Key Laboratory of Agricultural and Environmental Microbiology, Ministry of Agriculture, College of Life Science,

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1 AEM Accepts, published online ahead of print on 27 September 2013 Appl. Environ. Microbiol. doi: /aem Copyright 2013, American Society for Microbiology. All Rights Reserved Depth-Related Changes in Community Structure of Culturable Mineral Weathering Bacteria and in Weathering Patterns Caused by Them Along Two Contrasting Soil Profiles Jing Huang, Xia-Fang Sheng, Jun Xi, Lin-Yan He *, Zhi Huang, Qi Wang, Zhen-Dong Zhang Key Laboratory of Agricultural and Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agricultural University, Nanjing , PR China Downloaded from on August 19, 2018 by guest Corresponding author. address: xfsheng@njau.edu.cn (X.F. Sheng); helyan0794@njau.edu.cn (L.Y. He). 1

2 22 23 Abstract 24 Bacteria play important roles in mineral weathering and soil formation. However, few reports of mineral weathering bacteria inhabiting subsurfaces of soil profiles have been published, raising the questions whether the subsurface weathering bacteria are fundamentally distinct from those in surface communities. To address these questions, we isolated and characterized mineral weathering bacteria from two contrasting soil profiles with respect to their role in the weathering pattern evolution, their place in the community structure and their depth-related change in these two soil profiles. The effectiveness and pattern of bacterial mineral-weathering were different in the two profiles and among the horizons within the respective profiles. The abundance of highly effective mineral-weathering bacteria in the Changshu profile was significantly greater in the deepest horizon than in the upper horizons whereas in the Yanting profile it was significantly greater in the upper horizons than in the deeper horizons. Most of mineral-weathering bacteria from the upper horizons of the Changshu profile and from the deeper horizons of the Yanting profile significantly acidified the culture media in the mineral weathering process. The proportion of siderophore-producing bacteria in the Changshu profile was similar in all horizons except in the Bg2 horizon whereas the proportion of siderophore-producing bacteria in the Yanting profile was higher in the 41 upper horizons than in the deeper horizons. Both profiles existed different highly 42 depth-specific culturable mineral weathering community structures. The depth-related 43 changes in culturable weathering communities were primarily attributable to minor 2

3 44 45 bacterial groups rather than to a change in the major population structure. Keywords: Soil profile; Mineral weathering bacterial community; Mineral weathering pattern Mineral weathering plays an important role in soil and sediment formation (18); it represents a source of nutrients to terrestrial ecosystems and is also a major long-term sink for atmospheric CO 2 (4, 5, 11, 19, 30). So, there has been much interest in the weathering of silicate minerals (7, 18, 28). Mineral weathering is represented by biological processes associated with bacteria that inhabit a wide range of niches in surface and subsurface environments and influence various mineral transformation reactions (17). Microbes can accelerate mineral weathering reactions by producing organic and inorganic acids and metal-complexing ligands, changing redox conditions, or mediating formation of secondary mineral phases (5, 20). Increasing evidence points to an important role played by bacteria in mineral weathering (12, 13, 18). In addition, there is a large body of knowledge concerning the distribution, community structure, and the mineral weathering roles of bacterial communities in various environments such as forest soils, tree rhizosphere and ectomycorrhizosphere environments, rock surfaces and plant seeds (1, 10, 12, 25). However, previous studies on mineral weathering bacteria have focused on surface soil environments (20, 31, 32). Few studies have been published that provide an overview of mineral-weathering 63 populations throughout a soil profile and how these populations are distributed along a profile 64 of that soil. In general, the depth-related gradient of soil edaphic properties provides niches 65 for a wide variety of metabolically diverse microorganisms including mineral weathering 3

4 66 bacteria in a soil profile. We speculate that the mineral-weathering bacteria are specialized 67 with respect to their habitat and fundamentally distinct from those in surface communities (8, 68 16, 36), i.e., knowledge of the composition of mineral-weathering communities in a top soil does not necessarily predict the composition of the mineral-weathering communities in deeper layers beneath. To date, the culture-dependent approach remains the only way to study the mineral weathering ability of bacterial communities (3, 12, 20, 29). However, bacterial mineral weathering is really the result of a set of reactions (e.g., production of different corrosive acids, alkalis, and complexing agents), each of which is under control of a different gene or gene complexes, which are likely to be differently distributed among culturable and unculturable bacteria. For this reason, both of the culturable and unculturable bacteria play important roles in mineral weathering and soil evolution. In this study, we gave an insight into mineral weathering by the culturable portion of the active bacteria in the Changshu and the Yanting soil profiles. We hypothesize that the two different soil profiles harbour diverse and different culturable mineral weathering bacterial communities and that the weathering effectiveness and patterns of the culturable mineral weathering communities change with soil type and depth. To test these hypotheses we isolated culturable mineral-weathering bacteria to compare the abundance, weathering pattern, community structure and distribution between the two soil profiles. It is of great importance to determine whether depth affects the 85 populations and the effectiveness of the soil mineral-weathering bacteria. In this study, we 86 aim to establish relationships between bacterial taxa and the mineral weathering potential 87 along the soil profiles to further our understanding of the potential roles mineral-weathering 4

