Bacteria diversity, distribution and insight into their role in S and Fe biogeochemical cycling during black shale weathering.

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Environmental Microbiology (2014) 16(11), 3533–3547

doi:10.1111/1462-2920.12536

Bacteria diversity, distribution and insight into their role in S and Fe biogeochemical cycling during black shale weathering

Jiwei Li,1† Weimin Sun,2† Shiming Wang,3 Zhilei Sun,4 Sixiang Lin3 and Xiaotong Peng1* 1 Sanya Institute of Deep-Sea Science and Engineering, Chinese Academy of Sciences, Sanya 572000, China. 2 Department of Environmental Science, Rutgers University, New Brunswick, NJ 08901, USA. 3 Faculty of Geosciences and Environmental Engineering, Southwest Jiaotong University, Chengdu 610031, China. 4 Department of Marine Mineral Resources, Qingdao Institute of Marine Geology, Qingdao 266071, China. Summary A group of black shale samples, which were collected sequentially along a continuous depositional unit from bottom fresh zone toward the surface regolith of the weathering profile at Chengkou County, Southwest China, were examined using mineralogical, geochemical and pyrosequencing techniques. The mineralogical and geochemical analyses indicated that the black shale profile provided a series of extremely acidic and chemical species that changed microbial habitats following the process of weathering. This finding is in contrast with a previous hypothesis that a low-diversity bacterial community existed in these harsh environments; the pyrosequencing analyses showed extremely diverse microbial communities with 33 different phyla/groups in these samples. Among these phyla/groups, proteobacteria, actinobacteria and firmcutes were more dominant than other phyla, and the phylogenetic structures of the bacterial communities vary with the progressive process of weathering. Moreover, the canonicalcorrelation analysis suggested that pH and sulfur in sulfate, followed by total Fe and sulfur in pyrite, are the significant factors that shape the microbial community structure. In addition, a large proportion of Sand Fe-related bacteria, such as Acidithiobacillus, Received 19 October, 2013; accepted 9 June, 2014. *For correspondence. E-mail [email protected]; Tel. (+86) 898 88381109; Fax (+86) 898 88380102. †These authors contributed equally to this work.

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd

Sulfobacillus, Thiobacillus, Ferrimicrobium and Ferrithrix, may be responsible for pyrite biooxidation, as well as for S and Fe biogeochemical cycling, in the black shale weathering environments. Introduction Black shales are dark, fine-grained sedimentary rocks that are often deposited in stagnant aquatic environments with high organic productivity and oxygen deficiency at the bottom, creating sediments that are rich in organic matter and pyrite (Leventhal, 1991; Lipinski et al., 2003). Black shales are associated with ‘oceanic anoxia events’ (Lipinski et al., 2003) and have wide geographic and stratigraphic distributions around the world (e.g. the lower Cambrian in southern China (Peng et al., 2004), Devonian in the Yukon Territory of Canada and the central and eastern regions of the United States (Clayton and Swetland, 1978; Tuttle and Breit, 2009a). Tectonic activity, sea level changes and human activity may result in the exposure of black shale to near-surface weathering environments. Once the black shale strata comes into contact with exogenous perturbations (e.g. O2, meteoric waters and microbes), high concentrations of organic matter, low pH due to the oxidation of pyritic S and predominantly clay mineralogy render the strata susceptible to alteration (Peucker-Ehrenbrink and Hannigan, 2000). Black shale weathering has an important impact on water quality, global climate change, the biogeochemical cycles of C, S, Fe and various metal elements, and human health by redistributing elements and toxicants into the environment (Dalai et al., 2002; Pierson-Wickmann et al., 2002; Peng et al., 2004; 2007; Paikaray, 2012). Specifically, black shale might be one of the most important pyrite reservoirs on the earth’s crust (e.g. Zhu, 2010). Pyrite oxidation has been strongly suggested as the key step in the progress of weathering processes (Fischer et al., 2007; 2009; Tuttle and Breit, 2009a) due to its ability to produce sulfuric acid and to release trace metals and metalloids, such as As, Cd and Pb (Bednar et al., 2002; Joeckel et al., 2005; Zhu et al., 2008; Tuttle and Breit, 2009a; Tuttle et al., 2009b). Therefore, black shale weathering would not

3534 J. Li et al. only influence the geochemical cycle of elements but also lead to acid rock drainage and severe heavy metal damage to local environments (Carlson and Whitford, 2002; Joeckel et al., 2005; Lehner et al., 2007; Lehner and Savage, 2008; Lavergren et al., 2009). Recently, several studies have revealed that black shales can host intriguing microbial communities in spite of their low porosity and permeability (Petsch et al., 2003; 2005; Cockell et al., 2011). These studies all proposed that microbes can play a significant role in the weathering process (Petsch et al., 2003; Matlakowska and Skłodowska, 2009; 2011; Matlakowska et al., 2010; 2012; Cockell et al., 2011). For instance, Cockell and colleagues (2011) recently found a novel, low-diversity microbial community inhabiting the pyrite-containing receding shale cliffs on the coast of northeast England. These researchers suggested that these microorganisms might contribute to the early events of shale degradation (Cockell et al., 2011). Additionally, some studies further indicated S- and Fe-related bacteria were the vital and active players that influence black shale weathering because these bacteria can harness energy via redox reactions with the reduced chemical species (Fe2+, S2−) from this huge pyrite reservoir under surface atmospheric conditions. A direct evidence to support this conclusion was supplied by Zhu and colleagues (2008). They observed that preferred colonization of microorganisms, especially Fe-oxidizing bacteria (FOB)/S-oxidizing bacteria (SOB), is on the surface of pyrite mineral rather than on the shale matrix of the pyrite bearings and polished black shale thick sections during their incubation in subsurface groundwater at Newark Basin. The occurrence of pits and secondary minerals with cells suggests the likelihood of microbiological involvement in their formation. Hence, microbial biochemical reactions, particularly Sand Fe-related metabolism, were hypothesized to be an important approach to the erosion, decay and decomposition of black shale. If we want to deeply characterize the microbial functions in overall black shale weathering processes, detailed information regarding the community structure and spatial variations of these microorganisms is essential. However, until now, little has been known. Therefore, the comprehensive investigation of the microbial community in a black shale weathering profile has been strongly expected. For this purpose, we collected a group of samples from a black shale weathering profile in Chengkou County in south-western China. Geochemical methods were utilized to characterize the geochemical environmental changes within the black shale weathering profile from the surface weathering zone to the bottom fresh zone. In addition, a depth-resolved bacterial community analysis was performed utilizing pyrosequencing. The objective was to address the following four questions: (i) Is there a core

