Phylogeny of Microorganisms Populating a Thick, Subaerial, Predominantly Lithotrophic Biofilm at an Extreme Acid Mine Drainage S

Phylogeny of Microorganisms Populating a Thick, Subaerial, Predominantly Lithotrophic Bioﬁlm at an Extreme Acid Mine Drainage Site Downloaded from PHILIP L. BOND,* STEVEN P. SMRIGA, AND JILLIAN F. BANFIELD Department of Geology and Geophysics, University of Wisconsin—Madison, Madison, Wisconsin 53706

Received 14 December 1999/Accepted 6 June 2000

An unusually thick (ϳ1 cm) slime developed on a slump of ﬁnely disseminated pyrite ore within an extreme acid mine drainage site at Iron Mountain, near Redding, Calif. The slime was studied over the period of 1 year. The subaerial form of the slime distinguished it from more typical submerged streamers. Phylogenetic analysis

of 16S rRNA genes revealed a diversity of sequences that were mostly novel. Nearest relatives to the majority http://aem.asm.org/ of sequences came from iron-oxidizing acidophiles, and it appears that iron oxidation is the predominant metabolic characteristic of the organisms in the slime. The most abundant of the 16S rRNA genes detected were from organisms related to Leptospirillum species. The dominant sequence (71% of clones) may represent a new genus. Sequences within the Archaea of the Thermoplasmales lineage were detected. Most of these were only distantly related to known microorganisms. Also, sequences affiliating with Acidimicrobium were detected. Some of these were closely related to “Ferromicrobium acidophilus,” and others were affiliated with a lineage only represented by environmental clones. Unexpectedly, sequences that affiliated within the delta subdivision of the Proteobacteria were detected. The predominant metabolic feature of bacteria of this subdivision is anaerobic sulfate or metal reduction. Thus, microenvironments of low redox potential possibly exist in the predominantly oxidizing environments of the slime. These results expand our knowledge of the biodiversity of on November 11, 2015 by University of Queensland Library acid mine drainage environments and extend our understanding of the ecology of extremely acidic systems.

Dissolution of sulfide ores exposed to oxygen, water, and Our research is directed towards improving the understand- microorganisms results in acid production and environmentally ing of AMD microbial ecology and changes in microbial com- detrimental acid mine drainage (AMD) (35). For the aqueous munity structure as a function of geochemical conditions at dissolution of sulfide ores dominated by pyrite (FeS2)atlow major sites of acid generation. This information is relevant to pH, ferric ion is the predominant oxidant. The overall reaction constrain laboratory studies and for the modeling of AMD ϩ ϩ Ϫ is written: FeS ϩ 14Fe3 ϩ 8H O315Fe2 ϩ 2SO 2 ϩ 16 ϩ 2 2 4 generation. Understanding of the different iron-oxidizing H . The reaction is limited by the availability of ferric ion. At mechanisms is critical for the future development of assays for pH values of less than ϳ3.0, the inorganic rate of ferrous iron oxidation activity based on probes to conserved features of oxidation is slow, and acidophilic organisms can mediate pro- metabolic pathways. duction of ferric iron and conserve energy from this. Thus, it is Our field site at Iron Mountain, Redding, Calif., comprises not surprising that the oxidation of pyrite is greatly increased in an AMD system associated with a pyrite-dominated ore body the presence of iron-oxidizing species such as Thiobacillus fer- (up to 95% pyrite). Typically the drainage water pH measures rooxidans over the abiotic rate; see Nordstrom and Southham between 0.5 and 1.0, metal ion concentrations are in the deca- (36) for a discussion. Presently the understanding of biological grams per liter range (35), and temperatures range from 30 to enhanced pyrite oxidation is incomplete, but it is clear that 50°C over the course of a year. Presently, mine waters are microbial iron oxidation would replenish ferric ions for the treated to remove metals and raise pH. Ultimately, an im- above reaction. proved understanding of the mine subsurface microbiology, The best-studied organism with respect to microbial en- geochemistry, and resultant dissolution may provide informa- hancement of AMD is T. ferrooxidans. Models have been pro- tion useful for the development of strategies aimed at reducing posed for its energetic characteristics (28) and role in pyrite acid generation rates. dissolution (41), and several investigations have studied iron- Investigations of microbial communities have focused on oxidizing and respiratory enzymes (7, 11, 17). However, nu- samples taken from submerged sediment within the mine at merous microorganisms are known for their acidiphily and the Richmond 5-way and from the various tunnels extending iron-oxidizing capabilities, and it is apparent that different into the ore body (Fig. 1) (15, 43). Within the mine there are microorganisms have different mechanisms and perhaps differ- obvious macroscopic differences in the forms of microbial bio- ent abilities for iron oxidation (7). It has therefore been the mass occurring in different localities. Submerged slimes and endeavor of some investigations to increase the understanding streamers predominate in flowing water in the mine tunnels. In of the microbial ecology of AMD systems (29, 43). Such inves- addition to visible slimes, the surfaces of pyrite grains are often tigations are essential to understanding important microbial covered by microbial cells. T. ferrooxidans, the organism often processes that underpin the ecology of these environments. associated with AMD, has been detected at only low levels in samples collected within the ore body (43). Cells detected with * Corresponding author. Mailing address: Department of Geology a Leptospirillum ferrooxidans-specific probe were distributed and Geophysics, University of Wisconsin—Madison, Madison, WI throughout these samples but made up small proportions of 53706. Phone: (608) 262-0915. Fax: (608) 262-0693. E-mail: bond the cells attached to sediment particles (43). Analysis of 16S @geology.wisc.edu. rRNA gene sequences had previously detected Leptospirillum