5 88 89 communities could play in mineral weathering, elemental cycling and soil formation in the soil profile systems MATERIALS AND METHODS Sampling and sample analysis. Soil samples were collected in two different sites of Changshu ( N, E), jiangsu province and Yanting (31 16 N, E), sichuan province. Changshu and Yanting soils were classified as gleyosols developed from a lacustrine deposit and purple-orthic primosols developed from purple sand shale, respectively. According to the visual pedological characteristics, the Changshu soil profile contained five horizons: A1 (0-12 cm), A2 (12-21 cm), Bg1 (21-46 cm), Bg2 (46-65 cm) and Bg3 (65-85 cm). No significantly different pedological characteristics were observed in the Yanting soil profile. The Changshu soil profile was sampled according to its pedological characteristics. The depth increments for soil sampling used for the Yanting soil profile were 0-10 cm (P1 horizon), cm (P2 horizon), cm (P3 horizon), cm (P4 horizon), cm (P5 horizon), and cm (P6 horizon). Three replicate soil samples (about 600 g per sample) were collected at each depth (horizon) from randomly chosen locations within each horizon by vertically coring into the exposed soil profile and excluding exposed soil. Soil sampling tools were sterilized between sample collections. The soil samples were placed in sterile bags to prevent cross-contamination. All soil samples were immediately transported 107 back to laboratory. All visible root and fresh litter material were removed from the samples. 108 The soil samples were stored at 4 C for biological and physical-chemical analyses. 109 To determine the bioavailable elemental contents, 2.5 g of soil sample were mixed with 5

6 110 M3 soil test extractant (23) by shaking at 25 C, 200 rpm for 15 min and then filtering. The 111 filtrates were then used to analyze the major element (Fe, Si, Mn, Mg, Ca, Cu, Al, and K) 112 contents by ICP-OES (Inductively coupled plasma-optical emission spectrometer) (Optima DV, Perkin Elmer). For determinations of organic matter (OM) content and soil ph, subsamples were air-dried and sieved to < 2 mm. Soil OM was measured by dichromate wet oxidation procedure (37). Soil ph was measured with a digital ph meter by mechanically shaking soil in 1:2.5 (soil: H 2 O w/v) water suspension at 200 rpm for 1 h. The moisture content was measured gravimetrically using fresh samples (2). The clay mineralogical compositions of the < 2 m size fractions were determined using X-ray diffraction (XRD, Philips PW3020 diffractometer with CuK radiation target) according to method of Prakongkep et al. (24). Bacterial enumerations and isolation. Culture techniques were employed to determine the bacterial counts in the soil samples. Preliminary experiment showed that more bacterial colonies were obtained on sucrose-salts medium (SSM) agar plates than on Luria-Bertani (LB) (33) or half-strength LB agar plates, which are often used for bacterial isolation and counting. The solid SSM [containing 1% sucrose, 0.1% (NH 4 ) 2 SO 4, 0.001% NaCl, 0.05% MgSO4 7H2O, 0.2% K 2 HPO 4, 0.05% yeast extract, 0.001% CaCO 3, and 2% agar in distilled water, ph 7.2] was used to grow culturable bacteria from the above samples. To prevent the growth of fungi, the sterilized medium (autoclaved at 121 C for 30 min) was supplemented 129 with 10 mg fungicidin L -1 (USP, Amresco, USA). Soil samples (1.0 g, wet weight) were added 130 to Erlenmeyer flasks containing 100 ml of sterile physiological salt solution (0.85% NaCl) 131 and shaken at 200 rpm for 30 min to allow bacteria to detach from soil particles. The 6

7 132 suspensions were then allowed to stand for about 10 min. Serial 10-fold dilutions of sample 133 suspensions (10-3 and 10-4 ) were plated onto SSM agar plates [0.1 ml of bacterial suspension 134 was spread on a plate and suitable number of cfu (between 30 and 300) per agar plate] to determine total culturable bacteria. The plates were incubated for 7 days at 28 C, and then the numbers of colonies per gram of fresh soil were calculated from the colony count. One hundred bacterial colonies were picked randomly from the plates from each horizon of the two soil profile samples and then purified on the same medium by streaking on fresh medium. The obtained colonies were stored on slants for studying the solubilization of biotite. Mineral weathering potential of bacteria. A ubiquitous soil mineral biotite was used in this study for evaluating the mineral weathering potential of bacteria. The elemental composition of the mineral was as follows: SiO %, Al 2 O %, K 2 O 9.12%, Fe 2 O %, Na 2 O 0.28%, MgO 13.69%, and CaO 0.07%. Based on the low-nutrient Bushnell-Haas medium (BHm) (containing 0.002% KCl, 0.015% MgSO 4 7H 2 O, 0.008% NaH 2 PO 4 2H 2 O, 0.009% Na 2 HPO 4 2H 2 O, % (NH 4 ) 2 SO 4, 0.01% KNO 3, 0.002% CaCl 2, 0.2% glucose) (32), a modified BHm (free of KCl and KNO 3 ) was used to evaluate whether the bacteria could release Si, Al, K, and Fe from biotite under low-nutrient condition. To prepare the inoculum, bacterial isolates were cultivated overnight in sterilized liquid LB medium and harvested by centrifugation at 5,000 rpm for 10 min. Inoculum was washed two times in sterile distilled water to remove the nutrients and metabolic products (including acids) 151 in the medium. Cell pellets were then resuspended in sterile distilled water to a final 152 concentration of 10 8 cells ml -1 before the mineral weathering experiments were started. 153 Triplicate 100 ml polycarbonate Erlenmeyer culture flasks with vented caps (0.3 µm PTFE 7

