Members of the epsilonproteobacterial genus Arcobacter have been identified to be potentially important sulfide oxidizers in marine coastal, seep, and stratified basin environments. In the highly productive upwelling waters off the coast of Peru, Arcobacter cells comprised 3 to 25% of the total microbial community at a near-shore station where sulfide concentrations exceeded 20 μM in bottom waters. From the chemocline where the Arcobacter population exceeded 106 cells ml−1 and where high rates of denitrification (up to 6.5 ± 0.4 μM N day−1) and dark carbon fixation (2.8 ± 0.2 μM C day−1) were measured, we isolated a previously uncultivated Arcobacter species, Arcobacter peruensis sp. nov. (BCCM LMG-31510). Genomic analysis showed that A. peruensis possesses genes encoding sulfide oxidation and denitrification pathways but lacks the ability to fix CO2 via autotrophic carbon fixation pathways. Genes encoding transporters for organic carbon compounds, however, were present in the A. peruensis genome. Physiological experiments demonstrated that A. peruensis grew best on a mix of sulfide, nitrate, and acetate. Isotope labeling experiments further verified that A. peruensis completely reduced nitrate to N2 and assimilated acetate but did not fix CO2, thus coupling heterotrophic growth to sulfide oxidation and denitrification. Single-cell nanoscale secondary ion mass spectrometry analysis of samples taken from shipboard isotope labeling experiments also confirmed that the Arcobacter population in situ did not substantially fix CO2. The efficient growth yield associated with the chemolithoheterotrophic metabolism of A. peruensis may allow this Arcobacter species to rapidly bloom in eutrophic and sulfide-rich waters off the coast of Peru.
IMPORTANCE Our multidisciplinary approach provides new insights into the ecophysiology of a newly isolated environmental Arcobacter species, as well as the physiological flexibility within the Arcobacter genus and sulfide-oxidizing, denitrifying microbial communities within oceanic oxygen minimum zones (OMZs). The chemolithoheterotrophic species Arcobacter peruensis may play a substantial role in the diverse consortium of bacteria that is capable of coupling denitrification and fixed nitrogen loss to sulfide oxidation in eutrophic, sulfidic coastal waters. With increasing anthropogenic pressures on coastal regions, e.g., eutrophication and deoxygenation (D. Breitburg, L. A. Levin, A. Oschlies, M. Grégoire, et al., Science 359:eaam7240, 2018, https://doi.org/10.1126/science.aam7240), niches where sulfide-oxidizing, denitrifying heterotrophs such as A. peruensis thrive are likely to expand.


Communities of sulfide-oxidizing microorganisms in marine sulfide-rich environments, such as wood falls, seeps, and stratified water columns, often harbor large communities of the epsilonproteobacterial genus Arcobacter (18). The Arcobacter genus encompasses a diverse assemblage of species that include obligate and facultative chemolithoautotrophs as well as heterotrophs (see references 1, 9, and 10 and references therein). While much attention has focused on gut and food bearing pathogenic Arcobacter species, the Arcobacter genus also contains a wide variety of free-living, environmentally relevant species. For instance, the free-living species Arcobacter butzleri, which has been identified to be an enteropathogen, has also been isolated from aquatic environments (e.g., lakes) (1114). Arcobacter spp. have been enriched in engineered systems containing high levels of sulfide and high levels of organic matter (9, 1517), and Arcobacter spp. have been found in association with unicellular protists, where the Arcobacter spp. benefit from the transfer of labile organic matter and hydrogen (18). The metabolic capability to utilize reduced and intermediate-oxidation-state sulfur compounds for respiration as an electron donor (1, 11, 19) or even as an electron acceptor (10) appears to contribute to the ability of several Arcobacter spp. to thrive in marine, sulfur-rich (6, 20, 21) environments.
Sulfur-rich environments can be found in coastal upwelling systems, such as those off the coasts of Peru and Namibia, where large fluxes of organic carbon deposition drive enhanced rates of microbial sulfate reduction and sulfide production within the underlying sediments (2225). During periods of sluggish water column circulation, dissolved hydrogen sulfide (H2S and HS) diffuses out of the sediments and accumulates to concentrations often exceeding 20 μM in shelf bottom waters (6, 2529). These sulfidic events represent extreme manifestations of coastal deoxygenation processes and contribute to the formation of anoxic dead zones (3032). The occurrence of even low-micromolar concentrations of dissolved hydrogen sulfide restricts the availability of faunal habitat in hypoxic zones and eventually leads to a loss of faunal diversity and massive fish die-offs in otherwise productive, coastal upwelling systems (3134). Counteracting the accumulation of hydrogen sulfide is the rapid oxidation of dissolved sulfide at redoxclines mediated by a variety of bacteria that use dissolved oxygen or nitrate as the terminal electron acceptor (6, 26, 27, 35, 36). The detection of abundant CO2-fixing microbes in oxygen minimum zones (OMZs) and stratified basins, in concert with elevated rates of dark CO2 fixation at sulfide-nitrate redoxclines, has led to the conclusion that chemolithoautotrophic bacteria are primarily responsible for the oxidation of sulfide (6, 2629, 35, 37, 38). In addition to oxidizing sulfide to less toxic elemental sulfur or sulfate, many sulfide-oxidizing bacteria contribute to fixed nitrogen loss by coupling dissolved sulfide oxidation to denitrification (6, 2628, 39, 40). Arcobacter has been identified to be a potentially important sulfide-oxidizing, denitrifying bacterium in the sulfide-rich waters off the coast of Namibia (6) and is hypothesized to serve a similar role in the sulfide-rich waters off the coast of Peru.
To further elucidate the role of Arcobacter associated with sulfidic events in marine ecosystems, we isolated a marine Arcobacter strain from a site in coastal, sulfidic Peruvian shelf waters, where Arcobacter spp. may comprise a substantial fraction of the bacterial community typically associated with sulfide oxidation. We analyzed the genome of the isolated Arcobacter and tested its physiology under conditions mimicking the Peru shelf environment from which it was isolated. In order to place the isolated Arcobacter in its environmental context, we examined the phylogenetic relationship of our isolated Arcobacter to the in situ populations of Arcobacter spp. and with respect to other sulfide-oxidizing chemolithotrophs. Lastly, we quantified the activity at the single-cell level of in situ populations of Arcobacter spp.
(This research was conducted by C. M. Callbeck in partial fulfillment of the requirements for a Ph.D. from the University of Bremen, 2017 [41].)


Arcobacter distribution in sulfidic Peru shelf waters.

