Unexpected Dominance of Elusive Acidobacteria in Early Industrial Soft Coal Slags

13 Pages·2017·2.82 MB·English

by  Werner Liesack

ORIGINALRESEARCH
published:08June2017
doi:10.3389/fmicb.2017.01023
Unexpected Dominance of Elusive
Acidobacteria in Early Industrial Soft
Coal Slags
Carl-EricWegner1,2andWernerLiesack1*
1DepartmentofBiogeochemistry,MaxPlanckInstituteforTerrestrialMicrobiology,Marburg,Germany,2Aquatic
Geomicrobiology,InstituteofEcology,FriedrichSchillerUniversityJena,Jena,Germany
Acid mine drainage (AMD) and mine tailing environments are well-characterized
ecosystems known to be dominated by organisms involved in iron- and sulfur-cycling.
Here we examined the microbiology of industrial soft coal slags that originate from
alumleaching,anecosystemdistantlyrelatedtoAMDenvironments.Ourstudyinvolved
geochemical analyses, bacterial community profiling, and shotgun metagenomics. The
slags still contained high amounts of alum constituents (aluminum, sulfur), which
mediated direct and indirect effects on bacterial community structure. Bacterial groups
typicallyfoundinAMDsystemsandminetailingswerenotpresent.Instead,thesoftcoal
slagsweredominatedbyuncharacterizedgroupsofAcidobacteria(DA052[subdivision
2],KF-JG30-18[subdivision13]),Actinobacteria(TM214),Alphaproteobacteria(DA111),
andChloroflexi(JG37-AG-4),whichhavepreviouslybeendetectedprimarilyinpeatlands
Editedby: and uranium waste piles. Shotgun metagenomics allowed us to reconstruct 13
EtienneYergeau,
high-qualityAcidobacteriadraftgenomes,ofwhichtwogenomescouldbedirectlylinked
InstitutNationaldelaRecherche
Scientifique,Canada to dominating groups (DA052, KF-JG30-18) by recovered 16S rRNA gene sequences.
Reviewedby: Comparative genomics revealed broad carbon utilization capabilities for these two
JulienTremblay,
groups of elusive Acidobacteria, including polysaccharide breakdown (cellulose, xylan)
NationalResearchCouncilCanada
(NRC-CNRC),Canada andthecompetencetometabolizeC1compounds(ribulosemonophosphatepathway)
Nils-KaareBirkeland, and lignin derivatives (dye-decolorizing peroxidases). Equipped with a broad range of
UniversityofBergen,Norway
efflux systems for metal cations and xenobiotics, DA052 and KF-JG30-18 may have a
*Correspondence:
competitiveadvantageoverotherbacterialgroupsinthisuniquehabitat.
WernerLiesack
[email protected]
Keywords:softcoal,slags,mineralleaching,acidminedrainage,microbiome,acidobacteria,metagenomics,
microbialdarkmatter
Specialtysection:
Thisarticlewassubmittedto
TerrestrialMicrobiology, 1. INTRODUCTION
asectionofthejournal
FrontiersinMicrobiology
Mining frequently produces enormous amounts of tailings, which often pose the danger of
Received:15February2017 generatingacidminedrainage(AMD)(Rohwerderetal.,2003).AMDisaconsequenceofsulfur-
Accepted:22May2017
andiron-richminerals,primarilypyrite(FeS ),beingexposedtooxygenandhumidity(Druschel
2
Published:08June2017
etal.,1999).Pyriteoxidationleadstotheformationofsulfuricacidandferrousiron.Sulfuricacid
Citation: lowersthepHandferrousironisfurtheroxidizedyieldingferriciron.Thegraduallydecreasing
WegnerC-EandLiesackW(2017)
pH increases the solubility of ferric iron, which is a stronger oxidant of pyrite than oxygen
UnexpectedDominanceofElusive
(Druschel et al., 1999). Microbial activity accelerates AMD generation tremendously. Microbial
AcidobacteriainEarlyIndustrialSoft
CoalSlags.Front.Microbiol.8:1023. iron and sulfur oxidation are orders of magnitude faster than the abiotic oxidation of these
doi:10.3389/fmicb.2017.01023 two elements. The microbial activity easily leads to a release of acidic and metal-contaminated
FrontiersinMicrobiology|www.frontiersin.org 1 June2017|Volume8|Article1023
WegnerandLiesack ElusiveAcidobacteriainSoftCoalSlags
discharges.EarlystudiestargetingAMDmicrobialcommunities 2. MATERIALS AND METHODS
revealed the prevalence of a few taxonomic groups, including
2.1. Sampling
Leptospirillum and Acidithiobacillus among the bacteria and
Detailedinformationaboutthesamplingsite,samplingandalum
Ferroplasma and other Thermoplasma-related groups (A-, E-,
leaching from soft coal can be found in the Supplementary
and G-Plasma) within the archaea (Baker and Banfield, 2003).
Methods and Supplementary Figure 1. Three individual sites
Metagenomic (Tyson et al., 2004; Dick et al., 2009; Yelton
were sampled at the slag deposit “Red Hill” (abbreviated:
et al., 2011, 2013), metatranscriptomic (Lehembre et al., 2013;
RH1 [50◦ 44′21.45′′N, 7◦ 10′27.87′′E], RH2 [50◦ 44′21.85′′N,
Hua et al., 2015; Chen et al., 2015) and metaproteomic (Denef
7◦10′27.78′′E], RH3 [50◦ 44′22.23′′N, 7◦ 10′27.46′′E]) in
et al., 2010; Mueller et al., 2011) studies showed that iron- and
September 2014. After removing a cover layer (approx. 5 cm)
sulfur-cycling are the dominating microbial activities in AMD
of slowly humifying biomass, the underlying slag was collected
environments. Most AMD studies focused either on tailings as
in1Lplasticcontainers.Twoadditionalsitesweresampledfor
sourcematerial(RadevaandSelenska-Pobell,2004;Senkoetal.,
reference and comparison: Sediment of a nearby pond, which
2008; Urbanová et al., 2011; Korehi et al., 2014) or on AMD
collects the drainage water of slag deposits upon rain events
dischargesastheendproduct(BakerandBanfield,2003;Tyson
(abbreviated:RHP[50◦ 44′22.32′′N,7◦ 10′28.32′′E])andnearby
etal.,2004;Dicketal.,2009;Xieetal.,2011;Kuangetal.,2013).
undisturbed forest soil (abbreviated: Ref [50◦ 43′49.63′′N, 7◦
ThemostcommonsourcesofAMDareminetailings,andiron
10′30.44′′E). Three replicate samples were collected within a
andcoaldeposits.
