Community dynamics and functional characteristics of

Accepted Article

Community dynamics and functional characteristics of naphthalene-degrading populations in contaminated surface sediments and hypoxic/anoxic groundwater Running Title: “Dynamics of groundwater naphthalene biodegradation” Roland C. Wilhelm*1, Buck T. Hanson2, Subhash Chandra3 and Eugene Madsen Ѻ4 1

School of Integrative Plant Sciences, Cornell University, Ithaca, NY, 14853, USA Department of Microbiology and Ecosystem Science, University of Vienna, Vienna, Austria 3 Cornell SIMS Laboratory, Dept. of Earth & Atmospheric Sciences, Cornell University, Ithaca, NY, 14853 4 Department of Microbiology, Cornell University, Ithaca, NY, 14853, USA

Ѻ

2

Deceased Aug. 9th, 2017

*Corresponding Author:

Roland C. Wilhelm

Current address:

School of Integrative Plant Science, 306 Tower Road, Cornell University, Ithaca, NY 14853

Telephone: +1 607-262-1650 E-mail: [email protected]

Keywords: metagenomics, bioremediation, naphthalene, groundwater, trophic web, stable isotope probing, secondary ion mass spectrometry, single-cell imaging

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1462-2920.14309

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Originality-Significance Statement

Accepted Article

This study advances understanding of the ecological and genetic determinants of naphthalene-degradation using modern cultivation-independent methods. The study site has been the focus of twenty-five years of field-based research on the microbial processes governing natural attenuation of polyaromatic hydrocarbon (PAH) pollution in groundwater. We applied high-throughput sequencing for the first time, yielding results that expand upon and, in some cases, reshape earlier findings. Stable isotope probing (SIP) greatly improved the assembly of shotgun metagenomes, enabling the recovery of four high-quality metagenome-assembled genomes from active naphthalene-degrading species. The resulting genomic insights helped determine the ecological and functional differences between populations active in earlier and later stages of our time-course experiment. By integrating metagenomic data from both ground water and surface sediments, we were able to demonstrate the contiguity of naphthalenedegrading populations between both environments and identify taxa potentially active in situ under hypoxic/anoxic conditions. This study offers a rare multi-faceted, field-based characterization of PAH-degrading bacteria, informing our understanding of subsurface biodegradation. Additionally, we sought to identify trophic interactions between primary degraders and secondary feeders by tracking the fate of 13C from naphthalene over time with SIP and singlecell, isotopic imaging with secondary ion mass spectrometry (SIMS). The surprising lack of carbon transfer from primary degraders in the span of a week, led us to conclude that the common concerns over cross-feeding in SIP experiments may be overstated.


Summary

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Earlier research on the biogeochemical factors affecting natural attenuation in coal-tar contaminated groundwater, at South Glens Falls, NY, revealed the importance of anaerobic metabolism and trophic interactions between degrader and bacterivore populations. Field-based characterizations of both phenomena have proven challenging, but advances in stable isotope probing (SIP), single-cell imaging and shotgun metagenomics now provide cultivationindependent tools for their study. We tracked carbon from 13C-labeled naphthalene through microbial populations in contaminated surface sediments over six days using respiration assays, secondary ion mass spectrometry imaging and shotgun metagenomics to disentangle the contaminant-based trophic web. Contaminant-exposed communities in hypoxic/anoxic groundwater were contrasted with those from oxic surface sediments to identify putative features of anaerobic catabolism of naphthalene. In total, six bacteria were responsible for naphthalene degradation. Cupriavidus, Ralstonia and Sphingomonas predominated at the earliest stages of SIP incubations and were succeeded in later stages by Stenotrophomonas and Rhodococcus. Metagenome-assembled genomes provided evidence for the ecological and functional characteristics underlying these temporal shifts. Identical species of Stenotrophomonas and Rhodococcus were abundant in the most contaminated, anoxic groundwater. Apparent increases in bacterivorous protozoa were observed following exposure to naphthalene, though insignificant amounts of carbon were transferred between bacterial degraders and populations of secondary feeders.


Introduction The native capacity of microorganisms to degrade pollutants in soil and subsurface environments has motivated research in applied and environmental microbiology for decades.

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The long-term field-based characterization of coal-tar contaminated groundwater at ‘Site 24’ in South Glens Falls, NY, has served as a model for natural attenuation for over twenty-five years and represents one of the best characterized field systems for the impacts of polycyclic aromatic hydrocarbon (PAH) contamination. The bioremediation potential of subsurface hydrocarbondegrading bacteria and the biogeochemical / ecological impacts of contamination at the site have been described (Madsen et al., 1991; Bakermans et al., 2002; Yagi et al., 2010b). Yet, given the challenges of studying complex in situ microbial systems in a relatively inaccessible environment, there remain fundamental questions about the ecological, biological and genetic determinants that affect decontamination. In early research at Site 24, PAH contamination was found to increase the abundance of

heterotrophic bacteria and protozoa (Madsen et al., 1991). Protozoan populations were overrepresented in bacterivorous and anaerobic species that were absent at uncontaminated sites (Yagi et al., 2010a). These changes were indicative of trophic succession resulting from the accumulation of contaminant-fed bacterial biomass and the depletion of oxygen from heightened aerobic metabolism. The extent to which top-down predatory interactions feed back on hydrocarbon degrading populations is poorly understood. Several studies reported increased richness and abundance of protozoa in PAH- or hydrocarbon-contaminated sites (Kinner et al., 2002; Tso and Taghon, 2006; Gilbert et al., 2015; Moss et al., 2015) with varying impacts on bioremediation potential (Stoeck and Edgcomb, 2010). In a recent case, the expansion of bacterivorous protozoa diminished the rate of hydrocarbon degradation and mitigated changes in bacterial diversity caused by blooming degrader populations (Beaudoin et al., 2016). Stable


isotope probing (SIP) has been used to track the fate of carbon from artificially

13

C-labeled

bacterial biomass into grazer populations to great effect (Lueders et al., 2004; Frias-Lopez et al.,

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2009; Kuppardt et al., 2010; Anderson et al., 2013; Li et al., 2013). SIP has been used in myriad ways to identify primary catabolically active groups (see review by Coyotzi et al. 2016) and can be used to track secondary feeding by following the redistribution of 13C over time. The catabolism of PAHs in suboxic groundwater contributes substantively to natural

attenuation at Site 24, but the absence of enrichment cultures or isolates representing functional populations under hypoxic/anoxic conditions has limited understanding of the processes. The most heavily contaminated groundwater exhibits biogeochemical signatures of anaerobic microbial metabolism, such as increased concentrations of reduced by-products like ferrous iron, sulfide, ammonia, hydrogen gas and methane compared to reference sites (Bakermans et al., 2002; Yagi et al., 2010b). The detection of metabolites and mRNA transcripts corresponding to anaerobic catabolism of naphthalene indicates in situ biological degradation of the most persistent and concentrated PAH at the site (Yagi et al., 2010a; Yagi et al., 2010b). Yet, attempts to quantify the rate of anaerobic degradation of naphthalene or enrich for the functional populations in microcosm-based studies have yielded negative or weak results (Madsen et al., 1996; Bakermans et al., 2002; Yagi et al., 2010a

he responsible organisms are therefore li ely

to be sensiti e to changes in en ironmental conditions which is consistent with other studies of anaerobic naphthalene-degrading consortia

mmel et al., 2015). Under such circumstances,

the use of cultivation-independent, shotgun metagenomics-based methods have the highest likelihood of providing insights into the anaerobic degrader community and have not yet been attempted at Site 24.