The Peruvian shelf waters sampled onboard the RV Meteor Research Expedition M93 in February and early March 2013 (Fig. 1; see also Table S1 in the supplemental material) were characterized by high chlorophyll concentrations and the extreme depletion of dissolved oxygen (<5 μM) below a depth of 10 m and nitrate below a depth of 30 m (27). Dissolved hydrogen sulfide was present in the anoxic waters that covered the entire near-shore Peruvian shelf between 12°S, 78.3°W and 13.3°S, 77°W (Fig. 1). Bottom water sulfide concentrations were typically 5 to 10 μM in the near-shore waters and at station U1a reached up to 23 μM (Fig. 1 and 2a). Potential denitrification rates at station U1a were the greatest in the redoxcline (6.5 ± 0.4 μM N day−1) and decreased within the deeper sulfidic zone (0.9 ± 0.1 μM N day−1). In contrast, dark carbon fixation rates remained constant at 2.8 ± 0.2 μM C day−1 throughout the redoxcline and into the deep sulfidic bottom waters. The nitrogen loss and carbon fixation rates measured on the shelf were similar to the rates in the Peru-Chile and Namibia shelf waters and indicated active chemolithoautotrophic activity, consistent with the findings of previous studies (6, 26, 28).
FIG 1 Sampling area off the coast of Peru and distribution of maximum dissolved sulfide in the shelf oxygen minimum zone. Maximum dissolved sulfide concentrations are shown for the sampling period from February to March 2013. Dots indicate water column sampling locations. Key sampling stations for microbiologic and biogeochemical studies are indicated with bold filled circles (see Table S1 in the supplemental material). Isolate PSE-93 was obtained from station U1a. Sulfide data were plotted with Ocean Data View software (R. Schlitzer, 2018 [62]).
FIG 2 Depth distribution of concentrations, cell abundances, rates of dark carbon fixation and denitrification, as well as bacterial community diversity at station U1a (see Table S1 in the supplemental material). (a) Depth profiles of dissolved oxygen, sulfide, elemental sulfur, and nitrate. Nitrite concentrations were below the limit of detection throughout the water column, except at a 10-m depth (where they were up to 0.69 μM). (b) Arcobacter cell densities (Arc94 probe) and rates of dark carbon fixation and denitrification at 40-, 50-, and 60-m water depths are shown. Error bars represent the standard error. The asterisk indicates the depth from which the samples analyzed by nanoSIMS were collected. (c) Bacterial 16S rRNA gene diversity and relative abundances of key taxa; the top 18 most abundant taxa are shown.
Bacteria belonging to the genus Arcobacter, as identified and enumerated using the fluorescent in situ hybridization (FISH) probe Arc94, were also detected throughout these near-shore, highly productive, sulfide-rich Peru upwelling bottom waters. At near-shore station U1a, the nitrate-sulfide redoxcline supported a large Arcobacter population, exceeding 106 cells ml−1, that comprised 25% of the entire microbial community (Fig. 2b). At stations containing <10 μM dissolved sulfide in the bottom waters, Arcobacter was still present, but relative cell abundances were typically <3% of the microbial community, or below 105 cells ml−1 (Fig. S1). Offshore and in the absence of dissolved sulfide, Arcobacter abundances dropped below <0.1% of the microbial community, just at or below the limits of detection. Microbial 16S rRNA gene diversity analysis of station U1a redoxcline waters, using clone library and amplicon sequencing (pyrosequencing) techniques, identified common OMZ-occurring sulfide-oxidizing bacteria, such as the SUP05 (UThioglobus perditus, where the superscript “U” indicates an uncultivated taxon [27]) clade within the Gammaproteobacteria, as well as uncultured Sulfurovum and Arcobacter spp. within the Epsilonproteobacteria (Fig. 2c). Arcobacter abundances at station U1a, based on both catalyzed reporter deposition (CARD)-FISH cell counts and 16S rRNA gene abundances from amplicon pyrosequencing analysis (Fig. 2b and c), peaked at the middle of the broad redoxcline at 40 m, consistent with reports elsewhere of Arcobacter spp. occurring principally at oxic-sulfidic or nitrate-sulfidic redoxclines (4, 20, 42, 43).

Isolation and genome analysis of Arcobacter sp. strain PSE-93.

We isolated an Arcobacter bacterium from station U1a under anaerobic conditions using natural seawater amended with sulfide and nitrate. The isolate, Arcobacter sp. strain PSE-93, was enriched from Peruvian seawater containing sulfide and nitrate by continuous transfers until it reached 90 to 95% purity, as checked with CARD-FISH using the Arc94 probe (Fig. S2). PSE-93 had a slightly elongated morphology (0.9 μm in length and 0.6 μm in width) that was comparable to the morphology of Arcobacter spp. observed in situ. DNA extraction of the enrichment culture, followed by sequencing by use of the Pacific Biosciences RS II technology (PacBio), assembly, and polishing, resulted in 256 contigs. After the removal of short contigs (<10 kbp) from the assembly, only one contig (84-fold average coverage) that was circularized remained, yielding the complete genome of PSE-93. Analysis by the CheckM method (44), based on 338 conserved marker genes, indicated that the genome of Arcobacter PSE-93 was complete (99.6%) and free of contamination (1.0%) or strain heterogeneity (0.0%). Summary genome statistics for Arcobacter PSE-93 are shown in Table S2.
The closed circular genome (2.8 Mbp in size with a 27.8% GC content and 2,697 predicted protein-coding genes) of PSE-93 revealed that it has the potential to reduce nitrate to N2 via a complete denitrification pathway that includes periplasmic nitrate reductase (napAB), cd1-type nitrite reductase (nirS), nitric oxide reductase (norBC), and nitrous oxide reductase (nosZ) (Fig. 3). The presence of a cbb3-type terminal oxidase (fixNOQP) implies that Arcobacter PSE-93 might be able to respire with oxygen. The identification of flagellum- and chemotaxis-associated genes in the PSE-93 genome indicated, furthermore, that Arcobacter might be able to position itself within the redoxcline according to the gradients of dissolved sulfide and nitrate. A gene encoding a protein with a rhodanese-like domain that may act to shuttle zerovalent sulfur in between redox centers during sulfur metabolism was found (45). The Arcobacter PSE-93 genome contained a complete periplasmic sulfide oxidation Sox pathway (soxABCDXYZ) and a sulfide-quinone reductase gene (sqr) that allows for the oxidation of various reduced sulfur compounds (including sulfide, sulfur, and thiosulfate) to sulfate. The presence of soxCD is consistent with a lack of internal elemental sulfur storage, as missing soxCD genes have been shown in other sulfide oxidizers to be correlated with intracellular sulfur storage (46). Sulfur reduction genes, such as those for polysulfide reductase, tetrathionate reductase, and sulfide hydrogenases, some of which have been identified in A. anaerophilus (10), were absent from the Arcobacter PSE-93 genome.
FIG 3 Central metabolism of Arcobacter peruensis. Identified genes are based on the RAST annotation. NCBI gene accession numbers associated with key genes discussed in the text are indicated. +, the genes were present; ---, the genes were not found in the genome; TCA, trichloroacetic acid; rTCA, reverse trichloroacetic acid; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; TRAP, tripartite ATP-independent periplasmic transporter; ABC, ATP-binding cassette transporters.
Key genes associated with autotrophic CO2 fixation (e.g., ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] and succinyl coenzyme A [CoA] synthetase reverse trichloroacetic acid [rTCA]), which are frequently identified in sulfide-oxidizing bacteria and in other autotrophic members of the genus Arcobacter (1, 9, 10), were not found in the Arcobacter PSE-93 genome (Fig. 3), nor were genes responsible for nitrogen (N2) fixation present. Instead, genes encoding organic matter-dependent carboxylases involved in acetate and propionate assimilation were detected, as were genes encoding carboxylases involved in fatty acid biosynthesis and anaplerotic reactions. Genes encoding organic compound transporters, e.g., TonB-dependent receptors and TRAP (tripartite ATP-independent periplasmic transporter)-type C4 transport systems, including acetate permeases and ABC transporters associated with acetate and glucose metabolisms, were also detected in the genome. Alternative electron donors may also include formate (formate dehydrogenase) and hydrogen, for example, Ni/Fe hydrogenases, which have also been identified in marine Arcobacter species associated with unicellular protists (18).
The genome of Arcobacter PSE-93 contained six complete sets of rRNA genes (16S-23S-5S). The 16S rRNA gene sequences were almost identical and shared up to 7 mismatches (>99.53% identity). Multiple copies of rRNA genes appear to be a common feature of microorganisms within the genus Arcobacter (44) and might offer a competitive advantage because they confer a higher growth rate or competitive fitness (47). Furthermore, multiple copies of rRNA genes can lead to an overestimation of Arcobacter abundances in the environment and should be considered when interpreting 16S rRNA gene amplicon sequencing data.