4m2 areasurroundingeachindividualsamplingsite.Replicates
Though coal is nowadays almost exclusively mined for its
were pooled to obtain composite samples that were aliquoted
use as fossil fuel, mineral-rich coal is mined to win scarce and
andsubjectedtoindividualanalyses.Geochemicalanalyseswere
precious minerals such as germanium (Arroyo and Fernández-
carriedoutasdescribedintheSupplementaryMethods.
Pereira, 2008). In the nineteenth century, sulfur mineral-rich
soft coal was mined in Western Germany, in the surroundings
of Bonn, to extract these minerals by a smelting-like process 2.2. DNA Extraction
(Supplementary Figure 1). Among these minerals, especially DNA was extracted using the FastDNA(cid:13)R SPIN kit for soil
alum (KAl(SO ) ×12H O) was commonly used as mordant (MP Biomedicals, Eschwege, Germany). Quality and integrity
4 2 2
and precipitant in early textile and paper industry. Alum of the DNA was assessed by agarose gel electrophoresis.
was extracted from soft coal in a four-step process. Soft coal DNAconcentrationsweredeterminedfluorometrically.Extracts
was mined, sheared and subsequently smoldered to enrich were aliquoted and stored at −20◦ C for downstream
present alum. The resulting ash (one third of the original analyses, including qPCR, amplicon sequencing and shotgun
coal, alum content 15%) was leached and the leachate boiled metagenomics.
to yield crystalline alum. Overall, alum leaching was a low-
yield process. One ton of coal had to be processed to win 2.3. 16S rRNA Gene-Targeted qPCR
50 kg of crystalline alum, thereby leading to 300–400 kg
Bacterial16SrRNAgenecopiesweredeterminedbySybrGreen-
of leached slag as by-product. At peak year level, 500,000
based quantitative PCR (Stubner, 2004). Standard curves were
tons of coal were mined, producing 1,250 tons of alum and
generated using genomic DNA from E. coli (calibration range:
150,000–200,000 tons of slag. In the middle of the nineteenth 10–106 copies).QuantitativePCRwascarriedoutusingaCFX
century, the alum extraction plants and soft coal mines in the
Connect Real-Time PCR detection system (Bio-Rad, Munich,
surroundings of Bonn were the biggest of their kind in former Germany).ThePCRefficiencywasatleast85%(R2≥0.99).Melt
Prussia. Given a mining period of roughly 70 years (1805–
curveanalyseswerecarriedouttocheckforunspecificproducts.
1875), tremendous amounts of leached slag were produced
and dumped into the environment without precautions and
2.4. Amplicon Library Preparation and
remediation.
Here we investigated the microbiology of soft coal slag Sequencing
deposits. Given their industrial history and leached nature, PCR amplicons for subsequent amplicon library preparation
we hypothesized that the slags represent an endpoint AMD weregeneratedusingabacteria-specific(341f[S-D-Bact-0341-b-
environment that is characterized by a unique bacterial S-17,5′-CCTACGGGNGGCWGCAG-3′]/805r[S-D-Bact-0785-
community composition. In particular, we were interested a-A-21, 5′-GACTACHVGGGTATCTAATCC-3′]) primer pair
in exploring to which extent the remaining alum and other (Klindworth et al., 2013). The PCR assay is further described
metals affect the genome coding potential of the slag- in the Supplementary Methods. PCR products were checked
deposit-inhabiting microbial communities. An additional by agarose gel electrophoresis, gel-excised and purified using
challenge faced by these communities is the recalcitrant the Wizard(cid:13)R SV Gel and PCR clean-up system (Promega,
nature of their carbon sources. Our research involved a Mannheim, Germany). Library preparation was done using the
thorough geochemical analysis, bacterial community profiling, NEBNext(cid:13)R Ultra DNA Library Prep Kit for Illumina(cid:13)R (New
and shotgun metagenomics. In addition to samples from EnglandBioLabs,Frankfurt/Main,Germany),withafinallibrary
three slag deposit sites, sediment from a drainage pond size of 550 bp. The size distribution was checked by running
and nearby undisturbed forest soil were examined for DNA 12K chips on a Bio-Rad Experion(cid:13)R (Bio-Rad, Munich,
comparison. Germany). Sequencing was carried out at the Max Planck
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WegnerandLiesack ElusiveAcidobacteriainSoftCoalSlags
GenomeCentreCologneusinganIllumina(cid:13)R MiSeqplatformin Figure 4). The procedure is described in detail in the
paired-endmode(2×300bp). Supplementary Methods. MAGs were examined for
completeness and possible sequence contamination by
2.5. Shotgun Metagenome Library
CHECKM (v. 1.0.3) (Parks et al., 2015). MAGs more complete
Preparation and Sequencing than 85% were considered high-quality draft genomes, while
Extracts of total DNA were sheared to a fragment size of those more complete than 70% were considered good-quality
approximately200–250bpusingaBioruptor(cid:13)R Plus(Diagenode, draft genomes. The categorization was based on the presence
Denville, New Jersey, USA). Shearing involved 15 cycles of of lineage-specific single-copy marker genes and a sequence
30 s/30 s (on/off) at high intensity. Library preparation was contamination of less than 10%. High- and good-quality draft
done as described above. Size selection was modified to yield a genomesweresubjectedtogenomeannotation.MAGsoflesser
final library size of approximately 250–300 bp. Libraries were qualitywereprocessedtodeterminetheirtaxonomicaffiliation.
sequencedusinganIllumina(cid:13)R HiSeq2500deviceinpaired-end Genome annotations were done using PROKKA (v. 1.11)
mode(2×100bp)attheMaxPlanckGenomeCentreCologne. (Seemann,2014)andRAST(v.2.0)(Azizetal.,2008;Overbeek
etal.,2014)andvalidatedagainsteachother.Annotatedgenomes
2.6. Amplicon Data Processing
were analyzed using ARTEMIS (v. 16.0) (Carver et al., 2012).
