Respiration rates correlate with mRNA expression of G6PD and GLUT1 genes in individual bovine in vitro-produced blastocysts.

Quantification of embryo respiration is a promising procedure to assess embryonic metabolism and possibly select viable embryos. At the blastocyst stage, ATP is produced by glycolysis and oxidative phosphorylation, processes that require uptake of oxygen and glucose, which is regulated by the expression of GLUT1 and G6PD. The purpose of the present study was to investigate the relationship between respiration rates and relative abundances of G6PD and GLUT1 transcripts in individual bovine blastocysts produced in vitro. Respiration rates of 104 bovine in vitro-produced blastocysts were measured individually using the nanorespirometer technology. Real-time RT-PCR was employed to determine the relative abundance of G6PD and GLUT1 mRNA in individual embryos. The mean respiration rates were similar for male and female blastocysts of the same developmental stage, but the sex ratio was skewed towards males. GLUT1 expression was down-regulated in female versus male embryos. In contrast, a approximately 1.8-fold increase in the expression of G6PD mRNA was observed in female blastocysts when compared to male blastocysts, indicating that dosage compensation for this gene had not yet occurred. Both GLUT1 and G6PD expression levels were affected by morphological quality and stage of development. Expression of GLUT1 and G6PD mRNAs was correlated with respiration rates, indicating that, in metabolically active blastocysts, uptake of oxygen and glucose are jointly increased. These findings suggest that expression of genes for oxidative phosphorylation and glycolysis are both involved in oxygen demanding ATP production.

Investigation of respiration of individual bovine embryos produced in vivo and in vitro and correlation with viability following transfer.

BACKGROUND: Quantification of oxygen consumption by individual preimplantation embryos has the potential to improve embryo selection. This study investigated whether respiration rates of individual embryos are useful indicators of embryo viability. The effect of the Nanorespirometer on embryo viability was also evaluated. METHODS: The respiration rates of individual day 7 bovine in vivo- (n=44) and in vitro-produced (n=156) embryos were measured using the Nanorespirometer. In vivo-produced embryos were individually transferred to recipients. RESULTS: The respiration rates of in vivo-produced embryos increased with increasing morphological quality and stage of development (P < 0.05). Pregnancy rates on days 35 and 60 were 65 and 60%, respectively. The mean respiration rate did not differ significantly between embryos producing and not producing a pregnancy, but the transfer of embryos with respiration rates <0.78 nl/h, between 0.78 and 1.10 nl/h, and >1.10 nl/h resulted in 48, 100 and 25% pregnancy rate, respectively. The mean respiration rate of in vitro-produced embryos was higher than that of in vivo-produced embryos because of differences in the morphological quality and stage of development. CONCLUSION: The Nanorespirometer does not adversely influence embryo viability, but the sample size was too small to confirm the significance of the correlation observed between respiration rates and viability.

Micromanipulation and further identification of FISH-labelled microcolonies of a dominant denitrifying bacterium in activated sludge.

The activated sludge process relies on the formation of strong microbial flocs. The knowledge about dominant floc-forming bacteria is at present very limited, especially from a phylogenetic perspective. In this study, numerous microcolonies in the activated sludge flocs were found to be targeted by a Betaproteobacteria-group-specific oligonucleotide probe using fluorescence in situ hybridization (FISH). Some of these were micromanipulated and further identified by reverse transcriptase polymerase chain reaction (RT-PCR) and sequencing to belong to the Aquaspirillum genus in the Neisseriaceae family. A specific oligonucleotide probe, Aqs997, was designed to target the identified bacteria. A survey in nine different wastewater treatment plants with nutrient removal (WWTP) showed a high abundance of bacteria hybridizing to the oligonucleotide probe developed. Microautoradiography (MAR) combined with FISH on activated sludge incubated with radiolabelled substrate showed uptake of substrate with oxygen, nitrate and nitrite as electron acceptor demonstrating a denitrifying potential of the bacteria investigated. The Aquaspirillum-related bacteria seemed to be abundant denitrifiers in WWTPs with nitrogen removal and they were particularly numerous in plants mainly receiving domestic wastewater, where they constituted up to 30% of all bacteria.