Physiology of the PSE-93 isolate.

We tested the ability of isolates of Arcobacter PSE-93 to oxidize sulfide and reduce nitrate on various carbon substrates under anoxic conditions. In all subsequent physiology experiments, we used plate-grown colonies (colonies grown on PY-BROTH medium [DSMZ, Germany]) obtained from the isolate described above, which were then transferred to axenic liquid medium. The identity and purity of the transferred Arcobacter PSE-93 colonies were verified by full 16S rRNA sequencing. Enough dissolved sulfide was amended to reduce all added nitrate to N2, assuming the complete oxidation of sulfide to sulfate. In natural seawater experiments (in North Sea seawater [saNS] medium) under autotrophic conditions (i.e., in the absence of added organic carbon substrate), PSE-93 showed slightly enhanced rates of sulfide oxidation (1.29 ± 0.26 μM h−1) and nitrate reduction (1.00 ± 0.04 μM h−1) (Fig. 4a and d; Table 1). Arcobacter PSE-93 cell densities never exceeded 9 × 104 cells ml−1, and the cells exhibited negligible to nondetectable [13C]bicarbonate assimilation rates under these autotrophic conditions (Fig. 4d). Similarly, using synthetic seawater (Sas) medium under autotrophic conditions, cell numbers also never reached more than 3 × 105 cells ml−1 (Fig. S3). In both experiments, sulfide consumption occurred, and nitrate reduction rates were detectable (Fig. 4a and d; Table 1; Fig. S3). Neither sulfide nor nitrate was fully consumed; roughly half of the nitrate was reduced to N2, while the rest of the added nitrate was reduced only to nitrite.
FIG 4 Arcobacter peruensis activity and growth. Experiments were performed in saNS medium with sulfide, nitrate, and [13C]bicarbonate (a), sulfide, nitrate, and [13C]acetate (b), and nitrate and [13C]acetate (c). (d) Rates of C assimilation based on [13C]bicarbonate and [13C]acetate single-cell uptake determinations. Error bars represent the standard deviations from triplicate incubation experiments. BD, below the limit of detection (<9 × 104 cells ml−1).
TABLE 1 Arcobacter PSE-93 sulfide oxidation and nitrate reduction activity in amendment experimentsa
ExptActivity (μM h−1)Estimated no. of doublings/day
Sulfide oxidationNitrate reduction
Sas medium with NO3 + HS + HCO30.70 ± 0.200.50 ± 0.200.86
saNS medium with NO3 + HS + HCO31.29 ± 0.261.00 ± 0.04BD
saNS medium with NO3 + HS + glucose1.504.1ND
saNS medium with NO3 + HS + yeast extract2.604.7ND
saNS medium with NO3 + HS + acetate6.60 ± 0.676.20 ± 1.691.82
saNS medium with NO3 + acetate 1.3 ± 0.220.78
Experiments were performed in synthetic seawater (Sas) medium or North Sea seawater (saNS) medium, as indicated. The sulfide and nitrate turnover rates shown here were determined from the change in the substrate concentration divided by time (Fig. 4). Carbon assimilation rates are reported in the text. The estimated number of doublings per day is based on the 13C uptake rates. BD, below the limit of detection; ND, not determined.
Complete nitrate reduction (>4.1 μM h−1) to dinitrogen as well as enhanced sulfide oxidation (>1.5 μM h−1) did ensue when PSE-93 was grown heterotrophically on glucose, yeast extract, or acetate (Table 1). The nitrite that formed as an intermediate was completely reduced to N2 by the end of the experiment (Fig. 4b). Growth on acetate yielded the highest rates of sulfide oxidation (6.6 ± 0.67 μM h−1) and nitrate reduction (6.2 ± 1.69 μM h−1) for PSE-93 (Fig. 4b and d; Table 1). 13C-labeled acetate incorporation during growth on nitrate, dissolved sulfide, and acetate showed that acetate was assimilated into PSE-93 biomass at a rate of 1.55 ± 0.19 fmol C cell−1 day−1. Growth under these conditions yielded high cell densities of 4 × 106 cells ml−1 with a doubling rate of 1.4 to 1.8 per day (calculated from cell abundances and 13C assimilation rates). Arcobacter PSE-93 was also able to grow heterotrophically, coupling acetate oxidation to nitrate reduction (Fig. 4c and d); however, the nitrate reduction (1.3 ± 0.22 μM h−1) and acetate assimilation (0.38 ± 0.06 fmol C cell−1 day−1) rates were over 4-fold lower, and the calculated doublings per day (0.8 to 1.0) were nearly 2-fold lower than the rate of PSE-93 growth on dissolved sulfide, nitrate, and acetate (Fig. 4c and d; Table 1). These experiments demonstrated that the Peru upwelling Arcobacter PSE-93 strain is an obligate heterotroph capable of chemolithoheterotrophic and chemoorganoheterotrophic growth. More importantly, Arcobacter PSE-93 could couple sulfide oxidation to complete denitrification when low-molecular-weight organic carbon was made available for cell biomass synthesis.

Phylogeny of Arcobacter PSE-93.

