Microbial Community Composition in Acid Mine Drainage Lake of Xiang

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Geomicrobiology Journal Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ugmb20 Microbial Community Composition in Acid Mine Drainage Lake of Xiang Mountain Sulfide Mine in Anhui Province, China Chunbo Hao a b , Lina Zhang a b , Lihua Wang a b , Siyuan Li a b & Hailiang Dong a c a Geomicrobiology Laboratory, State Key Laboratory of Geobiology and Environmental Geology, China University of Geosciences, Beijing, China b School of Water Resources and Environment, China University of Geosciences, Beijing, China c Department of Geology and Environmental Earth Science, Miami University, Oxford, Ohio, USA Accepted author version posted online: 18 Apr 2012.Version of record first published: 14 Sep 2012.

To cite this article: Chunbo Hao , Lina Zhang , Lihua Wang , Siyuan Li & Hailiang Dong (2012): Microbial Community Composition in Acid Mine Drainage Lake of Xiang Mountain Sulfide Mine in Anhui Province, China, Geomicrobiology Journal, 29:10, 886-895 To link to this article: http://dx.doi.org/10.1080/01490451.2011.635762

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Microbial Community Composition in Acid Mine Drainage Lake of Xiang Mountain Sulﬁde Mine in Anhui Province, China

Chunbo Hao,1, 2 Lina Zhang,1, 2 Lihua Wang,1, 2 Siyuan Li,1, 2 and Hailiang Dong1, 3 1Geomicrobiology Laboratory, State Key Laboratory of Geobiology and Environmental Geology, China University of Geosciences, Beijing, China 2School of Water Resources and Environment, China University of Geosciences, Beijing, China 3Department of Geology and Environmental Earth Science, Miami University, Oxford, Ohio, USA

INTRODUCTION Acid mine drainage (AMD) lake of Xiang Mountain in An- Metalliferous mining activity has produced large quantities hui Province, China, was characterized by acidic waters (pH of sulfidic mineral wastes (such as pyrite or arsenopyrite) all around 2.8) containing high concentrations of soluble metals and over the world (Rowe et al. 2007). These mine wastes are con- sulfate. To investigate the function and dynamics of this extreme ecosystem, four water samples were collected from the tinuing sources of environmental contamination, mostly in the lake in the fall of 2010. The acidophilic community structure form of acid mine drainage (AMD), which is usually character- was analyzed by molecular approaches, and bacterial and ar- ized by low pH (below 3.0). Such waste waters generally contain chaeal clone libraries of 16S rRNA genes were constructed. In high levels of iron, toxic metals such as aluminum, manganese, contrast to dominance of chemolithotrophic acidophiles in typi- lead, cadmium, and zinc, as well as metalloids, of which ar- cal AMD environments, autotrophic iron/sulfur-oxidizing bacteria were detected in only one sample with low abundance. Un- senic is generally of the greatest concern (Bruneel et al. 2011). expectedly, the Cyanobacteria group was the predominant in all AMD is generated when waste sulfide ores come into contact four samples (54.9%−77%). Chemoheterotrophs Acidiphilium and with oxygenated water. The process can be greatly enhanced by Acidisphaera were also abundant. These two heterotrophic groups microbial activity because many acidophilic microorganisms contain bacteriochlorophyll that can perform photosynthesis, an can accelerate dissolution of sulfide minerals through oxidation advantage to grow and survive in such oligotrophic acidic environments. Only two clone sequences related to Legionella (2.8% of of ferrous iron and/or reduced forms of sulfur (Johnson and the total clones) were recovered from one sample in sharp contrast Hallberg 2003). to its higher abundance (12.7%) in the summer of 2009. All ar- The culture-independent molecular techniques for studying chaeal sequences were affiliated to the phylum Crenarchaeota.The microbial ecology has been well established and applied world- results of statistical analysis suggested that the water chemistry wide in the last decade. Many researchers have employed molec- of the AMD lake controlled microbial composition of the AMD

Downloaded by [Zhejiang University] at 17:31 11 April 2013 ecosystem. ular techniques to analyze acidophile populations and have described microbial community structures in various acidic en- Keywords acid mine drainage, acidophile, 16S rRNA, clone library, vironments (Demergasso et al. 2005; Gonzalez-Toril et al. 2003; Cyanobacteria Hao et al. 2010a; Rzhepishevska et al. 2005). The power of these techniques lies in their ability to identify and quantify microorganisms without cultivation, which may lead to biased conclusions about the structure and dynamics of microbial com- Received 19 July 2011; accepted 21 September 2011. munities. By the application of these cultivation-independent This research was supported by grants from the National Sci- molecular techniques, our understanding of the biodiversity of ence Foundation of China (40802059, 41030211), the Specialized Re- search Funds for the Doctoral Program of Higher Education of China acidophiles has been rapidly expanding, which has given more (200804911008), and the Fundamental Research Funds for the Central comprehensive insights into the role of acidophilic microorgan- Universities (2010ZD03, 2011YXL035). The authors are grateful to isms in mediating elemental cycling in acidic ecosystems (Bond two anonymous reviewers whose comments improved the quality of et al. 2000; Bruneel et al. 2006; Hallberg et al. 2006; He et al. the manuscript. 2007). Address correspondence to Hailiang Dong, Geomicrobiology Lab- oratory, State Key Laboratory of Geobiology and Environmental Geol- In a previous study, we characterized the planktonic aci- ogy, China University of Geosciences, Beijing 100083, China. E-mail: dophilic community in a water sample collected from the AMD [email protected] lake, Anhui Province, China, in the summer of 2009 using

