running seawater or at 32 m on the sea floor with a sediment core showed a decreased bacterial biomass with a different community structure and a decreased total microeucaryotic population of both grazers and algae. The quantitative differences in microbial biomass and community structure between the microcosms and the actual benthic population in the core were determined.