3842 VOL. 66, 2000 PHYLOGENY OF AN ACID MINE DRAINAGE BIOFILM 3843 Downloaded from http://aem.asm.org/

FIG. 1. Cartoon of mine tunnels within the mountain, showing various drifts and position of the slump in the A drift. on November 11, 2015 by University of Queensland Library sequences throughout the mine (40). More recently, extensive analysis of samples collected throughout 1997 indicated sub- stantial fluctuations in geochemical conditions and microbial community compositions and confirmed the scarcity of T. fer- FIG. 2. Thick subaerial slime covering part of the slump in the A drift. A rooxidans (15). In the high-ionic-strength conditions, archaea 15-ml Falcon centrifuge tube is positioned for scale. dominated microbial communities. Subsequently, an iron-oxidizing archaeon predominating in some microenvironments within the mine was isolated by us and tentatively named dium chloride, 10 mM sodium phosphate buffer [pH 7.2]) solution. The fixed “Ferroplasma acidarmanus” (14). Many of these organisms are samples were transported to the laboratory on ice, and within 30 h of fixation they were washed in PBS, resuspended in a PBS–96% ethanol solution (1:1, inferred to contribute significantly to pyrite dissolution. Pe- vol/vol), and stored at Ϫ20°C prior to hybridization (2). ripheral to the ore body, the pH values are higher (those Microscopy. For microscopic observations, sample smears were prepared on glass slides. Cells were stained with 4Ј,6-diamidino-2-phenylindole (DAPI) at 1.0 measured in the entrance tunnel are around pH 2.5), and ␮ distinct microbial communities exist. Assemblages include T. g/ml at room temperature for 5 min (25). Smears were then mounted in Vectorsheild (Vector Laboratories, Burlingame, Calif.) and viewed using a Leica ferrooxidans, but these are probably irrelevant to pyrite disso- DMRX microscope fitted with UV epifluorescence (Chromatech filter set 31000) lution (43). and transmitted differential interference contrast. In a region of the A drift, collapse of an overhead stope had DNA extraction and purification. Two methods were used for extraction of resulted in the formation of a slump on the tunnel floor. Hot nucleic acids from mine samples. Both were based on a bead-beating protocol described by Barns et al. (6) and included initial wash steps to exclude iron and air streams down from the stope, and water carrying biofilm to raise the pH prior to cell lysis. This treatment was necessary as the low pH may drips down onto the slump. The dripping slimes are referred to cause hydrolysis of DNA and a high iron concentration would have contaminated as snottites, a term coined to describe pendulous slime accu- the extracted DNA, having a deleterious effect on subsequent PCR. Extractions mulations from other subsurface environments (26). The were performed on 0.5-ml samples that were pelleted by centrifugation, washed once with PBS (pH 1.2), washed with one part 2ϫ buffer A (200 mM Tris [pH slump was typically covered in a subaerial slime up to ϳ1cm 8.0], 50 mM EDTA, 200 mM NaCl, 2 mM sodium citrate, 10 mM CaCl2) and one thick (Fig. 2). Both the slime and the snottite materials differed part 50% glycerol, and then resuspended in 0.5 ml of 2ϫ buffer A in 2-ml in appearance from other microbial growths in the mine. This screw-cap tubes. Pyrophosphate (0.2%), polyadenylic acid (200 ␮g/ml), and ly- paper describes the results of a molecular phylogenetic analysis sosyme (3 mg/ml) were added to the suspension, and this was incubated for 40 min at 37°C. Proteinase K (2 mg/ml) was added to the mixture, and this was of the slump slime and snottite materials. While the majority of incubated for 30 min at 50°C. Samples were treated by bead beating or by the sequences obtained were related to known acidophiles, freeze-thawing for cell lysis. For bead beating, samples were supplemented with most of them were novel. These results considerably extend the 50% (vol/vol) phenol-chloroform-isoamyl alcohol (24:24:1), 5% (wt/vol) sodium known diversity of microorganisms in AMD environments. dodecyl sulfate (SDS), and approximately 0.3 g of acid-washed zirconia-silica beads (0.1-mm diameter). The mixture was agitated on a Mini-Beadbeater (Bio- spec, Bartlesville, Okla.) at low speed for 2 min. For freeze-thawing, the cells MATERIALS AND METHODS were amended with 5% (wt/vol) SDS, subjected to three cycles of freezing in a dry ice-ethanol bath and heating for 3 min at 65°C, and mixed with 50% (vol/vol) Sample collection. Samples were collected from various locations within the phenol-chloroform-isoamyl alcohol (24:24:1). All cell lysates (bead beating and mine in November 1998 and in February and May 1999. Specimens for DNA freeze-thawing) were extracted with 50% (vol/vol) phenol-chloroform-isoamyl extraction were collected directly into sterile 15-ml Falcon tubes (Becton Dick- alcohol (24:24:1), and nucleic acids were precipitated in 50% (vol/vol) isopropa- inson, Paramus, N.J.) and kept on ice or at 4°C until processed (within 3 days). nol and 10% (vol/vol) 3 M sodium acetate (pH 5.2) on ice for 20 min. Nucleic Samples for fluorescence in situ hybridization (FISH) were fixed in freshly acids were pelleted by centrifugation, washed with 70% ethanol, and resus- prepared 3% paraformaldehyde–phosphate-buffered saline (PBS; 130 mM so- pended in TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]). DNA was 3844 BOND ET AL. APPL.ENVIRON.MICROBIOL. purified by passage through a ChromaSpinϩTE1000 column (Clonetech, Palo TABLE 1. Inventory of the slime (BA) and snottite (SC) 16S Alto, Calif.) and quantified by ethidium bromide-UV detection on an agarose rDNA cloned fragments arranged into groups according to RFLP gel. Multiple DNA extracts from a sample were combined prior to PCR ampli- patterns and sequence similarity fication. PCR and fractionation of 16S rRNA genes. Community 16S rRNA genes were Representative No. of Microbial group Postulated metabolism amplified by PCR in mixtures containing approximately 40 ng of purified DNA sequencea clonesb affiliation per ml, 1ϫ PCR buffer (Perkin Elmer, Norwalk, Conn.), a 200 ␮M concentration of each of the four deoxynucleoside triphosphates, 2.5 mM MgCl2, 350 mM BA18 1 ␦ Proteobacteria Sulfate/iron reduction (each) forward and reverse primers, and 0.025 U of AmpliTaq Gold (Perkin ␦ Proteobacteria ␮ Ј BA71 4 Sulfate/iron reduction Elmer) per l. In reactions, the reverse primer was the universal 1492R (5 -G BA29 69 Nitrospira group Iron oxidation/autotrophic GTTACCTTGTTACGACTT-3Ј) (31), and the forward primer was either Bac- Downloaded from BA24 4 Nitrospira group Iron oxidation/autotrophic teria-specific 27F (5Ј-AGAGTTTGATCCTGGCTCAG-3Ј) (31), Archaea-specific 21Fa (5Ј-TTCCGGTTGATCCYGCCGGA-3Ј) (12), or the universal 533F BA46 13 Acidimicrobium Iron oxidation/heterotrophic (5Ј-GTGCCAGCMGCCGCGGTAA-3Ј) (27). A Gene Amp 2400 (Perkin group Elmer) was used to incubate reactions through an initial denaturation at 94°C for BA48 1 Chimera 12 min, followed by 30 cycles of 94°C for 1 min, 45°C for 45 s, and 72°C for 1.5 BA50 1 Chimera min, and completed with an extension period of 20 min at 72°C. For all primer BA84 2 Acidimicrobium sets, products from four separate reactions were pooled and purified using group QIAquick PCR purification columns (Qiagen, Valencia, Calif.). These were quantified on an agarose gel (as above), ligated into the vector pGEM-T (Pro- SC02 13 Nitrospira group Iron oxidation/autotrophic mega, Madison, Wis.), and used to infect competent host cells following the manufacturer’s instructions. When possible for the ligation, we used an insert- SC07 10 Nitrospira group Iron oxidation/autotrophic http://aem.asm.org/ vector ratio of 3:1. SC17 3 Nitrospira group Iron oxidation/autotrophic RFLP and sequencing of inserted 16S rRNA genes. For restriction fragment SC28 3 Nitrospira group Iron oxidation/autotrophic length polymorphism (RFLP) and sequencing, the inserted fragment was ampli- SC12 4 Thermoplasmales fied by PCR as described above except that DNA was provided by contact of a group small pipette tip with a colony of cloned host cells which was immersed in the SC36 2 Thermoplasmales PCR mixture. Primers for the PCR were the vector-specific T7 and SP6. Re- group striction enzyme digestion of the unpurified PCR products was carried out, and SC38 3 Thermoplasmales the restricted fragments were observed, as previously described (27). RFLP patterns were grouped, and representative cloned fragments were sequenced. group Purified PCR products (QIAquick column; see above) were sequenced using SC42 1 Thermoplasmales Iron oxidation/autotrophic the Prism Big Dye terminator sequencing kit (Applied Biosystems, Foster City, group Calif.) with 50 to 100 ng of template DNA, according to the manufacturer’s on November 11, 2015 by University of Queensland Library a instructions. For initial analysis, partial sequences were obtained using the uni- Representative clone fragment sequenced for phylogenetic analysis. b Ն versal primer 533F. Extended sequences were obtained using additional primers Grouped by RFLP pattern and by having 98% sequence similarity to the 27F and 1492R in separate sequencing reactions. The extension products were