886 MICROBIAL COMMUNITY IN ACID MINE DRAINAGE LAKE 887

the molecular approaches, and found that heterotrophic aci- The geochemical samples were kept at 4◦C and shipped to dophiles were dominant (>50% of total bacterial population) the Geomicrobiology Laboratory of China University of Geo- while autotrophic organisms were scarce. Paucity of ferrous sciences in Beijing on the same day. The following analyses iron and turbid water may have accounted for low abundance were conducted within 2 days of collection. For sulfate and of chemoautotrophic and photosynthetic organisms. Abundant metal ions, the water samples were filtered through 0.45 µm heterotrophic acidophiles may be due to input of allochthonous filter prior to analyses. Sulfate concentration was determined dissolved organic carbon from soils and grass community in by ion chromatography; dissolved metals by inductively cou- the bank of the AMD lake (Hao et al. 2010b). Moreover, it pled plasma-atomic emission spectroscopy (ICP-AES, JYl- was unexpected that many sequences of Legionella, which can ULTIMA, France); dissolved ferrous iron titrated with ceric cause Legionnaires’ disease, were also detected in the AMD lake sulfate. Total organic carbon (TOC) was measured by an or- (Hao et al. 2010b). The high abundance of Legionella-related ganic carbon analyzer (Shimadzu, Japan). Total nitrogen (TN), + sequences was ascribed to the presence of a large number of total phosphorous (TP), and NH4 -N were determined in ac- protozoa in eukaryotic population. However, it was unclear if cordance with standard methods (Ministry of Environmental this community structure was transient in nature or stable over Protection 2002). long periods of time. The microbial samples (2.5 liter in volume in sterile plastic The objective of this study was therefore to examine if the bottles) were kept on regular ice for one day before they were community structure observed in the summer of 2009 remained filtered through a Millex-GS Millipore filter (pore size, 0.22 µm; stable over time. If the microbial structure was different, what diameter, 50 mm) in the lab. The filters were used immediately were the environmental conditions that were responsible for the for extraction of microbial genomic DNA. change? Similar molecular methods as those used in our earlier study (Hao et al. 2010b) were employed in this study. Our results DNA Extraction and Purification showed that the microbial community structure in the fall of The method used for extraction of nucleic acids from the 2010 was markedly different, and this change was apparently AMD samples was based on a protocol described previously caused by changes of the physicochemical conditions of the (Zhou et al. 1996) and included initial washing steps in order to AMD lake. remove any iron and to raise the pH to 8.0 prior to cell lysis. Each biomass-containing filter was cut and placed into a 15-ml Falcon tube. After thawing, the filter was washed twice with 5 ml 0.02 M MATERIALS AND METHODS H2SO4 and then with 5 ml STE buffer (sucrose 100 mg/ml, Tris·HCl 50 mM, EDTA 10 mM, NaCl 100 mM, pH 8.0). The Field Site Description samples were subsequently resuspended in 3 ml DNA extraction The study site is located at the Xiang Mountain, Anhui buffer (sucrose 100 g/L, Tris·HCl 50 mM, EDTA 10 mM, NaCl Province, China, and the climate of this region is subtropical 100 mM, 10 g/L CTAB, pH 8.0) followed by addition of 50 µl with an annual rainfall of about 1080 mm, mainly in summer. lysosyme (10 mg/L). This mixture was incubated at 37◦Cfor The dominant ore body of Xiang Mountain is composed of 40 min. pyrite (more than 90%), which has been mined for more than After incubation, 50 µL Proteinase K (10 mg/ml) and 1 ml 50 years. Waste products are disposed of in a nearby refuse 10 g/L SDS solutions were added to the mixture followed by dump. The exposed waste ore minerals are oxidized by aci- ◦ 3+ + 2− incubation at 50 C for 45 min. The supernatants were col- Downloaded by [Zhejiang University] at 17:31 11 April 2013 dophiles and large amounts of Fe ,H and SO4 are released, lected after centrifugation at 6,000×g for 10 min at room tem- which have resulted in the formation of an AMD lake since perature and transferred into new centrifuge tubes. The cell 1970s. The AMD lake is approximately 700 m long and 300 m lysates were extracted twice with an equal volume of chlo- wide. roform:isoamylalcohol (24:1, vol/vol). The nucleic acids were precipitated from the aqueous phase with 2 volumes of ethanol Geochemical Measurements and Sample Collection at −20◦C for 2 h. The pellet of nucleic acids was obtained by Temperature, pH, redox potential, dissolved oxygen (DO), centrifugation at 12,000×g for 20 min at 4◦C, washed with and total dissolved solids (TDS) of the lake water were measured ice-cold 70% ethanol, and dissolved in 50 µl sterile deionized in situ using a Hanna field analyzer (VWR, UK). Based on these water. measurements and in order to investigate the spatial variation of planktonic acidophilic community around the lake, four surficial water samples, named as AMD-1, AMD-2, AMD-3 and AMD- Polymerase Chain Reaction (PCR) Amplification 4, from the four corners of the lake were collected into 2.5-liter and Clone Library Construction bottles for geochemical and microbial analyses. AMD-2 was The 16S rRNA gene fragments of bacteria and archaea were collected from the same location as the sample studied in 2009 amplified with PCR system C1000 (Bio-Rad, USA). In the reac- (Hao et al. 2010b). The four sampling locations were about 0.5 m tions, bacterial primer pairs were universal 27F (5-AGA GTT away from the lake edge, and the water depths were 0.4–0.6 m. TGA TCM TGG CTC AG-3,M= C or A) and 1492R (5-TAC 888 C. HAO ET AL.