In this study, we applied a combination of SIP with

13

C-naphthalene and shotgun

metagenomic sequencing in two field-based experiments to characterize the composition and

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functional capabilities encoded by naphthalene degraders. First, we tracked the assimilation of carbon from

13

C-napthalene into microbial biomass over time in oxic surface sediments using

respiration assays, single-cell isotopic imaging, with secondary-ion mass spectrometry (SIMS), and SIP-shotgun metagenomics. A second approach was necessary to study groundwater, since the small quantities of

13

C-label used in SIP would rapidly dissipate (> 5 m below surface).

Therefore, we sampled groundwater across a gradient of naphthalene-contamination to identify populations associated with increased contaminant concentrations under hypoxic (dissolved oxygen < 2.0 mg/L) and suboxic/anoxic conditions (DO < 0.2 mg/L). We integrated both datasets, along with legacy data from Site 24, to i) determine the scope and scale of transfer of naphthalene-derived carbon through microbial communities and ii) to identify genes and taxa that predominate under oxic and hypoxic/suboxic conditions. Our study expands understanding of the ecological and genetic basis for naphthalene-degradation in subsurface environments, and builds upon the long-term characterization of Site 24.


Results Field work was conducted at the former coal-tar waste deposit site on the western edge of the

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Hudson River in South Glens Falls, New York. The buried source of PAH contamination was excavated in 1991, but had leached naphthalene and other aromatic hydrocarbons (e.g. xylenes, toluene, 2-methylnaphthalene, and acenaphthylene) for over 30-years into the shallow, sandy aquifer (Taylor et al., 1996). Shotgun metagenomic sequencing was performed on groundwater samples from an uncontaminated, upstream reference site and three contaminated groundwatermonitoring wells MW; Figure 1 including one hypoxic ‘shallow’ MW dissol ed oxygen < 2 0 mg/L and two suboxic/anoxic ‘mid’ and ‘deep ’ MWs DO < 0 2 mg/L Oxygen le els in MWs were consistent with previous reports (Bakermans et al., 2002; Hanson and Madsen, 2015) and are characterized by fluctuating low oxygen concentrations (Yagi et al., 2010a). Naphthalene contamination was 100-fold higher in the deep well compared to mid and shallow MW (Table 1). At a downstream seep, surface sediments chronically exposed to contaminated groundwater effluent were amended with either unlabeled ‘12C-control’ or 13C-labeled naphthalene in a SIP experiment and sampled over the course of 6 days (photos in Fig. S1). The biogeochemistry and microbial ecology of both sites have been characterized for over two decades (Madsen et al., 1991; Madsen et al., 1996; Bakermans et al., 2002; Jeon et al., 2003; Liou et al. 2008; Yagi et al., 2010a; Posman et al., 2017). The presence of active naphthalene-degrading populations in surface sediments was

confirmed by the mineralization of naphthalene in dosed samples, with respiration proportional to the total naphthalene added for both unlabeled and

13

C-naphthalene treatments (Fig. 2a).

Quantification of the 13C content of cells, using SIMS, revealed a small subset of the community (2-4% of total cells) had assimilated large quantities of 13C from naphthalene (Fig. 2b). The total proportion of cells that were unenriched or strongly enriched in

13

C did not significantly differ


over time in the 24-hr ‘3-dose’

3-day ‘5-dose’ and 6-day ‘8-dose’ dosing regimes see

Methods section). The dominance of specialized naphthalene-degrading bacteria was evident in

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the decreased species richness, 329 versus 115 (t-test; t=63, p < 0 001 and Shannon’s di ersity 4.56 versus 3.23 (t-test; t=62.5, p < 0.001), in metagenomes prepared from 12C-control and 13Clabeled DNA, respectively (Fig. S2). Exposure to naphthalene strongly altered the structure of microbial communities in naphthalene-amended sediments (Fig. 3ab) and in the deep groundwater well (Fig. 3c), where natural contaminant levels of naphthalene were highest. Six bacterial species were deemed responsible for the mineralization and assimilation of

13

C

based on a greater than three-fold increased relative abundance of 16S rRNA gene fragments in metagenomes derived from 13C-labeled versus 12C-control DNA (Fig 3 & Fig. S3). Naphthalenedegrading taxa were classified based on full-length 16S rRNA genes, assembled from metagenomes,

to

Proteobacteria

Cupriavidus

and

Ralstonia

(Burkholderiaceae),

Methylobacterium, Sphingomonas, and Stenotrophomonas and the Actinobacteria Rhodococcus (Fig. 4). Their substantially increased relative abundance aided metagenome assembly and the recovery

of

near-complete

metagenome-assembled

genomes

(MAGs)

for

all

but

Methylobacterium (Table 2). Separate Cupriavidus and Ralstonia MAGs were not obtained due to similar tetranucleotide signatures, resulting in one large heterogenous ‘Bur holderiaceae’

MAG. The most closely related isolate genomes were Cupriavidus sp. HMR-1, Ralstonia metallidurans CH34, Rhodococcus erythropolis, Stenotrophomonas maltophilia D457 and Sphingomonas UNC305MFCol5.2 (Fig. S4). The community in sediments exposed to the shortest and lowest dose of unlabeled

naphthalene ‘12C 3-dose’ was distinct from all other sediment metagenomes Fig 3a abundant taxa in the

12

he most

C 3-dose metagenome belonged to Desulfobulbaceae, Denitratisoma,


Melioribacter, Syntrophus, and Planctomycetia (Fig. 4a). The functional gene content resembled that of upstream, shallow and mid groundwater metagenomes (Fig. 4b). Thus, the 12C 3-dose was

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considered to be representative of the pre-amendment surface sediment community. A total of 119 assembled full length 16S rRNA genes, primarily from upstream, shallow and mid groundwater metagenomes, were deposited in GenBank under accessions: MF942608MF942726. There was significant overlap in naphthalene-degrading bacteria in surface sediments and

groundwater and with prior sequencing libraries from Site 24. The same Rhodococcus and