8 154 membranes) containing 30 ml of sterilized modified Bushnell-Haas medium (containing mg of 100- to 200- m particles of biotite) were each inoculated with 0.5 ml of a bacterial 156 suspension. Considering the fact that much work was needed for showing the release of the elements from the mineral over time by 1,100 bacterial strains, we focused on showing the releases of elements by the bacteria at one time point in this study. The flasks were incubated at 28 C on a rotary shaker at 150 rpm for 7 days. Controls with biotite but no bacterial cells to monitor the range of abiotic dissolution were treated in the same manner. The weathering of the mineral in the presence of bacteria was monitored at 7 days of incubation. Samples for chemical analyses were then filtered through a 5 m Millipore filter, and 20 ml of the filtrate from each flask were then centrifuged at 10,000 rpm for 10 min in order to remove cells from the suspension. Five ml of the supernatant were collected for ph determination and another 5 ml of the supernatant were acidified with HNO 3 (final concentration 2% v/v) to avoid precipitation of dissolved chemical species and then analyzed for Fe, Si, Al, and K contents by ICP-OES. DNA extraction and PCR. Using the standard lysozyme-sds-pronase protocol according to Molecular Cloning (26), genomic DNA was extracted from each mineral-weathering isolate after growth in SSM to late exponential phase. The obtained crude DNA was purified with DNA quick midi purification kit (TIANGEN Biotechnology Limited Company, Beijing), according to the manufacturer s instructions. Then the 16S rrna gene of 173 the isolates was amplified with the universal bacterial 16S rrna primers 27F 174 (5'-AGAGTTTGATCCTGGCTCAG) and 1492R (5'-ACGGCTACCTTGTTACGACTT) (21). 175 The PCR reaction system contained: 2 L of purified DNA extract, 0.2 M each primer,

9 176 L Premix Ex Taq (containing DNA polymerase, buffer, and dntp mixture) (TAKARA), and 177 sterile deionized water to a final volume of 50 L. The following cycle conditions were used: C for 5 min; 28 cycles of denaturation at 95 C for 30 s, annealing at 55 C for 30 s and extension at 72 C for 90 s; and a final extension at 72 C for 10 min. The presence of PCR products was confirmed by electrophoresis on 1.5% agarose gels and staining with ethidium bromide. Sequencing analyses. Purified PCR products from the 16S rrna genes of mineral weathering bacteria were sequenced on an ABI automated sequencer (Invitrogen) combined with a Sequencing Kit (BigDye Terminator) and the primers set 27f and 1492r as well as M13-47 and RV-M. The resulting nucleotide sequences were analyzed using the National Centre of Biotechnology Information (NCBI) database to obtain the closest species match. The phylogenetic affiliation was verified by using RDP classifier (34). The nucleotide sequences determined in this study have been deposited in the NCBI database. The accession numbers for the sequences of the cultured mineral weathering strains are shown in Tables S3 and S4. Siderophore production. The production of siderophores by the mineral weathering bacteria was determined according to the chrome azurol-s (CAS) analytical method (22, 27). Bacterial isolates were cultivated in liquid SSM medium at 28 C on a rotary shaker at 150 rpm for 48 h. Cells and supernatants were separated by centrifugation at 9000 g for 10 min. 195 One ml of supernatant was mixed with 1.0 ml of CAS Assay Solution (22). A control was 196 prepared by mixing 1.0 ml of the CAS Assay Solution with 1.0 ml of the uninoculated 197 medium used for culturing the bacterial strains. Absorbance at 630 nm was measured 1 h after 9

10 198 mixing, and values were compared with the optical density (O.D.) of the control (22). 199 Statistical analyses. One-way analysis of variance (ANOVA) and the Fisher s Least 200 Significant Difference test (Fisher s LSD) (p < 0.05) were used to compare the averages of soil parameters for the different layers of the soil profiles, the averages of ph determinations, and the averages of the concentrations of Fe, Si, Al, and K released in the bacterial treatments with those from the control of untreated medium. Statistical analyses were carried out using SAS 8.2 (Statistical Analysis System, USA). RESULTS Characteristics of soil samples collected from two profiles. Depth-related changes in biogeochemical properties of the two soil profiles are shown in Fig. 1. For the Changshu soil profile, the variation in the available Al, Si, Mn, Mg, and Ca contents showed similar trends, increasing with depth (Fig. 1A). However, available Fe content decreased with depth, reaching the lowest in the horizon of Bg2. Because of the high mobility, the available K in the soil profile exhibited irregular fluctuation and did not display any obvious depth-related effects. The ph increased with depth and became weakly alkaline. The OM content was high in the upper horizons A1 and A2 but decreased about 3-folds in the horizon Bg1 (Fig. 1A). High OM content was observed in the horizon Bg2 compared to the adjacent horizons Bg1 217 and Bg3 (Fig. 1A). The maximum and minimum moisture was found in the Bg3 and Bg1 218 horizons, respectively (Fig. 1A). 219 Contrary to the findings from the Changshu soil profile, the content of available Si, Mg, 10

11 220 Ca, and Mn in the Yanting soil profile fluctuated irregularly and did not display any obvious 221 depth-related trends. Available Al and OM contents decreased with depth. The ph also 222 increased with depth and became alkaline. The maximum and minimum moisture was found in the P1 and P3 horizons, respectively (Fig. 1B). The available Si, Al, Mg, and Ca content in the Changshu profile was higher than in the Yanting profile, however, higher OM content was observed in the Yanting profile than in the Changshu profile (Fig. 1). XRD analysis was conducted to identify the clay mineral components of the two soil profiles (Figs. S1 and S2). XRD of the soil samples revealed no significant differences in clay mineral components within the respective soil profiles. The clay mineral components of the Changshu soil profile samples included illite, montmorillinte, and kaolinite-smectite, while only illite was detected in the clay mineral of the Yanting soil profile samples. Bacterial counts and isolation of mineral weathering bacteria. The culturable bacterial counts (log cfu g -1 fresh soil) of the soil samples revealed appreciable tendency to change with increasing depth for both of the two soil profiles (Fig. 1). The bacterial counts decreased significantly with increasing depth, ranging from 4.25 (deepest horizon) to 7.27 (surface horizon) cfu g -1 fresh soil from the Changshu soil profile and from 4.13 (deepest horizon) to 6.50 (surface horizon) cfu g -1 fresh soil from the Yanting soil profile. More bacterial counts were obtained from the Changshu soil profile samples than from the Yanting soil profile samples. 239 Using agar plates, we obtained 1,100 bacterial strains, among which 500 and 600 strains 240 were isolated from the Changshu and Yanting soil profile samples respectively. In the mineral 241 weathering experiment, releases of major structural elements, dissolved Fe, Si, Al, and K for 11