microbial group in the microbial populations of the black shale weathering fields?, (ii) How do the changed geochemical parameters affect bacterial community structures in the black shale weathering profiles?, (iii) How does the phylogenetic structure of bacterial communities vary with the progressive process of the black shale weathering?, and (iv) What is the role that S- and Fe-related microorganisms play in S and Fe biogeochemical cycling during the progress of pyrite-rich black shale weathering?

Results Mineralogical and chemical compositions The X-ray diffraction (XRD) data showed that quartz, albite, illite and muscovite were the four dominant minerals in all these samples (Supporting Information Table S1). Pyrite, calcite, dolomite and gypsum were also detected; however, all of these minerals tended to appear in the lower region of the black shale weathering profile. The concentrations of the major elements are shown in Supporting Information Table S2. All samples had high concentrations of SiO2 and Al2O3, followed by Fe2O3, CaO and SO3, but relatively low concentrations of TiO2, BaO, Na2O, P3O5 and MnO2. Among these major elements, the concentration of Fe2O3 has low values in the middle zone but high values in the bottom and surface zones. CaO and SO3 both showed an increasing trend from the surface to the bottom in this profile. Environmental geochemical parameters and contents of various sulfur species in the black shale samples are listed in Supporting Information Table S3. In all the 11 black shale samples, the pH ranged from 2.68 to 6.88. The pH trend peaked in the bottom and surface zones and touched the bottom in the middle zone, minimized in sample A4 but maximized in samples A1 and A10. Generally speaking, the values of total inorganic carbon (TIC) (0.23% to 4.32%) were much higher than total organic carbon (TOC) (0.06% to 1.52%) in each sample. However, both of their changing curves showed an increasing trend from top to the bottom across this weathering profile. The inorganic reduced sulfur species AVS-S (sulfur in acid-volatile sulfide) and pyritic-S (sulfur in pyrite) ranged from 0.02% to 0.14%, and 0.17% to 3.92% respectively. The contents of Org-S (sulfur in organic matters) ranged from 0.06% to 0.29% in all the samples. AVS-S, pyritic-S and Org-S all showed an increasing trend from the top to the bottom across this weathering profile. SO4-S (sulfur in sulfate) ranged from 0.07% to 2.06% in all 11 samples. However, its distribution pattern had high values in the centre and low values in the bottom and surface zones. Pyritic-S accounted for

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 3533–3547

Bacteria in a black shale weathering profile 30.9–89.9% of the extracted total S in the 10 black shale samples.

General analyses of the pyrosequencing-derived dataset In total, 43 733 valid reads and 7473 operational taxonomic units (OTUs) were obtained from the nine black shale samples through 454 pyrosequencing analyses. These sequences/OTUs were assigned to 34 different phyla or groups and are available through the NCBI/ EBI/DDBJ Short Read Archive (accession number ERA043547). Each of the nine communities contained between 3974 and 5654 reads, with OTUs ranging from 474 to 1566 at genetic distances of 3% (Supporting Information Table S4). The rarefaction curves tended to approach the saturation plateau, except in the A1 and A2 samples (Fig. 1). Good’s coverage estimations revealed that 80.2% to 95.6% of the species were obtained in all of the samples, except for the A1 wherein only 73.8% of the species were determined. The Chao1, Ace, Simpson and Shannon indexes of diversity were also determined to evaluate the biodiversity and phylotype richness of bacterial communities in these samples (Supporting Information Table S4). At a genetic distance of 3%, the Shannon index ranged from 3.73 to 6.65. Ace and Chao1 had a value range from 1164 to 7134 and 944 to 3989 respectively. All of the three indexes showed the highest value in sample A1 and the lowest value in sample A7. The value of the Simpson index ranged from 0.0029 to 0.0966. However, the Simpson index had a reversing change trend when

Fig. 1. Rarefaction curves for bacterial libraries of these weathered black shale samples from Chengkou County. Phylotypes were defined at 97% sequence similarity.