Stenotrophomonas enriched in

13

C from naphthalene in surface sediments also dominated the

deep, anoxic groundwater metagenome. The clonality of these populations was evident in the identical full-length 16S rRNA genes (Fig. 4a) and in the near identical pairwise average nucleotide identities between corresponding MAGs (> 99.4%; Table S1). Two strains of benzene-degrading Rhodococcus previously isolated from the deep MW were identical across the full-length 16S rRNA gene ‘MA 2’ and ‘MA 4’; Posman et al., 2017). Similarly, near identical 16S rRNA gene clones for Stenotrophomonas (1261/1269 bases) and Ralstonia (1198/1201 bases) were present in an anaerobic SIP-benzene experiment from surface sediments (accessions: EU499720.1 & EU499693.1; Liou et al., 2008). Polaromonas and members of Desulfobacteraceae were previously identified as important

naphthalene-degrading taxa at Site 24, the former isolated from surface sediments (Jeon et al., 2004) and the latter found transcribing anaerobic catabolic genes, benzylsuccinate synthase (bssA), in groundwater (Yagi et al., 2010b). In the present study, a negligible proportion of

metagenomic reads mapped to the Polaromonas naphthalenivorans CJ2 genome (~ 0.07%) and no 16S rRNA gene fragments were classified to Polaromonas. However, 16S rRNA gene


fragments were assigned to an unclassified member of Comamonadaceae, the family to which Polaromonas belongs, was slightly increased in the

13

C-DNA pool from deep groundwater, but

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low in abundance (0.03% of total reads; Fig. S5). The relative abundance of Desulfobacteraceae was elevated in deep groundwater samples and were also low abundance (Fig. S5), and bssA genes were not encoded in any metagenome. The repeated dosing of naphthalene to surface sediments produced marked changes over the

6-day experiment. Microbial biomass increased with significantly higher DNA extracted from sediments treated to 8-doses compared to 3- and 5-doses (Table S2). The most contaminated, deep groundwater also had the highest microbial biomass of any groundwater sample (Table 1). In the shorter dosing periods, 3-dose (24-h) and 5-dose (3-day), the majority of 13C-labeled DNA belonged to Cupriavidus, Ralstonia and Sphingomonas. The rapid rise of early-stage taxa was evident in their increase from below, or marginally above, the sequencing depth in 12C 3-dose (0 - 0.04%) to between 4 – 15% of total reads in 13C 3-dose metagenomes. Methylobacterium were enriched within 24 hrs, but were most abundant after three days (5-dose). By the end of day six (8-dose), the 13C- DNA pool was dominated by Rhodococcus and Stenotrophomonas, leading to their designation as ‘late-stage’ taxa O erall the relati e abundance of bacterial and eu aryotic sequences increased in 13C-metagenomes over time, while archaeal sequences decreased. The expected transfer of

13

C from naphthalene-degrading bacterial populations to

bacterivorous protozoa did not produce strong or consistent trends in metagenomic data. Eukaryotes occupied a small proportion of the total DNA pool, with a median relative abundance of 1.2% of all SSU rRNA gene fragments (max = 8.3%; min = 0.03%) or 1.4% of all metagenome reads based on LCA classification (max = 3.0%; min = 0.6%). 18S rRNA gene fragments were 12-fold more abundant in

13

C-enriched samples relative to controls (Mann-


Whitney-Wilcoxon Test; p=0.03). This trend was driven by the predominance of 18S rRNA gene fragments classified to Chlorophyceae (Volvox) and Embryophyta (Chlamydomonas) in two of

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the 13C 8-dose samples (Fig. 5a). Removing these potential outliers reduced the effect to a 7-fold increase and reduced statistical support (p=0.08). Bacterivorous ciliates were found at highest relative abundances in

13

C 3-dose surface sediments and shallow and mid groundwater

metagenomes (Fig. S6). They were classified to Oligohymenophorea (Cyclidium), Armophorida (Metopus), Haptoria (Loxophyllum / Arcuospathidium) and Hypotrichia. Few eukaryotic genera were common across timepoints (Fig. 5c) or among groundwater samples (Fig. 5d). There are no known hydrocarbon-degrading protists (Stoeck and Edgcomb, 2010). The coincidence of Stenotrophomonas and Rhodococcus in deep groundwater and in the late-

stage of the SIP experiment, when oxygen had been depleted, indicated a potential competitive advantage under anoxic/suboxic conditions. Differences between early- and late-stage taxa were examined to identify the ecological and functional underpinning of potential trade-offs under oxic and suboxic conditions. Of the forty-two genes targeted for their involvement in anaerobic catabolism, thirty genes were present in the dataset, but mostly in low abundance (Fig. 6; Table S4). Genes encoding two key enzymes for the anaerobic degradation of naphthalene in sulfatereducing bacteria (naphthalene carboxylase and 2-naphthoate-coA ligase) were absent from all metagenomes. Genes involved in more general anaerobic degradation of aromatics were observed, such as phenylphosphate carboxylase, benzoate-CoA ligase and succinylbenzoate-CoA ligase, but their distribution in MAGs did not correspond with late-stage taxa (Fig. S7). The genetic basis for alternative redox coupling was explored in MAGs. Dissimilatory nitrate

(narG) and nitrite (nirB) reduction genes were present in the Burkholderiaceae MAG (Table 2) and were found in highest relative abundances in the 3-dose time-point (Fig. 7). Nitrate reduction


genes were absent in both Rhodococcus and Stenotrophomonas MAGs, though present in a close relative to the latter (S. maltophilia M30; Fig. S8). Dissimilatory sulfate reduction genes were

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absent from all naphthalene-affected metagenomes and MAGs (Fig. 7). Putative multi-haem ctype cytochromes involved in electron shuttling and dissimilatory metal reduction were also absent. All MAGs demonstrated the potential to aerobically catabolize aromatic compounds and

generate energy via oxidative phosphorylation. Each MAG encoded an A1 family terminal oxidase, while the Burkholderiaceae MAG also encoded a high-affinity cbb3-type terminal oxidase. Naphthalene dioxygenase genes were absent from all metagenomes except at very low abundances in shallow and mid groundwater samples (Fig. S9). The relative abundance of all dioxygenase encoding genes was greatest in the

13

C 3-dose samples (Fig. 6; Fig. S9), when

Ralstonia, Cupriavidus and Sphingomonas populations predominated. Yet, the greatest total

number of dioxygenase genes of any MAG occurred in Rhodococcus (Table 2). Two ring-

cleaving dioxygenases (2,3-dihydroxybiphenyl 1,2-dioxygenase and 3-hydroxyanthranilate 3,4dioxygenase) were exclusive to MAGs of late-stage taxa (Table 2) and were correspondingly most abundant in 13C 8-dose and deep metagenomes (Fig. 6). The presence of motility and chemotaxis genes was assessed to test whether other

ecophysiological differences might explain the succession of naphthalene degrading populations. Stenotrophomonas possessed the greatest set of capabilities, including flagellar and twitch motility, chemotaxis and biofilm formation (Table 2). All other MAGs possessed some form of motility except for Rhodococcus.