12 242 biotite, were used as an overall indicator of mineral weathering. Based on the mineral 243 weathering experiment, 53.4% (267/500) and 63.5% (381/600) of the strains from the 244 Changshu and Yanting profiles respectively were found to have the capacity to mediate biotite weathering (Tables S1 and S2). More mineral-weathering bacteria were obtained from the Yanting soil profile samples than from the Changshu soil profile samples. Among the 267 mineral-weathering bacteria from the Changshu profile, 43, 55, 49, 59, and 61 bacteria were obtained from the horizons A1, A2, Bg1, Bg2, and Bg3 respectively; among the 381 mineral-weathering bacteria from the Yanting profile, 71, 77, 50, 67, 69, and 47 bacteria were obtained from the horizons P1, P2, P3, P4, P5, and P6 respectively (Tables S1 and S2). More mineral-weathering bacteria were obtained in the deeper horizons Bg2 and Bg3 of the Changshu profile and in the upper horizons P1 and P2 of the Yanting profile. Mineral-weathering patterns by the isolates. The obtained mineral-weathering bacteria had different mineral weathering potentials and produced different weathering patterns. After 7 days of incubation, the dissolved Fe, Si, Al, and K contents in the cultures ranged from 3 to 457, 18 to 1439, 6 to 346, and 28 to 1015 M respectively for isolates from the Changshu soil profile and ranged from 8 to 663, 20 to 812, 6 to 693, and 29 to 1091 M respectively for isolates from the Yanting soil profile (Table 1). Dissolved Fe, Si, Al and K increased 1.9- to 305-fold, 1.1- to 89-fold, 1.2- to 72-fold, and 1.3- to 47-fold, respectively, in the presence of the mineral-weathering bacteria isolated from the Changshu profile, and increased 5.4- to fold, 1.3- to 50-fold, 1.2- to 144-fold, and 1.3- to 50-fold, respectively, in the presence of 262 mineral-weathering bacteria isolated from the Yanting soil profile when compared to the 263 respective concentrations in the uninoculated controls (Table 1). 12

13 264 Furthermore, among the mineral-weathering bacteria isolated from the Changshu profile, 265 the average Fe, Si, and Al releases by the bacteria were significantly lower in the Bg1 horizon 266 than in the other horizons (Fig. 2). The average Fe release by the bacteria was significantly higher in the deeper horizons Bg2 and Bg3 (ranging from 88 to 110 M Fe) than in the upper horizons A1, A2, and Bg1 (ranging from 57 to 73 M Fe). The bacteria from the deepest horizon Bg3 released significantly higher average amounts of Si than those from the upper horizons A1 and A2, while the bacteria from all the horizons, except Bg1 horizon, released similar average amounts of Al (Fig. 2). The bacteria from the horizons A1, Bg2, and Bg3 also released similar average amounts of K. Contrary to the findings from the Changshu soil profile, among the mineral-weathering bacteria isolated from the Yanting soil profile, the bacteria from the P2 horizon released significantly higher average amounts of Fe, Si, Al, and K than those from the other horizons (Fig. 2). The average amounts of Si, Al, and K releases by the bacteria were significantly higher in the upper horizons than in the deeper horizons, however, no significant changes in the average amounts of Fe, Si, Al, and K releases by the bacteria were observed in the deeper horizons (Fig. 2). According to the potential of bacterial influence on Fe, Si, Al, and K releases from biotite during the mineral weathering experiment (Tables S1 and S2), the mineral weathering bacteria could be grouped into three categories. The first group includes bacteria with low potential for element solubilization ( < 20 µm Fe, < 40 µm Si, < 20 µm Al, and < 100 µm K in the 283 culture); the second group includes bacteria with moderate potential for element solubilization 284 (20-40 µm Fe, µm Si, µm Al, and µm K in the culture); and the third 285 group includes bacteria with high potential for element solubilization ( > 40 µm Fe, > 80 µm 13

14 286 Si, > 40 µm Al, and > 300 µm K in the culture) (Tables S1 and S2). Among the 287 mineral-weathering bacteria from the two soil profiles, the proportion of the highly effective 288 mineral-weathering bacteria was significantly higher than that of the moderately and least effective mineral-weathering bacteria (Fig. 3). The proportion of the moderately effective mineral-weathering bacteria from the Yanting profile was also significantly higher than that of the least effective mineral-weathering bacteria (Fig. 3). Furthermore, the proportion of the highly effective mineral-weathering bacteria from the Changshu profile was significantly higher in the deeper horizons Bg2 and Bg3 than that in the upper horizons A1, A2, and Bg1 (Fig. 3); the proportion of the moderately effective mineral-weathering bacteria was significantly higher in the A2 horizon than that in the other horizons, and the proportion of the least effective mineral-weathering bacteria was significantly higher in the A1 and Bg1 horizons than that in the A2, Bg2, and Bg3 horizons. However, the proportion of the highly effective mineral-weathering bacteria from the Yanting profile was significantly higher in the upper horizons P1, P2, and P4 than that in the deeper horizons P5 and P6 (Fig. 3); the proportion of the moderately and least effective mineral-weathering bacteria was significantly higher in the deeper horizons P5 and P6 than that in the upper horizons (Fig. 3). Some of the strains isolated from the two soil profiles were highly effective in simultaneously solubilizing Fe, Si, Al, and K (Tables S1 and S2), while other strains were highly effective in solubilizing Si and K, but least effective in solubilizing Fe and moderately 305 effective in solubilizing Al (Table S1). Strain A2053 was the only highly effective Fe and Si 306 solubilizer (Table S1). In addition, strain 1075 was highly effective K, least effective Fe, and 307 moderately effective Si and Al solubilizer (Table S2). 14