PSE-93 formed a distinct clade that was different with respect to the four recently defined Arcobacter type strain clusters (48). The closest type strains to PSE-93 were in cluster 2, which contained A. venerupis, A. suis, A. ellisii, A. cloacae, and A. defluvii (Fig. 5). To place the PSE-93 identity in relation to the Arcobacter diversity in the Peruvian upwelling, we analyzed the 16S rRNA genes recovered from station U1a. From the nearly full-length 16S rRNA amplicon sequences obtained from clone library preparations at station U1a, we identified two Peruvian Arcobacter clones. ETSP (Eastern Tropical South Pacific) clone 3 and ESTP clone 5 formed two separate phylogenetic branches (Fig. 5). Sequences affiliated with ETSP clone 5 contained Arcobacter members capable of both CO2 fixation (e.g., A. sulfidicus and A. nitrofigilis) and acetate assimilation. In contrast, ETSP clone 3 and the nearby affiliated PSE-93 lineage contained, to the best of our knowledge, only members capable of heterotrophic growth.
FIG 5 Phylogeny of 16S rRNA genes of key sulfide-oxidizing bacteria recovered from Peruvian shelf waters. The unrooted consensus tree is based on the neighbor-joining, parsimony, and RAxML methods applying various Epsilonproteobacteria filters. The Arcobacter type strains (in blue) are organized into four distinct clusters, as defined previously (48). Included are partial and nearly full-length 16S rRNA sequences recovered from sulfidic station U1a (green; Fig. 1; see also Fig. S1 in the supplemental material) and the 16S rRNA gene of the Arcobacter peruensis (PSE-93) isolate enriched from sulfidic station U1a (red). Arcobacter sequences recovered from [13C]acetate RNA-stable isotope assimilation experiments are indicated by black stars (5, 8); conversely, strains capable of autotrophic carbon fixation are indicated by open stars (1, 9, 78). The scale bar represents the substitution rate per site.
Arcobacter PSE-93 was most closely affiliated with the Arcobacter clade that contained ETSP clone 3 (96.1% 16S rRNA gene identity). Within this clade, the sequence of PSE-93 was most closely related to a sequence recovered from the North Sea, that of Arcobacter sp. strain KT0913 (49), and nearly identical to that of Arcobacter sp. strain LPB0137 (the 16S rRNA gene from an environmental Korean strain). Arcobacter strain KT0913 shares a 16S rRNA gene identity value with PSE-93 above the species level (98.8%) (49); however, confirmation by comparison of the average nucleotide identity (ANI) values awaits the availability of the genome sequence of KT0913. Although Arcobacter PSE-93 enriched in our incubations was not identical to ETSP clone 3 or 5, our phylogenetic analysis suggests that PSE-93 is a close relative of ETSP clone 3, one of the most abundant Peruvian upwelling Arcobacter members. Additionally, the Arcobacter 16S rRNA genes recovered from 454 pyrosequencing (∼450 bp in length) were closely related to the clone library 16S rRNA genes, as well as to the 16S rRNA gene of the Arcobacter PSE-93 isolate (e.g., ETSP pyrotags 4, 5, and 6).

In situ single-cell analysis.

From isotope labeling experiments performed on samples obtained during RV Meteor Research Expedition M93 in Peru, we used nanoscale secondary ion mass spectrometry (nanoSIMS) to obtain single-cell measurements of the Arcobacter composition and the uptake of CO2 in the in situ population (Fig. 6). The analysis of single-cell 13CO2 assimilation rates showed that Arcobacter fixed 0.03 ± 0.01 fmol CO2 cell−1 day−1. This rate was significantly lower than the measured CO2 assimilation rates for Gammaproteobacteria clade SUP05 and other Epsilonproteobacteria (e.g., Sulfurovum spp.) of 0.19 ± 0.02 and 0.72 ± 0.03 fmol C cell−1 day−1, respectively (analysis of variance [ANOVA], degrees of freedom [df] = 2, P < 0.001 [considered a significant difference]; Fig. 6b). Arcobacter bacteria thus contributed to less than 1% of the overall dark CO2 fixation at station U1a, despite the large abundance of Arcobacter (25% of the total microbial community), whereas the Gamma- and Epsilonproteobacteria accounted for nearly one-quarter of the total dark carbon fixation rates at station U1a, consistent with their abundances (Table S3). The Arcobacter spp. present in the Peru upwelling waters also differentiated themselves from the SUP05 clade and other Epsilonproteobacteria, in that Arcobacter exhibited significantly lower sulfur contents than these other groups of sulfide-oxidizing bacteria (Fig. 6c).
FIG 6 Arcobacter single-cell activity and sulfur content at Peruvian upwelling station U1a (60-m depth). (a) The composite images show the CARD-FISH image and the single-cell isotope ratios. In the CARD-FISH image, cells in green and red are hybridized with the Arc94 and Epsy682 probes, respectively. The 19F signal indicates cells that hybridized with the Arc94 probe (also indicated by the white arrows). (b and c) The single-cell H[13C]CO3 assimilation rates (b) and cell sulfur contents (c) of Arcobacter, SUP05, and other Epsilonproteobacteria (excluding Arcobacter) are shown. The average (red line) and median (black line) as well as the 5th and 95th percentiles (closed circles) are indicated. Error bars represent the standard deviation. The numbers of cells analyzed by nanoSIMS were 58, 37, and 107 for Epsilonproteobacteria, Arcobacter, and SUP05, respectively. Note that data related to SUP05 (including the SUP05 single-cell assimilation rates and sulfur content) are presented elsewhere (27).