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compared with the above three indexes, with the lowest value in sample A1 and the highest value in sample A7. These results demonstrated that the top surface samples (A1, A2) had the highest bacterial diversity, which was followed by the bottom fresh sample (A10); in addition, the relative lower bacterial diversity appeared in the middle region of the black shale weathering profiles (A3–A8). Depth-resolved bacterial community analysis All the obtained bacterial sequences from these samples were classified from phylum to genus according to the program MOTHUR using the default setting. The sequences fell into the following 33 different phyla or groups: Acidobacteria, Actinobacteria, Armatimonadetes, BD1-5, Bacteroidetes, Chlorobi, Chloroflexi, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospirae, Candidate division OD1, Candidate division OP3, Candidate division OP11, Candidate division TM7, Planctomycetes, Proteobacteria, RF3, SM2F11, Spirochaetes, Synergistetes, Tenericutes, Thermotogae, TM6, Verrucomicrobia, WCHB1-60 and unclassified bacteria. Sequences that were affiliated with Proteobacteria, Actinobacteria and Firmicutes dominated these samples. Moreover, as indicated in Fig. 2 and Supporting Information Table S5, the nine clone libraries showed extremely dissimilar 16S rRNA profiles. Proteobacteria was the most abundant phylum in eight of nine samples and accounted for 23.61–60.26% of the total bacterial sequences, with an average value 43.71%. Actinobacteria was the second most abundant phylum in this study, ranging from 4.00% to 49.98% of the reads, with an average value at 26.11%. Particularly in samples A1, A2, A7 and A8, the relative concentrations of Actinobacteria were over 30%; however, its value was lower than 10% in samples A5 and A6. Firmicutes was another dominant phylum in these samples and accounted for 2.29–67.63% (average 22.53%) of the total bacterial sequences. Notably, this phylum was dominant in sample A6. Bacteroidetes and Chloroflexi were the major groups (abundance > 1%) in this study. The relative concentration of Bacteroidetes and Chloroflexi ranged from 0.51% to 13.89% (average 3.71%) and from 0% to 4.33% (average 1.0%) respectively. In addition to the above five phyla, a few other phyla were only major components in some individual samples, including Acidobacteria in A1 (1.51%) and A2 (1.06%); Candidate_division_TM7 in sample A1 (1.9%); Nitrospirae in A7 (1.3%); and Planctomycetes in sample A1 (1.1%). The abundance of other phyla was below 1% in all samples.


3536 J. Li et al.

Fig. 2. Bacterial community structures in black shale samples from Chengkou County.

Within Proteobacteria, ε- and δ-Proteobacteria only occurred at extremely low levels (0–0.22% and 0–1.94% respectively). Except for samples A7 and A8, the γ-subdivision was the most dominant class of Proteobacteria, which ranged from 4.09% to 51.43% (average value 24.97%). The β-subdivision had a range of 6.93% to 52.10% (average 15.25%) in all samples and was dominant in samples A7 and A8. The α-subdivision ranged from 0.30% to 17.22% (average 3.07%) in all samples. The most abundant genera within the different samples were also determined (Supporting Information Table S6). Sulfobacillus, Thiobacillus, Ferrimicrobium, Streptococcus, Serratia, Yonghaparkia, Bacillus and Nocardioides were the abundant genera within the black shale weathering profiles; however, these genera were not present in equal abundance. Specifically, Sulfobacillus was predominant in the middle region of the weathering profile (samples A3–A7), particularly in samples A6 (44.11%) and A7 (19.65%), and Thiobacillus commonly appeared in the lower region (sample A7–A10), particularly in samples A7 (11.49%) and A8 (48.79%). Furthermore, Nocardioides

frequently occurred in the surface samples A1 and A2, whereas Yonghaparkia appeared in the lower region (A8 and A10). Discussion Geochemical environments in the black shale weathering profile Results of XRD provided some typical mineralogical characteristics of these fresh and weathered black shale samples, which primarily consisted of quartz, albite, illite and muscovite. In addition to these minerals, pyrite, gypsum and carbonate minerals appeared in the middle and lower regions of this profile, but were nearly exhausted in the top region. The elemental analysis was generally consistent with the XRD analysis. All samples had significantly high concentrations of Si, Al and Fe, but relatively low concentrations of Ti, Ba, Ca, Na, P and Mn. The intensity of the weathering index Chemical Index of Alteration (Nesbitt and Young, 1984), which was calculated by element concentrations, confirmed that the extent of weathering increased from the bottom to the top.


Bacteria in a black shale weathering profile

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Fig. 3. The changes in geochemical environments in the black shales weathering profile at Chengkou County. A. Weathering intensity index CIA. B. Various sulfur species (AVS-S and Org-S using the top graduation line, SO4-S and Pyritic-S using the bottom graduation line). C. TOC (using top graduation line) and pH (using the bottom graduation line). D. The sketch map of four weathering zones in the black shale weathering profile.

The fact that abundant ancient organic matter is weathered in black shales has been widely supported by previous studies (e.g. Petsch et al., 2003; Matlakowska and Skłodowska, 2011) and is also consistent with this study. Here, the concentration of TOC decreased rapidly from top to the bottom within this weathering profile. The absence of pyrite diffraction patterns and the diminishment of pyritic sulfur in the upper region indicated that pyrite oxidation strongly occurred in the process of weathering. Furthermore, weathering could also contribute to AVS and organic sulfur oxidization, as indicated by their decreasing concentrations from the bottom to the top. Acidic solutions (sulfuric acid), which were produced by sulfide oxidation in the upper zone, seeped downward into the lower permeable shale and then dissolved carbonates and formed secondary minerals, such as gypsum in the middle region of this weathering profile. This finding is the reason why there are low pH values and high sulfate contents (mainly occurring as gypsum) present in the middle region of this weathering profile. The concentration of total Fe (TFe) showed a pattern with high values in two sides and low values in the middle region of this weathering profile. However, pyriticFe(II), which is indicated by the concentration of pyritic-S, was nearly exhausted in the upper region and showed a general increased pattern from the surface to the bottom through the whole transect. Thus, Fe in the surface region may be the primary Fe3+ bearing in the