15 308 Acid-producing patterns of the isolates during mineral weathering process. As shown 309 in Tables S1 and S2, mineral weathering bacteria exhibited different patterns of culture 310 medium acidification in the mineral weathering process. Among the mineral weathering bacteria isolated from the Changshu profile, 33, 44, and 23% of bacteria had poor (ph > 6 in the media), moderate (ph = 4-6), and high (ph < 4) ability to acidify the culture medium, respectively. Within the soil profile, the proportions of poorly, moderately, and highly effective acid-producing bacteria ranged from 9 to 52%, 20 to 51%, and 12 to 40%, respectively (Table S1). More highly effective acid-producing bacteria were observed in the A1 horizon while poorly effective acid-producing bacteria were found in the Bg3 horizon (Table S1). For the mineral weathering bacteria isolated from the Yanting soil profile, poorly, moderately, and highly effective acid-producing bacteria accounted for 22, 42, and 36% respectively (Table S2). The proportions of poorly, moderately, and highly effective acid-producing bacteria ranged from 10 to 34%, 26 to 64%, and 16 to 55%, respectively within the soil profile (Table S2). In addition, more poorly, moderately, and highly effective acid-producing bacteria were found in the horizons P1, P3, and P4 respectively (Table S2). Similar moderately effective acid-producing bacteria were detected among the mineral-weathering bacteria isolated from the two soil profiles, while more highly effective acid-producing bacteria were detected among the Yanting soil mineral-weathering bacteria 327 than were detected among those of the Changshu soil mineral-weathering bacteria. 328 Furthermore, more poorly effective acid-producing bacteria were observed among the 329 mineral-weathering bacteria isolated from the Changshu profile than from the Yanting profile. 15

16 330 Also, the proportion of highly effective acid-producing bacteria was significantly higher in the 331 horizon A1 of the Changshu soil profile and in the horizon P4 of the Yanting soil profile 332 (Table 1). The proportion of moderately effective acid-producing bacteria was significantly higher in the A2 horizon and in the horizons P3 and P5, while the proportion of poorly effective acid-producing bacteria was significantly higher in the horizons Bg2 and Bg3 and in the horizons P1 and P2 (Table 1). Siderophore-producing patterns of the isolates. As shown in Tables S1 and S2, the mineral weathering strains had different abilities to produce siderophores in the culture medium. The mineral weathering bacteria could be grouped into three categories on the basis of the patterns of siderophore production (Tables S1 and S2). The first category comprised 18-51% and 12-47% of the isolates from the Changshu and Yanting soil samples respectively which did not produce siderophores. The second category comprised 39-57% and 17-42% of the isolates from the Changshu and Yanting soil samples respectively which produced low concentrations of siderophores. The third category comprised 7-26% and 10-34% of the isolates from the Changshu and Yanting soil samples respectively which produced high concentrations of siderophores. The proportion of bacteria that did not produce siderophores was significantly higher in the Bg2 horizon than in the other horizons of the Changshu profile whereas in the Yanting profile it was significantly greater in the deeper horizons than in the upper horizons (Table 1). The proportion of bacteria that produced high concentrations of 349 siderophores was significantly larger in the upper horizons than in the deeper horizons of the 350 Changshu soil profile whereas in the Yanting profile it was significantly greater in the 351 horizons P2 and P3 than in the other horizons. The proportion of bacteria that produced low 16

17 352 concentrations of siderophores was not significantly different in the horizons from the two 353 profiles (Table 1). 354 Phylogenetic analysis of mineral weathering isolates. Sequencing of the 16S rrna and phylogenetic analysis showed that the mineral weathering bacteria were affiliated with 13 and 27 genera in the Changshu and Yanting soil profiles respectively (Figs. S3 and S4). Thirty different bacterial genera were obtained in the two soil profiles. Among the mineral weathering bacterial genera, Novosphingobium, Ochrobactrum, and Flavobacterium were specific to the Changshu soil samples while Agromyces, Brachybacterium, Microbacterium, Caulobacter, Sphingomonas, Phyllobacterium, Achromobacter, Massilia, Vogesella, Mitsuaria, Enterobacter, Pseudoxanthomonas, Serratia, Exiguobacterium, Solibacillus, Granulicatella, and Chryseobacterium were specific to the Yanting soil samples (Figs. S3 and S4). The most frequently isolated mineral weathering bacteria from the Changshu soil profile belonged to the Bacillus (14-49%), Arthrobacter (9-49%), and Pseudomonas (23-33%) species and were detected at all depths (Fig. 4). Members of Ochrobactrum and Acinetobacter with mineral weathering potential were specific to the horizon A1, while members of Flavobacterium, Stenotrophomonas, and Ensifer with mineral weathering potential were only present in the horizon A2. Mineral weathering Novosphingobium species were only obtained from the deepest horizon Bg3. No specific mineral weathering bacterial genera were detected 371 in the deeper horizons Bg1 and Bg2 (Fig. 4). Notably, an enrichment of Arthrobacter species 372 was observed in the deeper horizons, while Bacillus species were enriched in the subsurface 373 A2 horizon. In the Yanting soil profile, the composition of mineral weathering bacterial 17