The genotypic and physiologic characterizations of the isolated Arcobacter PSE-93 strain demonstrate that we have isolated a new Arcobacter species that we name Arcobacter peruensis. Both the chemolithoheterotrophic genotype and the physiology of A. peruensis suggest that it can thrive in the sulfide- and organic matter-rich environment in coastal Peru upwelling waters, where nitrate is supplied as an alternate electron acceptor. Our experiments employing environmentally relevant concentrations of substrates demonstrated that A. peruensis grows best on a combination of hydrogen sulfide, nitrate, and acetate. It appears to be uniquely suited to an environment where nitrate or microaerophilic concentrations of dissolved oxygen are present and where the fluxes of small dissolved organic compounds (e.g., acetate) and sulfide are great enough to enhance growth. The obligate organotroph A. peruensis thus differs from other environmentally relevant Arcobacter species, which are either facultative heterotrophs and which can also grow autotrophically, such as A. anaerophilus (10), or which are strict autotrophs, such as A. sulfidicus (1). The lack of genes for autotrophic CO2 fixation pathways and the presence of genes for organic carbon-dependent carboxylases in the genome indicate that the very low measured rates of CO2 assimilation might be attributable to anaplerotic CO2 uptake during organotrophy. In addition to being an obligate organotroph, A. peruensis, unlike A. anaerophilus and A. nitrofigilis, has no nitrogen fixation genes and therefore lacks the ability to fix N2 for growth.
In contrast to other Arcobacter species that can reduce nitrate to ammonium (e.g., A. butzleri and A. nitrofigilis) (11, 50), A. peruensis reduces nitrate to dinitrogen. The genes for a complete denitrification pathway are present in the genome of A. peruensis (Fig. 3), and the presence of this pathway was confirmed by the 15N labeling experiments. Complete denitrification by A. peruensis contrasts with the denitrification ability of A. anaerophilus, which is also capable of reducing nitrate to nitrite but lacks the nir and nrf genes and, thus, the ability to reduce nitrite and nitric oxide. On the other hand, A. anaerophilus can use reduced sulfur species, such as tetrathionate and zerovalent sulfur (in the form of elemental sulfur or polysulfides), to oxidize organic compounds. Genomic analysis indicates that A. peruensis does not appear to have the capacity to use intermediate-oxidation-state sulfur compounds as an electron acceptor. It is true that the A. peruensis isolate can also grow heterotrophically on acetate in the absence of sulfide, but it does so only poorly, as the estimated doublings per day are 2-fold lower than those in the presence of dissolved sulfide (Table 1). Our experiments suggest that A. peruensis grows best by coupling dissimilatory nitrate reduction to dinitrogen with the oxidation of dissolved sulfide (Fig. 4) and that environmental levels of sulfide, nitrate, and acetate could sustain the abundances of Arcobacter cells observed in situ (Fig. 2b).
The low rates of CO2 fixation determined by single-cell analysis of the in situ Arcobacter population (Fig. 6) are consistent with the conclusion that chemolithoheterotrophic A. peruensis probably dominates the in situ Arcobacter community. The rate of Arcobacter CO2-based growth, as measured by in situ single-cell 13C uptake, would be far too low (doubling once per 100 days) to support the large cell densities of Arcobacter observed in situ, if it were an autotroph. We ascribe the low per cell rates of CO2 fixation to anaplerotic reactions. In contrast, UT. perditus (SUP05) and Epsilonproteobacteria show active autotrophy in situ (27) (Fig. 6) and contribute substantially (11% to 51%) to the rates of dark carbon fixation on the Peru shelf (see Table S2 in the supplemental material).
Acetate, a short-chain fatty acid produced during the degradation of organic matter via fermentation, could support Arcobacter growth in the Peruvian OMZ waters. Permanently sulfidic marine basins exhibit not only elevated rates of dark carbon fixation but also high rates of both acetate production and acetate assimilation (0.05 to 0.5 μM day−1) (51). Acetate assimilation has been suggested to provide an important source of carbon for microbial growth on the highly productive northern Gulf of Mexico shelf (52). A. peruensis could assimilate acetate by converting it to acetyl-CoA using the combination of the high-affinity acetate permease (Km = 5.4 μM) and the acetyl-CoA synthetase (53, 54) (Fig. 3). Acetyl-CoA is a key precursor molecule for various biosynthesis pathways (55). Interestingly, the Arcobacter strain isolated and described here was initially enriched on Peru OMZ shelf water without the addition of extra organic substrates, suggesting that A. peruensis is very efficient at exploiting the residual dissolved organic pool in the Peru OMZ. The concentrations of dissolved organic matter in surface and benthic zone-influenced bottom waters of the shallow Peruvian OMZ shelf are high and in the range of 80 to 100 μmol liter−1 (56). Furthermore, other studies have indicated that Arcobacter species may be capable of assimilating acetate under sulfidic conditions within sediments and at redoxclines (3, 8).
Chemolithoheterotrophic growth may confer energetic advantages over a chemolithoautotrophic physiology in an organic matter-rich environment. For pyruvate, a key building block in biosynthesis, autotrophic CO2 fixation requires between 0.6 and 2.3 mol ATP per mol of pyruvate formed, while acetate assimilation requires only 0.5 mol ATP per mol of pyruvate formed (57). From the nanoSIMS-derived PSE-93 culture single-cell acetate assimilation rates (1.55 fmol C cell−1 day−1) obtained during experiments under sulfide-oxidizing, denitrifying conditions, we calculated an assimilation factor of 3.1 mol C per mol H2S oxidized. This rate of acetate carbon assimilation exceeds the rates of CO2 assimilation reported for cultivated chemolithoautotrophic sulfide-oxidizing bacteria, which range from 0.35 to 0.58 mol C assimilated per mol H2S oxidized (5860). The lower energy constraints of A. peruensis may enable A. peruensis (and, presumably, other sulfide-oxidizing chemolithoheterotrophic bacteria) to outcompete sulfide-oxidizing chemolithoautotrophs (e.g., SUP05 bacteria) when the production or flux of small organic compounds is sufficient. Thus, A. peruensis is most likely to bloom under highly sulfidic and organic matter-rich conditions, as observed in this study off the coast of Peru, and at other coastal, sulfidic environments, such as off the coast of Namibia (6).
A chemolithoheterotrophic physiology is not without drawbacks. The requirement for labile organic matter, in addition to sulfide and nitrate, places A. peruensis at a competitive disadvantage alongside chemolithoautotrophic bacteria that generate biomass (i.e., via CO2 fixation) independently of organic matter availability. In addition, compared to other co-occurring sulfide-oxidizing bacteria (e.g., SUP05), A. peruensis appears to lack the capacity to store intracellular sulfur. Genomic data showed the presence of the soxCD genes, which is often correlated with a lack of intracellular sulfur storage in sulfide oxidizers (46). NanoSIMS analysis of individual cells showed, moreover, that Peruvian OMZ Arcobacter spp. were significantly lower in sulfur content (ANOVA, df = 2, P < 0.001) than SUP05 and non-Arcobacter Epsilonproteobacteria cells (Fig. 4a and b). In contrast, SUP05 bacteria, which co-occur with Arcobacter in the sulfidic shelf waters, continue to denitrify using stored intracellular elemental sulfur and are, as a result of this ecophysiology, more broadly distributed within the Peru upwelling OMZ (27). In contrast, the Arcobacter dependency on sulfide and organic matter for growth ultimately constrains its distribution to sulfidic and highly productive shelf waters.
The metabolic flexibility inherent to the Arcobacter genus has enabled Arcobacter strains similar to A. peruensis to gain a foothold in a marine redox transition zone that is often assumed to be dominated by chemolithoautotrophs (6, 26, 28, 35, 37, 38). Despite its apparent restriction to sulfidic, eutrophic shelf systems, chemolithoheterotrophic metabolism, such as that exhibited by A. peruensis, may play an important role in nitrogen and sulfur cycling in environments similar to those on the Peru shelf, especially as hypoxic, sulfidic coastal regions expand (32). Organisms similar to the Peru upwelling A. peruensis bacterium may be more widely spread, as indicated by the very close similarity of a Korean environmental strain (LPB0137). How much these and similar Arcobacter species eventually contribute to sulfide oxidation and nitrogen loss through denitrification in increasingly eutrophic marine coastal ecosystems remains to be explored.

Description of Arcobacter peruensis sp. nov.

