Post-Sampling Changes in Microbial Community Composition and Activity in a Subsurface Paleosol

Abstract Laboratory storage of deep vadose zone sediments has previously resulted in an increase in the abundance of cultured microorganisms by as much as 10,000-fold, without concomitant increases in total microscopic counts. In the present study, factors contributing to the time-dependent stimulation of various microbiological parameters were examined during a 224 d post-sampling period, using a factorial-design experiment that partitioned the effects of storage time, sediment condition (intact blocks or homogenized) during storage, and O₂ concentration (0.5, 4.5, and 21%) during storage at 15°C. Stored samples were analyzed at selected intervals, to determine direct microscopic counts, viable biomass, lipid biomarker profiles, cultured aerobic heterotrophic microorganisms, and microbial activity. Time of storage prior to analysis of the samples was the most important factor affecting the microbiological response. Sediment condition influenced the stimulation response: microbial activity and the population of cultured microorganisms increased faster, and reached slightly higher values, in the homogenized samples, although maximum values were reached at similar times in the homogenized and intact samples. O₂ concentration also influenced the response, but was the least important of the factors evaluated. Total cells and viable biomass, measured as total phospholipid fatty acids, changed little during storage. Maximum cultured populations and activity were attained at 63 to 112 d, with culture counts approximating the total numbers of microscopically counted cells. At approximately the same time, unbalanced growth (evidenced by high ratios of polyhydroxybutyrate to phospholipid fatty acid) indicated that inorganic nutrients became limiting. Lipid biomarkers indicative of Gram-positive bacteria, including actinomycetes, became dominant components of the community profiles in samples maintained at 0.5% and 4.5% O₂. The shift in the microbial community from relatively inactive, predominantly uncultured microorganisms to metabolically active populations that were nearly all cultured highlights the need for rapid initiation of analyses after sample acquisition, if measurement of in situ microbiological properties is desired. The fact that these processes also occur in intact sediment blocks suggests that minor perturbations in the chemical or physical properties of subsurface sediments can result in major changes in the activity and composition of the microbial community. Revised: 22 October 1997; Accepted 20 November 1997     

Physiological Status and Community Composition of Microbial Mats of the Ebro Delta, Spain, by Signature Lipid Biomarkers

Abstract Physiological status of microbial mats of the Ebro Delta (Tarragona, Spain) based on the extraction of lipids considered ``signature lipid biomarkers' (SLB) from the cell membranes and walls of microorganisms has been analyzed. Data from a day–night cycle show significant differences in viable cells countings (PLFA cells counts) ranging from 1.5 × 10¹⁰ to 5.0 × 10¹⁰ cells g⁻¹ of sediment. Minimum values were observed at 18:00 and 6:00, when physicochemical conditions change drastically. The diversity of the microbial community was assessed by GC/MS analysis of phospholipid fatty acids (PLFA). The ratio of PLFA, representative of Gram-negative bacteria, comprises 47.8% of the total PLFA of the microbial mat community. The remaining PLFA was representative of Gram-positive (10.0%), anaerobic (5.7%), and eukaryotic microorganisms (5.7%), and other common lipids. Two different approaches were used as a comparative study to assess the physiological status of the microbial mats. Two parameters (cyclopropane fatty acids/ω7c monoenoic fatty acids, and measurement of the trans/cis monoenoic PLFA ratio) showed a minimum at midnight, suggesting the highest microbial activity. Higher values were observed at 18:00 and 6:00, coinciding with lower PLFA cell counts. Received: 14 May 1999; Accepted: 6 September 1999; Online Publication: 24 March 2000     

Determination of the sedimentary microbial biomass by extractible lipid phosphate

