Chemoautotrophic Carbon Fixation Research Articles

Database of nitrification and nitrifiers in the global ocean

Abstract. As a key biogeochemical pathway in the marine nitrogen cycle, nitrification (ammonia oxidation and nitrite oxidation) converts the most reduced form of nitrogen – ammonium–ammonia (NH4+–NH3) – into the oxidized species nitrite (NO2-) and nitrate (NO3-). In the ocean, these processes are mainly performed by ammonia-oxidizing archaea (AOA) and bacteria (AOB) and nitrite-oxidizing bacteria (NOB). By transforming nitrogen speciation and providing substrates for nitrogen removal, nitrification affects microbial community structure; marine productivity (including chemoautotrophic carbon fixation); and the production of a powerful greenhouse gas, nitrous oxide (N2O). Nitrification is hypothesized to be regulated by temperature, oxygen, light, substrate concentration, substrate flux, pH and other environmental factors. Although the number of field observations from various oceanic regions has increased considerably over the last few decades, a global synthesis is lacking, and understanding how environmental factors control nitrification remains elusive. Therefore, we have compiled a database of nitrification rates and nitrifier abundance in the global ocean from published literature and unpublished datasets. This database includes 2393 and 1006 measurements of ammonia oxidation and nitrite oxidation rates and 2242 and 631 quantifications of ammonia oxidizers and nitrite oxidizers, respectively. This community effort confirms and enhances our understanding of the spatial distribution of nitrification and nitrifiers and their corresponding drivers such as the important role of substrate concentration in controlling nitrification rates and nitrifier abundance. Some conundrums are also revealed, including the inconsistent observations of light limitation and high rates of nitrite oxidation reported from anoxic waters. This database can be used to constrain the distribution of marine nitrification, to evaluate and improve biogeochemical models of nitrification, and to quantify the impact of nitrification on ecosystem functions like marine productivity and N2O production. This database additionally sets a baseline for comparison with future observations and guides future exploration (e.g., measurements in the poorly sampled regions such as the Indian Ocean and method comparison and/or standardization). The database is publicly available at the Zenodo repository: https://doi.org/10.5281/zenodo.8355912 (Tang et al., 2023).

Potential response of dark carbon fixation to global warming in estuarine and coastal waters.

Dark carbon fixation (DCF), through which chemoautotrophs convert inorganic carbon to organic carbon, is recognized as a vital process of global carbon biogeochemical cycle. However, little is known about the response of DCF processes in estuarine and coastal waters to global warming. Using radiocarbon labelling method, the effects of temperature on the activity of chemoautotrophs were investigated in benthic water of the Yangtze estuarine and coastal areas. A dome-shaped thermal response pattern was observed for DCF rates (i.e., reduced rates at lower or higher temperatures), with the optimum temperature (Topt ) varying from about 21.9 to 32.0°C. Offshore sites showed lower Topt values and were more vulnerable to global warming compared with nearshore sites. Based on temperature seasonality of the study area, it was estimated that warming would accelerate DCF rate in winter and spring but inhibit DCF activity in summer and fall. However, at an annual scale, warming showed an overall promoting effect on DCF rates. Metagenomic analysis revealed that the dominant chemoautotrophic carbon fixation pathways in the nearshore area were Calvin-Benson-Bassham (CBB) cycle, while the offshore sites were co-dominated by CBB and 3-hydroxypropionate/4-hydroxybutyrate cycles, which may explain the differential temperature response of DCF along the estuarine and coastal gradients. Our findings highlight the importance of incorporating DCF thermal response into biogeochemical models to accurately estimate the carbon sink potential of estuarine and coastal ecosystems in the context of global warming.

Controls on the relative abundances and rates of nitrifying microorganisms in the ocean