GGY TAC CTT GTT ACG ACT T-3,Y= C or T). Archaea- Phylogenetic Analysis specific primers were Arch-21F (5 -TTC YGG TTG ATC CYG The clone sequences obtained in this study were compared CCR GA-3 ,R= A or C) and Arch-958R (5 -YCC GGC GTT with the 16S rRNA gene sequences deposited in GenBank using GAM TCC AWT T-3 ,W= A or T). PCR amplification con- the BLAST search program. The 16S rRNA gene sequences ditions were the same as previously described (Bruneel et al. of various microbes obtained from the GenBank database were 2006; Yin et al. 2008). aligned with the new sequences using BioEdit 7.0. Phylogenetic PCR products were purified using the EZ-10 spin column trees were constructed in MEGA 4.0 by the neighbor-joining DNA Gel Extraction kit (BBI, Canada), quantified by Nan- method with robustness of 1000 bootstrapping value. odrop (Thermo, USA), and ligated into the pGEM-T easy vector (Promega, USA). The resulting plasmids were transformed Statistical Analysis into competent E. coli DH5α cells following the manufacturer’s The similarities in the compositions of the bacterial and ar- instructions and then four bacterial and four archaeal clone li- chaeal clone libraries between the summer of 2009 and the fall braries were constructed. To select the positive clones, the in- of 2010 were examined by using the S-LIBSHUFF program as serted fragment in the competent E. coli cells was amplified by described elsewhere (Jiang et al. 2006; Schloss et al. 2004). PCR. Template DNA was provided by contact of a small pipette Principal-component analysis (PCA) was performed by using tip with a colony of cloned host cells which were immersed in the SPSS 13.0 program to group the four water samples collected the PCR mixture. Primers for the PCR were the vector-specific in 2010, which were either similar or different, based on the T7 (5 -TAA TAC GAC TCA CTA TAG GGC-3 ) and SP6 (5 - physicochemical parameters. Similarly, the correlation analysis ATT TAG GTG ACA CTA TAG AAT ACT C-3 ). The clones was also performed to the biological parameters, and the relative that generated 1.7 kb PCR product were selected to sequence. percentage of a given operational taxonomic units (OTU) in the The sequences of the eight clone libraries were determined by total number of OTUs was used as variables. These variables Shanghai Sangon Co., Ltd., China. were linearly combined with the eigenvectors of the correlation The clone sequences were checked for chimeric artifacts by matrix to generate a principal component axis. the program Bellerophon (Huber et al. 2004) and discarded if discovered. The obtained sequences were aligned and calculated Nucleotide Sequence Accession Numbers to generate distance matrix, which was used as the input file to Sequences reported in this paper have been submitted to the DOTUR program that can assign sequences to phylotypes GenBank with accession numbers JF817220-JF817253 and or operational taxonomic units (OTUs) for every possible dis- JF925133. tance (Schloss and Handelsman 2005). In this study, any 16S ≥ rRNA gene sequences with 97% similarity were assigned to RESULTS the same OTU. Rarefaction curves and Shannon-Weaver diversity index were also calculated by DOTUR. Dominance in Aqueous Geochemistry clone libraries was calculated as 1-Shannon evenness index (1- The geochemical properties of the four AMD waters around E), where evenness (E) is represented as follows: E = H/ln S the lake were similar (Table 1). The temperature and pH of the (H = Shannon-Weaver diversity index; S = total richness in a four AMD water samples was ∼20◦C and 2.8–2.9, respectively. sample). The acidic waters contained high levels of total dissolved Fe