representative sequence within that particular clone library. purified using Auto Seq G-50 columns (Amersham Pharmacia Biotech, Piscat- away, N.J.) and DNA sequences were determined on an automated sequencer (ABI 377XL) at the University of Wisconsin Biotechnology Center. Phylogenetic analysis. Phylogenetic affiliations of the partial sequences were covered in similar cells. Preliminary analysis of the microbial initially estimated using the program BLAST (basic local alignment search tool) communities by FISH using domain-specific oligonucleotide (1) and available nucleotide databases. Single primer sequences were aligned using the GDE (Genetic Data Environment) multiple sequence editor against probes indicated that the biofilms were mostly bacterial (re- close relatives in ARB (a software environment for sequence data) (45). Simi- sults not shown). Although the majority of cells were spirillum larity of partial sequences was determined using ARB, and those with more than shaped, these were not detected by FISH with the previously 98% similarity were grouped (Table 1). Extended sequences of representatives used Leptospirillum group probe LF581 (43) (results not from the groups were compiled on SeqEd (Applied Biosystems), checked for chimeras on the program CHECK_CHIMERA (34), and used in the phyloge- shown). To further analyze the community structures of slime netic analyses. Sequences for analysis were managed in ARB and reduced to and snottite biofilms, clone libraries of PCR-amplified 16S unambiguously alignable positions. For bacterial sequences, the Lane mask (31) rRNA genes were prepared. was used. Evolutionary analyses of alignments were performed by distance meth- Obtaining 16S rRNA sequences from snottites. During ex- ods using ARB and PAUP (47), parsimony in PAUP, and maximum likelihood using fastDNAml (34) essentially as described previously (5, 6). The program traction from AMD samples, we have noticed that the freeze- MODELTEST was used to determine parameter settings for analyses using thaw method produces a greater quantity and less sheared PAUP (38). DNA than is produced by bead beating. Thus, the freeze-thaw Nucleotide sequence accession numbers. Sequences (excluding potential chi- extraction method was used. meras) have been submitted to GenBank with accession numbers from AF225446 to AF225459. DAPI staining indicated that eukaryotic cells are not abundant in the slime or snottite samples. PCR amplification of the slime 16S rRNA genes was performed with the Archaea- and RESULTS Bacteria-specific primer sets. From the slime, no PCR amplifi- The slump slime and snottite materials were first noticed cates using the archaeon-specific primer pair were observed on during a sampling trip made to the Iron Mt. mine in November an agarose gel. Nevertheless, the archaeal primer amplificate 1998. Samples of the slump slime, snottite, and sediment on products were concentrated 10-fold for ligation into the clon- the surface of the slump were taken for analyses on this occa- ing vector. However, no ligated inserts could be detected. sion and on subsequent trips. Temperature and pH in water Therefore, it appeared there were very few or no archaea in associated with the slime measured in the ranges from 31.5 to the slime. PCR product from the Bacteria-specific primer pair 36.8°C and pH 0.77 to 1.21, respectively, throughout the course was observed, and this was ligated into the cloning vector. To of sampling. Similar biofilms have been observed in mine tun- obtain 16S rRNA gene sequence data, 96 clones were chosen nels D and C (Fig. 1). from the bacterial primer clone library (BA library). Extremely DAPI-stained smears indicated that the slime and snottite effective cloning occurred, as 95 of 96 clones chosen had in- were predominantly biological, as opposed to mineralogical serted fragments of the correct size. These clones were given (Fig. 3). The biofilms were made up mostly of an extracellular the prefix BA. polymeric substance infused with spirillum-shaped cells (ap- Evidence from other investigations indicated that while the proximately 70% of the slime cells and 50% of the snottite Archaea-specific primers failed to amplify 16S rRNA genes cells) and small cocci (approximately 1 ␮m in diameter). Sed- from archaea present in the mine, the universal primer set did iment particles sampled from the base of the slime layer were work (14). Consequently, the universal primer set was used to VOL. 66, 2000 PHYLOGENY OF AN ACID MINE DRAINAGE BIOFILM 3845 Downloaded from http://aem.asm.org/ on November 11, 2015 by University of Queensland Library