Abstract. Nitrification controls the oxidation state of bioavailable nitrogen. Distinct clades of chemoautotrophic microorganisms – predominantly ammonia-oxidizing archaea (AOA) and nitrite-oxidizing bacteria (NOB) – regulate the two steps of nitrification in the ocean, but explanations for their observed relative abundances and nitrification rates remain incomplete and their contributions to the global marine carbon cycle via carbon fixation remain unresolved. Using a mechanistic microbial ecosystem model with nitrifying functional types, we derive simple expressions for the controls on AOA and NOB in the deep, oxygenated open ocean. The relative biomass yields, loss rates, and cell quotas of AOA and NOB control their relative abundances, though we do not need to invoke a difference in loss rates to explain the observed relative abundances. The supply of ammonium, not the traits of AOA or NOB, controls the relatively equal ammonia and nitrite oxidation rates at steady state. The relative yields of AOA and NOB alone set their relative bulk carbon fixation rates in the water column. The quantitative relationships are consistent with multiple in situ datasets. In a complex global ecosystem model, nitrification emerges dynamically across diverse ocean environments, and ammonia and nitrite oxidation and their associated carbon fixation rates are decoupled due to physical transport and complex ecological interactions in some environments. Nevertheless, the simple expressions capture global patterns to first order. The model provides a mechanistic upper estimate on global chemoautotrophic carbon fixation of 0.2–0.5 Pg C yr−1, which is on the low end of the wide range of previous estimates. Modeled carbon fixation by AOA (0.2–0.3 Pg C yr−1) exceeds that of NOB (about 0.1 Pg C yr−1) because of the higher biomass yield of AOA. The simple expressions derived here can be used to quantify the biogeochemical impacts of additional metabolic pathways (i.e., mixotrophy) of nitrifying clades and to identify alternative metabolisms fueling carbon fixation in the deep ocean.

Carbon Assimilation by the Picoplanktonic Community Inhabiting the Secondary Chlorophyll Maximum of the Anoxic Marine Zones of the Eastern Tropical North and South Pacific

Anoxic marine zones (AMZs) constitute pelagic systems distinguished from the oxygen minimum zones (OMZs) by the complete absence of detectable oxygen and the accumulation of nitrite in mid-waters. At the top of the oxygen-depleted layer and below the oxycline, nutrients are abundant; light intensity is very much reduced (<1% of incident light) and a secondary chlorophyll maximum (SCM) is developed. The shoaling of the oxygen-depleted layer, product of the AMZ expansion, could enhance this SCM, which has little-known biogeochemical effects. Here, we show that the SCM is contributing a measurable signal in the particulate organic carbon (POC), enough to alter the δ13CPOC in the top of the oxygen-depleted layer. This data showed significant differences among stations with and without the development of a SCM, being 3.0‰ heavier when a SCM is developed, and indicating photosynthetic activity and/or remineralization in the top of the AMZ. More depleted δ13CPOC values were also found when no SCM was present indicating stronger chemoautotrophic activity, potentially driven by anammox and sulfur-oxidizing bacteria activity. Assimilation rate data show that when sufficient light and Prochlorococcus are present, photosynthesis exceeds chemoautotrophic carbon fixation, and can exceed heterotrophic assimilation of glucose or acetate. However, in the majority of the stations, assimilation rates of both glucose and acetate exceeded carbon fixation rates under light stimulation, suggesting that often the SCM is still a net heterotrophic system.

The Genomic Capabilities of Microbial Communities Track Seasonal Variation in Environmental Conditions of Arctic Lagoons.

In contrast to temperate systems, Arctic lagoons that span the Alaska Beaufort Sea coast face extreme seasonality. Nine months of ice cover up to ∼1.7 m thick is followed by a spring thaw that introduces an enormous pulse of freshwater, nutrients, and organic matter into these lagoons over a relatively brief 2–3 week period. Prokaryotic communities link these subsidies to lagoon food webs through nutrient uptake, heterotrophic production, and other biogeochemical processes, but little is known about how the genomic capabilities of these communities respond to seasonal variability. Replicate water samples from two lagoons and one coastal site near Kaktovik, AK were collected in April (full ice cover), June (ice break up), and August (open water) to represent winter, spring, and summer, respectively. Samples were size fractionated to distinguish free-living and particle-attached microbial communities. Multivariate analysis of metagenomes indicated that seasonal variability in gene abundances was greater than variability between size fractions and sites, and that June differed significantly from the other months. Spring (June) gene abundances reflected the high input of watershed-sourced nutrients and organic matter via spring thaw, featuring indicator genes for denitrification possibly linked to greater organic carbon availability, and genes for processing phytoplankton-derived organic matter associated with spring blooms. Summer featured fewer indicator genes, but had increased abundances of anoxygenic photosynthesis genes, possibly associated with elevated light availability. Winter (April) gene abundances suggested low energy inputs and autotrophic bacterial metabolism, featuring indicator genes for chemoautotrophic carbon fixation, methane metabolism, and nitrification. Winter indicator genes for nitrification belonged to Thaumarchaeota and Nitrosomonadales, suggesting these organisms play an important role in oxidizing ammonium during the under-ice period. This study shows that high latitude estuarine microbial assemblages shift metabolic capabilities as they change phylogenetic composition between these extreme seasons, providing evidence that these communities may be resilient to large hydrological events in a rapidly changing Arctic.