Amplicon data were quality-controlled and pre-processed as Gene predictions were examined by parsing the output from
described in the Supplementary Methods. OTUs (operational UBLASTsearchesinMEGAN(v.5)(Husonetal.,2011)andby
taxonomicunits)wereclusteredusingUSEARCH(v.7.0)(Edgar, generatingKEGG(KyotoEncyclopediaofGenomesandGenes)
2010) and a sequence identity threshold of 97%. A compatible annotations by subjecting coding genes to GHOSTKOALA
OTU table was assembled to facilitate amplicon data analysis (v. 2.0) (Kanehisa et al., 2015). The phylogenetic affiliation of
usingQIIME(v.1.8)(Caporasoetal.,2010)andPHYLOSEQ(v. recoveredMAGswasdeterminedbyidentified16SrRNAgenes
1.10)(McMurdieandHolmes,2013). andbyconcatenatedalignmentsof31single-copymarkergenes
using AMPHORA (v. 2.0) (Wu and Scott, 2012). A maximum-
2.7. Metagenome Data Processing and
likelihoodtreewascalculatedusingFASTTREE(v.2.1.3)(Price
Computational Analysis
etal.,2010)andtheWAGmodel(WhelanandGoldman,2001).
Quality control of metagenome reads was done as described Nearest neighbor interchange was applied for optimizing tree
for amplicon datasets (Supplementary Methods), except that topology and the CAT approximation was used to account for
initial paired-end assembly was omitted, and reads were not evolutionary rate heterogeneity (Stamatakis, 2014). Genes of
filtered based on expected errors but truncated from the first interest (e.g., genes encoding Czc efflux pumps) were extracted
position showing a quality score below Q20. The taxonomic from the genomes (nucleotide sequences and deduced amino
affiliation of quality-controlled reads prior to metagenome acidsequences)andqueriedagainstInterpro(v.62.0),Pfam(v.
assemblywasdeterminedusingKRAKEN(v.0.10.5)(Woodand 30.0)andTigrfam(v.15.0)formoredetailedanalyses(Haftetal.,
Salzberg, 2014) and UBLAST (an optimized BLAST (Altschul 2003;Mitchelletal.,2015;Finnetal.,2016).
et al., 1997) algorithm implemented in USEARCH) searches
againstNCBINR(e-value1e−3)(Pruittetal.,2012).Full-length 2.9. Statistical Analyses
16S rRNA gene sequences were reconstructed using EMIRGE Statistical analyses were carried out using the R software
(Miller et al., 2011) and matched against the SILVA database framework(v.3.2.2)(RDevelopmentCoreTeam,2008)andthe
(v. 123) (Quast et al., 2013) using ARB (v. 6.0.2) (Ludwig packagesAPE(v.3.3)(Paradisetal.,2004),METAGENOMESEQ
et al., 2004). The nearly complete 16S rRNA gene sequences (v. 1.13.02) (Paulson et al., 2016), PHYLOSEQ (v. 1.10)
allowed us to validate sufficient sequencing depth by detecting (McMurdieandHolmes,2013),andVEGAN(v.2.3.1)(Oksanen
abundant groups previously identified by community profiling etal.,2016),includingtheirrespectivedependencies.Individual
(Supplementary Figures 2, 3). Metagenome assembly was done analyses are described in more detail in the Supplementary
with MEGAHIT (v. 1.0.1) (Li et al., 2014). After examining a Methods.
variety of different k-mer lengths, a minimum k-mer length of
33 and a maximum k-mer length of 73 were applied with 10- 2.10. Figure Generation
meriterations.Contigsgreaterthan1000bpweretaxonomically FiguresweregeneratedusingtheRpackagesGGPLOT2(http://
binned using PHYLOPYTHIA S+ (Gregor et al., 2016). This ggplot2.org) (Wickham, 2009), as well as the python module
involved the analysis of marker genes and k-mer profiles. The MATPLOTLIB(http://matplotlib.org/)(Hunter,2007).
coverage of individual contigs was calculated using BBMAP (v.
35.02) (http://sourceforge.net/projects/bbmap/). Coding genes 2.11. Sequence Data Deposition
were predicted using PRODIGAL (v. 2.6.2) (Hyatt et al., Amplicon sequencing and shotgun metagenomics data,
2012). and genome bins of interest were deposited at NCBI SRA
and NCBI Genomes and are accessible under the following
2.8. Recovery of Metagenome-Assembled
BioProject/Genome accessions: PRJNA384238 (amplicon
Genomes sequencing data), PRJNA384362 (shotgun metagenomics
A two-step binning strategy was applied for the recovery of data), NFUN00000000 (RH2 MAG17b), NFUO00000000 (RH1
metagenome-assembled genomes (MAGs) (Supplementary MAG20).
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WegnerandLiesack ElusiveAcidobacteriainSoftCoalSlags
3. RESULTS previouslyrecoveredfromacidicforestsoil.DA052,KF-JG30-18
andTM214alsoshowedacloseaffiliationtounculturedbacteria
3.1. Bacterial Community Profiling
detected in volcanically impacted soil systems and, in addition,
Both 16S rRNA gene-targeted quantitative PCR and DNA
TM214 to populations in iron mineral-rich environments.
extraction efficiency showed that the recovery of microbial
UnculturedrelativesofDA052,DA111,andJG37-AG-4werealso
biomass was 10-fold lower for slag samples RH1-3 (2.20 −
detectedinmetal-contaminatedsites(SupplementaryTable2).