disorder of Fe-oxide minerals, whereas Fe in the lower region was Fe2+ bearing in the sulfide minerals. Overall, based on the analysis of mineralogical and geochemical characteristics, four zones within the black shale weathering profile with different geochemical environments can be defined in this study, which is similar to the findings by Chigira and Oyama (2000) (Fig. 3). (i) The surface oxidized zone (A1 and A2) was represented by sulfur species, carbonate and organic matter depletion, but an Fe(III)-rich neutral environment; (ii) the upper dissolved zone (A3–A6) was represented by an extremely acidic, oxidized and leached environment, which has low concentration of organic matter, carbonate and sulfur species; (iii) the lower weakly weathered illuviated zone (A7, A8 and A9) was represented by a relatively low pH, but relatively high levels of organic matter, carbonate and pyrite, as well as a maximum sulfate environment; and (iv) the bottom relatively fresh and reduced shale zone (A10 and A11) was represented by a relative neutral and reduced environment, with high amounts of organic matter, carbonate and pyrite, but low amounts of sulfate. Correlations between environmental data and bacterial communities Analysing the dynamic changes of these microbial communities, which were coupled with geochemical factors, will reveal how microbes can adapt to the changes of


3538 J. Li et al. Fig. 4. CCA of 16S gene data and geochemical parameters. Arrows indicate the direction and magnitude of geochemical parameters associated with bacterial community structures. A1, A2, A3, A4, A5, A6, A7, A8 and A10 represent the different bacterial community structures from the nine black shale samples.

physical-chemical environments in situ that result from weathering under surface atmospheric conditions. The canonic corresponding analysis (CCA) was useful to reach this goal (e.g. Wang et al., 2012). In the current study, the dominant genera (> 1%) and selected geochemical parameters (pH, Pyritic-S, SO4-S, AVS, TOC, TIC and TFe) were examined by CCA. As shown in Fig. 4, pH and SO4-S emerged as the most significant factors for structuring the bacterial community, which were followed by TFe and Pyritic-S, whereas org-S, TOC, TIC and AVS did not have an important impact on microbial populations. It is wide accepted that pH has a significant effect on the overall diversity and composition of microbial communities in a range of terrestrial and aquatic environments (Fierer and Jackson, 2006; Lauber et al., 2009; Kuang et al., 2012). This observation is because the intracellular pH of most microorganisms is usually within 1 pH unit of neutral, and any significant deviation in the environmental pH should impose stress on single-celled organisms (Fierer and Jackson, 2006; Wang et al., 2012). In addition to this explanation, Lauber and colleagues (2009) also stated that pH may not directly alter the bacterial community structure but may instead function as an integrating

variable that provides an integrated index of soil conditions. There are several environmental chemical parameters, such as nutrient availability and cationic metal solubility (Brady and Weil, 2007), that are often directly or indirectly related to soil pH, and these factors may drive the observed changes in community composition (Lauber et al., 2009). In this study, the flourishing acidophilic and acid tolerant bacteria (e.g. Ferrimicrobium and Sulfobacillus) in the acidic middle region of the black shale weathering profile also supported this observation. SO42− (represented by SO4-S) also emerged as one of the most significant factors. A reasonable explanation is that, as mentioned above, SO42− was closely related to pH in the black shale weathering profile (correlation coefficient = 0.7, data not shown). Pyritic-S [also related to pyrite or Fe(II)] and TFe were also significantly linked to bacterial community variance in this study. The Fe- and S-based metabolic processes have proven to be important in other natural environments (Campbell et al., 2006; Herbert Jr and Schippers, 2008; Li et al., 2013). With respect to the black shale weathering environments, the supply and transformation of Fe and S compounds would also influence all bacterial communities through controlling the distribution of


Bacteria in a black shale weathering profile S- and Fe-related bacteria. This observation is in accordance with the fact that a relatively high proportion of S- and Fe-related bacteria were more inclined to be present in the middle illuviated and bottom fresh reduced zones with a high concentration of pyritic-S compounds than the upper zones, which is pyrite exhausted in this study. TOC is also well recognized as an important parameter for shaping the microbial community structure in various natural environments. The indigenous bacteria in black shale can also use ancient organic matter as their sole carbon and energy source, as reported previously (Petsch et al., 2001; 2003; Matlakowska and Skłodowska, 2009). However, similar to TIC, CCA analysis of AVS and organic S determined that the influence of TOC on the bacterial community variance is much less than that of pH, SO4-S, pyritic-S and TFe in this study. Core bacterial groups in the black shale weathering profile Depth-resolved microbial community analysis was employed in this study can shed light on the detailed changes in bacterial communities from the surface weathering zone to the bottom fresh zone, which follow the progressive processes of weathering. The statistical analysis suggested that the diversity and richness of bacterial communities in different zones of the black weathering profile were distinct from each other. Generally speaking, samples in the middle dissolved zone and in the illuviated zone of the weathering profile have relative lower predicted bacterial diversity than that of the surface oxidized zone and bottom fresh reduced zone. This result could be related to the much harsher living environments that have extremely low pH and high sulfate. Among all observed phyla, proteobacteria, actinobacteria and firmicutes are more significant because of their dominant relative concentrations, despite considerable fluctuations in their relative recovery of 16S rRNA gene libraries. Members of Proteobacteria are metabolically versatile and have been reported to be predominant in various environments, such as in activated sludge from sewage treatment plants (Zhang et al., 2012), snottites of pyrite mine (Ziegler et al., 2009), acid mine drainage (Kuang et al., 2012) and in karst weathering regions (Tang et al., 2012). Actinobacteria are high-G+C (guanine + cytosine) Gram-positive bacteria, which are also composed of a wide range of physiologically diverse species (Tang et al., 2012). The high proportion of Actinobacteria could be attributed to their strong cell walls, high GC-contents and their capability of forming spores, which allows their survival in harsh environments (Horath and Bachofen, 2009; Tang et al., 2012). Similar to Actinobacteria, Firmicutes have also been frequently