FIG. 3. DAPI stain of the slump slime bioﬁlm occurring in the A drift. Curved and straight rods and cocci are evident.

obtain 16S rRNA gene sequences from the snottite, and 40 4). BA24 and SC28, representing four and three clones of their clones were analyzed from the snottite library. Of these, 39 had respective libraries, affiliated strongly with existing Leptospiril- fragments of the correct size that could be sequenced and were lum sequences, having Ͼ98% similarity to group II types. given the prefix SC. Clone sequences falling within the Nitrospira group made up Clones were grouped within each of the libraries according 76% of the total clone sequences. This result correlates some- to RFLP patterns and sequence similarity. Cloned inserts were what with the observed abundance of spirillum-shaped cells amplified by PCR and digested with restriction enzymes (see (Fig. 3). Considering problems associated with quantification Materials and Methods). RFLP banding patterns were clearly of microbial communities by PCR amplification of 16S rRNA distinguishable. The 95 BA clones consolidated into 18 groups, genes (46), this correlation could be fortuitous. However, the and the 39 SC clones reduced to 14 groups. Single-primer dominance of these clones likely reflects the abundance of sequencing was performed on representatives from the RFLP Leptospirillum in the sample. groups, and these ranged in length from 446 to 721 nucleo- Previously, phylogenetic analysis of Leptospirillum sequences tides. Sequence similarity then reduced the RFLP groups to defined two groups, I and II (16), that have approximately 93% eight representative BA sequences and eight representative SC 16S rDNA sequence similarity (Fig. 4). Results presented here sequences (Table 1). Efficacy of the grouping was confirmed by significantly extend the diversity of the Leptospirillum cluster partial sequencing of multiple representatives from the larger (Fig. 4). Sequences represented by group III clones have only RFLP groups. Partial sequences from the same groups were 89 to 93% similarity to sequences from both existing Leptospi- always greater than 99% similar. Only two clones were found rillum groups I and II. These likely represent a new group of to be strongly chimeric, and highest similarity scores of frag- organisms that are yet to be isolated in culture. ments occurred against sequences from this clone library. Considerable differences between the group III clone se- BA50 appeared to be a hybrid of clone types BA29 and BA46, quences and other Leptospirillum sequences are apparent. A and clone BA48 was possibly chimeric to fragments originating shorter stem in the V5 region is predicted by covariation anal- from sequence types BA29 and BA24. These possible chimeras ysis (23). Also, in the V3 region, from Escherichia coli base were excluded from further analyses. numbers 450 to 480, a string of base differences is apparent on Phylogenetic relationship of slime and snottite sequence both sides of the suggested stem structure. This includes nine types. The most abundant sequence type, representing some 69 consecutive tranversions from the Leptospirillum group II se- of the BA clones, was BA29. This sequence was positioned quence Lf30-A. Coincidentally, this sequence segment (differ- firmly in the Nitrospira group and formed a monophyletic ing V3 region) has been observed previously in a clone derived group (group III) with snottite clones SC17, SC02, and SC07 from an acidic bioleaching environment (21). While that se- (Fig. 4). These SC clones also collectively represented the quence was chimeric, it possibly indicates that Leptospirillum majority of the SC library. The group III clones clustered with group III types are not restricted to the Iron Mt. mine. Leptospirillum sequences from cultured organisms, and this After the Nitrospira-affiliated sequences, clone types cluster- topology was well supported by the phylogenetic analyses (Fig. ing with the Acidimicrobium group of the Actinomycete line of 3846 BOND ET AL. APPL.ENVIRON.MICROBIOL. Downloaded from http://aem.asm.org/

FIG. 5. Evolutionary distance dendogram of slime clones with Acidimicro- bium group sequences based on 1,107 nucleotides of 16S rDNA. Indications of branch point support and evolutionary distance are as described for Fig. 4. Database sequence accession numbers are as follows: Atopobium minutum, FIG. 4. Evolutionary distance dendogram of slime and snottite clones with M59059; Eubacterium lentum, AB011817; Microthrix parvicella, X89560; Rheims “Nitrospira” group sequences based on 781 nucleotides of 16S rDNA. Magneto- clone TM56, X92695; Rheims clone TM81, X92697; Rheims clone TM210, bacterium bavaricum was used as the outgroup (not shown). Branch points X92704; Acidimicrobium ferrooxidans TH3, M79433; Acidimicrobium ferrooxi- supported by distance, maximum-likelihood, and parsimony estimations (boot- dans ICP, U75647; Ferromicrobium acidophilus, unpublished data; and Rogers Ն strap values, 75%) are indicated by solid circles. Marginally supported branch clone TRA2-10, AF047642. on November 11, 2015 by University of Queensland Library points (supported by most phylogenetic analyses with bootstrap values of 50 to 74%) are indicated by open circles. Branch points without circles are not supported by the majority of analyses. Evolutionary distances are indicated by the sum of horizontal branch lengths. The scale bar represents changes per nucleo- the snottite clones (23%). These clones are only distantly re- tide. Database sequence accession numbers are as follows: Leptospirillum fer- Ͻ rooxidans L15, X86776; Rogers clone TRA1-10, AF047641; Goebel clone OS17, lated ( 93% sequence similarity) to other sequences within X86772; Leptospirillum sp. BU-1, M79383; Goebel clone OS4, X86770; Lepto- the Thermoplasmales group, and the phylogenetic position of spirillum sp. Lf30-A, X72852; and Nitrospira moscoviensis, X82558. these within the group could not be resolved further (Fig. 7).

