Rutger de Wit

Cyanobacterial Diversity in Natural and Artificial Microbial Mats of Lake Fryxell (McMurdo Dry Valleys, Antarctica): a Morphological and Molecular Approach

ABSTRACT Currently, there is no consensus concerning the geographic distribution and extent of endemism in Antarctic cyanobacteria. In this paper we describe the phenotypic and genotypic diversity of cyanobacteria in a field microbial mat sample from Lake Fryxell and in an artificial cold-adapted sample cultured in a benthic gradient chamber (BGC) by using an inoculum from the same mat. Light microscopy and molecular tools, including 16S rRNA gene clone libraries, denaturing gradient gel electrophoresis, and sequencing, were used. For the first time in the study of cyanobacterial diversity of environmental samples, internal transcribed spacer (ITS) sequences were retrieved and analyzed to complement the information obtained from the 16S rRNA gene. Microscopy allowed eight morphotypes to be identified, only one of which is likely to be an Antarctic endemic morphotype. Molecular analysis, however, revealed an entirely different pattern. A much higher number of phylotypes (15 phylotypes) was found, but no sequences from Nodularia and Hydrocoryne, as observed by microscopy, were retrieved. The 16S rRNA gene sequences determined in this study were distributed in 11 phylogenetic lineages, 3 of which were exclusively Antarctic and 2 of which were novel. Collectively, these Antarctic sequences together with all the other polar sequences were distributed in 22 lineages, 9 of which were exclusively Antarctic, including the 2 novel lineages observed in this study. The cultured BGC mat had lower diversity than the field mat. However, the two samples shared three morphotypes and three phylotypes. Moreover, the BGC mat allowed enrichment of one additional phylotype. ITS sequence analysis revealed a complex signal that was difficult to interpret. Finally, this study provided evidence of molecular diversity of cyanobacteria in Antarctica that is much greater than the diversity currently known based on traditional microscopic analysis. Furthermore, Antarctic endemic species were more abundant than was estimated on the basis of morphological features. Decisive arguments concerning the global geographic distribution of cyanobacteria should therefore incorporate data obtained with the molecular tools described here.

Distribution of phytoplankton pigments in nine European estuaries and implications for an estuarine typology

Phytoplankton pigments were studied by LiquidChromatography (HPLC) in nine West Europeanestuaries. Three estuaries, i.e. the Rhine,Scheldt and the Gironde were sampled four timesto cover the different seasons, whereas theother six estuaries were sampled once. Pigmentdistributions in estuaries reflect bothriverine inputs as well as autochthonousblooms. Fucoxanthin was the most commonaccessory photosynthetic pigment showing thatDiatoms were the most common group in thestudied estuaries and were particularlydominant during autumn and winter. In the veryturbid Gironde estuary, degradation processeswere predominant between salinities 1 and 20,while Diatoms, Dinoflagellates and Cryptophytesbloomed above 20 salinity during spring andsummer. This contrasted with the highlyeutrophic but less turbid Scheldt, wherephytoplanktonic blooms occurred at lowsalinities close to the city of Antwerp. In theScheldt, we observed both a tenfold fluctuationof phytoplankton biomass and a fluctuatingpigment diversity index. In contrast,chlorophyll a was always low in theGironde, but we observed large variations ofpigment diversity among samplings duringdifferent seasons. Distribution of pheopigmentsshowed that the maximum turbidity zone (MTZ)was a highly reactive region for heterotrophicphytoplankton degradation. The Scheldt and theThames were the most anthropogenic influencedestuaries contrasting with the Gironde estuarythat has a less urbanised watershed. Anestuarine typology is proposed based on threeclusters emerging from a correspondenceanalysis of pigment variables and variablescharacterising the anthropogenic impact andphysical forcing.

Effect of macrofaunal bioturbation on bacterial distribution in marine sandy sediments, with special reference to sulphur-oxidising bacteria