TABLE 1 Downloaded by [Zhejiang University] at 17:31 11 April 2013 Physicochemical characteristics of the AMD samples Temperature DO TDS TOC TIC TN TP Sample (◦C) pH (mV) (mg/L) (g/L) (mg/L) (mg/L) (mg/L) (mg/L) AMD-1 20.22.9 583.26.53 4.02 6.41.018.20.59 AMD-2 19.92.8 547.65.65 4.04 5.31.119.40.71 AMD-3 20.62.9 573.17.42 4.03 2.91.018.50.85 AMD-4 21.02.8 565.86.21 4.03 4.71.120.40.60 2+ 2− Salinity Fetot Fe Al Ca Cu Mn Na SO4 Sample (%) (mg/L) (mg/L) (g/L) (g/L) (mg/L) (g/L) (mg/L) (g/L) AMD-1 5.057.00.41.02 0.52 48.00.24 41.21.09 AMD-2 5.057.50.70.85 0.43 40.50.21 40.01.11 AMD-3 5.048.00.50.82 0.48 38.80.21 43.61.07 AMD-4 5.054.70.60.85 0.43 42.70.22 42.21.16 MICROBIAL COMMUNITY IN ACID MINE DRAINAGE LAKE 889

TABLE 2 respectively, and they were given the prefix “Bac.” The diver- Diversity indices calculated for the four bacterial clone sity rarefaction curves of the four bacterial clone libraries all libraries constructed from the four water samples from Xiang reached a plateau (data not shown), indicating that the num- Mountain mine, Anhui Province, China ber of clones sequenced was sufficient to cover the bacterial diversity. This observation was further supported by the high Clone Total Number Coverage Shannon coverage values (between 87.3% and 97.0%) for the four clone library clones of OTUs (C) (H) Dominance libraries. AMD-1 71 15 87.3% 1.76 0.35 The number of OTUs was 15, 8, 8, and 10 for AMD-1, AMD- AMD-2 63 8 93.7% 1.19 0.43 2, AMD-3 and AMD-4, respectively (Table 2), suggesting that AMD-3 66 8 97.0% 1.19 0.43 sample AMD-1 contained most diverse bacteria among the four AMD-4 61 10 90.2% 1.18 0.49 water samples. This result was consistent with the fact that the AMD-1 library showed higher diversity index (Shannon: 1.76) and lower Dominance (0.35) than the other three libraries (Shan- non ranging from 1.18 to 1.19; Dominance from 0.43 to 0.49). (48–57.5 mg/L) and sulfate (1.09–1.16 g/L) with high Eh values The AMD-4 library exhibited the highest Dominance (0.49) (565.8–583.2 mV). The dissolved oxygen level of the four water among the four bacterial libraries (Table 2), which provided ev- samples ranged from 5.7 to 7.4 mg/L, indicating the AMD was + idence that some species had more dominant status than others aerated. The Fe2 concentration was low, ranging from 0.4 to in this sample. 0.7 mg/L, suggesting that ferric iron was the dominant form of dissolved iron in this water body. The concentration of Al was the highest among all measured metals and was as high as Phylogenetic Analysis of Bacterial Sequences 1.02 g/L in AMD-1, which suggested that some Al-rich minerals The 16S rRNA gene sequences of the bacterial libraries fell may be associated with pyrite in the tailing and dissolved in the into nine phylogenetic divisions: Alphaproteobacteria, Betapro- acidic water. teobacteria, Gammaproteobacteria, Actinobacteria, Acidobac- Concentrations of Ca, Mn, Cu and Na were 0.43–0.52 g/L, teria, Bacteroidetes, Firmicutes, Cyanobacteria and Unclassi- 0.21–0.24 g/L, 38.8–48.0 mg/L, and 40–43.6 mg/L, respec- fied bacteria, four of which (Alphaproteobacteria, Actinobacte- tively. Among the four samples analyzed, TOC of AMD-1 was ria, Cyanobacteria and Unclassified bacteria) were ubiquitous the highest (6.4 mg/L), while the other three samples had TOC in all four samples. levels below 5.3 mg/L. TN, TP, and TDS of the four samples The Cyanobacteria-related sequences were the most abun- were 18.2–20.4 mg/L, 0.59–0.85 mg/L and 4.02–4.03 g/L, re- dant in all four bacterial 16S rRNA gene libraries, which ac- spectively. The salinity of AMD at Xiang Mountain was about counted for 54.9%, 66.7%, 74.3% and 77% of all sequences 5%. in AMD-1, AMD-2, AMD-3 and AMD-4, respectively (Fig- ure 1). The majority of the Cyanobacteria sequences, repre- Analysis of the Bacterial 16S rDNA Clone Libraries sented by Bac-1-13, were related to the cyanobacteria clones After removal of several chimeric sequences, 71, 63, 66, 61 detected in AMD, hot spring and Tibetan Lake (Figure 2). The valid sequences (1450 bp in length) were used to construct bac- Alphaproteobacteria group constituted the second largest group terial clone libraries for AMD-1, AMD-2, AMD-3 and AMD-4, (Figure 1). The abundant sequence type Bac-1-11 formed a Downloaded by [Zhejiang University] at 17:31 11 April 2013