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reported in some extreme environments (Orcutt et al., 2010; Emmerich et al., 2012). For instance, Orcutt and colleagues (2010) reported that Firmicutes dominated the subsurface microbial communities of the ocean crust at the eastern flank of the Juan de Fuca Ridge. Therefore, the dominance of the three phyla in the black shale environments could be related to their ability and adaptation to living in the metal-rich and extremely acidic black shale weathering environment. Deeper genus-level sequencing revealed that Sulfobacillus, Thiobacillus Streptococcus, Serratia, Ferrimicrobium, Yonghaparkia, Bacillus and Nocardioides were the most abundant genera within the black shale weathering profiles. In addition to these genera, certain genera, such as Acidithiobacillus, Acidiphilium, Anoxybacillus, Aquicella, Arthrobacter, Delftia, Ferrithrix, Flexibacter, Lactobacillus, Leptospirillum, Lysobacter, Pelomonas, Pseudomonas and Ralstonia also emerged as the dominant genera (> 1%) in some individual samples. This finding demonstrated that there was a core microbial community in the black shale weathering fields and that this community would be the key player actively participating in some crucial microbiological reactions such as organic matter biodegradation as well as Fe and S geochemical cycling in the weathering process. However, as mentioned above, these genera did not appear in equal abundance in the nine samples, and the majority of these genera showed an evident zonal distribution. For instance, the oxidized zone at the top harboured more aerobic and facultative bacteria. Nocardioides and Segetibacter, which are two strictly aerobic bacteria, both occurred in high abundance in the surface oxidized zone (A1 and A2) and were nearly absent in the middle region acidic zones of the black shale weathering profile. Moreover, many acidophilic or acidic tolerant microbes, such as Ferrimicrobium, Anoxybacillus, Sulfobacillus, Bacillus and Thiobacillus, thrived in the middle dissolved and illuviated zones. Additionally, it also should be noted that some microaerophiles/aerobes presented in the bottom fresh zones. This result could be due to a small amount of oxygen and run-off brought into the bottom fresh zone through the micro-fissures, which was caused by the early stage of weathering process in this zone, and the oxygen and run-off allowed microaerobic environments to form in isolated areas. Microbial S and Fe cycles during the black shale weathering process There is accumulating evidence that pyrite weathering is indisputably involved with, or even controlled by, biological activities in various natural environments (Ziegler et al., 2009; Johnson, 2012; Korehi et al., 2013). With respect to the black shale weathering environments, XRD


3540 J. Li et al.

Fig. 5. Map of the relative concentration of Fe- and S-related bacteria in the bacterial communities of the black shale weathering profile at Chengkou County.

and pyritic-S extraction analyses both indicated that pyrite appeared in a relative high concentration. Furthermore, pyrite was a crucial factor structuring the microbial community by CCA analyses in this study. Therefore, the microbial promotion of sulfur and iron biogeochemical cycling was hypothesized to be extremely active during the black shale weathering process. Fe and S oxidation. Our pyrosequencing analysis was in agreement with above hypothesis. This analysis revealed that there are many bacteria with S and Fe oxidation capabilities that exist in this black shale weathering profile. Among these bacteria, Sulfobacillus, Thiobacillus and Acidithiobacillus were the commonly detected acidophilic Fe- or S-oxidizing genera. Sulfobacillus also appeared in all nine samples but were apparently predominant in the acidic weathering zones, particularly in two relative-fresh shale samples, A6 (44.11%, pH = 3.4) and A7 (19.65%, pH = 4.24). In contrast, Thiobacillus were not detected in the upper acidic zones but were present from A6 to A10, and exhibited extremely high abundances in A8 (48.79%, pH = 5.88), as well as in high abundances in samples A7 (11.49%) and A10 (6.39%, pH = 6.88). Bedsides some neutrophilic species in Thiobacillus, both of the two genera contained acidophilic,

aerobic and disulfide-oxidizing species, and they can use reduced S and Fe as sole energy sources. Acidithiobacillus, which were acidophilic Fe(II)- and S-oxidizing and Fe(III)-reducing genera, were also present in relatively high abundance in sample A7 (1.14%). It is noteworthy that some of the species formerly classified into Thiobacillus have been reclassified to genus Acidithiobacillus, such as Acidithiobacillus thiooxidans, formerly known as Thiobacillus thiooxidans until its reclassification into the newly designated genus (Kelly and Wood, 2000). In addition to Acidithiobacillus, other SOB, including Sulfuricella, Sulfuritalea and Halothiobacillus, were also found but only in low abundance in individual samples. Acidophilic Fe(II)-oxidizing bacteria, including Acidiferrobacter, Leptospirillum, Ferrimicrobium and Ferruginibacter were also recognized in this study. Among these bacteria, Ferrimicrobium and Ferrithrix seem to act as the major players in Fe(II) oxidation because of their relative high abundance and frequency. Species of Ferrimicrobium were present in nearly all of these samples and showed extremely high abundances in intensively weathering zone (Fig. 5). Ferrithrix was detected with a high abundance in the upper intense weathered shale zone (5.3% in A3). Both of these two genera belong to Actinobacteria, which is a phylum that contains heterotrophic and extremely acidophilic FOB. Notably, species of the two genera can also reduce ferric iron, which is coupled with organic matter oxidation only under anaerobic conditions (Johnson et al., 2009). Leptospirillum tend to appear in the lower region of the black shale weathering profile, with a value range from 0.04% to 1.30% in samples A7–A10. Members of this genus all are obligate aerobes and autotrophs, which are limited to an electron transport chain from ferrous iron to oxygen (Hippe, 2000; Johnson, 2012). Some previous studies have reported that Leptospirillum dominated bacterial populations that are dependent on pyrite oxidation as a primary energy source (Rawlings et al., 1999; Ram et al., 2005). Additionally, Gram-positive iron-oxidizing acidophile SLC66 and Ferruginibacter were also present in low abundance in individual samples. Interestingly, some less acid tolerant and neutrophilic FOB, including Ferrovum myxofaciens, Gallionella, Sphaerotilus, Aquabacterium and Pseudoalteromonas, were also detected in this environment, but in a much lower abundance (Table 1). The investigations revealed that species of Gallionella being present with low abundance in sample A7 (e.g. Gallionella. ferruginea). Members of this species are described as autotrophic iron oxidizers of which habitats are sharply limited to a slightly acidic pH of 5.0–6.5 and to microaerophilic conditions. Therefore, the physico-chemical conditions in sample A7 could be more suitable for their survival than other