Abstract We have studied the impact of the bioturbating macrofauna, in particular the lugworm Arenicola marina and the bivalve Cerastoderma edule , on abundances and distribution patterns of total bacteria and of bacteria of selected functional groups in sandy intertidal sediments. The selected groups comprised the colourless sulphur-oxidising bacteria and the anoxygenic phototrophic bacteria, which are expected to occupy small zones at the oxygen–sulphide interface in stable (non-bioturbated) sediments. The presence of a wooden wreck buried in the sediment at 10 cm depth within a large area of intertidal sand flat colonised by lugworms provided a unique opportunity to confront field observations with laboratory simulations. The site with the wooden wreck, which was used as control site, was devoid of both A. marina and C. edule , while the composition of the rest of the zoobenthic community was rather similar to that of the surrounding area. In the field, the density of total bacteria was approximately one order of magnitude higher in the control site than in the natural (bioturbated) site. This can be explained by the higher contents of silt and clay particles (higher surface-area/volume ratio) and higher total organic-carbon contents found at the control site. It appears that the presence of macrofauna affects sedimentation processes, which indirectly influence bacterial dynamics. Samples from the control site have been incubated in the laboratory with A. marina and C. edule added (bioturbated core), while an unamended core served as a control. The laboratory experiments contrasted with the field observations, because it was found that total bacteria were actually higher in the deeper layers of the bioturbated core. Moreover, the populations were more homogeneous (less stratified) and colourless sulphur bacteria were on average less numerous in the bioturbated core. In general, laboratory incubations resulted in a decrease of total bacteria with a concomitant increase of colourless and phototrophic sulphur-oxidising bacteria and thus in modifications of the bacterial community structure. Hence, our results demonstrate that care must be taken in extrapolating results from laboratory experiments (e.g. mesocosm research) to field situations.

Microbial respiration and diffusive oxygen uptake of deep-sea sediments in the Southern Ocean (ANTARES-I cruise)

Abstract Benthic microbial respiration and diffusive oxygen uptake were measured, and used to calculate rates of aerobic mineralisation of organic matter and concomitant CO2 production at the sediment water interface in the abyssal region of the Crozet Basin. This study was part of the ANTARES-I cruise on the R.V. Marion Dufresne, in the Permanent Open Ocean Zone of the Southern Ocean on a south to north transect from 52° to 42°S in the Indian Ocean. At all stations, oxygen penetrated much deeper than 10 cm. Aerobic respiration was maximal in the top 1 cm (10–96 nmol cm−3 day−1), was always detectable down to 5 cm depth, and at some stations even to 10 cm depth. Total depth-integrated oxygen consumption corresponded to within ±25% of the diffusive oxygen uptake across the sediment water interface. The shape of the profile indicated that a diffusive downward flow of oxygen occurred below 10 cm depth. Thymidine incorporation experiments suggested that bacteria, present at depths of between 15 cm and 1 m in the sediment, were in a dormant state or growing extremely slowly. However, rapid DNA-synthesis started within 4 h after thymidine was added, indicating a deep bacterial biosphere in Southern Ocean sediments. It is proposed that the diffusive downward flux of oxygen below 10 cm depth sustains aerobic bacterial metabolism and survival at greater depths. Along the transect, the total depth-integrated oxygen uptake peaked at 48°S close to the Polar Front, and at the Subantarctic and Subtropical Convergence Frontal Zone. Nevertheless, in general, the differences were not very pronounced. The average value of depth-integrated microbial O2-consumption was 0.61 mmol m−2 day−1, which is equivalent to a carbon mineralisation rate of 2.3 g C m−2 year−1. These observations, together with relatively high Electron Transport System (ETS)-values (6.6 μl O2 g−1 h−1) and bacterial numbers (4 × 109 g−1 dry weight) in the top centimeter, imply that this region is less oligotrophic than previously assumed.

Characterization of the Population of the Sulfur-Oxidizing Symbiont of Codakia orbicularis (Bivalvia, Lucinidae) by Single-Cell Analyses†

ABSTRACT We investigated the characteristics of the sulfur-oxidizing symbiont hosted in the gills of Codakia orbicularis, a bivalve living in shallow marine tropical environments. Special attention was paid to describing the heterogeneity of the population by using single-cell approaches including flow cytometry (FCM) and different microscopic techniques and by analyzing a cell size fractionation experiment. Up to seven different subpopulations were distinguished by FCM based on nucleic acid content and light side scattering of the cells. The cell size analysis of symbionts showed that the symbiotic population was very heterogeneous in size, i.e., ranging from 0.5 to 5 μm in length, with variable amounts of intracellular sulfur. The side-scatter signal analyzed by FCM, which is often taken as a proxy of cell size, was greatly influenced by the sulfur content of the symbionts. FCM revealed an important heterogeneity in the relative nucleic acid content among the subclasses. The larger cells contained exceptionally high levels of nucleic acids, suggesting that these cells contained multiple copies of their genome, i.e., ranging from one copy for the smaller cells to more than four copies for the larger cells. The proportion of respiring symbionts (5-cyano-2,3-ditolyl-terazolium chloride positive) in the bacteriocytes of Codakia revealed that around 80% of the symbionts hosted by Codakia maintain respiratory activity throughout the year. These data allowed us to gain insight into the functioning of the symbionts within the host and to propose some hypotheses on how the growth of the symbionts is controlled by the host.