Summary The measurement of lipid phosphate is proposed as an indicator of microbial biomass in marine and estuarine sediments. This relatively simple assay can be performed on fresh, frozen or frozen-lyophilized sediment samples with chloroform methanol extraction and subsequent phosphate determination. The sedimentary lipid phosphate recovery correlates with the extractible ATP and the rate of DNA synthesis. Pulse-chase experiments show active metabolism of the sedimentary phospholipids. The recovery of added ¹⁴C-labeled bacterial lipids from sediments is quantitative. Replicate analyses from a single sediment sample gave a standard deviation of 11%. The lipid extract can be fractionated by relatively simple procedures and the plasmalogen, diacyl phospholipid, phosphonolipid and non-hydrolyzable phospholipid content determined. The relative fatty acid composition can be readily determined by gas-liquid chromatography.The lipid composition can be used to define the microbial community structure. For example, the absence of polyenoic fatty acids indicates minimal contamination with benthic micro-eukaryotes. Therefore the high content of plasmalogen phospholipids in these sediments suggests that the anaerobic prokaryotic Clostridia are found in the aerobic sedimentary horizon. This would require anaerobic microhabitats in the aerated zones.     

Microbial Transport, Survival, and Succession in a Sequence of Buried Sediments

Abstract Two chronosequences of unsaturated, buried loess sediments, ranging in age from <10,000 years to >1 million years, were investigated to reconstruct patterns of microbial ecological succession that have occurred since sediment burial. The relative importance of microbial transport and survival to succession was inferred from sediment ages, porewater ages, patterns of abundance (measured by direct counts, counts of culturable cells, and total phospholipid fatty acids), activities (measured by radiotracer and enzyme assays), and community composition (measured by phospholipid fatty acid patterns and Biolog substrate usage). Core samples were collected at two sites 40 km apart in the Palouse region of eastern Washington State, near the towns of Washtucna and Winona. The Washtucna site was flooded multiple times during the Pleistocene by glacial outburst floods; the Winona site elevation is above flood stage. Sediments at the Washtucna site were collected from near surface to 14.9 m depth, where the sediment age was approximately 250 ka and the porewater age was 3700 years; sample intervals at the Winona site ranged from near surface to 38 m (sediment age: approximately 1 Ma; porewater age: 1200 years). Microbial abundance and activities declined with depth at both sites; however, even the deepest, oldest sediments showed evidence of viable microorganisms. Same-age sediments had equal quantities of microorganisms, but different community types. Differences in community makeup between the two sites can be attributed to differences in groundwater recharge and paleoflooding. Estimates of the microbial community age can be constrained by porewater and sediment ages. In the shallower sediments (<9 m at Washtucna, <12 m at Winona), the microbial communities are likely similar in age to the groundwater; thus, microbial succession has been influenced by recent transport of microorganisms from the surface. In the deeper sediments, the populations may be considerably older than the porewater ages, since microbial transport is severely restricted in unsaturated sediments. This is particularly true at the Winona site, which was never flooded.     

Survival and Phospholipid Fatty Acid Profiles of Surface and Subsurface Bacteria in Natural Sediment Microcosms

Although starvation survival has been characterized for many bacteria, few subsurface bacteria have been tested, and few if any have been tested in natural subsurface porous media. We hypothesized that subsurface bacteria may be uniquely adapted for long-term survival in situ. We further hypothesized that subsurface conditions (sediment type and moisture content) would influence microbial survival. We compared starvation survival capabilities of surface and subsurface strains of Pseudomonas fluorescens and a novel Arthrobacter sp. in microcosms composed of natural sediments. Bacteria were incubated for up to 64 weeks under saturated and unsaturated conditions in sterilized microcosms containing either a silty sand paleosol (buried soil) or a sandy silt nonpaleosol sediment. Direct counts, plate counts, and cell sizes were measured. Membrane phospholipid fatty acid (PLFA) profiles were quantified to determine temporal patterns of PLFA stress signatures and differences in PLFAs among strains and treatments. The Arthrobacter strains survived better than the P. fluorescens strains; however, differences in survival between surface and subsurface strains of each genus were not significant. Bacteria survived better in the paleosol than in the nonpaleosol and survived better under saturated conditions than under unsaturated conditions. Cell volumes of all strains decreased; however, sediment type and moisture did not influence rates of miniaturization. Both P. fluorescens strains showed PLFA stress signatures typical for gram-negative bacteria: increased ratios of saturated to unsaturated fatty acids, increased ratios of trans- to cis-monoenoic fatty acids, and increased ratios of cyclopropyl to monoenoic precursor fatty acids. The Arthrobacter strains showed few changes in PLFAs. Environmental conditions strongly influenced PLFA profiles.     