FIG. 1. Relative proportions of major groups of bacteria in the four bacterial clone libraries. The proportion was calculated from the number of clone sequences belonging to a particular group divided by the total number of clone sequences. 890 C. HAO ET AL. Downloaded by [Zhejiang University] at 17:31 11 April 2013

FIG. 2. Neighbor-joining phylogenetic tree based on analysis of the 16S rRNA gene sequences of bacterial clones obtained from the AMD lake at Xiang Mountain in relation to reference sequences from the GenBank database. The sequences obtained in this study are indicated in bold. Scale bars indicate the Jukes-Cantor distances. Bootstrap values of >50% (for 1000 iterations) are shown. MICROBIAL COMMUNITY IN ACID MINE DRAINAGE LAKE 891

subdivision (Figure 2) with the Acidiphilium species, which con- cultures, but is not able to oxidize reduced forms of sulfur (John- sists of mixotrophic acidophiles capable of chemoorganotrophic son et al. 2009). The other two sequence types, Bac-1-23 and and photosynthetic metabolisms. Sequence type Bac-1-5, only Bac-1-39, were only detected in sample AMD-1, both of which detected in water sample AMD-1, was also related to genus clustered with heterotrophic acidophiles isolated from various Acidiphilium. sulfidic mine waste dumps (Figure 2). Sequence types Bac-1-10, Bac-1-22, and Bac-1-63 formed a One sequence type, Bac-1-90, from sample AMD-1 was af- cluster within the Alphaproteobacteria, and they were closely filiated with the Acidobacteria representing 1.4% of the total related (>97% similarity) to Acidisphaera sp. nju-AMDS1, clones. The sequence formed a branch with heterotrophic aci- which is an acidophilic heterotroph isolated from the sediment dophile Acidobacterium capsulatum.TheAcidobacterium spp. of an extremely acidic river (pH 2.81–2.93) in Jiguanshan mine are gram-negative, aerobic, mesophilic, non-sporeforming, cap- at Tongling, Anhui Province, China. The sequences related to sulated, saccharolytic, and rod-shaped bacteria. They can grow genus Acidisphaera were recovered from AMD-1, AMD-3, and between pH 3.0 and 6.0. They can use a variety of organic matter AMD-4, and their relative proportion was 16.9%, 3% and 1.6%, as the sole carbon source, but cannot use elemental sulfur and respectively. Sequence type Bac-1-64 related to a heterotrophic ferrous iron as energy source. acidophile, Acidocella was detected in all four water samples Certain sequences from all four samples could not be affili- and accounted for about 1.5%. Unlike Acidiphilium and Acidis- ated with any known phylogenetic groups. Bac-1-16 accounted phaera, Acidocella species does not contain photosynthetic pig- for 9.9%, 9.5%, 4.5% and 8.2% of all clones in the four clone ments and carotenoids. libraries. This sequence type had a low identity with most se- The Betaproteobacteria were detected only in sample AMD- quences in the GenBank (<90%). Its closest relative (95% sim- 1 and represented by only one sequence type, Bac-1-78, ac- ilarity) is an uncultured bacterium clone YTW-18–06 detected counting for 1.4% in the clone library. The sequence was af- in AMD at Dexing copper mine in Jiangxi Province, China. filiated with Ferrovum myxofaciens, which was isolated from an abandoned copper mine (Hallberg 2010). The species desig- Analysis of Archaeal 16S rDNA Clone Libraries nated as Ferrovum myxofaciens in Genbank (accession number After removal of several chimeric sequences, 17, 35, 18 and EF133508) has not been formally described, and it can not be 34 valid sequences were obtained for the AMD-1, AMD-2, phylogenetically associated with cultivated acidophiles. Limited AMD-3 and AMD-4 archaeal clone library, respectively. These physiological characterization carried out to date for this isolate sequences were grouped into 14 OTUs based on 97% sequence confirmed that it can solely use ferrous iron as an electron donor, similarity, and they were given the prefix “Arc.” The distribution has an obligately autotrophic metabolism, and appears to be less of the 14 OTUs was 3 in AMD-1, 8 in AMD-2, 5 in AMD-3 and acid tolerant than well-studied species Leptospirillum ferroox- 6 in AMD-4 (Table 3). idans and Acidithiobacillus ferrooxidans (Rowe and Johnson The rarefaction curves were nearly saturated (data not 2008). Its conspicuous occurrence has been reported in metal- shown), consistent with the high coverage values (between rich mine waters in North Wales and a mine water treatment 88.9% and 97.1%) for all four archaeal libraries. The AMD- plant in Germany (Hallberg et al. 2006; Heinzel et al. 2009). 2 library exhibited the highest diversity index (Shannon: 1.35) Similar to the Betaproteobacteria, the phylum Gammapro- and Dominance (0.35) among the four archaeal clone libraries teobacteria included just one sequence type, Bac-1-33, which (Table 3). was present in sample AMD-1 only. Sequence Bac-1-33 represented 2.8% of the total clones and clustered with Legionella Downloaded by [Zhejiang University] at 17:31 11 April 2013 species in the phylogenetic tree (Figure 2). Legionella species Phylogenetic Analysis of Archaeal Sequences are facultative intracellular gram-negative bacilli and ubiquitous All the archaeal sequences were affiliated with the phylum in natural and man-made aqueous environments. Some species Crenarchaeota, whereas Euryarchaeota was not present. The of genus Legionella can cause Legionnaires’ disease, a severe most abundant archaeal sequence type, Arc-1-1, represented and sometimes deadly pneumonia, the fatality rate of which can approach as high as 50% in immuno-compromised patients TABLE 3 (Diederen 2008). Diversity indices calculated for the four archaeal clone Of the three sequence types identified within the Actinobac- libraries constructed from the different water samples from teria, Bac-1-71 was ubiquitous in all four samples (1.4%, Xiang Mountain mine, Anhui Province, China 4.8%, 3.0% and 1.6% of clones in AMD-1, AMD-2, AMD- 3 and AMD-4, respectively). This sequence type was affiliated Clone Total Number Coverage Shannon with iron-oxidizing acidophile Ferrithrix thermotolerans strain library clones of OTUs (C) (H) Dominance Y005, which was isolated from a geothermal site in Yellowstone AMD-1 17 3 94.1% 0.85 0.23 National Park, USA. This species is an obligately heterotrophic AMD-2 35 8 91.2% 1.35 0.35 and moderately thermophilic and grows as long filaments, form- AMD-3 18 5 88.9% 1.27 0.21 ing macroscopic flocs in liquid media. This organism can ac- AMD-4 34 6 97.1% 1.25 0.30 celerate oxidative dissolution of pyrite in yeast extract-amended 892 C. HAO ET AL.