2.89×108 copies/gdryweight[DW]soil;4.45–8.24µgDNA/g
DW) than for the undisturbed forest soil (Ref: 2.21 × 109
copies/gDW,45.18µgDNA/gDW).Thecorrespondingvalues 3.2. Geochemical Characteristics
for RHP were as follows: 1.51 × 108 copies/g DW, 22.53 µg ThedifferencesinbacterialcommunitystructurebetweenRH1-
DNA/g DW (Supplementary Figure 5). Chao1 and Shannon 3 and RHP/Ref prompted us to collect various metadata to
indicesrevealedthatalphadiversityandevennessofthebacterial place the community profiles in a broader context. Given the
communitiesinslagdepositsandpondsedimentwerelowerthan unusualindustrialhistoryoftheslagdeposits,wewereespecially
inundisturbedforestsoil(SupplementaryFigure6).Lowestalpha interested in the metal content of the slag and the possible
diversityandevennessvalueswerecalculatedforRH2andRH3. presence of xenobiotics. Elemental analysis revealed that the
Betadiversityanalysisshowedthatthebacterialcommunitiesin contentofsulfur(37.33–46.31mg/gDWsoil),aluminum(21.57–
samplesRH1-3,RHP,andRefdifferedinstructuralcomposition 27.23 mg/g), and calcium (24.12–33.26 mg/g) was significantly
(SupplementaryFigure6).Inparticular,thecommunitiesinslag (ANOVA, p ≤ 0.05) increased in the slag deposits (RH1-3),
deposits (RH1-3) clustered clearly distinct from those in pond relative to the undisturbed forest soil (Ref) (sulfur: 0.77 mg/g,
sediment (RHP) and forest soil (Ref), as revealed by Jensen- aluminum: 9.85 mg/g, calcium: 3.32 mg/g) (Figure2A). The
Shannondivergences. pondsiteshowedastronglyelevatedlevelofiron(48.84mg/g)
Basic characteristics of amplicon datasets are given in (p ≤ 0.05) (RH1-3: 13.88–17.52 mg/g, Ref: 3.36 mg/g). BTEX
Supplementary Table1.Bacterialcommunitiesweredominated compounds(benzene,toluene,ethylbenzeneandxylene),PAHs
by four phyla (range of averaged relative abundances in (polycyclic aromatic hydrocarbons) and heavy metals were not
sample-specific amplicon sequencing datasets are given in detected.Samplesfromtheslagdeposits(RH1-3)hadalowpH
parenthesis): Acidobacteria (6.8–26.8%), Actinobacteria (14.6– of3.4–3.6.Thepondsediment(RHP)waslessacidic(Figure2B).
33.0%),Chloroflexi(5.3–26.8%)andAlphaproteobacteria(18.3– The attempt to relate the observed alpha diversity at the
42.0%) (Figure1A). Acidobacteria and Actinobacteria showed individualsamplingsitestoparticularenvironmentalparameters
a divergent pattern, with Acidobacteria being enriched in (aluminum,calcium,iron,manganese,sulfur)showednoorpoor
site RH1 (ANOVA, p ≤ 0.05), while Actinobacteria were correlations, with the exception of aluminum (correlation =
most prevalent in sites RH2 and RH3. Chloroflexi showed 0.44)(Figure2B).We,however,detectedsignificantcorrelations
increasedabundancesinslagdeposits(RH1-3,p≤0.05),while between the abundance of particular bacterial phyla and
Alphaproteobacteria were strongly enriched in pond sediment environmental parameters, such as sulfur, iron and aluminum
(RHP). Less-abundant phyla included Planctomycetes (1.8– (p ≤ 0.05, r2 ≥ 0.99), based on multiple regression analyses
7.6%),Cyanobacteria(0.1–1.6%),andBacteroidetes(0.1–6.0%). (Figures2C,D).ThepHwasnotanindependentparameter,but
The latter groups, however, were detected only in sites RHP significantly linked to the available sulfur and aluminum (p ≤
and Ref. A more detailed taxonomic analysis showed that all 0.05, r2 ≥ 0.99). As revealed by Jensen-Shannon divergences,
fourdominantphylawererepresentedmainlybyuncharacterized the correlations between the observed differences in bacterial
order- and family-level groups (Figure1B). These groups community structure and environmental parameters were in
included: DA052, KF-JG30-18 (Acidobacteria [subdivision 2, the range of 0.3–0.48 (Figure2C). Combining the different
subdivision 13]), TM214 (Actinobacteria [Acidimicrobiales]), environmentalparametersintoabest-fitmodel,however,yielded
DA111(Alphaproteobacteria[Rhodospirillales]),andJG37-AG- astrongcorrelationof0.96.
4(Chloroflexi).BothDA052andKF-JG30-18weresignificantly Given the dominance of yet-uncharacterized groups
enriched in all three RH sites (p ≤ 0.05), with DA052 being (Figure1B)andgreatestdifferencesinbothbacterialcommunity
most abundant in site RH1. TM214 was detected at all sites, profiles and environmental parameters between slag deposits
but most pronounced at sites RH2, RH3, and RHP (p ≤ 0.05). (RH1-3) and non-slag samples (RHP + Ref), we determined
Like DA052 and KF-JG30-18, DA111 and JG37-AG-4 were Kendall’s tau coefficients to identify links between particular
significantly enriched and showed high abundances in all slag taxonomic groups and single environmental parameters
samples. (Figure2D). Among the Acidobacteria, the higher relative
In order to gain a more resolved phylogenetic analysis, we abundance of most subphylum groups, including DA052 and
made the attempt to reconstruct full-length 16S rRNA gene KF-JG30-18, was found to be linked to aluminum, iron and
sequencesfromourmetagenomicdatasets.Werecoverednearly manganese (p ≤ 0.05 [corrected according to Benjamini and
complete(≥1200bp)16SrRNAgenesequencesforallthemost Hochberg,1995]).DA111(Alphaproteobacteria),butalsoJG30-
abundantgroups:DA052(9sequences),KF-JG30-18(2),DA111 KF-CM66andKD4-96(Chloroflexi),showedastrongcorrelation
(10), JG37-AG-4 (13), and TM214 (22). Phylogenetic analysis withaluminumandiron(p≤0.05).TheActinobacteriashowed
showedthatallfivegroups—DA052,KF-JG30-18,DA111,JG37- high correlations with all recorded environmental parameters.