Cultivation-independent, semiautomatic determination of absolute bacterial cell numbers in environmental samples by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has found widespread application for analyzing the composition of microbial communities in complex environmental samples. Although bacteria can quickly be detected by FISH, a reliable method to determine absolute numbers of FISH-stained cells in aggregates or biofilms has, to our knowledge, never been published. In this study we developed a semiautomated protocol to measure the concentration of bacteria (in cells per volume) in environmental samples by a combination of FISH, confocal laser scanning microscopy, and digital image analysis. The quantification is based on an internal standard, which is introduced by spiking the samples with known amounts of Escherichia coli cells. This method was initially tested with artificial mixtures of bacterial cultures and subsequently used to determine the concentration of ammonia-oxidizing bacteria in a municipal nitrifying activated sludge. The total number of ammonia oxidizers was found to be 9.8 x 10(7) +/- 1.9 x 10(7) cells ml(-1). Based on this value, the average in situ activity was calculated to be 2.3 fmol of ammonia converted to nitrite per ammonia oxidizer cell per h. This activity is within the previously determined range of activities measured with ammonia oxidizer pure cultures, demonstrating the utility of this quantification method for enumerating bacteria in samples in which cells are not homogeneously distributed.

Congruent phylogenies of most common small-subunit rRNA and dissimilatory sulfite reductase gene sequences retrieved from estuarine sediments.

The diversity of sulfate-reducing bacteria (SRB) in brackish sediment was investigated using small-subunit rRNA and dissimilatory sulfite reductase (DSR) gene clone libraries and cultivation. The phylogenetic affiliation of the most commonly retrieved clones for both genes was strikingly similar and produced Desulfosarcina variabilis-like sequences from the inoculum but Desulfomicrobium baculatum-like sequences from a high dilution in natural media. Related organisms were subsequently cultivated from the site. PCR bias appear to be limited (or very similar) for the two primersets and target genes. However, the DSR primers showed a much higher phylogenetic specificity. DSR gene analysis is thus a promising and specific approach for investigating SRB diversity in complex habitats.

Biogeochemical and molecular signatures of anaerobic methane oxidation in a marine sediment.

Anaerobic methane oxidation was investigated in 6-m-long cores of marine sediment from Aarhus Bay, Denmark. Measured concentration profiles for methane and sulfate, as well as in situ rates determined with isotope tracers, indicated that there was a narrow zone of anaerobic methane oxidation about 150 cm below the sediment surface. Methane could account for 52% of the electron donor requirement for the peak sulfate reduction rate detected in the sulfate-methane transition zone. Molecular signatures of organisms present in the transition zone were detected by using selective PCR primers for sulfate-reducing bacteria and for Archaea. One primer pair amplified the dissimilatory sulfite reductase (DSR) gene of sulfate-reducing bacteria, whereas another primer (ANME) was designed to amplify archaeal sequences found in a recent study of sediments from the Eel River Basin, as these bacteria have been suggested to be anaerobic methane oxidizers (K. U. Hinrichs, J. M. Hayes, S. P. Sylva, P. G. Brewer, and E. F. DeLong, Nature 398:802-805, 1999). Amplification with the primer pairs produced more amplificate of both target genes with samples from the sulfate-methane transition zone than with samples from the surrounding sediment. Phylogenetic analysis of the DSR gene sequences retrieved from the transition zone revealed that they all belonged to a novel deeply branching lineage of diverse DSR gene sequences not related to any previously described DSR gene sequence. In contrast, DSR gene sequences found in the top sediment were related to environmental sequences from other estuarine sediments and to sequences of members of the genera Desulfonema, Desulfococcus, and Desulfosarcina. Phylogenetic analysis of 16S rRNA sequences obtained with the primers targeting the archaeal group of possible anaerobic methane oxidizers revealed two clusters of ANME sequences, both of which were affiliated with sequences from the Eel River Basin.