FIG. 3. Neighbor-joining phylogenetic tree based on analysis of the 16S rRNA gene sequences of archaeal clones obtained from the AMD lake at Xiang Mountain in relation to reference sequences from the GenBank database. The sequences obtained in this study are indicated in bold. Scale bars indicate the Jukes-Cantor distances. Bootstrap values of >50% (for 1000 iterations) are shown.

58.8%, 54.3, 50.0% and 61.8% of all archaeal clones in AMD- Sequence type Arc-2-10 was the second most abundant type

Downloaded by [Zhejiang University] at 17:31 11 April 2013 1, AMD-2, AMD-3 and AMD-4, respectively. This sequence (22.7% of the total clones) in the AMD-2 archaeal clone library type had 98% homology with uncultured archaeon SAGMA- and had a relatively close relationship (near 97% similarity) 11 from waters in South African deep gold mines. The se- to Arc-1-10. These two sequence types formed a large clade quence type formed a cluster with Arc-2-21 (5.9% in both with related sequences from the GenBank database. Another AMD-2 and AMD-4 clone libraries) in the phylogenetic tree important sequence type was Arc-3-3, which was only detected (Figure 3). in sample AMD-3 accounting for 27.8% of the total clones. Sequence type Arc-1-10 was the second most abundant Arc-3-3 had a high identity with sequences from the acidic (35.3% of the total clones) in the AMD-1 archaeal clone li- subsurface water contaminated by AMD in the Kalahari Shield, brary, which was also detected in the sample AMD-2 with only South Africa and hot spring of the Colombian Andes. 2.9% of the total clones. Its closest relative (97% similarity) in the GenBank database was recovered from the acidic sub- Statistical Analysis surface water contaminated by AMD in the Kalahari Shield, S-LIBSHUFF analysis was used to characterize the relation- South Africa. The remaining sequence type of sample AMD-1, ships among the different AMD bacterial populations from the ARC-1-5, also appeared in AMD-2 and AMD-4 and its rela- 2009 and 2010 samples. The bacterial populations from the sum- tive proportion in the three clone libraries was 5.9%, 5.8% and mer of 2009 (AMD-2009) showed a signiﬁcant difference from 14.7%, respectively. ARC-1-5 was related to those sequences those (AMD-1 to AMD-4) of the fall of 2010 (Figure 4), in- detected in a hot spring of the Colombian Andes. dicating that the bacterial population structure changed greatly MICROBIAL COMMUNITY IN ACID MINE DRAINAGE LAKE 893