A. peruensis type strains: PSE-93, LMG 31510. Etymology: pe.ru.en′sis. N.L. masc. adj. peruensis, pertaining to Peru. Locality: isolated from coastal, sulfidic Peruvian shelf waters at 12.23°S, 77.18°W. Properties: Gram-negative, slightly elongated rods (0.9 μm in length and 0.6 μm in width). It grows chemolithoheterotrophically in sulfide-, nitrate-, and organic matter-rich environments in coastal upwelling water. It grows best by coupling dissimilatory nitrate reduction to dinitrogen to the oxidation of dissolved sulfide. Alternatively, heterotrophic growth on acetate in the absence of sulfide occurs, albeit at much slower doubling times. It grows aerobically on PY-BROTH medium (DSMZ, Germany) supplemented with 200 μM KH2PO4, 10 mM MgSO4·7H2O, 5 mM HEPES at a final pH of 7.8 and solidified with Bacto agar (1%, wt/vol). The median GC content of the type strain is 27.4 mol%.


Sampling and water chemistry.

Peru upwelling waters (12°S, 78.5°W and 13.5°S, 77°W) were sampled from 8 February to 4 March 2013 onboard the RV Meteor (Research Expedition M93; Fig. 1; see also Table S1 in the supplemental material). All seawater samples for this study were collected at an approximately 5-m resolution to within 5 m of the seafloor using a conductivity-temperature-depth (CTD) rosette equipped with 10-liter Niskin bottles. Oxygen was monitored with a Seabird sensor and calibrated against Niskin bottle samples by Winkler titration. Collected seawater nitrate and nitrite concentrations were determined with a QuAAtro autoanalyzer (Seal Analytical, UK) with precisions of ±0.1 μM. Sulfide concentrations were determined onboard as described by Cline (61) using 4 ml of seawater. The sulfide data in Fig. 1 were plotted with Ocean Data View software (62). For the determination of water column elemental sulfur, 50-ml samples of seawater were sampled using anaerobic techniques, fixed with 100 μl ZnCl2 (20%, wt/wt), and stored at −20°C. Elemental sulfur was extracted from the Zn-fixed samples using a chloroform-methanol procedure (63) and measured on a Waters Acquity H-class (Waters, Japan) ultra-high-pressure liquid chromatography (UPLC) system (1.7-μm-particle-size, 2.1- by 50-mm column with a methanol eluent at a flow rate of 0.4 ml min−1; Acquity UPLC BEH C18) equipped with a Waters photometric diode array detector (the absorbance wavelength was set to 265 nm, with the limit of detection being 50 nM). In this procedure, elemental sulfur can derive from suspended elemental sulfur, from elemental sulfur deposited on or within cells, or from the decomposition of dissolved polysulfides (63).

Microbial diversity analysis.

One to 2 liters of seawater was collected on polycarbonate filters (0.2-μm pore size; Millipore, Germany) for genomic DNA extraction. Genomic DNA was extracted using a Qiagen AllPrep DNA/RNA kit) and quantified spectrophotometrically by use of a NanoDrop spectrophotometer (Thermo Scientific). Universal bacterial barcoded PCR primers (primers Bakt341F [CCTACGGGNGGCWGCAG] and 805R [GACTACHVGGGTATCTAATCC], targeting the V3-V4 regions [59]) were used to generate amplicons for 454 pyrosequencing (454 FLX+ Titanium chemistry; Max Planck Genome Center, Cologne, Germany [https://mpgc.mpipz.mpg.de/home/]). The PCR conditions were initial denaturation at 95°C for 5 min, followed by 25 cycles of 95°C for 40 s, 55°C for 2 min, and 72°C for 1 min and a final extension of 72°C for 7 min, with a ramp rate of 3°C s−1. The PCR products were purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany). The recovered 16S rRNA sequences were trimmed to remove the primer adaptors before submission to the SILVAngs pipeline, which provided automated alignments, quality management, dereplication, and taxonomic classification (60).
The microbial diversity at station U1a was also analyzed by clone library sequencing, which produced nearly full-length 16S rRNA gene sequences. The clone library preparation procedure, including raw read quality controls and taxonomic classification using ARB software, was performed by a method similar to that described by Callbeck et al. (27). Universal bacterial primers GM3f (5′-AGA GTT TGA TCM TGG C-3′) and GM4r (5′-TAC CTT GTT ACG ACT T-3′) were used to generate 16S rRNA PCR amplicons from DNA samples taken at station U1a. The PCR conditions were initial denaturation at 95°C for 5 min, followed by 25 cycles of 95°C for 1 min, 50°C for 1 min, and 72°C for 2 min and a final extension of 72°C for 10 min, with a ramp rate of 3°C s−1. DNA was visualized by gel electrophoresis. The 16S rRNA PCR amplicons were purified and then ligated into a TOPO TA vector (Invitrogen). Escherichia coli clones were picked and screened for the vector insert by PCR. The plasmids were extracted from vector-positive colonies (using a plasmid extraction kit [MoBio]), and then the plasmid vector was PCR amplified using forward and reverse M13 primers (primers M13f [5′-CCC AGT CAC GAC GTT GTA AAA CG-3′] and M13r [5′- AGC GGA TAA CAA TTT CAC ACA GG-3′]). The PCR products were purified using Sephadex (G-50 Superfine; Amersham Bioscience) and then sequenced (Sanger sequencing with a BigDye kit; Applied Biosystems). Raw sequence data were quality controlled, and vector ends were trimmed and then assembled into nearly full-length 16S rRNA contigs using Sequencher (version 4.6) software (Gene Codes Corporation, Ann Arbor, MI). The nearly full-length and partial 16S rRNA sequences recovered from the clone library preparations and from pyrosequencing were aligned by use of the SINA aligner (64) and then imported into the SILVAref115 curated 16S rRNA reference database (65) using ARB software (66). A consensus 16S rRNA gene tree of neighbor-joining (distance matrix), parsimony (Phylip DNApars program), and maximum likelihood (RAxML, version 7, software) phylogenetic trees applying various epsilonproteobacterial filters was calculated from the nearly full-length sequences (67). Partial 16S rRNA genes were added to the consensus tree using RAxML (version 7) software, applying an epsilonproteobacterial filter.

Cell identification and enumeration.

Seawater samples collected from Niskin bottles were filtered over polycarbonate filters (0.2-μm pore size; Millipore, Germany) for analysis of cell abundances and identification. The filters were fixed in paraformaldehyde (final concentration, 1 to 2% [wt/vol]) for 12 h at 4°C prior to filtration. Catalyzed reporter deposition (CARD)-FISH analysis was performed using probes targeting Arcobacter and general Epsilonproteobacteria: Arc94 (5′-TTAGCATCCCCGCTTCGA-3′) (68) and Epsy682 (5′-CGGATTTTACCCCTACACM-3′) (43). The Arc94 and Epsy682 probes were incubated in 20% formamide containing hybridization buffer at 46°C for 3 h. The total microbial community was stained with 4′,6-diamidino-2-phenylindole (DAPI), and together, DAPI-stained and Arc94- and Epsy682-hybridized cells were visualized and quantified using epifluorescence microscopy. Hybridized cells in 10 fields of view and up to 1,000 DAPI-stained cells were counted. Negative and positive controls were performed using the NON338 and EUB3381-III probes, respectively, as described previously (69).