Diverse nitrogen cycling pathways across a marine oxygen gradient indicate nitrogen loss coupled to chemoautotrophic activity.

Genetic markers and geochemical assays of microbial nitrogen cycling processes, including autotrophic and heterotrophic denitrification, anammox, ammonia oxidation, and nitrite oxidation, were examined across the oxycline, suboxic, and anoxic zones of the Cariaco Basin, Venezuela. Ammonia and nitrite oxidation genes were expressed through the entire gradient. Transcripts associated with autotrophic and heterotrophic denitrifiers were mostly confined to the suboxic zone and below but were also present in particles in the oxycline. Anammox genes and transcripts were detected over a narrow depth range near the bottom of the suboxic zone and coincided with secondary NO2 - maxima and available NH4 + . Dissolved inorganic nitrogen (DIN) amendment incubations and comparisons between our sampling campaigns suggested that denitrifier activity may be closely coupled with NO3 - availability. Expression of denitrification genes at depths of high rates of chemoautotrophic carbon fixation and phylogenetic analyses of nitrogen cycling genes and transcripts indicated a diverse array of denitrifiers, including chemoautotrophs capable of using NO3 - to oxidize reduced sulfur species. Thus, results suggest that the Cariaco Basin nitrogen cycle is influenced by autotrophic carbon cycling in addition to organic matter oxidation and anammox.

Dark carbon fixation in the Arabian Sea oxygen minimum zone contributes to sedimentary organic carbon (SOM)

AbstractIn response to rising CO2 concentrations and increasing global sea surface temperatures, oxygen minimum zones (OMZ), or “dead zones”, are expected to expand. OMZs are fueled by high primary productivity, resulting in enhanced biological oxygen demand at depth, subsequent oxygen depletion, and attenuation of remineralization. This results in the deposition of organic carbon‐rich sediments. Carbon drawdown is estimated by biogeochemical models; however, a major process is ignored: carbon fixation in the mid‐ and lower water column. Here, we show that chemoautotrophic carbon fixation is important in the Arabian Sea OMZ; and manifests in a 13C‐depleted signature of sedimentary organic carbon. We determined the δ13C values of Corg deposited in close spatial proximity but over a steep bottom‐water oxygen gradient, and the δ13C composition of biomarkers of chemoautotrophic bacteria capable of anaerobic ammonia oxidation (anammox). Isotope mixing models show that detritus from anammox bacteria or other chemoautotrophs likely forms a substantial part of the organic matter deposited within the Arabian Sea OMZ (~17%), implying that the contribution of chemoautotrophs to settling organic matter is exported to the sediment. This has implications for the evaluation of past, and future, OMZs: biogeochemical models that operate on the assumption that all sinking organic matter is photosynthetically derived, without new addition of carbon, could significantly underestimate the extent of remineralization. Oxygen demand in oxygen minimum zones could thus be higher than projections suggest, leading to a more intense expansion of OMZs than expected.

Nutrient Acquisition, Rather Than Stress Response Over Diel Cycles, Drives Microbial Transcription in a Hyper-Arid Namib Desert Soil.

Hot desert surface soils are characterized by extremely low water activities for large parts of any annual cycle. It is widely assumed that microbial processes in such soils are very limited. Here we present the first metatranscriptomic survey of microbial community function in a low water activity hyperarid desert soil. Sequencing of total mRNA revealed a diverse and active community, dominated by Actinobacteria. Metatranscriptomic analysis of samples taken at different times over 3 days indicated that functional diel variations were limited at the whole community level, and mostly affected the eukaryotic subpopulation which was induced during the cooler night hours. High levels of transcription of chemoautotrophic carbon fixation genes contrasted with limited expression of photosynthetic genes, indicating that chemoautotrophy is an important alternative to photosynthesis for carbon cycling in desiccated desert soils. Analysis of the transcriptional levels of key N-cycling genes provided strong evidence that soil nitrate was the dominant nitrogen input source. Transcriptional network analyses and taxon-resolved functional profiling suggested that nutrient acquisition processes, and not diurnal environmental variation, were the main drivers of community activity in hyperarid Namib Desert soil. While we also observed significant levels of expression of common stress response genes, these genes were not dominant hubs in the co-occurrence network.