Thermodynamic and Kinetic Requirements in Anaerobic Methane Oxidizing Consortia Exclude Hydrogen, Acetate, and Methanol as Possible Electron Shuttles.

Anaerobic methane oxidation (AMO) has long remained an enigma in microbial ecology. In the process the net reaction appears to be an oxidation of methane with sulfate as electron acceptor. In order to explain experimental data such as effects of inhibitors and isotopic signals in biomarkers it has been suggested that the process is carried out by a consortium of bacteria using an unknown compound to shuttle electrons between the participants. The overall change in free energy during AMO with sulfate is very small (?22 kJ mol-1) at in situ concentrations of methane and sulfate. In order to share the available free energy between the members of the consortium, the concentration of the intermediate electron shuttle compound becomes crucial. Diffusive flux of a substrate (i.e, the electron shuttle) between bacteria requires a stable concentration gradient where the concentration is higher in the producing organism than in the consuming organism. Since changes in concentrations cause changes in reaction free energies, the diffusive flux of a catabolic product/substrate between bacteria is associated with a net loss of available energy. This restricts maximal inter-bacterial distances in consortia composed of stationary bacteria. A simple theoretical model was used to describe the relationship between inter-bacterial distances and the energy lost due to concentration differences in consortia. Key parameters turned out to be the permissible concentration range of the electron shuttle in the consortium (i.e., the concentration range that allows both participants to gain sufficient energy) and the stoichiometry of the partial reactions. The model was applied to two known consortia degrading ethanol and butyrate and to four hypothetical methane-oxidizing consortia (MOC) based on interspecies transfer of hydrogen, methanol, acetate, or formate, respectively. In the first three MOCs the permissible distances between producers and consumers of the transferred compounds were less than two times prokaryotic cell wall diameters. Consequently, it is not possible that a MOC can be based on inter-species transfer of hydrogen, methanol, or acetate. Formate, on the other hand, is a possible shuttle candidate provided the bacteria are attached to one another. In general the model predicts that members of consortia thriving on low energy such as the MOC must adhere to each other and utilize a compound for the exchange of electrons that has a high permissible concentration range and a high diffusion coefficient and transfers as many electrons as possible per molecule.

Desulfomusa hansenii gen. nov., sp. nov., a novel marine propionate-degrading, sulfate-reducing bacterium isolated from Zostera marina roots.

The physiology and phylogeny of a novel sulfate-reducing bacterium, isolated from surface-sterilized roots of the marine macrophyte Zostera marina, are presented. The strain, designated P1T, was enriched and isolated in defined oxygen-free, bicarbonate-buffered, iron-reduced seawater medium with propionate as sole carbon source and electron donor and sulfate as electron acceptor. Strain P1T had a rod-shaped, slightly curved cell morphology and was motile by means of a single polar flagellum. Cells generally aggregated in clumps throughout the growth phase. High CaCl2 (10 mM) and MgCl2 (50 mM) concentrations were required for optimum growth. In addition to propionate, strain P1T utilized fumarate, succinate, pyruvate, ethanol, butanol and alanine. Oxidation of propionate was incomplete and acetate was formed in stoichiometric amounts. Strain P1T thus resembles members of the sulfate-reducing genera Desulfobulbus and Desulforhopalus, which both oxidize propionate incompletely and form acetate in addition to CO2. However, sequence analysis of the small-subunit rDNA and the dissimilatory sulfite reductase gene revealed that strain P1T was unrelated to the incomplete oxidizers Desulfobulbus and Desulforhopalus and that it constitutes a novel lineage affiliated with the genera Desulfococcus, Desulfosarcina, Desulfonema and 'Desulfobotulus'. Members of this branch, with the exception of 'Desulfobotulus sapovorans', oxidize a variety of substrates completely to CO2. Strain P1T (= DSM 12642T = ATCC 700811T) is therefore proposed as Desulfomusa hansenii gen. nov., sp. nov. Strain p1T thus illustrates the difficulty of extrapolating rRNA similarities to physiology and/or ecological function.