DISCUSSION descent were the next most abundant of the BA clones (Table 1). BA46, representing 13 clones, was well positioned in the Microbial diversity and community variability in low-pH group and clustered with “Ferromicrobium acidophilus,” having biofilms. Previous culture-based analyses and some molecular 99% similarity to this (Fig. 5). Sequence BA84 was closely analyses have suggested that microbial populations from highly related to the Acidimicrobium group and affiliated with clone acidic environments tend to have low diversity and be domi- sequences originating from a peat bog (Fig. 5) (39), having 92 nated by culturable organisms (20, 48). Consequently, organ- to 93% similarity to these. This lineage contains only environ- isms implicated in AMD generation are typically the chemo- mental clone sequences from a number of different sites (39). lithoautotrophs T. ferrooxidans and L. ferrooxidans and a As yet no cultured organism of this group has been obtained. variety of acidophilic heterotrophs or facultative heterotrophs Sequence related to the Acidimicrobium group has been de- (29). Most of the clone sequences from this study affiliate with tected previously from Iron Mt. mine samples (16). However, recognized, cultured acidophiles. This was the expected result that sequence, TRA2-10, was not particularly closely related to and indicates that contamination was not a problem during those obtained here (Fig. 5). construction of the clone libraries. However, only clones from Sequences representing five slime clones were positioned Acidimicrobium and Leptospirillum, comprising 10% of the to- within the delta subdivision of the Proteobacteria (Fig. 6). Se- tal clone libraries, could be considered closely related to cul- quences BA18 and BA71 were most similar to each other tured organisms. Most of the sequences detected here repre- (91%) and ranged from 82 to 78% similarity with other rep- sent organisms that constitute new taxa. resentative delta Proteobacteria sequences. After the phyloge- New organisms have been detected at this site previously. netic analyses (Fig. 6), it was not possible to affiliate the clone An archaeon, “F. acidarmanus,” is the dominant prokaryote in sequences with any particular representatives of the subdivi- some submerged slime streamers. It also colonizes pyrite sur- sion. Nonetheless, to our knowledge, this is the first occasion faces associated with more concentrated solutions (14). In that microorganisms from the delta subdivision have been de- combination, this prior study (14) and our current results in- tected in such acidic environments. dicate that microorganisms not previously reported from AMD Other clone types detected only in the snottite library were sites probably contribute significantly to pyrite dissolution and those affiliating within the Thermoplasmales of the domain acid production. The detection of novel microorganisms could Archaea (Table 1 and Fig. 7). One clone, SC42, had high relate to the characteristics of the study site. Other investiga- sequence similarity with “F. acidarmanus.” This archaeon has tions have sampled AMD from regions peripheral to the sul- been isolated from and detected in other localities within the fide ore bodies, where the pH is higher and culturable bacteria Iron Mt. mine (14). Other SC clones grouped firmly within the such as T. ferrooxidans thrive (see the report by Schrenk et al. Thermoplasmales and constituted a considerable proportion of [43]). Furthermore, the novel microorganisms detected in this VOL. 66, 2000 PHYLOGENY OF AN ACID MINE DRAINAGE BIOFILM 3847 Downloaded from http://aem.asm.org/

FIG. 7. Evolutionary distance dendogram of snottite clones with representative sequences of the Thermoplasmales and relatives based on 870 nucleotides of FIG. 6. Evolutionary distance dendogram of slime clones within the delta 16S rDNA sequences. Sulfolobus solfataricus (not shown) was used as the out- subdivision of the Proteobacteria based on 1,121 nucleotides of 16S rDNA se- group. Indications of branch point support and evolutionary distance are as quences. Desulfovibrio desulfuricans (not shown) was used as the outgroup. In- described for Fig. 4. Database sequence accession numbers are as follows: Ther- dications of branch point support and evolutionary distance are as described for