—

—

— 0.03 — — — — — — —

Leptospirillum (%) Sulfobacillus (%) Sulfuricella (%) Sulfuritalea (%) Sulfurospirillum (%) Shewanella (%) Sphaerotilus (%) Thiomonas (%) Thiobacillus (%)

—, Not detected in this study.

— 0.27 — 0.06 0.75 — —

— 0.05 — — — — —

Acidithiobacillus (%) Aquabacterium (%) Desulfovibrio (%) Ferrimicrobium (%) Ferrithrix (%) Gallionella (%) Iron-oxidizing acidophile SLC66 (%) Halothiobacillus (%) — 0.04 — — — — — — —

— 0.18

— —

Acidiferrobacter (%) Acidiphilium (%)

A2

A1

Groups

— 1.78 — — — — — — —

—

— 1.08 0.02 10.9 5.30 — —

— —

A3

— 7.22 — — — — — — —

—

— 1.10 — 7.27 — — —

— —

A4

— 3.66 — — — — — — —

—

— 0.56 — 6.14 — — —

— —

A5

— 44.11 — — — 0.07 0.02 — 0.19

—

— 0.07 — 1.57 — — —

— —

A6

1.30 19.65 0.05 — — — — 0.05 11.49

0.13

1.14 0.07 — 0.27 0.04 0.02 —

0.05 0.02

A7

0.36 0.16 — — — — — — 48.79

—

0.14 0.08 — — — — —

0.02 —

A8

Table 1. S- and Fe-related bacteria in the black shale samples from Chengkou County.

0.04 0.04 — 0.29 0.04 — — 0.02 6.39

0.51

0.38 0.40 — — — — 0.02

— 7.76

A10

Obligate aerobic, autotrophs, acidophilic, Fe oxidation Halotolerant, acidophilic, Fe and S oxidation Anaerobic, S oxidation Facultatively anaerobic and autotrophic, S oxidation Anaerobic, alkalitolerant, S reduction; Facultative anaerobic, Fe reduction Aerobic, neutrophilic, Fe oxidation Chemoautotrophic, acidophilic, S oxidation Halotolerant, acidophilic, Fe and S oxidation

Halophilic, obligatory arrobic, S oxidation

Acidophilic, Fe and S oxidation, and Fe reduction Neutrophilic, Fe oxidation Aerotolerant, S reducing Heterotrophic, acidophilic, Fe oxidation Heterotrophic, acidophilic, Fe oxidation Lithoautotrophic, neutrophilic, Fe oxidation Lithoautotrophic, acidophilic, Fe oxidation

Acidophilic, facultatively anaerobic, Fe and S oxidation Heterotrophic, Acidophilic, Fe reduction

Microbial metabolism

Emmerich et al., 2012; Headd and Engel, 2013 Kimura et al., 2011; Johnson, 2012 Korehi et al., 2013 Kojima and Fukui, 2010. Kojima and Fukui, 2010. Sorokin et al., 2013 Jiang et al., 2013 Seder-Colomina et al., 2014 Headd and Engel, 2013 Davis-Belmar et al., 2008

Hallberg et al., 2011 Johnson and McGinness, 1991; Küsel et al., 1999; Bridge and Johnson, 2000 Kimura et al., 2011; Korehi et al., 2013 Roden et al., 2012 Keller and Wall, 2011 Johnson et al., 2009 Johnson et al., 2009 Sobolev and Roden, 2001 Lu et al., 2010

Reference



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3542 J. Li et al.

Fig. 6. Geomicrobiological model of the black shale weathering profile. The model shows the roles of the S- and Fe-related bacteria identified in the S and Fe cycles. FeOB, Fe(II)-oxidizing bacteria.