Discussion The combination of SIP and shotgun metagenomics was effective at identifying and

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recovering genomic information for active, in situ naphthalene-degrading organisms. Each of the six identified taxa had strong prior evidence for PAH and naphthalene catabolism, confirming that degrader populations were primarily targeted (Patel et al., 1982; Boonchan et al., 1998; Juhasz and Naidu, 2000; Stolz, 2009; Perez-Pantoja et al., 2012; Herbs et al., 2013; Mukherjee et al., 2014; Ventorino et al., 2014). The existence of relative few naphthalene-degrading taxa

would have simplified the challenge of tracking carbon from biomass by constraining the number of variables related to growth, physiology and ecology. Despite this advantage, we did not observe strong trends that support the transfer of substantial amounts of carbon from primary to secondary feeders over the six-day period. SIMS did not reveal any cross-feeding of the slight, but insignificant, increase in the relative abundance of eukaryotes in

13

13

C and

C-enriched

DNA was further evidence that C-transfer to higher trophic levels was negligible. This could have resulted from insufficient incubation time and/or sequencing depth, though similar experiments tracking

13

C from bicarbonate through bacteria to protozoa were able to detect

transfer within a 6-hr period (Moreno et al., 2010) and within five days using

13

C-labeled

benzene (Bastida et al., 2009). Despite the limited labeling of secondary-populations, temporal

changes were pronounced in active naphthalene-degrading bacterial populations. The rise of Ralstonia, Cupriavidus and Sphingomonas within 24 hrs of exposure to

naphthalene revealed the rapid stimulation of metabolic and reproductive activity from a relatively low concentration of naphthalene (< 25 ppm). The swift growth of Betaproteobacteria following PAH contamination in soils is well-documented, particularly for members of Comamonadaceae and Burkholderiaceae, like Ralstonia and Cupriavidus (Martin et al., 2012; Guazzaroni et al., 2013), and is consistent with the broader copiotrophic characteristics of that


Class of bacteria (Eilers et al., 2010; Morrissey et al., 2016). The succession of late-stage Rhodococcus and Stenotrophomonas in the six-day, 8-dose treatment indicated potential

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differences in the functional and ecological attributes of early- and late-stage taxa. Multiple factors could underlie the succession of naphthalene-degrading populations, such as

inherent differences in motility or growth rate, changes in the bioavailability of naphthalene with time (Madsen et al., 1996), and the accumulation or depletion of key chemical species by earlystage taxa, such as electron acceptors. Differences in growth rate among early- and late-stage taxa could be inferred from a previous study of deep groundwater at Site 24, where the absence of sequences from Rhodococcus isolates in Betaproteobacteria-dominated amplicon libraries corresponded with a shorter incubation period in sequencing versus culturing experiments (40 versus 7 days, respectively; Posman et al., 2017). In an unrelated study, Stenotrophomonas were highly abundant in undisturbed naphthalene-contaminated field soil, but absent in short-term naphthalene enrichments dominated by Comamonadaceae and Burkholderiaceae (Guazzaroni et al. 2013). The inherent difference in growth rate among early- and late-stage taxa is a plausible explanation for succession, yet may be confounded by co-occurring changes in oxygen availability. This was evident in a study of naphthalene-degrading enrichment cultures under oxygen-limitation, where Betaproteobacteria declined as conditions became anoxic and members of Nocardiaceae increased (Martirani-Von Abercron et al., 2017). The predominance of late-stage taxa in the most contaminated, oxygen-limited groundwater

indicates that adaptations to suboxic/anoxic conditions was a factor. The highest relative abundance of dioxygenases occurred at the earliest time-point (3-dose), suggesting a heightened potential for aerobic degradation. Yet, MAGs from both early- and late-stage taxa, namely Burkholderiaceae and Rhodococcus, contained several diverse ring-cleaving dioxygenases. To


date, no characterized strains of Rhodococcus have exhibited anaerobic growth (Goodfellow et al., 2014), suggesting Rhodococcus may, instead, outcompete early-stage taxa under

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microaerophilic conditions. In our study, a high-affinity 2,3-catechol dioxygenase (catE) gene was exclusive to the Rhodococcus MAG (82% identity) and is known to differentiate aromatic degraders under oxygen-limiting conditions (Kukor et al., 1996; Balcke et al., 2007; Táncsics et al., 2013). The dbd gene cluster, found in other low-oxygen adapted aromatic degrading bacteria, was absent from all MAGs (Hirano et al., 2006; Martirani-Von Abercron et al. 2017). The Burkholderiaceae MAG also possessed a high-affinity cbb3-type terminal oxidase, absent in Rhodococcus, suggesting competing potentials for activity at low oxygen concentration. The lack of evidence for pathways involved in the anaerobic degradation and the presence of several ring-cleaving dioxygenases supports the designation of Rhodococcus as microaerophilic

naphthalene degraders in suboxic/anoxic conditions. The Stenotrophomonas MAG also lacked genes involved in anaerobic catabolism, yet there

was considerable circumstantial evidence to support a putative role in the anaerobic degradation of naphthalene. The Stenotrophomonas MAG was, by far, the most abundant species where naphthalene concentrations were highest and oxygen lowest, accounting for 20% of all 16S rRNA gene fragments in deep groundwater and 8-dose samples (~ 2-fold more than Rhodococcus). The same Stenotrophomonas species was previously found to catabolize benzene under strict anaerobic conditions in a two-week incubation of surface sediments from Site 24 (Liou et al., 2008). Stenotrophomonas spp. are known to be facultatively anaerobic nitrate-

reducers (Ryan et al., 2009) and are frequently prominent members of anode-respiring communities in hydrocarbon degrading microbial fuel cells (Venkidusamy et al., 2016; Venkidusamy and Megharaj, 2016) and in electrochemically-stimulated remediation of


hydrocarbon contaminated ground water (Morris et al., 2009) and soil (Lu et al., 2014). The mechanism for electron shuttling to solid surfaces in Stenotrophomonas is not yet understood,

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but the capacity of Stenotrophomonas to reduce gold particles is indicative of a capacity for diverse redox coupling (Nangia et al., 2009). While the Stenotrophomonas MAG did not encode a nitrate reductase or homologs to c-type cytochromes common in model exo-electrogenic

bacteria (Shi et al., 2016), it did encode filamentous haemagglutinin which facilitates cell–cell

aggregation, biofilm production and surface attachment (Ryan et al., 2009; Gottig et al., 2009).