Potential effect of pesticides currently used in salmon farming on photo and chemoautotrophic carbon uptake in central – southern Chile

Aquaculture has become an important component of Chilean economy, especially in the southern region, where salmon farming is an active industry. However, high density in salmon cages can increase stress and susceptibility to parasitic outbreaks of the copepod Caligus rogercresseyi. The pesticides used against sea lice can have potential effects on non-target microbiota and on the structure and functioning of aquatic ecosystems. The objective of this study was to investigate the response of natural microbial communities to the addition of the anti-lice pesticides azamethiphos, deltamethrin and emamectin benzoate and their potential impact on photoautotrophic and chemoautotrophic carbon fixation in central-southern Chile (37°S to 42°S). The addition of pesticides on primary production samples was related to changes in carbon uptake, which were significant if a single pesticide was applied, mainly emamectin benzoate and azametiphos. In surface waters of central Chile, emamectin benzoate produced a 60–90% decrease for both photo and chemoautotrophic carbon fixation. Enhanced rates were also observed for in situ primary production as a result of azamethiphos addition in northern Patagonia. Such stimulation, although limited, was possibly related to the supply of nitrogen and phosphate for phytoplankton requirements by this organophosphate compound.

Diversity of Total Bacterial Communities and Chemoautotrophic Populations in Sulfur-Rich Sediments of Shallow-Water Hydrothermal Vents off Kueishan Island, Taiwan.

Shallow-water hydrothermal vents (HTVs) are an ecologically important habitat with a geographic origin similar to that of deep-sea HTVs. Studies on shallow-water HTVs have not only facilitated understanding of the influences of vents on local ecosystems but also helped to extend the knowledge on deep-sea vents. In this study, the diversity of bacterial communities in the sediments of shallow-water HTVs off Kueishan Island, Taiwan, was investigated by examining the 16S ribosomal RNA gene as well as key functional genes involved in chemoautotrophic carbon fixation (aclB, cbbL and cbbM). In the vent area, Sulfurovum and Sulfurimonas of Epsilonproteobacteria appeared to dominate the benthic bacterial community. Results of aclB gene analysis also suggested involvement of these bacteria in carbon fixation using the reductive tricarboxylic acid (rTCA) cycle. Analysis of the cbbM gene showed that Alphaproteobacterial members such as the purple non-sulfur bacteria were the major chemoautotrophic bacteria involving in carbon fixation via the Calvin-Benson-Bassham (CBB) cycle. However, they only accounted for <2% of the total bacterial community in the vent area. These findings suggest that the rTCA cycle is the major chemoautotrophic carbon fixation pathway in sediments of the shallow-water HTVs off Kueishan Island.

Contribution of ammonia oxidation to chemoautotrophy in Antarctic coastal waters.

There are few measurements of nitrification in polar regions, yet geochemical evidence suggests that it is significant, and chemoautotrophy supported by nitrification has been suggested as an important contribution to prokaryotic production during the polar winter. This study reports seasonal ammonia oxidation (AO) rates, gene and transcript abundance in continental shelf waters west of the Antarctic Peninsula, where Thaumarchaeota strongly dominate populations of ammonia-oxidizing organisms. Higher AO rates were observed in the late winter surface mixed layer compared with the same water mass sampled during summer (mean±s.e.: 62±16 versus 13±2.8 nm per day, t-test P<0.0005). AO rates in the circumpolar deep water did not differ between seasons (21±5.7 versus 24±6.6 nm per day; P=0.83), despite 5- to 20-fold greater Thaumarchaeota abundance during summer. AO rates correlated with concentrations of Archaea ammonia monooxygenase (amoA) genes during summer, but not with concentrations of Archaea amoA transcripts, or with ratios of Archaea amoA transcripts per gene, or with concentrations of Betaproteobacterial amoA genes or transcripts. The AO rates we report (<0.1–220 nm per day) are ~10-fold greater than reported previously for Antarctic waters and suggest that inclusion of Antarctic coastal waters in global estimates of oceanic nitrification could increase global rate estimates by ~9%. Chemoautotrophic carbon fixation supported by AO was 3–6% of annualized phytoplankton primary production and production of Thaumarchaeota biomass supported by AO could account for ~9% of the bacterioplankton production measured in winter. Growth rates of thaumarchaeote populations inferred from AO rates averaged 0.3 per day and ranged from 0.01 to 2.1 per day.