counterparts. In addition to these bacteria, Aquabacterium and Pseudoalteromonas, which were also detected in this study, contained putative species, which could participate in the Fe oxidation process (Roden et al., 2012; Li et al., 2013). Generally, the distribution of FOB and SOB showed a positive correlation with the concentration of pyrite in the weathering profile (Fig. 5). Fe and S reduction. Additionally, Acidithiobacillus, Ferrimicrobium and Ferrithrix might participate in iron reduction in this study. Acidophilic Fe(III)-reducing organisms of the genus Acidiphilium were also detected in high abundance in the sample A10 (7.76%). Species belonging to this genus are well known for their ability to oxidize organic carbon sources and to transfer the electrons onto ferric iron (Johnson and McGinness, 1991; Küsel et al., 1999). Coupled with the FOB found here, these results suggest that complete and active Fe biogeochemical cycles were occurring in the black shale weathering profile. However, only a trace amount of sequences that belonged to Desulfovibrio and Sulfurospirillum, which were identified here, were affiliated with sulfate-reducing bacteria. This result was not in agreement with the high amount of sulfate anions in the black shale weathering profile. However, this result is consistent with those results of some previous acidic mineral drainage and hypersaline sediments studies (Emmerich et al., 2012; Johnson, 2012). A reasonable explanation for this result is that microbial Fe (III) reduction should be more energeti-

cally favourable than sulfate reduction from a thermodynamic point of view (Emmerich et al., 2012). Therefore, as long as bioavailable Fe(III) is not limiting, the ironreducing bacteria may outcompete sulfate reducers for organic electron donors. Overall, one significant implication of these results was that these S and Fe bacteria are mainly responsible for the oxidative dissolution of pyrite and for the biogeochemical cycles of Fe and S in the black shale weathering environments (Fig. 6). Microorganisms in the black shale weathering environments can achieve this goal by a variety of metabolic approaches, such as acidophilic chemolithoautotrophic S and Fe oxidation, acidophilic heterotrophic S and Fe oxidation, and neutral chemolithoautotrophic Fe oxidation, as well as acidophilic heterotrophic Fe reduction. Experimental procedures Site and sample description Chengkou County is in the Northern Sichuan Basin. This region has a subtropical mountain climate and belongs to subtropical monsoon climate zone. The average annual temperature is 13.8°C, and the average annual precipitation is 1261.4 mm. The widely distributed black shale outcrops, favourable natural climatic conditions and frequent human activities (e.g. mining and construction) have created the strong weathering of the black shale in this region and developed the typical black shale weathering profile. The sample site for this study is a fresh road cut on the east-facing slop of a ridge, which is near the town of



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Fig. 7. A. Location of the research area; B. Geostructure picture in the black box that is shown in A. C. Sample site at Chengkou County. Black circles indicate the locations of the two sample sites.

Chengkou County (Fig. 7, N31°57′27.92″, E108°38′10.71″). An appropriately 300 cm weathering profile was developed on a natural exposure of the Shuijingtuo Formation. This profile is ideal for the weathering study because this group was horizontal at this area and well exposed for at least 10 years old after the road construction. Eleven samples, including A11, A10, A9, A8, A7, A6, A5, A4, A3, A2 and A1, were orderly collected along a continuous depositional unit from the bottom fresh parent shale toward the surface regolith of the weathering profile. The weathering extent generally increased from A11 to A1 through field observation.

When collecting, the surface 3 cm of each sample was removed to avoid the mixture of exogenous matters, and the inner shale was collected carefully by the antiseptic chasing punch. The 11 samples were stored in sterile plastic bags and then kept in dry ice during the transport to the laboratory.

Mineralogical and element analyses For the inorganic geochemical analysis, the bulk samples were dried at approximately 60°C and thoroughly ground


3544 J. Li et al. using a mortar and pestle before passing through a 200 mesh sieve. The XRD pattern analysis was performed using an X-ray diffractometer (D/max2550VB3, Rigaku Corporation, Tokyo, Japan) with Cu Ka radiation at 35 kV and 30 mA. Diffraction angles (called ‘2θ’) corresponding to the crystal structure unique to each mineral were measured. Bulk chemical analyses were performed using X-ray fluorescence spectrometry (XRF Shimadzu XRF-1800) at 40 kV and 95 mA for the major elements and for some trace elements (e.g. Cu, Zn, Cr and Ba). The detection limit was below 0.01%. In this analysis, 1 g ground sample was combusted at 900°C for 2 h, and the difference in sample weight before and after combustion was reported as loss on ignition. The major elements were analysed quantitatively after the fusion of 0.1 g combusted sample with 3.6 g dilithium tetraborate at 1050°C for 16 min.

Environmental parameters analysis and sulfur species extraction To measure the pH in the weathered shales, 10 g powder samples were put into a 50 ml glass tube and mixed with 25 ml of distilled water. The mixture was left to equilibrate for 20 min. The pH was measured using a calibrated HACH HQ30d pH meter (USA). The measurement was repeated on three separate rock samples. Different sulfur species in these black shale samples were also analysed. Simply, AVS-S, FeS2 (pyritic-S), Sulfate (SO4-S) and organic matter (Org-S) were separated and gravimetrically quantified using an analytical scheme that was modified from Tuttle and colleagues (1986) and Chu and colleagues (1993). AVS-S and pyritic-S produced H2S when treated with chloride-acid and acidified chromous chloride solution, respectively, under an inert atmosphere (N2). H2S was collected as Ag2S. SO4-S was dissolved during the HCl extraction and precipitated as BaSO4. Org-S was extracted by calcining residues with a Eschka mixture (w : w, MgO : Na2CO3 = 3:2) in a muffle furnace in air at 800°C for 2 h, then dissolved by HCl extraction and precipitated as BaSO4. The results are reported on a ration between the weights of S in different phases (e.g. Pyritic-S) to the whole sample weight. The accuracy was determined by analysing standards, which were composed of known amounts of AVS (Na2S•9H2O), pure pyrite, sulfate (Na2SO4) and organic sulfur (C12H10O4S). The Na2S•9H2O recovery was in the range of 95–105%. The pure pyrite and Na2SO4 reference material recovery was 90–98%. The organic sulfur, C12H10O4S, recovery was 90–96%. The precision was determined by calculating the relative standard deviation (RSD) of duplicate samples and was considered acceptable when the RSD was no greater than 10%.