Highly ordered vertical structure of Synechococcus populations within the one-millimeter-thick photic zone of a hot spring cyanobacterial mat.

A variety of contemporary techniques were used to investigate the vertical distribution of thermophilic unicellular cyanobacteria, Synechococcus spp., and their activity within the upper 1-mm-thick photic zone of the mat community found in an alkaline siliceous hot spring in Yellowstone National Park in Wyoming. Detailed measurements were made over a diel cycle at a 61 degrees C site. Net oxygenic photosynthesis measured with oxygen microelectrodes was highest within the uppermost 100- to 200-microm-thick layer until midmorning, but as the day progressed, the peak of net activity shifted to deeper layers, stabilizing at a depth of 300 microm from midday throughout the afternoon. Examination of vertical thin sections by bright-field and autofluorescence microscopy revealed the existence of different populations of Synechococcus which form discrete bands at different vertical positions. Denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA gene segments from horizontal cryosections obtained at 100-microm-thick vertical intervals also suggested vertical stratification of cyanobacterial, green sulfur bacterium-like, and green nonsulfur bacterium-like populations. There was no evidence of diel migration. However, image analysis of vertical thin sections revealed the presence of a narrow band of rod-shaped Synechococcus cells in which the cells assumed an upright position. These upright cells, located 400 to 800 microm below the surface, were observed only in mat samples obtained around noon. In mat samples obtained at other time points, the cells were randomly oriented throughout the mat. These combined observations reveal the existence of a highly ordered structure within the very thin photic zone of this hot spring microbial mat, consisting of morphologically similar Synechococcus populations that are likely to be differentially adapted, some co-occurring with green sulfur bacterium-like populations, and all overlying green nonsulfur bacterium-like populations.

Sulfate-reducing bacteria and their activities in cyanobacterial mats of solar lake (Sinai, Egypt).

The sulfate-reducing bacteria within the surface layer of the hypersaline cyanobacterial mat of Solar Lake (Sinai, Egypt) were investigated with combined microbiological, molecular, and biogeochemical approaches. The diurnally oxic surface layer contained between 10(6) and 10(7) cultivable sulfate-reducing bacteria ml-1 and showed sulfate reduction rates between 1,000 and 2, 200 nmol ml-1 day-1, both in the same range as and sometimes higher than those in anaerobic deeper mat layers. In the oxic surface layer and in the mat layers below, filamentous sulfate-reducing Desulfonema bacteria were found in variable densities of 10(4) to 10(6) cells ml-1. A Desulfonema-related, diurnally migrating bacterium was detected with PCR and denaturing gradient gel electrophoresis within and below the oxic surface layer. Facultative aerobic respiration, filamentous morphology, motility, diurnal migration, and aggregate formation were the most conspicuous adaptations of Solar Lake sulfate-reducing bacteria to the mat matrix and to diurnal oxygen stress. A comparison of sulfate reduction rates within the mat and previously published photosynthesis rates showed that CO2 from sulfate reduction in the upper 5 mm accounted for 7 to 8% of the total photosynthetic CO2 demand of the mat.

Light-induced motility of thermophilic Synechococcus isolates from Octopus Spring, Yellowstone National Park.

This study demonstrates light-induced motility of two thermophilic Synechococcus isolates that are morphologically similar but that belong to different cyanobacterial lineages. Both isolates migrated away from densely inoculated streaks to form fingerlike projections extending toward or away from the light source, depending on the light intensity. However, the two isolates seemed to prefer widely different light conditions. The behavior of each isolate was controlled by several factors, including temperature, preacclimation of inocula, acclimation during the experiment, and strain-specific genetic preferences for different light conditions (adaptation). Time-lapse microscopy confirmed that these projections were formed by actively gliding cells and were not simply the outcome of directional cell division. The observed motility rates of individual cells of 0.1 to 0.3 micrometers s-1 agreed well with the distance traversed by the projections, 0.3 to 0.5 mm h-1, suggesting that most cells in each projection are travelling in the same direction. The finding of motility among two phylogenetically unaffiliated unicellular cyanobacteria suggests that this trait may be widespread among this group. If so, this would have important implications for experiments on colonization, succession, diel positioning, and photosynthetic activity in hot spring mats dominated by Synechococcus-like cyanobacteria.