Community structure and soil pH determine chemoautotrophic carbon dioxide fixation in drained paddy soils.

Previous studies suggested that microbial photosynthesis plays a potential role in paddy fields, but little is known about chemoautotrophic carbon fixers in drained paddy soils. We conducted a microcosm study using soil samples from five paddy fields to determine the environmental factors and quantify key functional microbial taxa involved in chemoautotrophic carbon fixation. We used stable isotope probing in combination with phospholipid fatty acid (PLFA) and molecular approaches. The amount of microbial (13)CO2 fixation was determined by quantification of (13)C-enriched fatty acid methyl esters and ranged from 21.28 to 72.48 ng of (13)C (g of dry soil)(-1), and the corresponding ratio (labeled PLFA-C:total PLFA-C) ranged from 0.06 to 0.49%. The amount of incorporationof (13)CO2 into PLFAs significantly increased with soil pH except at pH 7.8. PLFA and high-throughput sequencing results indicated a dominant role of Gram-negative bacteria or proteobacteria in (13)CO2 fixation. Correlation analysis indicated a significant association between microbial community structure and carbon fixation. We provide direct evidence of chemoautotrophic C fixation in soils with statistical evidence of microbial community structure regulation of inorganic carbon fixation in the paddy soil ecosystem.

Microbial carbon metabolism associated with electrogenic sulphur oxidation in coastal sediments.

Recently, a novel electrogenic type of sulphur oxidation was documented in marine sediments, whereby filamentous cable bacteria (Desulfobulbaceae) are mediating electron transport over cm-scale distances. These cable bacteria are capable of developing an extensive network within days, implying a highly efficient carbon acquisition strategy. Presently, the carbon metabolism of cable bacteria is unknown, and hence we adopted a multidisciplinary approach to study the carbon substrate utilization of both cable bacteria and associated microbial community in sediment incubations. Fluorescence in situ hybridization showed rapid downward growth of cable bacteria, concomitant with high rates of electrogenic sulphur oxidation, as quantified by microelectrode profiling. We studied heterotrophy and autotrophy by following 13C-propionate and -bicarbonate incorporation into bacterial fatty acids. This biomarker analysis showed that propionate uptake was limited to fatty acid signatures typical for the genus Desulfobulbus. The nanoscale secondary ion mass spectrometry analysis confirmed heterotrophic rather than autotrophic growth of cable bacteria. Still, high bicarbonate uptake was observed in concert with the development of cable bacteria. Clone libraries of 16S complementary DNA showed numerous sequences associated to chemoautotrophic sulphur-oxidizing Epsilon- and Gammaproteobacteria, whereas 13C-bicarbonate biomarker labelling suggested that these sulphur-oxidizing bacteria were active far below the oxygen penetration. A targeted manipulation experiment demonstrated that chemoautotrophic carbon fixation was tightly linked to the heterotrophic activity of the cable bacteria down to cm depth. Overall, the results suggest that electrogenic sulphur oxidation is performed by a microbial consortium, consisting of chemoorganotrophic cable bacteria and chemolithoautotrophic Epsilon- and Gammaproteobacteria. The metabolic linkage between these two groups is presently unknown and needs further study.

Spatial differences in East scotia ridge hydrothermal vent food webs: influences of chemistry, microbiology and predation on trophodynamics.

The hydrothermal vents on the East Scotia Ridge are the first to be explored in the Antarctic and are dominated by large peltospiroid gastropods, stalked barnacles (Vulcanolepas sp.) and anomuran crabs (Kiwa sp.) but their food webs are unknown. Vent fluid and macroconsumer samples were collected at three vent sites (E2, E9N and E9S) at distances of tens of metres to hundreds of kilometres apart with contrasting vent fluid chemistries to describe trophic interactions and identify potential carbon fixation pathways using stable isotopes. δ13C of dissolved inorganic carbon from vent fluids ranged from −4.6‰ to 0.8‰ at E2 and from −4.4‰ to 1.5‰ at E9. The lowest macroconsumer δ13C was observed in peltospiroid gastropods (−30.0‰ to −31.1‰) and indicated carbon fixation via the Calvin-Benson-Bassham (CBB) cycle by endosymbiotic gamma-Proteobacteria. Highest δ13C occurred in Kiwa sp. (−19.0‰ to −10.5‰), similar to that of the epibionts sampled from their ventral setae. Kiwa sp. δ13C differed among sites, which were attributed to spatial differences in the epibiont community and the relative contribution of carbon fixed via the reductive tricarboxylic acid (rTCA) and CBB cycles assimilated by Kiwa sp. Site differences in carbon fixation pathways were traced into higher trophic levels e.g. a stichasterid asteroid that predates on Kiwa sp. Sponges and anemones at the periphery of E2 assimilated a proportion of epipelagic photosynthetic primary production but this was not observed at E9N. Differences in the δ13C and δ34S values of vent macroconsumers between E2 and E9 sites suggest the relative contributions of photosynthetic and chemoautotrophic carbon fixation (rTCA v CBB) entering the hydrothermal vent food webs vary between the sites.