This collection of evidence raises the hypothesis that Stenotrophomonas respiration may be driven by minerology and surface interactinos, potentially explaining previously negative results for anaerobic microcosm experiments based on the addition of soluble forms of sulfate, nitrate, iron and manganese (Madsen et al., 1996; Bakermans et al., 2002; Yagi et al., 2010b). Based solely on our experimental evidence, we can conclude that Stenotrophomonas possess a strong

competitive advantage for the suboxic degradation of naphthalene, though lack an apparent genetic basis for anaerobic catabolism. The expectation that predation pressure would affect bacterial degrader populations was

weakly substantiated in our data. The most abundant members of the eukaryotic community were Chlamydomonas and Volvox, both are mixotrophic photosynthetic organisms with no reported bacterivore activity (Tittel et al., 2005; Forbes and Hammill, 2013). However, the relative abundance of bacterivorous ciliates was increased in early-stages of SIP. These included anaerobic bacterivores Cyclidium and Metopus, previously found on-site (Yagi et al., 2010b), and aerobic Loxophyllum and Hypotrichia Fenchel and Finlay 1990; Šime et al., 1994; Song et al., 2001; Wu et al., 2014). The capacity of Stenotrophomonas to resist ciliate predation with Reb proteins (Ryan et al., 2009), encoded in our MAG, offers one hypothesis for their late-stage


emergence contingent on predation exerting a strong selection pressure on other naphthalenedegrading populations. We conclude that the minor changes in ciliate abundances in early-stages

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of the incubation were likely insufficient to cause substantive changes in bacterial populations. Previously collected clone-library data from Site 24 provided useful context for our

analyses, yet high-throughput metagenomic sequencing significantly altered our understanding of naphthalene-degrading populations. Members of the genus Polaromonas were previously identified as the major naphthalene degraders on site (Jeon et al., 2003; Jeon et al., 2004), but were entirely absent, including their associated naphthalene dioxygenases, from the present dataset (Yagi et al., 2009). This difference highlights the potential long-term variability in functional populations during natural attenuation or the spatial-temporal variability in which copiotrophic, betaproteobacterial populations respond to naphthalene amendments. The diversity of dioxygenases present in MAGs indicated that horizontal gene transfer of naphthalene dioxygenase is less important than previously hypothesized (Herrick et al., 1997). The identification of Rhodococcus and Stenotrophomonas as naphthalene-degraders represents an important advancement in characterising the ongoing process of decontamination, given their abundances is deep groundwater where contamination has been slowest to attenuate (Yagi et al. 2010a). The recovery of identical strains of Rhodococcus and Stenotrophomonas from both groundwater and surface sediments demonstrated the contiguity of populations throughout the contaminated aquifer, indicating that future characterizations of their ecology and functional capabilities may be performed from a more accessible vantage. Our study highlighted the value of collecting time-dependent information during SIP

experiments. The temporal shifts we observed demonstrate that short incubations, commonly employed in SIP experiments to avoid cross-feeding, disproportionately favour the


characterization of fast-growing organisms. We suspect that concerns over cross-feeding may be overstated given the lack of 13C transfer to higher trophic levels in our experiment. SIMS offers

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an effective method to resolve the dynamics of cross-feeding in SIP experiment, particularly with advances in the use of higher resolution NanoSIMS technology in microbiology (Musat et al., 2016). The succession of naphthalene degraders revealed the rapid feedback from microbial activity in soil environments, which may broadly differ from aquatic environments that can be dominated by a single strain over long periods (Fuentes et al., 2016). To advance understanding of natural attenuation, we need a better understanding of the factors governing the maintenance or succession of naphthalene-degraders under fluctuating subsurface conditions. Broadening our knowledge of the subsurface environment and ecology will yield insights into undescribed taxa and unresolved metabolic potential which comprised the majority our metagenomic data.

Experimental Procedures Description of Groundwater Sample Collection and SIP Experiment In August of 2011 groundwater samples were ta en from an uncontaminated ‘upstream’

groundwater monitoring well (MW60B, 5.3 m depth) and three down-gradient contaminated wells at depths termed ‘shallow’ MW46 4 3 m ‘mid’ MW37 5 8 m and ‘deep’ MW36 6 7 m) for shotgun metagenomic sequencing (N43.293034, W73.603835). At each MW, twenty-five litres of groundwater were filtered through a set of five replicate 142 mm diameter 0.2-μm pore size filters (5 L per filter; Millipore Corp., Bedford, MA, USA) at a flow rate of 300 ml min-1 using a Geopump-2 (Geotech Environmental, Denver, CO, USA). Filters were stored in Whirlpak bags (NASCO, Modesto, CA, USA) in liquid nitrogen until long-term storage at 80°C. Oxygen was measured with a YSI Model 85 probe (YSI, Yellow Springs, OH, USA) with a sensitive range of 0 – 20 mg/L and accuracy of +/- 0.3 mg/L. Definitions of hypoxic (dissolved


oxygen < 2.0 mg/L) and anoxic (DO < 0.2 mg/L) were based on Hagy et al., (2004). Oxygen concentration in groundwater at Site 24 have been observed to fluctuate between hypoxic and

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anoxic (Yagi et al., 2010a). Cell counts, naphthalene concentrations and other geochemical data were measured from groundwater as previously described (Bakermans et al., 2002; Neuhauser et

al., 2009; Hanson and Madsen, 2015).

Description of Surface Sediment SIP Experiment On the same sampling expedition, a field-based SIP experiment was conducted with fully

13

C-labeled (99% atom 13C10, Cambridge Isotope Laboratories) and unlabeled (Fisher Scientific)

naphthalene at a surface seep, downstream from the monitoring wells, where contaminated groundwater emerges at the base of a hill (N43.291927, W73.601465). The separated treatments were created by inserting open-ended chambers (9 x 2.75 cm diameter sections of stainless steel pipe) into contaminated sediment to a depth such that a 10-ml headspace was retained (~ 5-cm deep; photos in Fig. S1). Oxygen concentrations were previously recorded at 4 ppm in surface sediments to a depth of 8 cm (Madsen et al., 1996). Solutions of

12

C- or

13

C-naphthalene-

saturated water (~24 ppm) were prepared according to the method outlined in Padmanabhan et

al., (2003) and dripped evenly across the surface of sediments according to three dosing regimes details in Supplementary Methods

he longest and highest dosing regime termed ‘8-dose’

consisted of daily 1-mL applications for five consecutive days, followed by three 1-mL doses on the sixth day (7 AM, 5 PM and 7 AM

he next regime ‘5-dose’ recei ed daily 1-mL doses for

two consecutive days followed by three doses on the third day. The lowest and shortest exposure, ‘3-dose ’ recei ed three 1-ml doses within a 24-hour period (7 AM, 5 PM and 7 AM). Immediately after the final dosing, chambers were sealed with rubber septa (SubaSeal No. 57, Sigma-Aldrich) and headspace gasses were sample at 0, 4 and 8 hrs hence. A volume of twoand-a-half millilitres of headspace was sampled and pressurized in evacuated 1.8 ml glass vials