Biodiversity within hot spring microbial mat communities: molecular monitoring of enrichment cultures.

We have begun to examine the basis for incongruence between hot spring microbial mat populations detected by cultivation or by 16S rRNA methods. We used denaturing gradient gel electrophoresis (DGGE) to monitor enrichments and isolates plated therefrom. At near extincting inoculum dilutions we observed Chloroflexus-like and cyanobacterial populations whose 16S rRNA sequences have been detected in the 'New Pit' Spring Chloroflexus mat and the Octopus Spring cyanobacterial mat. Cyanobacterial populations enriched from 44 to 54 degrees C and 56 to 63 degrees C samples at near habitat temperatures were similar to those previously detected in mat samples of comparable temperatures. However, a lower temperature enrichment from the higher temperature sample selected for the populations found in the lower temperature sample. Three Thermus populations detected by both DGGE and isolation exemplify even more how enrichment may bias our view of community structure. The most abundant population was adapted to the habitat temperature (50 degrees C), while populations adapted to 65 degrees C and 70 degrees C were 10(2)- and 10(4)-fold less abundant, respectively. However, enrichment at 70 degrees C favored the least abundant strain. Inoculum dilution and incubation at the habitat temperature favored the more numerically relevant populations. We enriched many other aerobic chemoorganotrophic populations at various inoculum dilutions and substrate concentrations, most of whose 16S rRNA sequences have not been detected in mats. A common feature of numerically relevant cyanobacterial, Chloroflexus-like and aerobic chemorganotrophic populations, is that they grow poorly and resist cultivation on solidified medium, suggesting plating bias, and that the medium composition and incubation conditions may not reflect the natural microenvironments these populations inhabit.

Structure and function of a nitrifying biofilm as determined by in situ hybridization and the use of microelectrodes.

Microprofiles of O2 and NO3- were measured in nitrifying biofilms from the trickling filter of an aquaculture water recirculation system. By use of a newly developed biosensor for NO3-, it was possible to avoid conventional interference from other ions. Nitrification was restricted to a narrow zone of 50 microns on the very top of the film. In the same biofilms, the vertical distributions of members of the lithoautotrophic ammonia-oxidizing genus Nitrosomonas and of the nitrite-oxidizing genus Nitrobacter were investigated by applying fluorescence in situ hybridization of whole fixed cells with 16S rRNA-targeted oligonucleotide probes in combination with confocal laser-scanning microscopy. Ammonia oxidizers formed a dense layer of cell clusters in the upper part of the biofilm, whereas the nitrite oxidizers showed less-dense aggregates in close vicinity to the Nitrosomonas clusters. Both species were not restricted to the oxic zone of the biofilm but were also detected in substantially lower numbers in the anoxic layers and even occasionally at the bottom of the biofilm.

Morphological and metabolic responses to starvation by the dissimilatory metal-reducing bacterium Shewanella alga BrY.

The response of the dissimilatory metal-reducing bacterium Shewanella alga BrY to carbon and nitrogen starvation was examined. Starvation resulted in a gradual decrease in the mean cell volume from 0.48 to 0.2 micron 3 and a dramatic decrease in Fe(III) reductase activity. Growth of starved cultures was initiated with O2, ferric oxyhydroxide, Co(III)-EDTA, or Fe(III)-bearing subsurface materials as the sole electron acceptor. Microbially reduced subsurface materials reduced CrO(4)2-. Starvation of dissimilatory metal-reducing bacteria may provide a means of delivering this metabolism to contaminated subsurface environments for in situ bioremediation.

Distribution of bacterial populations in a stratified fjord (mariager fjord, denmark) quantified by in situ hybridization and related to chemical gradients in the water column.