Computation of metabolic fluxes and efficiencies for biological carbon dioxide fixation

With rising energy prices and concern over the environmental impact of fossil fuel consumption, the push to develop biomass derived fuels has increased significantly. Although most global carbon fixation occurs via the Calvin Benson Bassham cycle, there are currently five other known pathways for carbon fixation; the goal of this study was to determine the thermodynamic efficiencies of all six carbon fixation pathways for the production of biomass using flux balance analysis. The three chemotrophic pathways, the reductive acetyl-CoA pathway, the 3-hydroxypropionate/4-hydroxybutyrate cycle and the dicarboxylate/4-hydroxybutyrate cycle, were found to be more efficient than photoautotrophic carbon fixation pathways. However, as hydrogen is not freely available, the energetic cost of hydrogen production from sunlight was calculated and included in the overall energy demand, which results in a 5 fold increase in the energy demand of chemoautotrophic carbon fixation. Therefore, when the cost of hydrogen production is included, photoautotrophic pathways are more efficient. However, the energetic cost for the production of 12 metabolic precursors was found to vary widely across the different carbon fixation pathways; therefore, different pathways may be more efficient at producing products from a single precursor than others. The results of this study have significant impact on the selection or design of autotrophic organisms for biofuel or biochemical production. Overall biomass production from solar energy is most efficient in organisms using the reductive TCA cycle, however, products derived from one metabolic precursor may be more efficiently produced using other carbon fixation pathways.

Molecular characterization and endosymbiotic localization of the gene encoding D-ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) form II in the deep-sea vestimentiferan trophosome.

To better understand the contribution of micro-organisms to the primary production in the deep-sea gutless tubeworm Lamellibrachia sp., the 16S-rDNA-based phylogenetic data would be complemented by knowledge of the genes that encode the enzymes relevant to chemoautotrophic carbon fixation, such as D-ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO; EC 4.1.1.39). To phylogenetically characterize the autotrophic endosymbiosis within the trophosome of the tubeworm Lamellibrachia sp., bulk trophosomal DNA was extracted and analysed based on the 16S-rRNA- and RuBisCO-encoding genes. The 16S-rRNA- and RuBisCO-encoding genes were amplified by PCR, cloned and sequenced. For the 16S rDNA, a total of 50 clones were randomly selected and analysed directly by sequencing. Only one operational taxonomic unit resulted from the 16S rDNA sequence analysis. This may indicate the occurrence of one endosymbiotic bacterial species within the trophosome of the Lamellibrachia sp. used in this study. Phylogenetic analysis of the 16S rDNA showed that the Lamellibrachia sp. endosymbiont was closely related to the genus Rhodobacter, a member of the alpha-Protebacteria. For the RuBisCO genes, only the form II gene (cbbM) was amplified by PCR. A total of 50 cbbM clones were sequenced, and these were grouped into two operational RuBisCO units (ORUs) based on their deduced amino acid sequences. The cbbM ORUs showed high amino acid identities with those recorded from the ambient sediment bacteria. To confirm the results of sequence analysis, the localization of the symbiont-specific 16S rRNA and cbbM sequences in the Lamellibrachia sp. trophosome was visualized by in situ hybridization (ISH), using specific probes. Two types of cells, coccoid and filamentous, were observed at the peripheries of the trophosome lobules. Both the symbiont-specific 16S rDNA and cbbM probes hybridized at the same sites coincident with the location of the coccoid cells, whereas the filamentous cells showed no cbbM-specific signals. The RuBisCO form I gene (cbbL) was neither amplified by PCR nor detected by ISH. This is the first demonstration of chemoautotrophic symbiosis in the deep-sea gutless tubeworm, based on sequence data and in situ localization of both the 16S-rRNA- and RuBisCO-encoding genes.