A comparison of microbial community characteristics among petroleum-contaminated and uncontaminated subsurface soil samples

Measurements of microbial community size, including total cell counts and specific degrader enumerations, were conducted on subsurface soil samples from both petroleum-contaminated and pristine aquifers. Samples were collected from both uncontaminated and contaminated areas of the petroleum-contaminated sites. In pristine and uncontaminated samples, total cell counts (acridine orange direct counts) were related to depth. The deeper samples contained smaller total microbial populations. However, indices of microbial activity varied considerably from sample to sample and probably reflect soil and site heterogeneity. Exposure to petroleum contamination apparently altered the microbial community structure. In samples exposed to low levels of contaminants as vapors and/or dissolved phases (ppb concentrations), and not free product, the toluene-specific degrader populations were larger at greater depths, and the numbers of amino acid-specific degraders were highly correlated to the numbers of decane-specific degraders, indicating that petroleum-adapted microbial communities were present in the contaminated samples. In highly contaminated samples, total microbial population densities decreased with increasing depth; however, microbial activity tended to increase with depth. These results indicate that petroleum contaminants exert toxic effects on the active microbial community at high exposures and enrich specific degraders at ppb levels of dissolved contaminants. Correspondence to: S.C. Long     

Distribution and Composition of Microbial Populations in a Landfill Leachate Contaminated Aquifer (Grindsted, Denmark)

Abstract To investigate whether landfill leachates affected the microbial biomass and/or community composition of the extant microbiota, 37 samples were collected along a 305-m transect of a shallow landfill-leachate polluted aquifer. The samples were analyzed for total numbers of bacteria by use of the acridine orange direct count method (AODC). Numbers of dominant, specific groups of bacteria and total numbers of protozoa were measured by use of the most probable number method (MPN). Viable biomass estimates were obtained from measures of ATP and ester-linked phospholipid fatty acid (PLFA) concentrations. The estimated numbers of total bacteria by direct counts were relatively constant throughout the aquifer, ranging from a low of 4.8 × 10⁶ cells/g dry weight (dw) to a high of 5.3 × 10⁷ cells/g dw. Viable biomass estimates based on PLFA concentrations were one to three orders of magnitude lower with the greatest concentrations (up to 4 × 10⁵ cells/g dw) occurring at the border of the landfill and in samples collected from thin lenses of clay and silt with sand streaks. Cell number estimates based on ATP concentrations were also found to be lower than the direct count measurements (<2.2 × 10⁶ cells/g dw), and with the greatest concentrations close to the landfill. Methanogens (Archaea) and reducers of sulfate, iron, manganese, and nitrate were all observed in the aquifer. Methanogens were found to be restricted to the most polluted and reduced part of the aquifer at a maximum cell number of 5.4 × 10⁴ cells/g dw. Populations of sulfate reducers decreased with an increase in horizontal distance from the landfill ranging from a high of 9.0 × 10³ cells/g dw to a low of 6 cells/g dw. Iron, manganese, and nitrate reducers were detected throughout the leachate plume all at maximum cell numbers of 10⁶ cells/g dw. Changes in PLFA profiles indicated that a shift in microbial community composition occurred with increasing horizontal distance from the landfill. The types and patterns of lipid biomarkers suggested that increased proportions of sulfate- and iron-reducing bacteria as well as certain microeukaryotes existed at the border of the landfill. The presence of these lipid biomarkers correlated with the MPN results. There was, however, no significant correlation between the abundances of the specific PLFA biomarkers and quantitative measurements of redox processes. The application of AODC, MPN, PLFA, and ATP analyses in the characterization of the extant microbiota within the Grindsted aquifer revealed that as distance increased from the leachate source, viable biomass decreased and community composition shifted. These results led to the conclusion that the landfill leachate induced an increase in microbial cell numbers by altering the subsurface aquifer so that it was conducive to the growth of methanogens and of iron-and sulfate-reducing bacteria and fungi. Received: 11 June 1998; Accepted: 10 December 1998     