18 374 populations differed from that of the Changshu profile (Fig. 5). The most frequently isolated 375 mineral weathering bacteria belonged to the Bacillus (35-83%) species and were detected at 376 all depths (Fig. 5). Pantoea species were the dominant mineral weathering bacteria in the upper horizons P1 and P2, while Pseudomonas species were the dominant mineral weathering bacteria in the deeper horizons P4 and P5. Members of Caulobacter, Agromyces, Solibacillus, Achromobacter, and Pseudoxanthomonas with mineral weathering potential were specific to the horizon P1, while members of Granulicatella, Mitsuaria, Stenotrophomonas, Arthrobacter, Chryseobacterium, and Vogesella with mineral weathering potential were only present in the deeper P5 horizon (Fig. 5). Phyllobacterium and Brachybacterium species were obtained only from the horizon P4. No specific mineral weathering bacterial genera were observed in the horizons P2, P3, and P6. Notably, an enrichment of Bacillus species was observed in the P3 and P6 horizons, while Pseudomonas species were enriched in the deeper horizons P4 and P5 (Fig. 5). Depth-related change of the effective mineral-weathering bacteria. Among the highly effective mineral weathering bacterial species isolated from the Changshu soil profile, the most frequently isolated taxon from the horizons A1, A2, Bg1 and Bg2 was Pseudomonas frederiksbergensis, whereas Arthrobacter globiformis and Arthrobacter oryzae were most frequently isolated from the deeper horizons Bg2 and Bg3. Horizons A1 and A2 harbored specifically 45.5% and 35.7% of the highly effective mineral-weathering bacteria, 393 respectively (Fig. 6a). Bacillus flexus was specific to the horizon Bg2, while Arthrobacter 394 nicotianae, Pseudomonas lundensis, and Pseudomonas libanensis were only present in the 395 horizon Bg3. No specific highly effective mineral weathering bacterial species were detected 18

19 396 in the Bg1 horizon. Arthrobacter globiformis, dominant in the deeper horizons, was absent in 397 the upper A1 and A2 horizons. Bacillus simplex was present in the horizons Bg1 and Bg3 but 398 absent in the horizons A1, A2, and Bg2, while Pseudomonas arsenicoxydans was only present in the horizons A1 and Bg3. Horizon Bg3 showed maximum bacterial diversity, represented by 23 different species, whereas the horizon Bg1 showed minimal diversity, represented by only eleven species. Only P. frederiksbergensis and B. aryabhattai were common to 5 horizons of the profile (Fig. 6a). Among the moderately effective mineral weathering bacterial species isolated from the Changshu soil profile, the most frequently isolated taxon from all horizons but the Bg3 horizon was Arthrobacter oryzae, whereas Arthrobacter globiformis was most frequently isolated from the deeper horizons Bg1, Bg2, and Bg3. Bacillus aryabhattai and Pseudomonas frederiksbergensis were most frequently isolated from the upper horizons A1, A2, Bg1 or Bg2. Horizons A1, A2, Bg1, Bg2, and Bg3 harbored specifically 43%, 67%, 63%, 57% and 60% of the moderately effective mineral-weathering bacteria, respectively (Fig. 6a). Bacillus cereus was only present in the horizons A1 and Bg3. Among the least effective mineral weathering bacterial species isolated from the Changshu soil profile, the most frequently isolated taxon from the deeper horizons A2, Bg1, and Bg2 was Arthrobacter globiformis, whereas Arthrobacter phenanthrenivorans was most frequently isolated from the upper horizons A1 and Bg1. Horizons A1, A2, Bg1, Bg2, and Bg3 harbored specifically 71%, 75%, 40%, 50% and 100% of the least effective mineral-weathering bacteria, respectively 415 (Fig. 6a). Bacillus flexus and Rhizobium giardinii were specific to the horizons Bg2 and Bg3 416 respectively, while Bacillus aryabhattai, Pseudomonas frederiksbergensis, Pseudomonas 417 gessardii, Pantoea brenneri, and Pseudomonas azotoformans were only present in the horizon 19

20 418 A1. Pseudomonas benzenivorans, Ensifer adhaerens, and Arthrobacter nicotinovorans, 419 dominant in the horizon A2, were absent in the horizons A1, Bg1, Bg2, and Bg3. Horizon A1 420 showed maximum bacterial diversity, represented by 7 different species, whereas horizon Bg showed minimal diversity, represented by only one species. Among the highly effective mineral weathering bacterial species isolated from the Yanting soil profile, the most predominant taxon isolated from the horizons P1, P2, P3, P5, and P6, was Bacillus aryabhattai, while Pantoea vagans was the most predominant taxon in the horizons P2, P3, and P4. Pseudomonas umsongensis and Bacillus safensis were the most predominant taxon in the horizons P4 and P6 respectively. Of the highly effective mineral-weathering bacteria, 52%, 40%, and 40% were specific to the horizons P1, P2, and P4, respectively. Enterobacter ludwigii and Sphingomonas molluscorum were specific to the horizon P3, while Bacillus koreensis was only present in the horizon P6. No specific highly effective mineral weathering bacteria were observed in the P5 horizon. The dominant Enterobacter hormaechei was present in the upper horizons P1 and P2 but absent in the deeper horizons. Pseudomonas putida was present in the horizons P1, P2, P4, and P5, but absent in the horizons P3 and P6, while Exiguobacterium indicum was only present in the deeper horizons P4 and P5. Horizon P2 showed maximum diversity represented by 25 different species, while horizon P5 showed minimal diversity represented by only four species. Only Bacillus aryabhattai was common to 6 horizons of the profile and only Bacillus simplex 437 and Pantoea vagans were common to 4 horizons. Among the moderately effective mineral 438 weathering bacterial species isolated from the Yanting soil profile, the most frequently 439 isolated taxon from all the horizons but the P1 horizon was Bacillus aryabhattai, whereas 20

