Craig D. Taylor

Characterization of an Autotrophic Sulfide-Oxidizing Marine Arcobacter sp. That Produces Filamentous Sulfur

ABSTRACT A coastal marine sulfide-oxidizing autotrophic bacterium produces hydrophilic filamentous sulfur as a novel metabolic end product. Phylogenetic analysis placed the organism in the genus Arcobacter in the epsilon subdivision of the Proteobacteria. This motile vibrioid organism can be considered difficult to grow, preferring to grow under microaerophilic conditions in flowing systems in which a sulfide-oxygen gradient has been established. Purified cell cultures were maintained by using this approach. Essentially all 4′,6-diamidino-2-phenylindole dihydrochloride-stained cells in a flowing reactor system hybridized with Arcobacter-specific probes as well as with a probe specific for the sequence obtained from reactor-grown cells. The proposed provisional name for the coastal isolate is “Candidatus Arcobacter sulfidicus.” For cells cultured in a flowing reactor system, the sulfide optimum was higher than and the CO2 fixation activity was as high as or higher than those reported for other sulfur oxidizers, such as Thiomicrospira spp. Cells associated with filamentous sulfur material demonstrated nitrogen fixation capability. No ribulose 1,5-bisphosphate carboxylase/oxygenase could be detected on the basis of radioisotopic activity or by Western blotting techniques, suggesting an alternative pathway of CO2 fixation. The process of microbial filamentous sulfur formation has been documented in a number of marine environments where both sulfide and oxygen are available. Filamentous sulfur formation by “Candidatus Arcobacter sulfidicus” or similar strains may be an ecologically important process, contributing significantly to primary production in such environments.

Evidence for Autotrophic CO2 Fixation via the Reductive Tricarboxylic Acid Cycle by Members of the ε Subdivision of Proteobacteria

Based on 16S rRNA gene surveys, bacteria of the epsilon subdivision of proteobacteria have been identified to be important members of microbial communities in a variety of environments, and quite a few have been demonstrated to grow autotrophically. However, no information exists on what pathway of autotrophic carbon fixation these bacteria might use. In this study, Thiomicrospira denitrificans and Candidatus Arcobacter sulfidicus, two chemolithoautotrophic sulfur oxidizers of the epsilon subdivision of proteobacteria, were examined for activities of the key enzymes of the known autotrophic CO(2) fixation pathways. Both organisms contained activities of the key enzymes of the reductive tricarboxylic acid cycle, ATP citrate lyase, 2-oxoglutarate:ferredoxin oxidoreductase, and pyruvate:ferredoxin oxidoreductase. Furthermore, no activities of key enzymes of other CO(2) fixation pathways, such as the Calvin cycle, the reductive acetyl coenzyme A pathway, and the 3-hydroxypropionate cycle, could be detected. In addition to the key enzymes, the activities of the other enzymes involved in the reductive tricarboxylic acid cycle could be measured. Sections of the genes encoding the alpha- and beta-subunits of ATP citrate lyase could be amplified from both organisms. These findings represent the first direct evidence for the operation of the reductive tricarboxylic acid cycle for autotrophic CO(2) fixation in epsilon-proteobacteria. Since epsilon-proteobacteria closely related to these two organisms are important in many habitats, such as hydrothermal vents, oxic-sulfidic interfaces, or oilfields, these results suggest that autotrophic CO(2) fixation via the reductive tricarboxylic acid cycle might be more important than previously considered.

Sulfate reduction rates and low molecular weight fatty acid concentrations in the water column and surficial sediments of the Black Sea