Volumn 62, no. 4, p. 1392, lines 37 and 38: "Aluminum oxide filters (0.2 mm pore size; Anopore; Millipore GmbH, Eschborn, Germany)" should read "Aluminum oxide filters (0.2 (mu)m pore size; Anopore; Whatman, Inc., Clifton, N.J.)." Page 1403, reference 15: "Fenchel, T." should read "Fenchel, T., L. D. Kristensen, and L. Rasmussen." [This corrects the article on p. 1391 in vol. 62.].

Distribution of bacterial populations in a stratified fjord (mariager fjord, denmark) quantified by in situ hybridization and related to chemical gradients in the water column.

Volume 62, no. 4, p. 1391-1404: after publication of this article, it was brought to the attention of the authors that a more extensive treatment of the hydrodynamics of Mariager Fjord and the vertical distribution of bacteria and protozoa therein was published in a recent article by Fenchel et al. This work contains important information about the studied ecosystem, but unfortunately, the existence of this work was realized only after publication of our article. Thus, the following reference should have been cited in our article: [This corrects the article on p. 1391 in vol. 62.].

Community Structure of Filamentous, Sheath-Building Sulfur Bacteria, Thioploca spp., off the Coast of Chile.

The filamentous sulfur bacteria Thioploca spp. produce dense bacterial mats in the shelf area off the coast of Chile and Peru. The mat consists of common sheaths, shared by many filaments, that reach 5 to 10 cm down into the sediment. The structure of the Thioploca communities off the Bay of Concepcion was investigated with respect to biomass, species distribution, and three-dimensional orientation of the sheaths. Thioploca sheaths and filaments were found across the whole shelf area within the oxygen minimum zone. The maximum wet weight of sheaths, 800 g m(sup-2), was found at a depth of 90 m. The bacterial filaments within the sheaths contributed about 10% of this weight. The highest density of filaments was found within the uppermost 1 cm of the mat. On the basis of diameter classes, it was possible to distinguish populations containing only Thioploca spp. from mixed populations containing Beggiatoa spp. Three distinct size classes of Thioploca spp. were found, two of which have been described previously as Thioploca araucae and Thioploca chileae. Many Thioploca filaments did not possess a visible sheath, and about 20% of the sheaths contained more than one Thioploca species. The three-dimensional sheath structure showed that Thioploca filaments can move from the surface and deep into the sediment.

Distribution of bacterial populations in a stratified fjord (Mariager Fjord, Denmark) quantified by in situ hybridization and related to chemical gradients in the water column.

The vertical distribution of major and intermediate electron acceptors and donors was measured in a shallow stratified fjord. Peaks of zero valence sulfur, Mn(IV), and Fe(III) were observed in the chemocline separating oxic surface waters from sulfidic and anoxic bottom waters. The vertical fluxes of electron acceptors and donors (principally O2 and H2S) balanced within 5%; however, the zones of oxygen reduction and sulfide oxidation were clearly separated. The pathway of electron transfer between O2 and H2S was not apparent from the distribution of sulfur, nitrogen, or metal compounds investigated. The chemical zonation was related to bacterial populations as detected by ethidium bromide (EtBr) staining and by in situ hybridization with fluorescent oligonucleotide probes of increasing specificity. About half of all EtBr-stained cells were detectable with a general oligonucleotide probe for all eubacteria when digital image analysis algorithms were used to improve sensitivity. Both EtBr staining and hybridization indicated a surprisingly uniform distribution of bacteria throughout the water column. However, the average cell size and staining intensity as well as the abundance of different morphotypes changed markedly within the chemocline. The constant overall cell counts thus concealed pronounced population shifts within the water column. Cells stained with a delta 385 probe (presumably sulfate-reducing bacteria) were detected at the chemocline at about 5 x 10(4) cells per ml, and this concentration increased to 2 x 10(5) cells per ml beneath the chemocline. A long slim rod-shaped bacterium was found in large numbers in the oxic part of the chemocline, whereas large ellipsoid cells dominated at greater depth. Application of selective probes for known genera of sulfate-reducing bacteria gave only low cell counts, and thus it was not possible to identify the dominant morphotypes of the sulfate-reducing community.