PLFA Analyses of Microbial Communities Associated with PAH-Contaminated Riverbank Sediment

Sediment contaminated with polycyclic aromatic hydrocarbons (PAHs) is widely distributed in aquatic ecosystems. The microbial community structure of riverbank PAH-contaminated sediments was investigated using phospholipid-derived fatty acid (PLFA) analysis. Surface and subsurface riverbank sediment was collected from a highly contaminated site and from an uncontaminated site along the Mahoning River, OH. PAH concentrations, physical sediment characteristics, and other microbial community parameters (biomass as phospholipid phosphate (PLP) and activity) were also measured. PAHs were detected in all samples but were only quantifiable in the contaminated (250?μg/g?g(-1)) subsurface sediment. Subsurface samples from both locations showed very similar PLP values and distribution of PLFAs, with 27-37?% of the microbial community structure being composed of sulfate reducing and other anaerobic bacteria. Principal components analysis indicated no correlation between PAH contamination and PLFA diversity. Although PLP and phospholipid fatty acid measurements of bacterial communities did not reflect the environmental differences among sites, the highly PAH-contaminated sediment showed the highest measured microbial activity (reduction of 1,200?nmol?INT?g(-1)?h(-1)), likely from a population adapted to environmental pollutants, rates that are much higher than measured in many uncontaminated soil and sediment systems. These data warrant further investigation into community structure at the genetic level and indicate potential for bioremediation by indigenous microbes.     

Spectral imaging detection and counting of microbial cells in marine sediment

Semiautomated detection and counting techniques for microbial cells in soil and marine sediment using microscopic-spectral-imaging analysis were developed. Microbial cells in microscopic fields were selectively detected from other fluorescent particles by their fluorescent spectrum, based on the spectral shift between the conjunction and nonconjunction of DNA fluorochrome (SYBR Green II) with nucleic acids. Using this technique, microbial cells could be easily detected in soil and 30-cm deep sediment samples from Tokyo Bay, both of which contain particles other than microbial cells. Total cell density was semiautomatically estimated at 1-6 x 10(9) cells cm(-3) of sediment sampled at different depths in Tokyo Bay, which corresponded to 65-106% (mean 88%) of visual direct counting. This technique may be useful for detecting microbial cells in soil and sediment samples from the deeper subsurface environment.     

Archaeal phylotypes in a metal-rich and low-activity deep subsurface sediment of the Peru Basin, ODP Leg 201, Site 1231

Site 1231 of the Ocean Drilling Project (ODP) was characterized by low concentrations of organic carbon, as well as low cell numbers and biological activity rates. A 16S rRNA survey was performed in order to analyse the microbial community composition of these central oceanic sediments. Archaeal 16S rRNA genes from subsurface sediments at Site 1231 (1.8, 9.0, and 43 mbsf) were affiliated with uncultured lineages from subsurface or hydrothermal vent habitats. Members of the Marine Group I (MGI) found in the 1.8 mbsf sediment formed distinct clusters, some dominated by phylotypes from Site 1231 and other subsurface environments. The archaeal community survey at Site 1231 indicated that several archaeal lineages were widespread in subsurface environments, marine sediments as well as hydrothermal habitats.     

Microbial growth and resuscitation alter community structure after perturbation

Abstract: An increase in the number of culturable organisms and a decrease in the diversity of recoverable microbiota have been reported in deep subsurface materials after storage perturbation. The magnitude of the microbial community shift in stored samples was more pronounced at 4°C compared to −20°C. Phospholipid fatty acid analyses and acridine orange direct counts indicated that biomass did not increase significantly throughout storage. Changes in the types of fatty acid methyl esters determined over the time course indicated that some of the microbial community shift was due to bacterial proliferation. However, the recovery of new bacterial types only after the storage process suggested that some of the increase in culturable cell count was due to the resuscitation of dormant microorganisms, possibly activated by some aspect of sampling, sample handling, and/or storage. Comparison of acridine orange direct counts with phospholipid and diglyceride fatty acid content suggested that much of the biomass may have been non-living at early time points; however, after 30 days of storage most of the bacterial biomass was viable.     