DNA extraction Nine samples, including A1, A2, A3, A4, A5, A6, A7, A8 and A10, were chosen for molecular biological analysis. The bulk samples were freeze dried. The microbial genomic DNA was extracted directly from the samples utilizing an E.Z.N.A Soil DNA kit (OMEGA, USA) following the manufacturer’s protocol and stored at −80°C until analysis.

Polymerase chain reaction (PCR) amplification, amplicon quantitation, pooling and pyrosequencing A region of ∼ 506 bp in the 16S rRNA gene, which covered the V1–V3 region, was selected to construct a community library through tag pyrosequencing. The bar-coded, broadly conserved primers 27F and 533R, which contained the A and B sequencing adaptors (454 Life Sciences), were used to amplify this region. The forward primer (B27F) was 5′-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG AGAGTT TGATCCTGGCTCAG -3′, where the sequence of the B adaptor is shown in italics and underlined. The reverse primer (A533R) was 5′-CCATCTCATCCCTGCGTGTCTCCGACTC AGNNNNNNNNNNTTACCGCGGCTGCTGGCAC-3′, where the sequence of the A adaptor is shown in italics and underlined, and the Ns represent an eight-base sample specific barcode sequence (Wu et al., 2012). In a final volume of 20 μl, the PCR reaction mixture contained: 2 μM each of the primer, 10 ng of template DNA, 4 μl of 5× FastPfu buffer and 2 U of TransStart FastPfu DNA Polymerase (TransGen Biotech, Beijing, China). The amplification conditions were as follows: an initial denaturation step (95°C for 2 min), which was followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. The final extension was for 5 min at 72°C. Negative controls were also performed. For each sample, three PCR products were pooled, purified on a 2% (w/v) agarose gel and extracted using a Gel-extraction kit (Omega) following the manufacturer’s instructions. Before sequencing, the DNA concentration of each PCR product was determined using a Quant-iTTM PicoGreen double-stranded DNA assay (Invitrogen, Germany) and was quality-controlled on an Agilent 2100 bioanalyzer (Agilent, USA). Following quantitation, the amplicons from each reaction mixture were pooled in equimolar ratios, which were based on concentration, and subjected to an emulsion PCR to generate amplicon libraries, as recommended by 454 Life Sciences. Amplicon pyrosequencing was performed from the A-end using a 454/Roche A sequencing primer kit on a Roche Genome Sequencer GS FLX Titanium platform obtained from Majorbio Bio-Pharm Technology, Shanghai, China.

Statistical and bioinformatics analysis The valid reads were picked by complying with the following rules: each pyrosequencing read containing a primer sequence should be 350–600 bp in length, have no ambiguous bases, match the primer and one of the used barcode sequences, and present at least an 80% match to a previously determined 16S rRNA gene sequence. These pyrosequencing reads were simplified using the ‘unique.seqs’ command to generate a unique set of sequences, aligned using the ‘align.seqs’ command and compared with the Bacterial Silva database (SILVA VERSION SSU111; http://www.arb-silva.de/). The aligned sequences were further trimmed, and the redundant reads were eliminated using the ‘screen.seqs’, ‘filter.seqs’ and ‘unique.seqs’ commands in order. The ‘chimera.slayer’ command was used to determine chimeric sequences. The ‘dist.seqs’ command was performed, and unique sequences were clustered into OTUs defined by 97% similarity. Rarefaction analysis, Good’s coverage, Chao, Ace, Simpson and Shannon indexes for the


Bacteria in a black shale weathering profile nine libraries were determined. In the present study, data preprocessing, OTU-based analysis and hypothesis testing were performed using the program MOTHUR (Schloss et al., 2009).

Acknowledgements This research was financially supported by the National Basic Research Program of China (Grant No. 2013CB429703), the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB06020200), the National Natural Science Foundation of China (Grant No. 41272370 and 41202042), the Knowledge Innovation Program of the Chinese Academy of Sciences (Grant No. SIDSSE-QN2O13O3) and the Open Fund of Key Laboratory of Marine Spill Oil Identification and Damage Assessment Technology, SOA (Grant No. 201307). We are greatly indebted to two anonymous journal reviewers and the Editor, Prof Michael Wagner, for their constructive remarks. The authors declare that they have no conflicts of interest in this research.

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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Mineral composition in the black shale samples from Chengkou County. Table S2. Major elemental content in the black shale samples from Chengkou County. Table S3. Geochemical parameters and various sulfur species in the black shale samples from Chengkou County. Table S4. Diversity and richness indexes for the bacterial communities in the black shale samples from Chengkou County. Table S5. Bacterial community structures in the black shale samples from Chengkou County in phylum level. Table S6. The dominant genera of the bacterial communities in the black shale samples from Chengkou County.


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Insight into sulfur reactions in Li-S batteries.

Insight into thermophiles and their wide-spectrum applications.

Differential leaflet mortality may influence biogeochemical cycling following tropical cyclones.

Hydraulic fracture extending into network in shale: reviewing influence factors and their mechanism.

Distribution and Diversity of Bacteria and Fungi Colonization in Stone Monuments Analyzed by High-Throughput Sequencing.

Prevalence of hemoglobin S in black and non-black children.

Insight into post-transcriptional gene regulation: stress-responsive microRNAs and their role in the environmental stress survival of tolerant animals.

Understanding Biogeochemical Cycling of Trace Elements and Heavy Metals in Estuarine Ecosystems.

Insight into the Diversity of Penicillin-Binding Protein 2x Alleles and Mutations in Viridans Streptococci.

Insight into S-RNase-based self-incompatibility in Petunia: recent findings and future directions.