Sulfate reduction rates and concentrations of low molecular weight organic acids were measured in the water column and surficial sediments at two sites in the central Black Sea. Water column sulfate reduction rates were much lower than previously reported. The highest rate measured was 3.5 nM day−1 and on a depth integrated basis values of 1.2 and 0.22 mmol m−2 day−1 were obtained for the two sites. Sediment sulfate reduction rates were within the ranges previously reported but were higher than some for comparable abyssal sites. Rates were about 21 μM day−1 in the flocculent layer at the sediment-water interface, decreasing to 2–3μM day−1 at 20 cm depth. On an areal, depth integrated basis, rates at the two sites were 1.45 and 1.29 mmol m−2 day−1. Thus, the water column and sediments have comparable areal rates, but on a volume basis the sediment rates are several thousand times higher than the water column rates. Organic acid concentrations in the anoxic Black Sea water column were surprisingly high, reaching several μM in some cases. One deep sample contained 60μM acetate. Lactate, acetate and formate were the only acids detected in the water column. Some propionate was seen in sediment porewaters. Apparent turnover times of the organic acids in the water column, calculated for utilization solely by sulfate reducing bacteria, are tens to hundred of years. This suggests that sulfate reduction rates in the water column were not limited by organic substrate supply. In the sediments, apparent acid turnover times calculated in this way are generally less than one day, suggesting that sulfate reduction may be limited to by the supply of these substrates through fermentation reactions.

Use of microbial enrichments for the study of the anaerobic degradation of cholesterol

Abstract To demonstrate the potential of model microbial assemblages in studies of the biogeochemistry of complex organic molecules, anaerobic microbial populations capable of degrading cholesterol (cholest-5-en-3β-ol) have been enriched from marine sediment sources. The bacterial enrichments actively mineralized the C-4 nucleus of the cholesterol ring system to carbon dioxide when nitrate was present as the terminal electron acceptor. Nitrite was found as an intermediate in the reduction of nitrate, indicating the presence of denitrifying bacteria in the enrichments. When sulfate was supplied as the sole electron acceptor, active dissimilation of cholesterol was not observed. In enrichments containing 5 mM nitrate, 95–98% of the added cholesterol was recovered as carbon dioxide (2–5%), transformation products (20–30%), or as the unmodified sterol (70–80%). Cholesterol transformation products thus far identified include 5α- and 5β-cholestan-3β-ol, cholest-4-en-3-one, 5α-androstan-3, 17-dione and androst-4-en-3, 17 dione.

Validated methods for sampling and headspace analysis of carbon monoxide in seawater

Abstract A headspace analysis system with well demonstrated precision and accuracy for measuring carbon monoxide (CO) in natural waters and for CO incubation experiments is described. High water/gas volume ratios are accurately set by injecting known volumes of CO-free air into known volumes of water in glass syringes. CO in equilibrated headspace gas is separated chromatographically and quantified by a mercuric oxide reduction detector. A water/gas ratio of ∼7 is sensitive and precise enough for determining low-level CO; sensitivity can be increased by raising the water/gas ratio. At a water/gas ratio of 7 (40 ml total), the analytical blank, precision, and accuracy are 0.02 nM (nanomolar), ±0.018 nM±2%, and better than ±10%, respectively. Recovery of CO from the water phase is ∼88%. The system is efficient, simple, convenient, rapid and robust; it responds linearly up to ∼12 nM, and can process ∼8–12 samples/h. Several applications are illustrated: studies elucidating subtle CO-contamination artifacts, microbial oxidation incubations, and an oceanic profile. Validated low-contamination sampling methods are presented, and contamination control measures are recommended. A detailed 0–200-m profile at BATS in summer shows less “deep” CO than previously reported, but there is CO well below the seasonal mixed layer (ML) and even at the 1% light level.

Deep-Sea Bacteria: Isolation in the Absence of Decompression

Sampling and pure culture isolation of deep-sea bacteria without loss of in situ pressure is required in order to determine the viability of decompressionsensitive strains. This was achieved by using a pressure-retaining sterilizable seawater sampling system in connection with a prepressurized hyperbaric isolation chamber. Rates of growth and substrate uptake of the majority of isolates showed highly barotolerant characteristics, while the remainder (4 out of 15) exhibited barophilic characteristics.

Class Cariacotrichea, a novel ciliate taxon from the anoxic Cariaco Basin, Venezuela

The majority of environmental micro-organisms identified with the rRNA approach have never been visualized. Thus, their reliable classification and taxonomic assignment is often difficult or even impossible. In our preliminary 18S rRNA gene sequencing work from the worlds largest anoxic marine environment, the Cariaco Basin (Caribbean Sea, Venezuela), we detected a ciliate clade, designated previously as CAR_H [Stoeck, S., Taylor, G. T. & Epstein, S. S. (2003). Appl Environ Microbiol 63, 5656-5663]. Here, we combine the traditional rRNA detection method of fluorescent in situ hybridization (FISH) with scanning electron microscopy (SEM) and confirm the phylogenetic separation of the CAR_H sequences from all other ciliate classes by showing an outstanding morphological feature of this group: a unique, archway-shaped kinety surrounding the oral apparatus and extending to the posterior body end in CAR_H cells. Based on this specific feature and the molecular phylogenies, we propose a novel ciliate class, Cariacotrichea nov. cl.