AG-4, and TM214—were related to 16S rRNA gene sequences The significance of these correlations, however, has to be
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WegnerandLiesack ElusiveAcidobacteriainSoftCoalSlags
FIGURE1|Bacterialcommunityprofilingbasedonampliconsequencingofbacterial16SrRNAgenes. Communityprofileswereobtainedforthreeslagdepositsites
(RH1-RH3),sedimentofapondcollectingtheslagdrainagewaterofrainfallevents(RHP)andnearbyundisturbedforestsoil(Ref).(A)Phylum-levelanalysisisshown
forthetenmostabundantbacterialgroups.Panelslinkedtothefourphylashowinghighestabundancesareemphasizedingray.Orangebars=RH1-3,light-orange
bars=RHP,graybars=Ref.(B)Taxonomicallymoreresolvedcommunityprofilesforthefourmostabundantphyla.*Significantenrichment(p≤0.05)basedon
one-wayANOVA.
considered low, given that members of this phylum showed in terms of completeness (8 out of 13, >80% complete) and
no distinct distribution pattern between the different sampling contamination (9 out of 13, <10% contaminated) (Figure3).
sites (Figures1A,B). The high aluminum content at the three Based on treeing analysis of a concatenated alignment of
slag deposition sites (RH1-3) (Figure2A) and the relatively 31 single-copy marker genes, a group of 5 MAGs (RH2
lowpH(Figure2B)maypointtoahighbioavailabilityoftoxic MAG 17b, RH2 MAG 17c, RH1 MAG 19a, RH1 MAG
aluminumspecies(SupplementaryFigures7,8). 19b, and RH2 MAG21) had an uncertain affiliation with
no close relationship to any described genus. 16S rRNA
3.3. Reconstruction of
gene sequences could be identified for 6 MAGs. Based on
Metagenome-Assembled Genomes identified 16S rRNA gene sequences it was possible to link
An overview of processed metagenome datasets is given in two genomes to uncharacterized taxonomic groups (DA052
Supplementary Table 3. Due to the co-dominance of yet- and KF-JG30-18) that were found to be dominant in the
uncharacterized Acidobacteria groups on subphylum level bacterial community profiles (Figure1B). These two genomes
(Figure1), we were interested in elucidating both their were analyzed in greater detail (RH1 MAG 20 [completeness:
metabolicpotentialandtheirspecificadaptationsfavoringtheir 94%]: DA052 [Acidobacteria, Subdivision 2]; RH2 MAG 17b
persistence in slag deposits. Application of a two-step binning [completeness: 89%]: KF-JG30-18 [Acidobacteria, Subdivision
strategy (Supplementary Figure 4) allowed us to reconstruct 13 13]).Thefocusofourgenomeanalysiswasoncarbonutilization
AcidobacteriaMAGs(RH1:6,RH2:5,RHP:2)(Supplementary capabilitiesandadaptationsfavoringcolonizationofRedHillslag
Table 4). A major proportion of MAGs was of high quality deposits.
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WegnerandLiesack ElusiveAcidobacteriainSoftCoalSlags
FIGURE2|Correlationanalysesbetweentheabundanceofparticularbacterialtaxaandelements.Detectionandquantitationoftheexaminedelementsinvolved
combustionanalysisandinductivelycoupledplasmamassspectrometry,*significantenrichment(p≤0.05)basedonone-wayANOVA,DW=dryweight(A).
CorrelationanalysisbetweenbacterialspeciesrichnessandparticularelementsbasedonPearsoncorrelationcoefficients(corr=correlation)(B).Multipleregression
analysisbasedonlog-transformedelementaldata(left)andcorrelationsbetweeneitherindividualenvironmentalparametersorthecompletesetofallfive
environmentalparametersandbacterialcommunitystructureasdeterminedbyrelatingtheenvironmentalparameterstodeterminedJensen-Shannondivergences
(right;envp=environmentalparameter,commd=communitydissimilarity)(C).Kendall’staucoefficientswerecalculatedtolinktheoccurrenceofspecifictaxawithin
phylaofinteresttoselectedenvironmentalparameters.Determinedp-valueswerecorrectedformultiplecomparisons(BenjaminiandHochberg,1995).Asteriks
indicate*p≤0.05and**p≤0.01(D).
3.4. MAG Analysis of KF-JG30-18 and and starch as carbon source and nitrite as additional nitrogen
DA052 source(Figure4A).Whilenosulfateadenylkinase(EC2.7.1.25)
Both Acidobacteria MAGs encode key pathways for central gene is present, KF-JG30-18 carries all the other genes for
carbohydrate metabolism, most pathways for the synthesis of assimilatory sulfate reduction. The presence of genes encoding
proteinogenic amino acids and complete respiratory chains, CusS/CusR and HydH/HydG two-component systems enables
therebysuggestingaheterotrophiclifestyle(Figure4).Incaseof sensingofextracellularmetalcations.BaeS/BaeRandLiaS/LiaR
KF-JG30-18,thefailuretodetectthecompletegenesetsencoding two-component systems allow detection of cell envelope stress.
glycolysisandTCAcyclecanbeattributedtotheincompleteness KF-JG30-18possesses11geneslinkedtoCzcCBAeffluxsystems,
ofitsMAG(SupplementaryTable4). whichbelongtothemultifunctionalfamilyoftypeVIsecretion
systems. It also contains various transporter genes including
3.4.1.KF-JG30-18
those for molybdate, cobalt/nickel, branched-chain amino acid
RH2 MAG 17b showed a total length of 2.8 Mb with a GC uptake systems, and heme export systems. In addition, genes
content of 58% (Table1). The KF-JG30-18 representative has encoding high-affinity phosphate uptake systems and genes
the genetic potential to utilize the polysaccharides cellulose encoding enzymes facilitating polyphosphate formation were
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WegnerandLiesack ElusiveAcidobacteriainSoftCoalSlags
FIGURE3|PhylogenetictreeofMAGs. AvailableAcidobacteriagenomesofknownphylogeneticaffiliationwereretrievedfromtheNCBIgenomerepository.
Phylogenetictreeingwasbasedonaconcatenatedalignmentof31single-copymarkergenesusingAMPHORA(v.2)(WuandScott,2012).UsingFASTTREE(v.