limiting step, the oxidation of Fe2+ to Fe3+, which is the most effective oxidant in the oxidation of sulfide minerals at low pH (Baker and Banfield 2003). Chemolithotrophic iron/sulfur- oxidizing acidophiles, such as Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans, and Ferroplasma acidiphilum, and sometimes facultatively autotrophic Sulfobacillus species usually dominate AMD environments (Bond et al. 2000; Druschel et al. 2004; Hallberg 2010; Kock and Schippers 2008). Surpris- ingly, in the AMD Lake at Xiang Mountain, our results from both FIG. 4. Clustering of the bacterial clone libraries of 2009 (AMD-2009) and 2010 (from AMD-1 to AMD-4) based on Cxy values determined from the 2009 and 2010 sampling seasons consistently demonstrated that S-LIBSHUFF analysis. The UPGMA tree was constructed with MEGA 4.0. these chemolithotrophic iron/sulfur-oxidizing acidophiles were The parameter Cxy in the LIBSHUFF analysis represents the difference in only minor members in the bacterial community (Hao et al. coverage of any two clone libraries (the larger Cxy, the greater dissimilarity 2010b; this study). Again the reasons can be ascribed to low between the given clone libraries). The software for the analysis was available concentrations of Fe2+ and availability of allochthonous organic at http://www.plantpath.wisc.edu/joh/s-libshuff.html. matter in this particular AMD environment (Hao et al. 2010b). There were important differences between the two sam- from the summer of 2009 to the fall of 2010. The LIBSHUFF pling seasons, however. Whereas mixotrophic Acidiphilium result of archaeal populations was similar to that of bacterial spp. (28.6%), and heterotrophic Ferrithrix spp. (22.3%) and populations (data not shown). Legionella (12.7%) were most abundant members in bacterial PCA was employed to assess the similarity and difference community in 2009, these species were only minor in 2010. among the four AMD samples collected in October 2010. The Temperature change may be the main reason for the decline of results showed that AMD-2 and AMD-4 were grouped together the genus Acidiphilium because its optimal growth is around in the profile while AMD-1 and AMD-3 were separated notably ◦ 30 C, which was the measured temperature in summer 2009. from the other samples (Figure 5A), suggesting that AMD-2 and ◦ The in-situ temperature in the fall of 2010 was ∼20 C, and AMD-4 had similar geochemistry. Correspondingly, PCA based therefore the growth of Acidiphilium species is expected to be on the microbial community compositions including both bacte- slow (Harrison 1981). Similarly, Ferrithrix species are moder- ria and archaea revealed that the microbial community structure ately thermophilic and obligately heterotrophic with an optimal between AMD-2 and AMD-4 were similar to each other, but growth temperature of 43o C (Johnson et al. 2009). Thus, the different from those of AMD-1 and AMD-3 (Figure 5B). These low in-situ temperature of the fall sampling season of 2010 may data suggest the importance of geochemical conditions in shap- be the main reason for the low abundance of Ferrithrix species. ing the microbial community structure. The reasons for the high abundance of Legionella sequences in the summer of 2009 were ascribed to high levels of proto- DISCUSSION zoa that served as the replication host of Legionella species. The oxidation and dissolution of sulfide ores, when exposed However, in the fall 2010 samples, the protozoa abundance to oxygen and water, result in generation of acid mine drainage, including ciliate and ameba sharply decreased (unpublished and the process can be greatly enhanced by the activity of data). This decrease may have been accounted for the low abun- chemolithotrophic microorganisms through catalysis of the rate- dance of the Legionella-related sequences in 2010. These data Downloaded by [Zhejiang University] at 17:31 11 April 2013

FIG. 5. PCA proﬁles of the four water samples based on the physicochemical characteristics (A) and microbial compositions (B). 894 C. HAO ET AL.