Biochemical markers for measurement of predation effects on the biomass,community structure,nutritional status,and metabolic activity of microbial biofilms

Chemical measures for the biomass, community structure, nutritional status, and metabolic activities of microbes in biofilms attached to detrital or sediment surfaces based on analysis of components of cells and extracellular polymers represent a quantitative and sensitive method for the analysis of predation. These methods require neither the quantitative removal of the organisms from the surfaces nor the efficient culture of each group of microbes for analysis of predation effects on the biofilm. The biomass of microbes can be determined by measuring the content of cellular components found universally in relatively constant amounts. If these components have a high natural turnover or are rapidly lost from viable cells, they can be utilized to measure the viable cell mass. The membrane phospholipids have a naturally high turnover, are found in all cellular membranes, are rapidly hydrolyzed on cell death, and are found in reasonably constant amounts in bacterial cells as they occur in nature. Estimates of the viable biomass by phospholipid content correspond to estimates from the content of muramic acid, ATP, several enzyme activities, direct cell counts, and in some cases viable counts of subsurface sediments. The analysis of the ester-linked fatty acids of the phospholipids (PLFA) using capillary gas chromatography/mass spectrometry (GC/MS) provides sufficient information for the detection of specific subsets of the microbiota based on patterns of PLFA. With this technique shifts in community structure can be quantitatively assayed. Some of the microbiota form specific components such as poly beta-hydroxyalkanoate (PHA) under conditions of unbalanced growth. Others form polysaccharide glycocalyx when subjected to mechanical or chemical stress. The combination of analysis of phospholipids, PLFA, PHA, and glycocalyx provides a definition of the biomass, community structure, and metabolic status of complex microbial communities. These methods involve chromatographic separation and analysis so rates of incorporation or turnover into specific components can be utilized as measures of metabolic activities. With these methods it has proved possible to show that amphipod grazing can induce shifts in biofilm community structure, nutritional status, and metabolic activities. With this technology it proved possible to show resource partitioning amongst sympatric detrital feeding amphipods, prey specificity of feeding of benthic microvores, effects of sedimentary microtopology on predation, and shifts in the microbiota by exclusion of top epibenthic predators.     

Microbiological Comparisons within and across Contiguous Lacustrine, Paleosol, and Fluvial Subsurface Sediments

Twenty-six subsurface samples were collected from a borehole at depths of 173.3 to 196.8 m in the saturated zone at the Hanford Site in south-central Washington State. The sampling was performed throughout strata that included fine-grained lacustrine (lake) sediments, a paleosol (buried soil) sequence, and coarse-grained fluvial (river) sediments. A subcoring method and tracers were used to minimize and quantify contamination to obtain samples that were representative of subsurface strata. Sediment samples were tested for total organic carbon, inorganic carbon, total microorganisms by direct microscopic counts, culturable aerobic heterotrophs by plate counts, culturable anaerobes by most-probable-number enumeration, basal respiration rates, and mineralization of (sup14)C-labeled glucose and acetate. Total direct microscopic counts of microorganisms were low, ranging from below detection to 1.9 x 10(sup5) cells g (dry weight)(sup-1). Culturable aerobes and anaerobes were below minimum levels of detection in most samples. Direct microscopic counts, basal respiration rates, and (sup14)C-glucose mineralization were all positively correlated with total organic carbon and were highest in the lacustrine sediments. In contrast to previous subsurface studies, these saturated-zone samples did not have higher microbial abundance and activities than unsaturated sediments sampled from the same borehole, the fine-textured lacustrine sediment had higher microbial numbers and activities than the coarse-textured fluvial sands, and the paleosol samples did not have higher biomass and activities relative to the other sediments. The results of this study expand the subsurface microbiology database to include information from an environment very different from those previously studied.     