Differential anaerobic decomposition of seagrass (Zostera noltii) and macroalgal (Monostroma obscurum) biomass from Arcachon Bay (France)

Arcachon Bay is characterized by extensive meadows of the seagrass Zostera noltii. Moreover, as a consequence of eutrophication, massive proliferations of the macroalga (Monostroma obscurum) have occurred since the beginning of 1990s.This paper describes the anaerobic decomposition of biomass of both species under experimental conditions by two methods. Firstly, the dynamics of decomposition were studied in situ using litter bags. The remaining biomass and the elemental composition of the decomposing macrophytes were monitored. Secondly, degradation was studied in experimental containers under anoxic conditions in which the release of inorganic nutrients and the development of fermentative and sulfate-reducing bacterial populations were followed.The decomposition rate of total biomass was faster for macroalgae than for the vascular plants, thus corroborating previous observations. However, both in situ and laboratory experiments showed that the anaerobic decomposition of the seagrass Z. noltii resulted in rapid release of inorganic N and P, and increasing C/N and C/P ratios of the residual biomass. As a result, the recycling of inorganic nitrogen and phosphorus compounds was slightly more efficient for Z. noltii than for M. obscurum. Recycling of inorganic nutrients appears to be of a great importance to the whole ecosystem, because of the extensive spreading of Z. noltii in the bay.

Disentangling the relative influence of bacterioplankton phylogeny and metabolism on lysogeny in reservoirs and lagoons

Previous studies indicate that lysogeny is preponderant when environmental conditions are challenging for the bacterial communities and when their metabolism is reduced. Furthermore, it appears that lysogeny is more frequent within certain bacterial phylogenetic groups. In this comparative study from 10 freshwater reservoirs and 10 coastal lagoons, we aim to disentangle the influence of these different factors. In eight reservoirs and four lagoons, lysogeny was detected by induction assays with mitomycin C, and induction significantly modified the bacterial community composition (BCC), whereas community composition remained constant in ecosystems in which lysogeny was not observed. Among the phylogenetic groups studied, the most abundant ones were Bacteroidetes and α-proteobacteria in lagoons, and β-proteobacteria and Bacteroidetes in reservoirs. These dominant groups comprised the highest proportions of inducible lysogens. In order to unravel the effects of bacterial metabolism from phylogeny on lysogeny, we measured bacterial community physiology and the specific activities of selected phylogenetic groups. The proportion of inducible lysogens within the α- and the β-proteobacteria decreased with increasing group-specific metabolism in lagoons and reservoirs, respectively. In contrast, this relationship was not observed for the other lysogen-containing groups. Hence, both host physiology and phylogeny are critical for the establishment of lysogeny. This study illustrates the importance of lysogeny among the most abundant phylogenetic groups, and further suggests its strong structuring impact on BCC.

Diversity and Function of Chloroflexus-Like Bacteria in a Hypersaline Microbial Mat: Phylogenetic Characterization and Impact on Aerobic Respiration

ABSTRACT We studied the diversity of Chloroflexus-like bacteria (CLB) in a hypersaline phototrophic microbial mat and assayed their near-infrared (NIR) light-dependent oxygen respiration rates. PCR with primers that were reported to specifically target the 16S rRNA gene from members of the phylum Chloroflexi resulted in the recovery of 49 sequences and 16 phylotypes (sequences of the same phylotype share more than 96% similarity), and 10 of the sequences (four phylotypes) appeared to be related to filamentous anoxygenic phototrophic members of the family Chloroflexaceae. Photopigment analysis revealed the presence of bacteriochlorophyll c (BChlc), BChld, and γ-carotene, pigments known to be produced by phototrophic CLB. Oxygen microsensor measurements for intact mats revealed a NIR (710 to 770 nm) light-dependent decrease in aerobic respiration, a phenomenon that we also observed in an axenic culture of Chloroflexus aurantiacus. The metabolic ability of phototrophic CLB to switch from anoxygenic photosynthesis under NIR illumination to aerobic respiration under non-NIR illumination was further used to estimate the contribution of these organisms to mat community respiration. Steady-state oxygen profiles under dark conditions and in the presence of visible (VIS) light (400 to 700 nm), NIR light (710 to 770 nm), and VIS light plus NIR light were compared. NIR light illumination led to a substantial increase in the oxygen concentration in the mat. The observed impact on oxygen dynamics shows that CLB play a significant role in the cycling of carbon in this hypersaline microbial mat ecosystem. This study further demonstrates that the method applied, a combination of microsensor techniques and VIS and NIR illumination, allows rapid establishment of the presence and significance of CLB in environmental samples.