effectively demonstrate that changes in environmental condi- than TN values in eutrophic lakes (1.09–3.08 mg/L) during tions were responsible for the observed changes in bacterial cyanobacterial bloom (Havens et al. 2001). Concentrations of community composition. total phosphorus (TP) in the studied samples (0.59–0.85 mg/L) In contrast to heterotroph-dominated bacterial community in were more than three orders of magnitude higher than those 2009, sequences related to photoautotrophic cyanobacteria were in similar AMD lakes in Germany (typically around 5 µg/L) abundant in 2010 (54.9%–77% of the total clones in the four (Nixdorf et al. 1998). These high nutrient levels may be a result bacterial clone libraries). Cyanobacteria are most prominent in of allochthonous input and outwash of shore materials by heavy habitats with neutral to alkaline pH and should be completely rain during the rainy season. absent from habitats in which the pH was less than 4 or 5 (Brock Finally, the obvious temperature decline from 30o C in June 1973). 2009 to ∼20o C in October 2010 should result in an increase Therefore, the general consensus has been that cyanobacteria of the dissolved inorganic carbon (DIC) content in the AMD can not grow at acidic pH, especially in the environment with lake, which would have favored the flourish of photoautotrophic pH<3 (Waterbury 2006). Gonzalez-Toril´ et al. (2003) only de- cyanobacteria. The values of DIC in the four samples (around tected three cyanobacterial sequences in the acidic Tinto River 1 mg C/l) were much higher than the theoretical CO2 concentra- (Spain). Two populations of filamentous cyanobacteria resem- tions at the in-situ pH and temperature (∼0.16 mg C/l according bling Oscillatoria/Limnothrix and Spirulina spp. were detected to Henry’s law) (Satake and Saijo 1974). This high level of DIC in an acidic lake (pH 2.9) in Germany using flow cytometry and values, despite a high photosynthetic activity, suggested that light microscopy (Steinberg et al. 1998), but the proportion of there are sources of supply to the lake. One such source is the the cyanobacteria in total bacterial population was not deter- atmospheric CO2. mined. Thus, the absolute dominance of cyanobacteria in the Acidic pH leads to rapid equilibrium between the dissolved AMD lake with pH<3, as observed in this study, has not been CO2 and atmospheric CO2 (Brinkman et al. 1934). Other reported previously. The cyanobacterial sequences detected in sources of CO2 may be CO2-rich groundwater or low lay- this research should not be from algal chloroplast since most ers of the AMD lake through decomposition of organic com- of the eukaryotic populations in these four water samples were pounds by heterotrophic acidophiles. There may be symbiotic mainly made up of a variety of fungi (unpublished data). relationships between phototroph (cyanobacteria) and chemo- There may be several reasons for the dominance of cyanobac- heterotroph, such as Acidiphilium spp., which can favor survival teria in the samples collected in 2010. First of all, the concen- of both organisms in such an extreme environment (Baffico et al. tration of total dissolved iron in the lake sharply decreased from 2004). 100.6 mg/L in June 2009 to 48.0–57.5 mg/L in October 2010 Genus Acidisphaera was also detected in the Xiang Mountain (Hao et al. 2010b). Summer (from June to August) is the typi- AMD lake, achemoheterotrophic bacterial group that contained cal rainy season in this region and the wastewater in the AMD photosynthetic pigments, similar to the Acidiphilium spp. How- lake rose to a high level in October 2010, which would have ever, there are important differences between these two groups. greatly diluted the total iron content. In addition, precipitation Whereas Acidiphilium species contains typical bacteriochloro- of Fe(III) minerals may have contributed to the decline of sol- phyll a chelated with zinc (Zn-BChl a) that is thought to be uble iron concentration as well. In AMD ecosystem, Fe(II) is stable under the acidic conditions (Hiraishi et al. 2000), Acidis- rapidly oxidized to Fe(III) by acidophiles, and Fe(III) is subse- phaera species produces magnesium-chelated bacteriochloro- quently hydrolyzed and precipitated at pH 2.5–3.5 in the form of phyll (Mg-BChl) a. The presence of Acidisphaera species in hydrous iron oxides and/or oxyhydroxy sulfate minerals, such the AMD lake at pH < 3 suggested that the occurrence of Zn- Downloaded by [Zhejiang University] at 17:31 11 April 2013 as schwertmannite (Bigham et al. 1996). BChl a is not always essential for photosynthesis in obligately The pH of the lake water in October 2010 was about 2.9, acidophilic bacteria (Hiraishi et al. 2000). Moreover, a previous which was appropriate for hydrolysis and precipitation of Fe(III) study reported that the Acidisphaera could grow in the pH range minerals. Through this natural process, up to 70% of dissolved of 3.5–6.0, but not at pH 3.0 or 6.5 (Hiraishi 2005), whereas the iron can be removed from AMD solution (Espana˜ et al. 2007). pH of the four water samples in this research was below 2.9, Visual inspection indicated that the red-color of the AMD water indicating that Acidisphaera can survive in the more acidic en- of Xiang Mountain, which was present in the summer of 2009 vironment than previously thought. was not present in the fall of 2010, apparently because of pre- The dominant archaeal sequence types were also different cipitation of Fe(III) minerals. The resulting clear water would from those detected in the summer of 2009. However, all of have favored light transmission to the lake and promoted the the archaeal sequences retrieved in the present study could not rapid growth of phytoplanktonic cyanobacteria. be closely related to any cultured organisms, and thus their Second, in contrast to the oligotrophic nature of most AMD physiological role in the AMD ecosystem remains uncertain. lakes, the important nutrients of the AMD lake at Xiang Moun- In summary, the observed major differences in the bac- tain may be sufficient to sustain rapid growth of cyanobacte- terial community between the two sampling seasons were ria. Total nitrogen (TN) concentrations in the four water sam- largely caused by changes in environmental conditions from the ples from the AMD lake (18.2–20.4 mg/L) were even higher summer of 2009 to the fall of 2010. This correlation was MICROBIAL COMMUNITY IN ACID MINE DRAINAGE LAKE 895

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