Microbial colonization of aquifer sediment exposed in a groundwater well in northern Germany.

Microbial growth within the water-saturated subsurface environment was investigated by exposing sandy sediments to groundwater for 12 weeks at a depth of 10 or 20 m in a stainless-steel groundwater well. Washing and heating the sediment to 600 degrees C (removal of organic carbon) prior to the exposure did not prevent the natural microbial community from colonizing the sterilized sediment samples. Total cell counts of more than 10(7) or 10(8) per g of dried sediment were obtained. Viable cell counts of 10(5) cells per g on oligotrophic media indicated the presence, within the exposed sediment, of a highly active and multiplying biota. Microscopic analysis of enrichments inoculated with exposed sediment samples revealed a total of 45 different morphotypes, approximately 42% of the microbial community observed in previous studies of this site. The interstitial water running off of the retrieved sediment contained only 17 morphotypes and had up to 6 x 10(5) viable cells per ml.     

Microbial colonization of aquifer sediment exposed in a groundwater well in northern Germany

Microbial growth within the water-saturated subsurface environment was investigated by exposing sandy sediments to groundwater for 12 weeks at a depth of 10 or 20 m in a stainless-steel groundwater well. Washing and heating the sediment to 600 degrees C (removal of organic carbon) prior to the exposure did not prevent the natural microbial community from colonizing the sterilized sediment samples. Total cell counts of more than 10(7) or 10(8) per g of dried sediment were obtained. Viable cell counts of 10(5) cells per g on oligotrophic media indicated the presence, within the exposed sediment, of a highly active and multiplying biota. Microscopic analysis of enrichments inoculated with exposed sediment samples revealed a total of 45 different morphotypes, approximately 42% of the microbial community observed in previous studies of this site. The interstitial water running off of the retrieved sediment contained only 17 morphotypes and had up to 6 x 10(5) viable cells per ml.     

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.     

Polar lipid ester-linked fatty acid composition of Lake Vechten seston: an ecological application of lipid analysis

Abstract In order to relate the benthic lipid composition to possible sources in the water column, the sestonic communities of a monomictic lake were profiled using their saponifiable polar lipid fatty acids, which were identified by capillary gas chromatography-mass spectrometry (GC-MS). The epilimnion, dominated by the dinoflagellate alga Ceratium hirundella , was characterized by C_20:5 and C_22:6 polyunsaturated fatty acids. The photic anoxic metalimnion supported a radically different community, dominated by photosynthetic sulfur-oxidizing bacteria ( Chromatium and Chloronema spp.) and a Synechococcus -like cyanobacterium, and was characterized by high concentrations of C₁₆ and C₁₈ monounsaturated fatty acids. The fatty acid compositions of the hypolimnetic seston and the sediment were qualitatively similar to that of the metalimnion. Methyl-branched acids, commonly found in eubacteria, increased with depth in the water column. The concentrations of several unusual fatty acids found in Desulfovibrio spp. Desulfobacter spp. and Desulfotomaculum spp. were inversely related to sulfate concentration in the metalimnion. After the water column mixed in the winter, steep gradients of respiratory terminal electron acceptors developed in the surface sediment and were reflected in the polar lipid fatty acid compositions. The results show that fatty acids derived from the membranes of epilimnetic phytoplankton were efficiently metabolized in the oxic portion of the water column. The fatty acids synthesized by prokaryotic microorganisms at and below the oxycline dominated the sediment. The polar lipid fatty acid composition of the sediment showed seasonal changes which can be associated with concentrations of terminal electron acceptors of microbial respiration, and thus with physiologically distinct bacterial groups.     