21 440 Acinetobacter johnsonii was most frequently isolated from the horizons P2 and P4. Horizons 441 P1, P2, P3, P4, P5, and P6 harbored specifically 64%, 33%, 25%, 43%, 63%, and 33% of the 442 moderately effective mineral-weathering bacteria, respectively (Fig. 6b). Paenibacillus borealis, Bacillus idriensis, and Paenibacillus xylanexedens were only present in the horizon P2, while Paenibacillus jilunlii, Bacillus anthracis, and Pseudomonas monteilii were specific to the horizon P6. Among the least effective mineral weathering bacterial species isolated from the Yanting soil profile, the most frequently isolated taxon from the horizons P3, P4, and P6 was Bacillus aryabhattai. Horizons P1, P3, P4, P5, and P6 harbored specifically 100%, 50%, 80%, 100%, and 67% of the least effective mineral-weathering bacteria, respectively (Fig. 6b). No least effective mineral-weathering bacteria were observed in the horizon P2 (Fig. 6b). Bacillus anthracis was only present in the horizons P3 and P6. DISCUSSION Understanding the depth-related changes in weathering effectiveness and pattern, community structure of mineral weathering bacteria in the soil systems should allow us to better comprehend the roles of bacteria in mineral weathering, element cycling, and soil formation along the soil profiles. In this study, we focused on the culturable mineral-weathering bacteria. A large number of mineral-weathering bacteria were obtained 459 from two contrasting soil profiles (Tables S1 and S2) and were used for comparative analyses 460 of mineral weathering and community structure of culturable bacteria. To our knowledge, this 461 is the first time that the impacts of soil type and depth on the effectiveness and pattern of 21

22 462 culturable mineral-weathering bacterial communities have been tested. 463 The effectiveness and pattern of bacterial mineral-weathering were different in the two 464 profiles and among the horizons within the respective profiles (Figs. 2 and 3). The average Fe and Si releases by the bacteria from the Changshu profile were significantly higher in the deeper horizons than in the upper horizons, while the average Al and K releases by the bacteria from the Changshu profile (except for Bg1 or A2 horizon) were similar (Fig. 2). However, the average Si, Al, and K releases by the bacteria from the Yanting soil profile were significantly higher in the upper horizons than in the deeper horizons, while no significant changes in average Fe, Si, Al, and K releases by the bacteria were observed in the deeper horizons (Fig. 2). Although more than half of the bacteria isolated from the two soil profiles belonged to the highly effective mineral-weathering bacteria, the depth-related change in the proportion of the highly, moderately, and least effective mineral-weathering bacteria was different between the two soil profiles (Fig. 3). Furthermore, the extent of mineral weathering was dependent on the isolates and their origin (Table 1, Tables S1 and S2). Under laboratory conditions the mineral weathering effectiveness of dominant Pseudomonas and Arthrobacter species was greater in the deeper horizons than in the upper horizons of the Changshu profile based on the average Si and Al releases from biotite by the bacteria. However, the mineral weathering effectiveness of dominant Bacillus species at all horizons of the Yanting profile was similar. Furthermore, the mineral weathering effectiveness of the Bacillus or 481 Pseudomonas species isolated from the Changshu profile was greater than that of the same 482 bacterial species isolated from the Yanting profile. The differences in physical/chemical 483 characteristics between the two horizons or profiles may cause the difference in the level of 22

23 484 mineral weathering by the same species. 485 Despite the difficulties associated with attempting to interpret complex processes 486 occurring in the natural environment based on simple laboratory experiments, it is possible to glean information about the basic principles of bacterially mediated mineral weathering from experimental work. Interestingly, the proportion of highly effective mineral-weathering bacteria to total mineral weathering bacteria was higher (75.4%) in the deepest horizons of the Changshu profile, while the proportion of highly effective mineral weathering bacteria was higher (64.9%) in the upper horizon P2 of the Yanting profile, suggesting that the highly effective mineral weathering bacteria had distinct distribution pattern between the two profiles. Several studies also have demonstrated that the distribution and potential effectiveness of culturable mineral-weathering bacteria vary in relation to the ecological niche they occupy (10, 29). Uroz et al. (29) demonstrated that the bacterial communities from the oak-scleroderma citrinum mycorrhizosphere were characterized by a higher ability to weather minerals compared to the ones from the surrounding bulk soil. However, no significant differences were observed between the bacterial isolates from the Scleroderma citrinum hyphosphere and ectomycorrhizosphere (31). It is generally assumed that mineral weathering in the presence of bacteria is caused by proton- and/or ligand-promoted mineral dissolution (6, 9, 31, 35). The obtained mineral weathering bacteria varied in ability to produce acids and siderophores. For the Changshu soil profile, 87-91% of bacteria from the A1 and A2 horizons 503 produced moderate and large ph changes, while 46-53% of bacteria from the Bg2 and Bg3 504 horizons produced small ph changes (Table S1), suggesting that most bacteria from the upper 505 horizons could weather biotite by proton-or/and ligand-promoted dissolution mechanisms, 23