15N- and 13C-labeled incubation experiments.

Isotope labeling experiments to determine the rates of nitrate reduction (15N labeling) and CO2 fixation (13C labeling) were performed as described previously (70). Briefly, denitrification, as well as bulk and single-cell carbon fixation rates, were determined from 12-ml Exetainer incubation experiments, as follows: in experiment 1, 15NO3 plus H13CO3; in experiment 2, 15NO2 plus 14NH4+ plus H13CO3; and in experiment 3, 15NH4+ plus 14NO2 and H13CO3. The concentrations of labeled substrates were 25 μM, 5 μM, and 5 μM for NO3-, NO2-, and NH4+, respectively. Isotopic ratios of 15N15N, 15N14N, and 14N14N nitrogen gas were measured on a gas chromatography (GC) isotope-ratio mass spectrometer (IRMS) (VG Optima, Manchester, UK), while bulk carbon fixation rates were measured separately in 13C incubation experiments performed in gas-tight 4.5-liter bottles (26). The 13C/12C isotope ratio was measured on an element analyzer (EA) IRMS (FlashEA 1112 series coupled with an IRMS; Finnigan Delta Plus XP; Thermo Scientific).

Single-cell analysis.

In addition to the sample for IRMS measurements, extra samples from the last time point of the stable isotope incubation experiments were filtered onto gold-palladium-precoated polycarbonate filters (0.2-μm pore size; Millipore, Germany) for FISH-secondary ion mass spectrometry (SIMS) analysis. Select field of views containing hybridized cells were marked using laser dissection microscopy (DM 6000 B, Leica, Germany) and analyzed by nanoSIMS (nanoSIMS 50L; Cameca, France), which was used to measure simultaneously the single-cell isotopic composition of Arc94-hybridized cells. Secondary ions 12C, 13C, 19F, 12C14N, 12C15N, 31P, and 32S were measured on 7 mass detectors. Samples were presputtered with a Cs+ primary ion beam of ∼300 pA, and thereafter, the instrument was tuned on a 50- by 50-μm-field-of-view raster size (yielding a mass resolution of over 8,000). Final image acquisition was performed with a primary beam between 1 and 2 pA at a 10- by 10-μm raster size (256 × 256 pixels) with a dwell time of 1 ms per pixel for 40 planes.
The 13C/12C and 32S/(12C + 13C) ratios were calculated using look@nanoSIMS software as outlined elsewhere (71). The 19F signal (of the halogenated oligonucleotide Arc94 probe) was used to specifically identify Arcobacter hybridized cells (Fig. 6a). Single-cell carbon assimilation rates were determined using 13C/12C enrichment, the labeling percentage, the cell volume (based on a rounded rod consisting of two half spheres), and the cell carbon content (calculated using an allometric model, in which the number of femtograms of C cell−1 is equal to 133.754 × V0.438, where V is the cell biovolume [in cubic micrometers] [72]) and divided over the incubation period.

Enrichment of Arcobacter.

The Arcobacter culture used in this study was enriched from sulfidic station U1a (Fig. 1; Table S1) at a 50-m depth onboard the research vessel. We used sterile-filtered Peru seawater with additions of sodium nitrate and either sodium sulfite, sodium thiosulfate, or elemental sulfur as electron donors to a final concentration of 100 μM. A total of three transfers were made (with a 1% [vol/vol] inoculum) into new medium over the course of the research campaign, and samples were incubated in the dark at 14°C onboard the research vessel. Samples at ∼4°C along with additional filter-sterilized Peruvian seawater were express delivered (arriving within 2 to 3 days) to the laboratory at the Max Planck Institute for Marine Microbiology in Bremen, Germany. The enrichment cultures were maintained using 0.2-μm-pore-size filtered and autoclaved anaerobic (N2-CO2 atmosphere) Peruvian seawater. After autoclaving, the medium was supplemented with sulfide and nitrate (∼80 μM), inoculated (with a 1% [vol/vol] inoculum), and then incubated at close to ambient water temperatures (14°C). Sulfide and nitrate concentrations were monitored, and once the medium was consumed, the cultures were transferred to fresh medium. After five transfers (1% [vol/vol] inoculum) on nitrate and dissolved sulfide, based on the CARD-FISH screening using the Arc94 probe, Arcobacter cells made up 90 to 95% of the microbial community. Genomic DNA was then extracted from the nearly pure Arcobacter culture using a QIAamp genomic DNA kit (Qiagen, Netherlands).

Genome sequencing and analysis.

Whole-genome sequencing on culture-extracted genomic DNA was performed using the Pacific Biosciences RS II technology (P4-C2 chemistry; Max Planck Genome Center, Cologne, Germany). The collected data were processed and filtered using SMRT analysis software (v.2.1). For genome assembly, SMRT analysis routine HGAP2 was applied. From the polished assembly of the sequences obtained by the use of PacBio, contigs shorter than 10 kbp were excluded, resulting in a single, long contig. For genome circularization and polishing, identical sequences on both contig ends were identified by blastn analysis and trimmed from the genome. Annotation was performed using the RAST program (73), and the annotation of the key metabolic genes was manually inspected and refined. Further analysis of the annotated genome was performed using both the RAST and Pathway tools (74). Genome completeness and contamination were estimated using the CheckM (v.1.0.11 11) tool (44) running the lineage-specific workflow using 338 marker genes.


The enriched Arcobacter strain (PSE-93) was further maintained on sterile anaerobic North Sea seawater (saNS medium) prepared in a Widdel flask, in which the medium was filter sterilized, autoclaved, and then allowed to cool under an N2-CO2 (90:10) atmosphere. The saNS medium was buffered to a final concentration of 2 mM HCO3. Sulfide and nitrate (∼80 μM) were amended to the medium, which was eventually dispensed into smaller serum bottles under an N2-CO2 atmosphere and inoculated with a 1% (vol/vol) culture from Peruvian seawater medium. The isolation of Arcobacter strain PSE-93 was performed by plating on DSMZ 1071 PY-BROTH medium (DSMZ, Germany) supplemented with 200 μM KH2PO4, 10 mM MgSO4·7H2O, 5 mM HEPES at a final pH of 7.8 and solidified with Bacto agar (1%, wt/vol). The plates were incubated at 14°C under oxic conditions. After a few weeks of incubation, single colonies were picked and restreaked on new plates until an axenic culture of PSE-93 was obtained, as confirmed by full 16S rRNA in-house Sanger sequencing and microscopic evaluation. Colonies from the axenic PSE-93 culture were thereafter used for inoculation of fresh sterile saNS medium with 50 to 100 μM nitrate and sulfide. PSE-93 was also grown on synthetic seawater (Sas) medium. Sas medium was prepared in a Widdel flask and contained, per liter of deionized water, 27.5 g NaCl, 5 g MgCl2·6H2O, 4.1 g MgSO4·7H2O, 0.66 g CaCl2·2H2O, and 1.02 g KCl (75). The medium was autoclaved and allowed to cool under an N2-CO2 (90:10) atmosphere. After cooling, sterile trace element, vitamin, and mineral solutions were added as described by Kamp et al. (75). The medium was buffered by the addition HCO3 (to a final concentration of 2 mM), and the pH was adjusted to 7.5.