2.1.3)(Priceetal.,2010),amaximum-likelihoodtreewascalculatedapplyingtheWAGmodel(WhelanandGoldman,2001).Nearestneighborinterchangeandthe
CATapproximationwereusedtooptimizetreetopologyandtoconsiderevolutionaryrateheterogeneity(Stamatakis,2014).Coloredcirclesindicatethetaxonomic
affiliationbasedonavailable16SrRNAgenesequences.Black-whitespheresandsquaresshowthedegreeofcompletenessandcontamination,whichwasderived
fromthepresence/absenceofsingle-copymarkergenesandtheiractualcopynumberintheMAGs(Parksetal.,2015).MAGsanalyzedinmoredetailarehighlighted
inbold.Thescalebarindicates0.1substitutionsperaminoacidposition.
detected for KF-JG30-18. Finally, two CRISPR arrays were for flagellar motility and type IV secretion was identified
identified(Table1). in RH1 MAG 20. It also encodes high-affinity phosphate
uptake systems, is equipped with the potential to form and
3.4.2.DA052
release polyphosphate, and contains multiple genes that code
The RH1 MAG 20 genome was substantially bigger in for phosphate-binding subunits. Genes involved in sensing
comparisontoKF-JG30-18andshowedatotallengthof4.9Mb extracellularmetalcationsweredetected,suchasthoseencoding
featuringaGCcontentof59%(Table1).IncontrasttoKF-JG30- CusS/CusR and HydH/HydG two-component systems. DA052
18, the DA052 representative (RH1 MAG 20) has the capacity containsevenmoregenesassociatedwithCzcCBAeffluxsystems
to express the ribulose monophosphate pathway, suggesting (29genes)thanKF-JG30-18(11).Worthmentioningisalsothe
the potential to metabolize C1 compounds by formaldehyde high number of transposase genes and genes encoding mobile
fixation. The RH1 MAG 20 also possesses genes involved in elementproteins(29)(Table1).
the breakdown of cellobiose, starch and xylan. In addition,
genes that encode dye-decolorizing peroxidases were detected.
Like KF-JG30-18, DA052 has the potential to utilize oxidized 4. DISCUSSION
nitrogenspeciessuchasnitriteandnitrateasadditionalnitrogen
sources (Figure4B). Albeit no adenyltransferase (EC 2.7.7.4) The slag samples contained high amounts of iron and sulfur
gene could be identified, DA052 is likely able to carry out (Figure2A),presumablyinmineralform.Thiswasunexpected
assimilatorysulfatereduction.Thecompletesetofgenesrequired given the age (150 years) and leached nature of the slags.
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FIGURE4|MetabolictraitsofKF-JG30-18(A)andDA052(B)asderivedfromthegeneticpotentialofRH2MAG17bandRH1MAG20. Functionalannotationsare
basedonPROKKA(Seemann,2014)andRAST(Azizetal.,2008;Overbeeketal.,2014).Bothannotationresultswerethoroughlyvalidatedagainsteachother.
PathwayswerereconstructedbyUBLAST(Edgar,2010)searchesofpredictedcodinggenesagainstNCBInr.Predictedgeneswereadditionallyclassifiedagainstthe
KEGGreferencedatabaseusingKEGGMAPPER.Redcrossesindicatemissinggenesintherespectivepathways.Co/Zn/Cd(cobalt/zinc/cadmium),Czcefflux
pumps;Mo,molybdate;Ni,nickel;BCAA,branched-chainaminoacids;YadG/YadH,uncharacterizedABCtransporter;MsbA+MdlB,multi-drugresistantABC
transporter;MacB+YddA,macrolide-bindingABCtransporter;CusS/CusR,two-componentsystemsensingextracellularcopper;HydH/HydG,two-component
systemsensingextracellularmetalions;LiaS/LiaR,two-componentsystemsensingcellenvelopestress;BaeS/BaeR,two-componentsysteminvolvedinantibiotic
sensing;TXSS,typeXsecretionsystem;CxI-IV,complexItoIVoftherespiratorychain;G3P,glycerate3-phosphate;PEP,phosphoenolpyruvate;Citr,citrulline;Orn,
ornithine;PolyP,polyphosphate;Oxa,oxaloacetate;Mal,malate;Fum,fumarate;Suc,succinate;Suc-CoA,succinyl-CoA;2Oxo,2-oxoglutarate;Iso,isocitrate;Cit,
citrate;APS,adenosinephosphosulfate;PAPS,phosphoadenosinephosphosulfate;Cel,cellulose;CelB,cellubiose;Sta,starch;Xyl,xylan;F6P,fructose
6-Phosphate;GA3P,glyceroaldehyde3-phosphate;G3P,glycerol-3-phosphate;RuMP,ribulosemonophosphatepathway;Dyp,dye-decolorizingperoxidases.
Assuming a continuous exposure to oxygen and humidity, high aluminum content highlights the inefficiency of the early-
an oxidation of present sulfide-mineral components was industrial leaching process. Given the low pH (3.4–3.6) of the
anticipated.Sulfide-richminetailingsshowazonationovertime: slags, aluminum was likely present as cations (Supplementary
asulfide-depletedzone;anactive,transitoryoxidationzone;and Figure7).Cationicaluminumishighlytoxicbecauseofpotential
aprimaryzonerepresentingtheoriginalconditions(Diabyetal., interferences with ATP metabolism, lipid metabolism and
2007;Tanetal.,2008;Huangetal.,2011).Inourstudy,theslags membrane transport processes (Supplementary Figure 8). In
originatedfromsoftcoalwithahighcontentofpyrite.Organic soils, aluminum toxicity can be mitigated by clay minerals and
carbon in soft coal and, as a consequence, in the studied slags organic acids, which act as a buffer system due to their cation
primarily consists of humic and fulvic acids (Peuravuori et al., exchangecapacity(Turpaultetal.,1996).
2006). Humic acids were repeatedly reported to be efficient in Thechelatingcapabilitiesofhumicandfulvicacidsmaylead
pyritepassivation(Lalvanietal.,1996;Belzileetal.,1997;Chen to the immobilization of aluminum cations as described above
et al., 1999), either by chelating released ferric iron with their for iron-sulfur minerals. Mohan and Chander (2006) showed
negatively charged functional (carboxylic, phenolic) groups or that brown coal is an efficient adsorbent for metal cations in
bydirectpyriteadsorption.Bothprocessesmayexplainwhythe AMDdischarges.Givenapassivationofiron-sulfurmineralsand
oxidationofsulfidemineralsintheslagdepositswasmitigated. aluminum,theirroleasdriversforbacterialcommunitystructure
This low oxidation level clearly differentiates the Red Hill slag canbeconsideredindirect,albeittoxiceffectsofaluminumdue
depositsfromminetailingsandotherAMDenvironments. to minor releases may occur. Previous research had revealed
Aluminum was found to be a major driver of bacterial thattheretentionofmetalcationscanaltertheaccessibilityand
community structure in the leached slags (Figures2B–D). The utilizabilityofhumicandfulvicacidsaspotentialcarbonsources
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TABLE1|CharacteristicsofRH1MAG20(DA052)andRH2MAG17b clearly differentiates the slag deposits from AMD systems and
(KF-JG30-18). minetailings.