moplasma acidophilum, M20822; Picrophilus oshimae, X84901; Ferroplasma aci- on November 11, 2015 by University of Queensland Library Fig. 4. Database sequence accession numbers are as follows: Bdellovibrio bacte- diphilum, AJ224936; Ferroplasma acidarmanus, AF145441; unidentified ar- riovorus, M59297; Myxococcus xanthus, M34114; Pelobacter acetylenicus, X70955; chaeon, AB019742; uncultured archaeon, AF068817; aquifer clone WCHD3-33, Desulfuromonas palmitatis, U28172; Desulfuromonas acetoxidans, M26634; AF050619; and Methanothermus fervidus, M32222. Geobacter metallireducens, L07834; Desulfobacter postgatei, M26633; Desulfobac- terium niacini, U51845; Desulfosarcina variabilis, M34407; Desulfobacterium ce- tonicum, AJ237603; Syntrophus gentianae, X85132; uncultured bacterium SJA-63, AJ009471; Desulfomonile tiedjei, M26635; Desulfobacca acetoxidans, AF002671; Syntrophobacter sp. X94911; and uncultured bacterium SJA-162, AJ009498. much less extreme environments (8, 18) in that the vast majority of these acidic biofilm clones affiliate with only a handful of species. In situ hybridization studies (7a; P. L. Bond, G. K. Druschel, and J. F. Banfield, submitted for publication) sup- study are not readily isolated and thus may have been over- port the conclusion that, at the scale of sampling, the subaerial looked previously. biofilms studied have a relatively simple microbial composi- Based on the proximity and general similarity in biogeo- tion. This is probably a consequence of a limited supply of chemical conditions of the slime and snottite, similar microbial electron donors and acceptors and the multiple challenges communities were expected. Group III type Leptospirillum se- presented by the very high hydrogen ion activity, high metal quences predominate in both libraries. However, Thermoplas- activities, and elevated temperature. males-related clones were only detected in the snottite. It is Metabolic inferences and microbial ecology. Although mi- important to note that the primer sets used to construct librar- crobial diversity in individual samples is low, microbial growth ies from the slime would amplify the 16S rRNA genes from this is prolific. Water entering the mine workings is provided by group poorly. Thus, it is probable that Thermoplasmales-re- precipitation on a steep, moderately vegetated hillslope a few lated archaea are present in the slime as well. Also, a greater hundred feet above the ore body. The levels of organic carbon diversity of bacterial clones was detected in the slime library, in mine solutions are low and are attributed to degradation of suggesting a more complex community there (however, this lithotrophic cells and not to externally derived organic carbon. result may be somewhat biased by the size of the clone library Consequently, carbon fixation in this ecosystem is based on analyzed). Although the snottites and slimes are exposed to chemolithoautotrophic oxidation of iron and sulfur compounds. essentially the same solutions, some differences in chemical Although microorganisms of the biofilms have not been iso- conditions could occur and result in variation in the microbial lated, it may be possible to predict their metabolic capabilities population compositions. The snottites are exposed to a con- based on those of phylogenetically related organisms. We con- tinuous flow of solutions in contact with air, whereas slimes sider this valid because the organisms tend to fall within clus- form a more coherent biofilm and associated fluids are prob- ters that utilize the same substrate(s) and have distinct, shared ably less oxygenated. Thus, there is the possibility that micro- physiological characteristics. Consequently, we use phylogeny- environments exist within the slime for the support of anaer- based metabolic and physiological inferences to correlate geo- obes and microaerophilic bacteria. chemical with microbial characteristics so as to develop an Annual changes in microbial population compositions have initial understanding of the ecology of these environments. been observed at specific sites within the mine (43), and these Leptospirillum species are acidophilic, gram-negative bacte- variations show a strong correspondence with ambient physical ria that are morphologically helical curved rods (37). Different and chemical conditions. Thus, it appears that microorganisms types of Leptospirillum have been detected in different acidic are finely adapted to suit specific temperature-, pH-, or solu- environments. Leptospirillum has been formally recognized as tion chemistry-defined niches. The clone library reported in a coherent bacterial genus (25a), and its members form a phy- this study is distinct from those in cloning studies of other, logenetically distinct cluster within the taxon Nitrospira (32). 3848 BOND ET AL. APPL.ENVIRON.MICROBIOL.

Genomic diversity, as shown by DNA hybridization analyses, play a similar synergistic role in the biofilm microbial commu- has been demonstrated for some isolates (24). Based on phy- nities described here. logeny, two possibly genus-level groups were suggested initially Clones falling within the delta Proteobacteria could represent (16). These are referred to here as group I (the original isolate) anaerobic sulfate-reducing heterotrophs, as sulfate reduction is and group II. Groups I and II are autotrophic (4), oxidize iron the dominant physiological trait of this group (13). They could for energy (7, 37), and have optimum growth temperatures of also be iron-reducing bacteria, as similar anaerobic respiration 26 to 30°C (group I) and 30 to 40°C (group II) (19). Group III involving metal (iron) reduction has also been detected in this sequences (this paper) have low similarity with the existing group (30, 33). As well, the clones may represent aerobic sequences and probably represent a new genus within the Lep- bacteria of the Myxobacteria and Bdellovibrio genera. These Downloaded from tospirillum. Because Leptospirillum group III clusters with genera also fall into the delta Proteobacteria. Although sulfate groups I and II, we suggest that group III members are also and ferric iron concentrations are high in solutions, sulfate autotrophic iron oxidizers. An additional species, referred to as and/or iron reducers have not been previously observed in such Leptospirillum ferrooxidans, has a higher (ϳ45 to 50°C) tem- acidic environments. It is possible that local concentrations of perature optimum (22). However, the phylogenetic placement bioavailable carbon overlap with oxygen-limited microenviron- of L. thermoferrooxidans, and thus its relationship to group III ments and that iron- or sulfate-reducing microbes can operate organisms, is not known. The predominance of the Leptospi- in more highly acidic environments than has been previously rillum clones (78%) likely reflects the fact that the prevalent recognized. Within the microbial consortium, sulfate- or iron- metabolism within the biofilms is iron oxidation. Phenotypic reducing heterotrophs could have the same synergistic effect as http://aem.asm.org/ variability of Leptospirillum groups requires further consider- mentioned for Acidimicrobium and “Ferromicrobium.” ation. Implications for future studies. Presently there is some un- It was suggested that group I Leptospirillum organisms are derstanding of metabolic and physiological characteristics of the less abundant type (21). Our initial work at Iron Mt. mine acidophiles pertinent to AMD production (29). However, the detected only group I type Leptospirillum associated with sed- results of this study emphasize that understanding of microbial iments and pore fluids (16). However, in the biofilms, only diversity and proportions comprising communities where sequences from group II and group III types were detected. AMD production occurs is incomplete, if not inaccurate. Ul- We speculate that group III is distinct in that it is optimized for timately, we aim to present a more complete picture of micro-