(VWR International, Radnor, PA) secured with crimp-sealed septa (ThermoFisher Scientific, Pittsburgh, PA) and analyzed within 24 hrs using GC/MS. After gas sampling was completed,

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chambers were unsealed, and 0.1 g of sediment was aseptically removed from the upper 1 mm and preserved in 4% formaldehyde for SIMS single-cell imaging. Another 0.5 g of sediment was removed and frozen in liquid nitrogen and stored at -80°C for DNA extraction. Measuring the Respiration and Microbial Assimilation of 13C-Naphthalene Total respired

13

CO2 was measured by GC/MS as according to the procedures outlines in

(DeRito et al., 2005) with a complete description available in the Supplementary Methods. Background natural abundance

13

CO2 (measured at 1.11% v/v) was subtracted to calculate net

13

CO2 yield. Quantification of assimilated

13

C in individual cells was performed using SIMS

according to the procedures of Chandra et al. 2008

In brief a small drop ~2 μl of

formaldehyde-fixed sediment slurry was evenly spread on a polished sterile silicon wafer (Silicon Quest International, Santa Clara, CA, about 1 cm2 surface area), air-dried, heat-fixed

over a flame, and washed for 20 s in deionized distilled water, to reduce the salt deposits, and coated with a thin film of Au/Pd to enhance conductivity. A CAMECA IMS-3f SIMS ion microscope (CAMECA, France) was used to image cells and was capable of producing secondary ion images revealing isotopic gradients at 500 nm spatial resolution (Chandra et al.,

2000). The isotopic enrichment of a cell was quantified based on the negative secondary ion image ratio of

13

C14N (27 Da) to

12

C14N (26 Da) and converted to

13

C-content with a standard

curve generated from isotopically labeled Pseudomonas putida cells as previously described (Pumphrey et al., 2009). A complete description of SIMS methodology, including the standard

curve, is provided in the Supplementary Methods.


Shotgun Metagenomic Sequencing, Assembly and Genome Binning Total nucleic acids were extracted from groundwater filters and surface sediments according to the method of Griffiths et al. (2000), following one round of bead beating. For SIP samples,

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the

13

C-labeled DNA was separated from whole community DNA by cesium chloride density

gradient ultracentrifugation according to the method described by Neufeld et al. (2007), with the

following exceptions: a force of 146,000 g was applied for 48 hrs and the resulting gradient was collected in 0.4 ml fractions. Fractions with a density between 1.616 – 1.576 g/ml, measured with an AR200 digital refractometer (Reichert Analytical Instruments, Germany), were deemed 13

C-enriched DNA and combined for downstream processing. To enhance the separation of

DNA from

12

13

C-

C-DNA, the ultracentrifugation and purification steps were repeated. Parallel

incubations amended with natural abundance naphthalene (~1.11%

13

C) were identically

processed, yielding low concentrations of background and GC-rich DNA in the high density CsCl fractions (Youngblut and Buckley, 2014), which was sequenced to provide a baseline comparison or ‘12C-control ’ to

13

C-enriched DNA. Our method for recovering

13

C-enriched

DNA was validated using DNA prepared from P. naphthalenivorans str. CJ2 grown on unlabeled or 13C-labeled glucose (Supplementary Methods). Triplicate 13C-enriched and single 12C-control DNA samples for each dosing time-point were

sequenced by the Joint Genome Institute (Walnut Creek, CA) as part of the Community Science Program (proposal ID 44). Paired-end, 150-bp Illumina sequencing libraries were generated for all samples and raw data can be accessed via IMG/ER (Markowitz et al., 2009) or the European Sequencing Archive (Study: PRJEB22302) using accessions provided in Table S3. Sequences were quality filtered using fastX (v. 0.7; Gordon and Hannon, 2010) and Trimmomatic (v. 0.32; Bolger et al., 2014 and merged using FLASH

2; Magoč and Salzberg 2011 A targeted

assembly of full length 16S rRNA genes was performed using reago (v1.1; Yuan et al., 2015),


while the recovery of all partial 16S rRNA gene fragments was performed using infernal (v. 1 1 2; Nawroc i and Eddy 2013 Metagenomes were assembled using SPAdes with option ‘-

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meta’ flagged

3 10 1; Ban e ich et al., 2012). Individual assemblies were binned into

metagenome-assembled genomes (MAGs) based on tetranucleotide frequency and phylogenetic marker genes using myCC (Lin and Liao, 2016). MAGs were further improved via additional mapping, re-assembly and manual curation (Supplementary Methods). The quality and completeness of MAGs were assessed by QUAST (Gurevich et al., 2013) and checkM (Parks et al., 2015). Bioinformatic and Statistical Analyses Taxonomic classifications of small subunit ribosomal gene fragments were performed using

the RDP Classifier (Wang et al., 2007) using either the Greengenes database (gg_13_8_99) or SILVA database (silva.nr_v128) for partial 16S or 18S rRNA genes, respectively. Maximum parsimony phylogenetic trees were prepared with full length 16S rRNA genes by placing sequences

into

the

pre-aligned

‘SSURef_NR99_123_SILVA_12_07_15’

SILVA with ARB

tree

(Pruesse

et

al.,

2007;

Westram et al., 2011). Pairwise

comparisons of the average nucleotide identity of all MAGs were calculated using Mummer (Kurtz et al., 2004) with pyani (Pritchard et al., 2016). The functional content and genomic

structure of MAGs were explored in KBase (Arkin et al., 2016) using annotations from RAST (Aziz et al., 2008). The phylogeny of MAGs was determined by multi-locus sequence alignment and reconstructed using the maximum likelihood method in KBase. Average rRNA gene copy numbers for each MAG was determined using genus-level information in rrnDB (Stoddard et al., 2015). Annotations of KEGG Orthology (KO) were performed by the JGI on the unassembled read sets and with ghostKOALA for MAGs (Kanehisa et al., 2016). The relative abundances of 104 dioxygenases, toluene monooxygenases and 42 genes reportedly involved in anaerobic


degradation of aromatic compounds were determined based on KO numbers (Table S4; Tan et al., 2015). The presence of genes involved in biofilm formation, motility, and electron shuttling

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were also assessed (Supplementary Methods). All functional genes and phylogenetic gene markers from previous research at Site 24 were recovered using accessions for surface sediment studies: Jeon et al. (2003) and Liou et al. (2008), and groundwater studies: Yagi et al. (2009), Yagi et al. (2010a), and Posman et al. (2017). Statistics were performed using R v. 3.3.1 (R Core Team, 2016) with general dependency on

the following packages: reshape2, ggplot2, plyr (Wickham, 2007; Wickham, 2009; Wickham, 2011), Hmisc (Harrell and Dupont, 2015) and phyloseq (McMurdie and Holmes, 2014). Complete lin age hierarchical clustering was performed with ‘hclust’ based on Bray-Curtis

dissimilarities calculated using ‘ egdist’ from the R-pac age ‘ egan’ (Oksanen et al., 2015). All analyses can be reproduced using R scripts and data available at https://github.com/RoliWilhelm/Wilhelm_SIP_Naphthalene.