Quantitative analysis of bacterial communities from Mediterranean sapropels based on cultivation-dependent methods

Microbial communities of ancient Mediterranean sapropels, buried sediment layers of high organic matter, were analyzed by most probable number (MPN) approaches. Mineral media containing different carbon sources in sub-millimolar concentrations were used. MPN numbers were elevated in sapropels and at the sediment surface, which mirrored total cell count distributions. Highest MPN counts were obtained with a mixture of different monomeric and polymeric substrates, with amino acids or with long-chain fatty acids as sole carbon sources. These values reached up to 2 x 10(7) cm(-3), representing 3.3% of the total cell count. A total of 98 pure cultures were isolated from the highest positive dilutions of the MPN series, representing the most abundant microorganisms culturable by the methods used. The strains were identified by molecular biological methods and could be grouped into 19 different phylotypes. They belonged to the alpha-, beta-, gamma-, and delta-Proteobacteria, to the Actinobacteria and the Firmicutes. However, about half of the number of isolates was closely related to the genera Photobacterium and Agrobacterium. Regarding the high cultivation success, these organisms can be assumed to be typical sapropel bacteria, representing a substantial part of the culturable indigenous microbial community.     

Changes in Ester-Linked Phospholipid Fatty Acid Profiles of Subsurface Bacteria during Starvation and Desiccation in a Porous Medium

Ester-linked phospholipid fatty acid (PLFA) profiles of a Pseudomonas aureofaciens strain and an Arthrobacter protophormiae strain, each isolated from a subsurface sediment, were quantified in a starvation experiment in a silica sand porous medium under moist and dry conditions. Washed cells were added to sand microcosms and maintained under saturated conditions or subjected to desiccation by slow drying over a period of 16 days to final water potentials of approximately - 7.5 MPa for the P. aureofaciens and - 15 MPa for the A. protophormiae. In a third treatment, cells were added to saturated microcosms along with organic nutrients and maintained under saturated conditions. The numbers of culturable cells of both bacterial strains declined to below detection level within 16 days in both the moist and dried nutrient-deprived conditions, while direct counts and total PLFAs remained relatively constant. Both strains of bacteria maintained culturability in the nutrient-amended microcosms. The dried P. aureofaciens cells showed changes in PLFA profiles that are typically associated with stressed gram-negative cells, i.e., increased ratios of saturated to unsaturated fatty acids, increased ratios of trans- to cis-monoenoic fatty acids, and increased ratios of cyclopropyl fatty acids to their monoenoic precursors. P. aureofaciens starved under moist conditions showed few changes in PLFA profiles during the 16-day incubation, whereas cells incubated in the presence of nutrients showed decreases in the ratios of both saturated fatty acids to unsaturated fatty acids and cyclopropyl fatty acids to their monoenoic precursors. The PLFA profiles of A. protophormiae changed very little in response to either nutrient deprivation or desiccation. Diglyceride fatty acids, which have been proposed to be indicators of dead or lysed cells, remained relatively constant throughout the experiment. Only the A. protophormiae desiccated for 16 days showed an increase in the ratio of diglyceride fatty acids to PLFAs. The results of this laboratory experiment can be useful for interpreting PLFA profiles of subsurface communities of microorganisms for the purpose of determining their physiological status.     

The microbial community structure of different permeable sandy sediments characterized by the investigation of bacterial fatty acids and fluorescence in situ hybridization

This study describes the microbial community structure of three sandy sediment stations that differed with respect to median grain size and permeability in the German Bight of the Southern North Sea. The microbial community was investigated using lipid biomarker analyses and fluorescence in situ hybridization. For further characterization we determined the stable carbon isotope composition of the biomarkers. Biomarkers identified belong to different bacterial groups such as members of the Cytophaga-Flavobacterium cluster and sulfate-reducing bacteria (SRB). To support these findings, investigations using different fluorescent in situ hybridization probes were performed, specifically targeting Cytophaga-Flavobacterium, gamma-Proteobacteria and different members of the SRB. Depth profiles of bacterial fatty acid relative abundances revealed elevated subsurface peaks for the fine sediment, whereas at the other sandy sediment stations the concentrations were less variable with depth. Although oxygen penetrates deeper into the coarser and more permeable sediments, the SRB biomarkers are similarly abundant, indicating suboxic to anoxic niches in these environments. We detected SRB in all sediment types as well as in the surface and at greater depth, which suggests that SRB play a more important role in oxygenated marine sediments than previously thought.