different conditions. Confirmation of the factors controlling bially mediated AMD production. At this point it is not ap- on November 11, 2015 by University of Queensland Library distribution of Leptospirillum species awaits in situ character- propriate to use the sequence data to quantify relative ization of their habitats in a variety of acidic environments. proportions of taxa within these samples. Variation in the Sequence data reported here have important implications ability of the primers to amplify target genes during PCR will for the development of oligonucleotide probes for character- bias the pool of sequences obtained. An extreme case of this ization of microbe distributions in environmental samples. was observed here when archaeal genes were not amplified Prior studies used a Leptospirillum group probe (LF581) de- with the Archaea-specific primers, but these were obtained veloped on the group I and II sequences available at the time using the universal primers. More importantly, the sequence (43). However, two mismatches occur between the target re- data obtained here will be used to design specific oligonucle- gion for LF581 and group III Leptospirillum. Under the hy- otide probes for in situ quantification of the microbial com- bridization conditions used here, group III Leptospirillum cells munities. Results of this work will be reported separately (7a; would not be detected with LF581. Comparison of the se- Bond et al., submitted). quence data indicates that probes could be developed for each Understanding of the ecology of highly acidic environments group, as well a probe for all Leptospirillum species. This would requires investigation of metabolic pathways and understand- allow resolution of the geochemical habitats for group I versus ing of microbial survival strategies. In the long term, an assay group II versus group III Leptospirillum. for the level of iron oxidation in the environment is needed. In Clones that positioned in the Archaea were placed in the order to develop such an assay, understanding of the diversity Thermoplasmales and, except for SC42, were only distantly of iron-oxidizing organisms and the biochemistry of their met- related to known organisms. Thermoplasmales lack a cell wall abolic pathways is critical. Thus, culturing, isolation, and char- and are hyperacidophilic (42, 44). Most Thermoplasmales are acterization of newly detected species are essential. Results of aerobic heterotrophs. However, T. acidophilum can respire this study provide information about species habitats that can anaerobically using sulfur compounds other than sulfate (44). be used to direct future isolation and culturing strategies. “F. acidarmanus” is an iron oxidizer that is almost certainly autotrophic as well as heterotrophic (14). The detection of new ACKNOWLEDGMENTS Thermoplasmales taxa extends our understanding of archaeal We thank Norman Pace (UC Berkeley) and Michael Tanner for biodiversity in acid microbial communities and emphasizes the assistance with the ARB software and for helpful discussion. We also probable importance of these organisms in AMD production. thank Brett Goebel and Katrina Edwards for ongoing discussion of Both Acidimicrobium and “Ferromicrobium” species are acidophile diversity and many helpful suggestions. Thomas Gihring iron-oxidizing, heterotrophic, acidophilic bacteria. In this en- and Greg Druschel are thanked for their help with sample collection. vironment their source of organic carbon is almost certainly This project received NSF support via a supplement to grant CHE the slime-associated biomass. Acidimicrobium ferrooxidans is 9521731, as well as support from NSF CHE 9807598. Assistance from capable of autotrophic growth (10). However, “Ferromicro- Bob Hamers is also gratefully acknowledged. bium acidophilus” requires organic carbon (3). Strains of A. REFERENCES ferrooxidans and “F. acidophilus” also possess iron-reducing 1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. H. Zhang, Z. Zhang, W. capabilities, using ferric iron as an electron acceptor during Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new low redox potential (3, 9). From its phylogenetic placement, generation of protein database search programs. Nucleic Acids Res. 25: BA46 may represent iron-oxidizing, heterotrophic bacteria. It 3389–3402. 2. Amann, R. I., L. Krumholz, and D. A. Stahl. 1990. Fluorescent-oligonucle- has been suggested that heterotrophic growth of “F. acidophi- otide probing of whole cells for determinative, phylogenetic, and environ- lus” removes dissolved organic carbon inhibitory to coexisting mental studies in microbiology. J. Bacteriol. 172:762–770. autotrophs (3). Acidimicrobium and “Ferromicrobium” may 3. Bacelar-Nicolau, P., and D. B. Johnson. 1999. Leaching of pyrite by acido- VOL. 66, 2000 PHYLOGENY OF AN ACID MINE DRAINAGE BIOFILM 3849

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