Impact of macroalgal dredging on dystrophic crises and phototrophic bacterial blooms (red waters) in a brackish coastal lagoon

Abstract The Prevost lagoon (Mediterranean coast, France), was subject to annual dystrophic crises caused by the biodegradation of opportunistic macroalgae (Ulva lactuca) in the past. These crises result in anoxic waters with subsequent blooms of Purple Sulphur Bacteria (red waters) which, by oxidizing sulphide, contribute to the reestablishment of oxic conditions in the water column. Mechanical dredging of the macroalgal biomass has been carried out in the lagoon since 1991 with the aim of preventing the ecological and economic disturbances caused by such crises. Dredging began just before the phototrophic bloom when the water was already hypoxic (O2 = 0.7 mg·L−1) and contained sulphilde (H2S = 7.3 mg·L−1) and purple patches of phototrophic bacteria (Thiocapsa sp.) that were beginning to develop on decaying macroalgae at the sediment surface. The dredging prevented red water formation and drastically modified both phototrophic community structure and activity and biogeochemical sulphur cycling. The dredging permitted the reestablishment of oxic conditions for a short period only (1–13 August). Resuspension of the superficial sediment layers disturbed the phototrophic bacterial community, whose numbers decreased by one order of magnitude (from 2 × 106 to 3.9 × 105 CFU.mL−1). The phototrophic community was no longer effective in reoxidizing the reduced sulphur compounds remaining in the sediments, as shown by a drastic sulphate depletion in the superficial sediment layers. Moreover, the increase in the specific bacteriochlorophyll a concentration of the phototrophic purple bacteria and the rapid development of Green Sulphur Bacteria (Prosthecochloris-like microorganisms) indicated that the phototrophic community was growing under severe light-limiting conditions due to the resuspension of sediment particles in the water. These conditions did not allow the phototrophic bacterial community to efficiently reoxidize the reduced sulphur compounds originating from the sediments. In consequence, hypoxic conditions (O2 = 4.7 to 4.8 mg·L−1) and low sulphide concentrations (H2S = 0.4 to 0.7 mg·L−1) were detected in the water column until September. The ecological balance in the lagoon was reestablished only in October, whereas, in previous years it had been restored in August.

Heterotrophic dinitrogen fixation (acetylene reduction) in phosphate-fertilised Microcoleus chthonoplastes microbial mat from the hypersaline inland lake 'la Salada de Chiprana' (NE Spain)

Microcoleus chthonoplastes dominated microbial mats are conspicuous along the shallow littoral zone in Lake Chiprana, a hypersaline lake located in the Ebro river basin in north-eastern Spain. Pigment data show that these mats included diatom species and anoxygenic phototrophs, Chloroflexus-type bacteria and purple bacteria. In situ, these mats showed low rates of dinitrogen fixation (acetylene reduction). Acetylene reduction was stimulated about 30-fold in excised mats after moderate phosphate fertilisation during 2 weeks incubation in a mesocosm. Pigment analyses showed that this treatment had little impact on the phototrophic community structure, except that it induced a decrease of Chloroflexus-type bacteria. The use of metabolic inhibitors indicated that methanogenic archaea and aerobic heterotrophic bacteria were the major dinitrogen fixers in this system. This is in agreement with the fact that the mat-building cyanobacterium M. chthonoplastes lacks the dinitrogenase reductase nifH gene and with the fact that acetylene reduction rates were strongly stimulated by additions of H2/CO2, methanol, fructose and sucrose, but not by lactate, acetate, formate and glucose. No significant differences where found for acetylene reduction rates when comparing light and dark incubations of these microbial mats. However, acetylene reduction rates were enhanced in the light when the near infrared (NIR) light was filtered out, which arrested anoxygenic photosynthesis. We suggest, therefore, that the chemoheterotrophic dinitrogen fixing bacteria were in competition with anoxygenic phototrophic bacteria for organic substrates, while the latter did not contribute to dinitrogen fixation in the mat.