Acknowledgments The authors acknowledge the assistance provided by Dr. James P. Shapleigh in revising this

manuscript. This research was supported by funding from an O.P.U.S. grant from the National Science Foundation and with DNA sequencing support from the U.S. Department of Energy Joint Genome Institute. Work by S. Chandra and the Cornell SIMS Laboratory was supported by a NIH grant 5RO1 CA129326. Dr. Eugene Madsen died on August 9th, 2017 and his co-authors would like to acknowledge his efforts in sustaining the long-term research program at Site 24 as well as his pioneering contributions to the field of en ironmental microbiology Dr Madsen’s passionate curiosity and exemplary character were an inspiration to all.


Author Contributions R. Wilhelm performed data analysis and writing. B. Hanson performed all field work and

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experiments with assistance from E. Madsen. S. Chandra performed all single-cell SIMS imaging analyses. E. Madsen guided all research efforts, but was not present to approve the final version of this manuscript.


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Table Legends

Accepted Article

Table 1. Geochemistry and total cell counts for groundwater samples collected from monitoring wells outside ‘upstream’ and inside the plume of coal-tar contamination. Representative geochemical data for surface sediment were obtained from Madsen et al. (1992) and Madsen et al. 1996 Abbre iations for total organic carbon OC total inorganic carbon IC ‘below detection’ BD and ‘not determined ND ha e been used Values contained in brac ets represent the standard error based on triplicate measurements. Chemicals denoted by superscripta are reported from Bakermans et al. (2002). Table 2. Information on the quality and composition of four metagenome-assembled genomes (MAGs) and the complete genome of Polaromonas naphthalenivorans str. CJ2 isolated from surface sediments at Site 24 (Jeon et al., 2004). The designation of motility and chemotaxis was based on sets of multiple genes (see Supplementary Methods). All other functional annotations were based on the presence (+) or absence (-) of genes in either RAST (KBase) or KEGG (ghostKOALA) annotations. The quality and completeness of MAGs were assessed with checkM. The rRNA gene copy number presented was based on the average for either the genus or family in rrnDB. The alpha-beta-bond-cleaving (*), ring-hydroxylating (§) and ring-cleaving ‡ acti ity of dioxygenases has been denoted


Figure Legends

Accepted Article

Figure 1. A map of the study site indicating the location of the uncontaminated upstream groundwater sampling well and neighbouring contaminated sampling wells located in the contaminant plume. The location of the downstream and downslope surface seep, where the SIPexperiment was conducted, is also shown (photos available in Fig. S1). Figure 2. The activity of naphthalene-degrading populations displayed as (a) the net 13C-CO2 respired from 13C-naphthalene-amended sediments and (b) the proportion of cells (y-axis) that contained varying degrees of 13C-label (x-axis) according to SIMS imaging of cells recovered from 13C-naphthalene-amended sediments. Doses correspond to a 24-hr incubation treated with three 1-mL doses of naphthalene ('3-dose'); a three-day incubation treated with a total of 5 doses ('5-dose'); and a six-day incubation treated with a total of 8-doses ('8-dose'). Doses were comprised of either 13C-labeled naphthalene or unlabeled naphthalene (i.e. natural abundance 13 C In b ‘off-scale’ denotes levels of enrichment that saturated the detector during exposures needed to detect low level 13C-enrichment. Each sample was measured in triplicate using independent field replicates. Error bars correspond to the standard error of the mean. Figure 3. The average relative abundance of 16S rRNA gene fragments classified at (a) the phylum and (b & c) family level, demonstrating differences in community composition in metagenomes from 12C-control and 13C-labeled naphthalene-amended sediments and among groundwater samples. Classification were provided at the family level to manage the number of taxonomic categories displayed and only taxa that occupied greater than 2.5% of total 16S rRNA gene fragments were shown. Consult Figure S3 for a genus level breakdown of naphthalenedegrading taxa. Triplicate metagenomes were sequenced for 13C-naphthalene amended samples, while 12C-controls and subsurface metagenomes are represented by a single sequencing library.

Figure 4. Community structure and function of samples according to (a) the phylogenetic relatedness of full-length 16S rRNA genes assembled from metagenomes and (b) the hierarchical clustering of functional gene profiles (KO annotations), of unassembled read sets, using BrayCurtis dissimilarity. Full-length 16S rRNA genes from upstream, shallow and mid samples were not included due to the extensive number and diversity of those sequences. Labels were coloured to emphasize the difference between 12C 3-dose (red) and 13C libraries (blue), with later 12C doses 5- and 8- shaded purple to reflect the similarity with 13C libraries. Figure 5. The average relative abundance of 18S rRNA gene fragments classified at the taxonomic rank of Class from (a) naphthalene-amended sediment and (b) groundwater metagenomes. The total overlap of genera among (c) dosing treatments and (d) groundwater metagenomes are inset. Figure 6. A heatmap of the average relative abundance of dioxygenase and anaerobic degradation genes present in unassembled metagenomes. Genes with fewer than a total of 60 counts per million reads across all samples were removed. Samples were clustered based on the


Bray-Curtis dissimilarity of functional gene content (KO annotations) prior to filtering low abundance genes. Squares were blacked out when a gene was not detected. The full list of dioxygenase and anaerobic degradation genes queried are presented in Table S4 and the full dataset of all gene annotations is available in the Supplementary Data package.

Accepted Article

Figure 7. The average relative abundance of nitrate (narG), nitrite (nirB) and sulfate (dsrA) reductases among metagenomes. Error bars correspond to one standard error of the mean and are absent when only one replicate metagenome contained the genes. Dissimilatory nitrate reductase gene, napA, exhibited a similar relative abundance pattern as narG, while dissimilatory nitrite reductase, nrfA, was only detected at low abundance in mid and shallow metagenomes.