Physiology experiments.

For the time course experiments, the medium was dispensed anaerobically under N2-CO2 using Hungate techniques into sterile Duran bottles that were mounted and sealed at the top with a 50-ml glass syringe (SGE Analytical Science, Australia). Duran bottles, including the connected syringe, were filled without a headspace. In addition to the PSE-93 inoculum (1%, vol/vol), organic matter compounds glucose (1%, wt/vol), yeast extract (1%, wt/vol), and [13C]acetate (100 μM), as well as inorganic substrates sulfide (100 μM), nitrate (100 μM; 50% 15N labeling), and bicarbonate (2 mM; 10% 13C labeling), were added via a sidearm port in different combinations (Table 1). Experiments were run at 14°C, mimicking in situ conditions. Regular subsamples were taken via the sidearm port over the course of the experiment for the analysis of sulfide, nitrate, and nitrite production (1 ml subsample fixed in 500 μl 5% ZnCl2) as well as for the analysis of labeled N2 production (1 ml subsample in 12-ml Exetainer glass vials with a helium atmosphere and 50 μl saturated HgCl2 solution). Additionally, 1 ml subsample was fixed in 100 μl 20% paraformaldehyde (PFA) for cell count analysis. At the end of the experiment, the remaining contents of the incubation were filtered onto a precombusted Whatman glass microfiber grade GF/F filter (GE Healthcare Life Sciences, UK) and stored at −20°C until further analysis. Samples for N2 measurements were stored cap down in the dark at room temperature.
Dissolved sulfide concentrations in ZnCl2-fixed samples were determined photometrically as described above. Nitrate and nitrite concentrations were determined with a CLD 60 chemiluminescence NO/NOx analyzer (Eco Physics AG, Switzerland) after reduction to NO with acidic sodium iodide (NaI) and acidic vanadium(II) chloride, respectively (76, 77). Isotopic ratios of 15N15N, 15N14N, and 14N14N nitrogen gas were measured on a gas chromatography (GC) isotope-ratio mass spectrometer (IRMS) (VG Optima, Manchester, UK). Cell counts were obtained by flow cytometry on a BD FACSCalibur system (BD Biosciences, CA, USA) after the 2% PFA-fixed cells were stained for 20 min with SYBR green DNA stain. The instrument flow rate was calibrated with saNS medium. The sampling time was 1 min, and the background was determined with samples from the uninoculated control bottles. To quantify the amounts of 15N and 13C incorporated into biomass, the GFF filters were decalcified overnight, dried at 60°C for 1 h, pelletized into tin cups, and analyzed by use of a Thermo Flash EA 1112 elemental analyzer coupled to an isotopic-ratio mass spectrometer (Finnigan Delta Plus XP; Thermo Fisher Scientific, USA).

Data availability.

This Whole Genome Shotgun project and the 16S rRNA gene sequences (clone library) have been deposited in the DDBJ/ENA/GenBank databases under accession numbers CP032363 and MH916845 to MH916849, respectively. Water column nutrients and physical data for the RV Meteor M93 Research Expedition are available at Pangaea (https://doi.pangaea.de/10.1594/PANGAEA.860727), while station sulfur chemistry, Arcobacter cell densities, and rate process measurements have been submitted to Pangea (https://doi.pangaea.de/10.1594/PANGAEA.894324). Additional data regarding SUP05 cell densities can be found at https://doi.pangaea.de/10.1594/PANGAEA.876062.


We are grateful to the Peruvian authorities for access to their national waters and to the captain and crew of RV Meteor Research Expedition M93.
C.M.C., G.L., R.A.S., and M.M.M.K. designed the study; C.M.C., C.P., B.V., H.S., S.L., P.F.H., and T.K. performed the experiments; C.M.C., C.P., G.L., B.V., T.G.F., B.M.F., P.F.H., S.L., T.K., and J.S.G. analyzed the data; C.M.C. and T.G.F. wrote the manuscript with substantial input from G.L., B.M.F., J.S.G., B.V., R.A.S., and M.M.M.K.
We thank G. Klockgether, D. Tienken, A. Schwedt, S. Haas, and K. Latham for analytical and laboratory assistance and C. Schelten for administrative support. A. Oren graciously assisted with the derivation of the species name A. peruensis. We thank the Max Planck Genome Centre Cologne (http://mpgc.mpipz.mpg.de/home/) for performing the genomic analysis in this study.
This work was supported by the Max Planck Society; the German National Science Foundation (DFG) Sonderforschungsbereich (SFB754) GEOMAR, Kiel, Germany; and a Natural Sciences and Engineering Research Council of Canada (NSERC) scholarship to C.M.C. B.V. was supported by a European Research Council Starting Grant (640422).

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Information & Contributors


Published In

cover image Applied and Environmental Microbiology
Applied and Environmental Microbiology
Volume 85Number 24December 2019
eLocator: e01344-19
Editor: Harold L. Drake, University of Bayreuth
PubMed: 31585991


Received: 14 June 2019
Accepted: 5 September 2019
Published online: 27 November 2019


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  1. chemolithoheterotrophy
  2. denitrification
  3. epsilonproteobacteria
  4. nanoSIMS
  5. sulfide oxidation
  6. upwelling



Max Planck Institute for Marine Microbiology, Bremen, Germany
Present address: Cameron M. Callbeck, Swiss Federal Institute of Aquatic Science and Technology (Eawag), Kastanienbaum, Switzerland.
Chris Pelzer
Max Planck Institute for Marine Microbiology, Bremen, Germany
Department of Microbiology, IWWR, Radboud University Nijmegen, Nijmegen, Netherlands
Gaute Lavik
Max Planck Institute for Marine Microbiology, Bremen, Germany
Max Planck Institute for Marine Microbiology, Bremen, Germany
Max Planck Institute for Marine Microbiology, Bremen, Germany
Bram Vekeman
Max Planck Institute for Marine Microbiology, Bremen, Germany
Harald Schunck
Insitute for General Microbiology, University of Kiel, Kiel, Germany
Sten Littmann
Max Planck Institute for Marine Microbiology, Bremen, Germany
Max Planck Institute for Marine Microbiology, Bremen, Germany
Philipp F. Hach
Max Planck Institute for Marine Microbiology, Bremen, Germany
Tim Kalvelage
Max Planck Institute for Marine Microbiology, Bremen, Germany
Insitute for General Microbiology, University of Kiel, Kiel, Germany
Marcel M. M. Kuypers
Max Planck Institute for Marine Microbiology, Bremen, Germany


Harold L. Drake
University of Bayreuth


Address correspondence to Timothy G. Ferdelman, [email protected].

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