Unexpected diversity of bacteria capable of carbon monoxide oxidation in a coastal marine environment, and contribution of the Roseobacter-associated clade to total CO oxidation.

ABSTRACT The species diversity, phylogenetic affiliations, and physiological activity rates of carbon monoxide-oxidizing microorganisms were investigated, using new isolates from surface waters collected from the coast of New England and type strains from established collections. A direct isolation method allowed the simultaneous recovery of organisms with different growth rates and nutritional requirements and the identification of marine microorganisms that oxidize CO at an environmentally relevant concentration (42 nM CO). Isolates that oxidized CO at environmentally relevant rates (>4.5 × 10−11 nmol CO oxidized cell−1 h−1) were taxonomically diverse, with representatives in the alpha and gamma subclasses of the Proteobacteria and the phylum Bacteroidetes, and represent a hitherto unreported metabolic function for several diverse microbial types. Isolates and type strains having the greatest specific rates of CO metabolism (1.1 × 10−10 to 2.3 × 10−10 nmol CO oxidized cell−1 h−1) belonged to the Roseobacter-associated clade (RAC) of the alpha subclass of the Proteobacteria. By using triple-labeled slide preparations, differential counts of active CO-oxidizing RAC cells, total RAC cells, and total bacterial cell counts in environmental samples were obtained. RAC organisms were a major component of total cell numbers (36%). Based on the density of active CO-oxidizing RAC cells in natural samples and RAC-specific metabolic activities determined for pure cultures, active CO-oxidizing RAC cells may contribute up to 15% of the total CO oxidation occurring in coastal waters.

Accessing marine protists from the anoxic Cariaco Basin

The earliest microbial cells evolved in an anoxic ocean (Martin and Muller, 1998), and many argue that the earliest Eukarya arose in the absence of oxygen as well. The microbe-mediated biogeochemical processes taking place below oxic/anoxic interfaces are of undeniable importance and interest, and the Cariaco Basin off the coast of Venezuela is a superb model ecosystem in this regard. It is the world’s largest body of truly marine anoxic waters and has been almost continuously anoxic for at least the last 2M years (Schubert, 1982). Consequently, the Basin became the site of many investigations, including a long-term biogeochemical time series initiated in 1995 (Muller-Karger et al., 2001; Taylor et al., 2001) and an NSF-funded Microbial Observatory established in 2004. This Observatory was a multi-year, multi-investigator international effort. Its principal goals were: to combine molecular and cell-based approaches to survey microbial Eukarya among geochemically diverse habitats in the Basin; to analyze the community structure and its dynamics across time and space; to discover novel organisms, and to gain insights into what governs their distribution dispersal and biogeography.

Submersible incubation device (SID), autonomous instrumentation for the in situ measurement of primary production and other microbial rate processes

Abstract A Submersible Incubation Device (SID) is described that will conduct three sequential time-course incubation experiments directly in situ . Each incubation involves a cleaning cycle, procurement of a 400 ml sample at depth, with simultaneous introduction of fresh tracer, and the time-course preservation of four subsamples for analysis upon retrieval of the instrument (12 subsamples total). The instrument records elapsed time, pressure (depth), light intensity, temperature and battery voltage throughout the deployment. The instrument has been used successfully for measurements of phytoplankton production in the open ocean and coastal wates. Three modes of operation have been employed: (a) a mode for obtaining daily rate information by summation of data from multiple short-term incubations, (b) a rapid profiling mode for measurement of microbial rate processes at several depths during a single deployment, and (c) an unattended time series mode for obtaining representative short-term activity measurements at several day intervals. The described instrument is a prototype for a long-term (months) time series version deployable in the open ocean or in remote locations.