Given that cultured representatives are not yet available for
RH1MAG20 RH2MAG17b
the dominant groups identified in the Red Hill slag deposits,
(DA052) (KF-JG30-18)
their metabolic capabilities remained elusive. Therefore, high
Totallength[bp] 4,885,294 2,798,462 abundancesofAcidobacteriasubdivisions2(DA052)and13(KF-
No.ofrecruitedreads 404,720 254,272 JG30-18) prompted us to conduct a metagenomic approach in
406,649 254,736 ordergetfirstinsightsintotheirgeneticpotential.Acidobacteria
Coverage 18 18 are known to be abundant members of terrestrial microbiomes
No.contigs 760 169 (Janssenetal.,2002;Saitetal.,2006)andtobephylogenetically
N50[bp] 58,735 25,655 highly diverse (Barns et al., 2007). A total of 22 genera have
GCcontent[%] 59 58 beendescribed,whichcoveronly8of26subdivisionsproposed
Completeness[%] 93.97 89.39 until today. Genome sequences are available for members of 5
Contamination[%] 5.15 2.14 subdivisions(1,3,4,8,and23;Kielaketal.,2016).Acidobacteria
Classification* Nearlycompletegenome Substantialgenomewith arebelievedtohaveanoligotrophic(K-strategist)lifestyle.This
withmediumcontamination lowcontamination perception, however, may be biased due to the low number
No.ofcodinggenes 5,311 2,822 of acidobacterial isolates of which most belong to subdivision
No.ofrRNAoperons 1 1 1. Members of subdivisions 2 and 13 have repeatedly been
No.oftRNAs 44 35 detectedinhigherabundancesinU-contaminatedenvironments
Mobileelements** 29 3 (Selenska-Pobelletal.,2001;Barnsetal.,2007).Theoccurrence
CRISPRarrays – 2 of subdivision 2 populations in Amazonian forest soils was
No.ofencodedCzcefflux 29 11 correlated to the microbial availability of CO2, Fe, and Al3+.
pumps Thus, it was hypothesized that members of subdivision 2 are
No.ofencodedAcriflavine 5 6 toleranttoaluminum(Navarreteetal.,2013;Catãoetal.,2014).
resistanceproteins AdaptationandtolerancetoAl3+couldalsobeonemajorreason
Recruited reads refer to the number of reads incorporated during the assembly of forthehighrelativeabundanceofDA052inoursamplingsites,
respective contigs. Numbers represent forward and reverse reads. Coverage was especially in RH1. Amazonian forest soils are commonly rich
determined by determining the average coverage of genome bin contigs. Genome in biochar whose chemical characteristics are remotely similar
completeness and contamination have been assessed using CHECKM(Parks et al.,
to those of studied soft coal slags. However, a recent study of
2015). The number of coding genes, rRNA operons, tRNAs, mobile elements,
CRISPR/CASrepeatsandrepeatregionsisgivenbasedongenomeannotationsdone biochar microbiomes and metagenomes revealed a rather low
withPROKKA(v.1.11)(Seemann,2014)andRAST(v.2.0)(Azizetal.,2008;Overbeek abundance of Acidobacteria (Noyce et al., 2016). Given the
etal.,2014)followedbymanualrefining.*AccordingtoParksetal.(2015);**transposons
limitedknowledgeofyetuncharacterizedAcidobacteriagroups,
andinsertionsequences.
theMAGsobtainedforsubdivisions2(DA052)and13(KF-JG30-
18) allowed us to infer valuable information on the physiology
for microbes (Jones and Edwards, 1998; Jones et al., 2003). andmetabolicpotentialofthesetwoAcidobacteriasubdivisions.
Consideringthathumicandfulvicacidsarenaturallyratherinert As of November 2016, 27 genome sequences were available
and recalcitrant carbon sources (Kirk and Farrell, 1987), metal for Acidobacteria, with most of them being affiliated with
complexation would increase the supposed oligotrophic nature subdivision 1 Acidobacteria. This number has recently more
ofthestudiedslagdeposits. than doubled by deep metagenomic sequencing of an aquifer
Intheleachedslags,bothmicrobialbiomassandbacterialcell system (Anantharaman et al., 2016). However, to the best of
numbers were significantly lower than in the undisturbed our knowledge, no genomes of Acidobacteria subdivisions 2
forest soil (Supplementary Figure 5), but in the range and 13 have yet been reported, highlighting the novelty of the
previously determined for sulfidic mine tailings (Korehi draft genomes reconstructed for DA052 (RH1 MAG20) and
et al., 2014). Five yet-uncultured subphylum groups were KF-JG30-18 (RH2 MAG 17b). The genome size of DA052 is
identified to be dominant in the slag deposits. These belonged comparable to those of subdivision 1 Acidobacteria such as
to Acidobacteria, Actinobacteria, Alphaproteobacteria, and Terriglobus roseus (4.9 Mb, 60% GC) (Rawat et al., 2014).