Supplementary Table Legends

Accepted Article

Table S1. The pairwise average nucleotide identity among metagenome-assembled genomes recovered from individual metagenomes, demonstrating high degree of similarity between populations from subsurface groundwater and surface sediments. Representative MAGs were not recovered from every metagenome. Table S2. A comparison of the total DNA extracted from surface sediment samples. Table S3. Data accessions for all raw and assembled metagenomic reads, polished metagenomeassembled genomes and full-length 16S rRNA genes reconstructed from metagenomic data. Raw and assembled metagenome data was uploaded via the European Nucleotide Archive and 16S rRNA genes to GenBank (NCBI). Table S4. A complete list of genes targeted in characterizing aerobic (dioxygenase) and anaerobic catabolism of naphthalene along with those involved in motility and chemotaxis.


Supplementary Figure Legends

Accepted Article

Figure S1. Photographs taken at a groundwater outflow showing (a) the appearance of PAHcontaminated sediments and (b and c) how experimental chambers were established for gas sampling. The image in (c) is provided to illustrate how the containers were sealed, but was taken at an unrelated field site. Figure S2. Shannon diversity estimates of bacterial diversity of l 16S rRNA gene fragments from (a) surface sediments and (b) groundwater wells. Two hundred estimates were calculated for each sample based on rarefication without replacement. Figure S3. The relative abundance of 16S rRNA gene fragments classified to all six naphthalene-degrading bacterial taxa, as well as all reads assigned to the family Comamonadaceae to which the genus Polaromonas belongs. Figure S4. A maximum likelihood phylogeny tree of MAGs and all available genomes within each genus based on a multi-locus sequence alignment produced in KBase.

Figure S5. Relative abundances of unclassified Comamonadaceae and Desulfobacteraceae, predominantly related to Desulfobacca, in 16S rRNA gene fragment libraries from groundwater well samples.

Figure S6. The relative abundances of all observed genera within Intramacronucleata, a subphylum of ciliates, based on 18S rRNA gene fragment libraries from (a) surface sediment and (b) groundwater well metagenomes. Figure S7. The relative abundances of three predominant genes involved in anaerobic catabolism of aromatic compounds in unassembled read sets. Error bars correspond to one standard error of the mean and are absent when only present in one replicate metagenome. Figure S8. Gene synteny in the Stenotrophomonas MAG and the genome of its closest relative, S. maltophilia M30, demonstrating the absence of a nitrate reduction gene cluster. The contigs presented are ‘Stenotrophomonas_SIP_Madsen’ and ‘Ga0055024_1115 ’ respecti ely Figure S9. The relative abundances of (a) all dioxygenases, (b) naphthalene dioxygenase and (c) biphenyl-2,3-diol 1,2-dioxygenase in unassembled read sets. Statistically significant differences using TukeyHSD were marked by lettering (p < 0.05). Error bars correspond to one standard error of the mean and are absent when only present in one replicate metagenome.


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Accepted Article

Naphthalene (mg/L) TOC (mg/L) TIC (mg/L) O2 (mg/L) NH4+ (mg/L) NO3- (mg/L) S2- (mg/L)a SO2 (mg/L)a Fe (mg/L)a Fe3+ (mg/L)a H2 (nM)a Methane (mg/L) pH Salinity (ppt) Alkalinity Temperature (°C) Cells (per L)

MW60B

MW46

MW37

‘upstream’ BD 1.6 (0.45) 22.4 (1.2) 2.79 0.15 (0.00) 0.1 (0.00) 0.004 (0.002) 43 (11) BD 0.03 (0.01) BD 2.2 (3.8) 5.7 0.2 0.61 10.5

'shallow' 0.002 1.4 (0.03) 28.4 (0.3) 1.33 0.06 (0.02) 0.05 (0.07) BD 5.8 0.2 0.85 11.4

‘mid’ 0.002 1.5 (0.06) 28.8 (0.2) 0.17 0.04 (0.02) BD 38 (9.7) 5.8 0.2 0.81 ND

0.2 1.7 (0.27) 31.8 (1.2) 0.14 0.12 (0.02) BD 0.028 (0.006) 43 (14) BD 0.04 (0.02) >30 57 (18) 5.9 0.3 0.9 10.7

7

7

7

7

3.5 x 10

3.7 x 10

MW36

3.6 x 10


Surface Sediment

‘deep’

7.1 x 10

1.0 4.0 15 - 26 5.0 x 109

Accepted Article

Table 2 Burkholderiaceae Taxonomy Total Length (bp) N50 Median Read Depth GC (%) Number of Contigs Contigs > 10,000 bp Genome Contigs > 500,000 bp Statistics Largest Contig Estimated Contamination (%) Estimated Completeness (%) Predicted Genes Average Read Coverage Ribosomal Copy Number* Total # of Mobile elements (Counts per million bp) Flagellar motility Twitch motility Motility / Chemotaxis Morphology Biofilm adhesin production Filamentous haemagglutinin Benzoate-CoA-ligase Anaerobic Benzoyl-CoA reductase Aromatic 4-carboxymuconolactone decarboxylase Degradation Succinylbenzoate CoA ligase Genes Benzylsuccinate synthase Lignostilbene-alpha,beta-dioxygenase* Catechol 1,2-dioxygenase‡ 2,3-dihydroxybiphenyl 1,2-dioxygenase‡ 3-hydroxyanthranilate:oxygen 3,4-oxidoreductase‡ Homogentisate 1,2-dioxygenase‡ Aerobic Extradiol ring-cleavage dioxygenase‡ Aromatic Intradiol ring-cleavage dioxygenase‡ Degradation 3,4-dihydroxyphenylacetate 2,3-dioxygenase‡ Genes Gentisate 1,2-dioxygenase‡ 4-hydroxyphenylpyruvate dioxygenase§ 3-phenylpropionate dioxygenase§ Naphthalene dioxygenase§ Other putative dioxygenases Nitrate reductase (narG) Alternative Nitrite reductase (nirB) TEAs Sulfate reductase (dsrA)

Cupriavidus/Ralstonia 77,51,494 2,14,970 266 63.33 77 63 37 4,20,138 65.1 98.3 7,529 286x 4.1 43 (5.5) + + + + + + + + + + + 2 + + -


Table 2

Accepted Article

Sphingomonas Sphingomonas 44,73,753 46,778 136 67.42 174 105 24 1,89,486 1.83 97.06 4,457 146x 2.2 18 (4.0) + + + + + + 1 -

Stenotrophomonas Stenotrophomonas 55,81,712 49,37,700 408 65.82 19 10 5 49,37,700 14.2 97.8 5,251 388x 3.7 11 (2.0) + + + + + + + + + + 0 -

Rhodococcus Rhodococcus 73,60,966 64,16,447 155 62.31 44 28 8 64,16,447 3.52 99.8 7,253 171x 4.2 33 (4.5) + + + + + + + + + 4 + -

P. naphthalenivorans CJ2 Polarmonas 53,66,140 44,10,291 12 61.66 9 9 NA 53,66,140 0 100 4,879 ~ 2x 1.5 65 (12.1) + + + + + + + + + + + 1 + + -


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