Distribution of sulfate-reducing bacteria in a stratified fjord (Mariager Fjord, Denmark) as evaluated by most-probable-number counts and denaturing gradient gel electrophoresis of PCR-amplified ribosomal DNA fragments.

The sulfate-reducing bacterial populations of a stratified marine water column, Mariager Fjord, Denmark, were investigated by molecular and culture-dependent approaches in parallel. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA and DNA encoding rRNA (rDNA) isolated from the water column indicated specific bacterial populations in different water column layers and revealed a highly differentiated pattern of rRNA- and rDNA-derived PCR amplificates, probably reflecting active and resting bacterial populations. Hybridization of DGGE patterns with rRNA probes indicated the increased presence and activity (by at least 1 order of magnitude) of sulfate-reducing bacteria within and below the chemocline. Parallel to this molecular approach, an approach involving most-probable-number (MPN) counts was used, and it found a similar distribution of cultivable sulfate-reducing bacteria in the water column of Mariager Fjord, Approximately 25 cells and 250 cells per ml above and below the chemocline, respectively, were found. Desulfovibrio- and Desulfobulbus-related strains occurred in the oxic zone. DGGE bands from MPN cultures were sequenced and compared with those obtained from nucleic acids extracted from water column samples. The MPN isolates were phylogenetically affiliated with sulfate-reducing delta subdivision proteobacteria (members of the genera Desulfovibrio, Desulfobulbus, and Desulfobacter), whereas the molecular isolates constituted an independent lineage of the delta subdivision proteobacteria. DGGE of PCR-amplified nucleic acids with general eubacterial PCR primers conceptually revealed the general bacterial population, whereas the use of culture media allowed cultivable sulfate-reducing bacteria to be selected. A parallel study of Mariager Fjord biogeochemistry, bacterial activity, and bacterial counts complementing this investigation has been presented elsewhere (N.B. Ramsing, H. Fossing, T. G. Ferdelman, F. Andersen, and B. Thamdrup, Appl. Environ.

Distribution of sulfate-reducing bacteria, O2, and H2S in photosynthetic biofilms determined by oligonucleotide probes and microelectrodes.

The vertical distribution of sulfate-reducing bacteria (SRB) in photosynthetic biofilms from the trickling filter of a sewage treatment plant was investigated with oligonucleotide probes binding to 16S rRNA. To demonstrate the effect of daylight and photosynthesis and thereby of increased oxygen penetration, we incubated two 4-mm-thick biofilm samples in darkness or exposed to light at natural intensity. Gradients of O2, H2S, and pH were examined with microelectrodes during incubation. The samples were subsequently frozen with liquid nitrogen and sliced on a cryomicrotome in 20-microns vertical slices. Fluorescent-dye-conjugated oligonucleotides were used as "phylogenetic" probes to identify single cells in the slices. Oligonucleotide sequences were selected which were complementary to short sequence elements (16 to 20 nucleotides) within the 16S rRNA of sulfate-reducing bacteria. The probes were labeled with fluorescein or rhodamine derivatives for subsequent visualization by epifluorescence microscopy. Five probes were synthesized for eukaryotes, eubacteria, SRB (including most species of the delta group of purple bacteria), Desulfobacter spp., and a nonhybridizing control. The SRB were unevenly distributed in the biofilm, being present in all states from single scattered cells to dense clusters of several thousand cells. To quantify the vertical distribution of SRB, we counted cells along vertical transects through the biofilm. This was done in a blind experiment to ascertain the reliability of the staining. A negative correlation between the vertical distribution of positively stained SRB cells and the measured O2 profiles was found. The distribution differed in light- and dark-incubated samples presumably because of the different extensions of the oxic surface layer. In both cases the SRB were largely restricted to anoxic layers.