Chloroflexi (Figures1A,B). None of the five groups have The KF-JG30-18 genome is comparably small, being in the
previouslybeenreportedtobehighlyabundantinAMDsystems. rangeoftheonlysubdivision23genome(Thermoanaerobaculum
Theirfirstdetectionwasinpeatlands(DA052,DA111,TM214) aquaticum 2.7 Mb) (Stamps et al., 2014). Apparently, members
(Rheims et al., 1996; Felske et al., 1998) and uranium waste of DA052 and KF-JG30-18 are able to utilize polysaccharides
piles (KF-JG30-18, JG37-AG-4, DA111) (Selenska-Pobell et al., such as cellulose and xylan (Figure4). Comparative genomics
2001; Selenska-Pobell, 2002). Phylogenetic analysis of nearly suggests that the ability of polysaccharide breakdown is widely
complete 16S rRNA gene sequences also revealed relationships distributed among the Acidobacteria (Kielak et al., 2016), but
to populations present in acidic, metal-rich, and contaminated todateexperimentalevidenceforpolysaccharideutilizationwas
soilenvironments(SupplementaryTable2).Inaccordancewith obtainedonlyformembersofsubdivisions1,3,and4(Dedysh
thegeochemicaldifferencesdiscussedabove,non-occurrenceof et al., 2012; Pascual et al., 2015; Garcia-Fraile et al., 2016; Jiang
bacteriaknowntobeinvolvediniron-andsulfur-cyclingagain et al., 2016; Lladó et al., 2016). Xylan degradation appears to
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WegnerandLiesack ElusiveAcidobacteriainSoftCoalSlags
bearathercommonmetabolictraitoftheAcidobacteria,while thatthenumberofacriflavineresistancegenescanhaveastrong
cellulose breakdown has been shown only for Telmatobacter effectontheirfitnesswhenbeingexposedtometals,metalloids,
bradus (Pankratov et al., 2008). Plant cell wall constituents or hazardous organic compounds (Emerson et al., 2013). It is
such as cellulose or xylan are commonly identified in soft coal thustemptingtohypothesizethatthepresenceofsuchsubstances
in varying quantities dependent on the degree of coalification in the leached slags selects for acriflavine-tolerant bacteria. In
(del Río et al., 1994; Rumpel et al., 1998). The DA052 draft consequence,thepresenceofmultiplegenesencodingacriflavine
genome RH1 MAG20 contains all the genes required for resistanceproteinscouldthusrepresentacompetitiveadvantage,
formaldehyde fixation via the cyclic ribulose monophosphate favoring the colonization of studied slags by DA052 and KF-
pathway (Figure4). Thus, members of DA052 may be able to JG30-18.
utilize C1 compounds as carbon source. These are present in In summary, our analyses revealed that the microbiology
coal as methyl- and methoxyl-groups or as formate (Stafford, of the studied slags greatly differs from commonly studied
1988; Stout et al., 1988; Hatcher and Clifford, 1997). A survey AMDandminetailingenvironments.Theunusualgeochemistry,
of the available Acidobacteria genomes revealed key genes of including the proposed mitigation of mineral oxidation and
the ribulose monophosphate pathway (3-hexulose-6-phosphate subsequent metal release, increases the oligotrophic nature
synthase and 6-phospho-3-hexuloisomerase) in Edaphobacter of the Red Hill slag deposits. Members of the elusive
aggregansandTerriglobusroseus,butbothorganismshavenotyet Acidobacteria subdivisions 2 (DA052) and 13 (KF-JG30-
beenshowntoutilizeC1compounds(Eichorstetal.,2007;Koch 18) showed a dominant occurrence not observed in any
etal.,2008).IntheDA052representative,wealsoidentifiedthe other previously studied environment. Metagenome-assembled
genetic potential to produce lignin-degrading dye-decolorizing genomes allowed us to identify broad carbon utilization
peroxidases. This family of enzymes is of increasing interest capabilities and, more intriguingly, pronounced metal and
as it represents the bacterial counterpart of lignin-cleaving xenobiotic detoxification mechanisms. These may explain the
peroxidases and laccases known from fungi (de Gonzalo et al., dominant occurrence of DA052 and KF-JG30-18. Our findings
2016). The ability to utilize rather inert, lignin-derived carbon call for further research into the microbiology of the Red
sourceswouldgiveDA052aselectiveadvantageaslignin-derived Hillslagdeposits,inparticularforactivity-centeredapproaches
carbon is assumed to be abundantly present in the studied suchasmetatranscriptomicsand-proteomicsthatallowtolink
slags. both genome coding potential and actual metabolic activity of
Although most aluminum in the Red Hill slag deposits identified groups in this unique, early-industrial, man-made
is assumed to be immobilized by humic and fulvic acids, habitat.
some bioavailability cannot be excluded. Both draft genomes,
DA052 and KF-JG30-18, contain genes encoding high-affinity AUTHOR CONTRIBUTIONS
phosphate uptake systems, while the potential to form and
release polyphosphate was detected specifically for DA052. The CW and WL designed research. CW carried out research,
intracellularcleavageofpolyphosphateandreleaseofphosphate analyzed data, wrote code. CW and WL wrote the manuscript
moieties was described for bacteria and archaea (Remonsellez together.
et al., 2006; Rao et al., 2009; Navarro et al., 2013) as a measure
toinactivatetoxicmetalcationsbyprecipitation.Sinceinorganic FUNDING
phosphate has manifold roles in cellular metabolism, the sole
presence of these genes cannot be exclusively attributed to Funding was provided by the German Research Foundation
metal resistance. The draft genomes of both DA052 and KF- (DFG SFB 987, Microbial Diversity in Environmental Signal
JG30-18, however, harbor an unexpectedly high number of Response,ProjectA05toWL).
genes encoding Czc efflux pumps (Figure4). These mediate
resistance to cobalt, zinc, and cadmium (Nies, 1995; Chen ACKNOWLEDGMENTS
et al., 2015). Using InterPro and PFAM, a closer inspection
of the encoded proteins revealed that 6 and 5 sequences for, The authors wish to express their sincere gratitude to
respectively, KF-JG30-18 and DA052 are closely related to Walter Wilhelm Jungmann (Department of Geology) for
acriflavine resistance proteins, which, like Czc efflux pumps, experimentaladvice,andtoChristophWeihrauch(Department
belong to the resistance/nodulation/division superfamily of of Geology), Daniel Schindler (SYNMIKRO) and Heike
solutetransporters(Table1).Acriflavineresistanceproteinsare Mallinger (Department of Chemistry) for excellent technical
multi-drug efflux systems with a tremendously broad substrate assistance.AllofthemaremembersofthePhilipps-Universität
range, including different classes of antibiotics, detergents and Marburg (Germany). In addition, the authors would like to
small organic molecules (Blair and Piddock, 2009; Pos, 2009). thankHorstWolfgartenforin-depthinformationaboutsoftcoal
Acriflavine was first extracted from smoldered coal tar by mininginthesurroundingsofBonn.
precipitation with diluted sulfuric acid, a procedure that is
reminiscent of the early industrial mineral leaching from soft SUPPLEMENTARY MATERIAL
coal (Supplementary Figure 1). Paul Ehrlich found acriflavine
to be bacteriocidal (Wainwright, 2001; Kumar et al., 2012) and The Supplementary Material for this article can be found
intercalation into DNA was identified as its mode of action online at: http://journal.frontiersin.org/article/10.3389/fmicb.
(Lerman, 1961). It has been shown for iron-oxidizing bacteria 2017.01023/full#supplementary-material
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