24 506 while nearly half of the bacteria from the deeper horizons promoted biotite weathering by 507 ligand-promoted dissolution mechanism. In the Yanting soil profile, 80-90% of the mineral 508 weathering bacteria from the horizons P3, P4, P5, and P6 produced moderate and large ph changes (Table S2), suggesting that most of the bacteria from the deeper horizons could weather biotite by proton and/or ligand-promoted dissolution. Furthermore, the proportion of siderophore-producing bacteria was similar in all horizons except in the Bg2 horizon for the Changshu profile, while the proportion of siderophore-producing bacteria from the Yanting profile was higher in the upper horizons P1, P2, and P3 than in the deeper horizons P4, P5, and P6. The proportions of siderophore-producing bacteria of the upper horizons P1 and P2 and of the deeper horizons P4, P5, and P6 were similar, respectively. Phylogenetic analysis has revealed that all the mineral weathering bacterial isolates from forest soils belong to 16 genera within -, -, -Proteobacteria and Firmicutes lineages (11). In our study, a similar highly diverse mineral weathering bacterial community was found in the two soil profiles (Figs. S3 and S4). The cultured mineral-weathering bacteria were represented by many genera (Figs. S3 and S4), among which Arthrobacter, Pseudomonas, and Bacillus were dominant in the Changshu profile whereas Bacillus and Pseudomonas were dominant in the Yanting profile. It is noteworthy that the cultured mineral weathering communities from the two profiles were distinct. Greater diversity at the species level was detected in the Yanting profile than in the Changshu profile (Fig. 6). Furthermore, some 525 identical bacterial species were observed in the least, moderately, and highly effective 526 mineral-weathering bacteria, while some bacterial species were specific to the least or to the 527 moderately or to the highly effective mineral-weathering bacteria (Fig. 6). Higher diversity of 24

25 528 the highly effective mineral-weathering bacteria from the two profiles was observed. The species with mineral weathering ability have not been previously reported, suggesting that 530 these weathering bacteria were specific in their soil environments. Interestingly, the most efficient mineral weathering Burkholderia and Collimonas genera which have been frequently found in acidic and nutrient-poor soils were not detected in the soil profiles (31). The above results indicate significant differences in the mineral weathering bacterial communities between surface and subsurface soil environments. The distribution pattern of the cultured mineral weathering bacterial communities along the two profiles differed significantly (Figs. 4, 5, and 6). Although Bacillus and Pseudomonas species from both profiles displayed an even distribution along the soil profiles, bacterial species of the weathering communities showed different depth-specific distribution along the two vertical profiles (Figs. 4 and 5). For instance, rare and specific species in the two profiles tended to be limited to the upper or deeper horizons, while the Rhizobium and Paenibacillus species were less abundant in the Changshu and Yanting profiles respectively but could be found at greater depths than the Curtobacterium and Ensifer species isolated from the Changshu and Yanting profiles respectively (Figs. 4 and 5). Even among the common Arthrobacter and Acinetobacter species in the two profiles, highly depth-specific distributions were found. Arthrobacter species were detected in all horizons of the Changshu profile, whereas they were detected only in one horizon of the Yanting profile. Similarly, Pantoea 547 species from the Changshu profile and Ensifer species from the Yanting profile peaked in the 548 surface horizons, but were absent or just a minor component at other horizons (Figs. 4 and 5). 549 Furthermore, the Arthrobacter and Bacillus species were enriched in the deeper horizons Bg1, 25

26 550 Bg2, and Bg3 of the Changshu profile and in the horizons P3 and P6 of the Yanting profile 551 respectively (Figs. 4 and 5). These observations suggest that the depth-related changes in the 552 composition of mineral weathering bacterial populations and the level of weathering activity of individual species of bacteria between the soil profiles and among the horizons within respective profiles may be shaped by distinct edaphic factors (14, 15). ACKNOWLEDGMENT This work was supported by the Chinese National Natural Science Foundation ( ). REFERENCES 1. Abdulla, H Bioweathering and biotransformation of granitic rock minerals by Actinomycetes. Microb. Ecol. 58: Angel, R., M. I. M. Soares, E. D. Ungar, and O. Gillor Biogeography of soil archaea and bacteria along a steep precipitation gradient. The ISME J. 4: Balland, C., A. Poszwa, C. Leyval, and C. Mustin Dissolution rates of phyllosilicates as a function of bacterial metabolic diversity. Geochim. Cosmochim. Acta 74: Balogh-Brunstad, Z., C. K. Keller, J. T. Dickinson, F. Stevens, C.Y. Li, and B. T. Bormann Biotite weathering and nutrient uptake by ectomycorrhizal fungus, 569 Suillus tomentosus, in liquid-culture experiments. Geochim. Cosmochim. Acta : Barker, W. W., S. A. Welch, S. Chu, and J. F. Banfield Experimental 26

27 572 observations of the effects of bacteria on aluminosilicate weathering. Am. Mineral : Bennett, P. C., F. K. Hiebert, and W. J. Choi Microbial colonization and weathering of silicates in a petroleum-contaminated groundwater. Chem. Geol. 132: Bennett, P. C., J. R. Rogers, F. K. Hiebert, and W. J. Choi Silicates, silicate weathering, and microbial ecology. Geomicrobiol. J. 18: Blume, E., M. Bischoff, J. Reichert, T. Moorman, A. Konopka, and R. Turco Surface and subsurface microbial biomass, community structure and metabolic activity as a function of soil depth and season. Appl. Soil Ecol. 592: Buss, H. L., A. Lüttge, and S. L. Brantley Etch pit formation on iron silicate surfaces during siderophore-promoted dissolution. Chem. Geol. 240: Calvaruso, C., M. P. Turpault, E. Leclerc, J. Ranger, J. Garbaye, S. Uroz, and P. Frey-Klett Influence of forest trees on the distribution of mineral weathering-associated bacterial communities of the Scleroderma citrinum mycorrhizosphere. Appl. Environ. Microbiol. 76: Cockell, C. S., N. Kennerley, M. Lindstrom, J. Watson, V. Ragnarsdottir, E. Sturkell, S. Ott, and A. G. Tindle Geomicrobiology of a weathering crust from an impact crater and a hypothesis for its formation. Geomicrobiol. J. 24: Collignon, C., S. Uroz, M-P. Turpault, and P. Frey-Klett Seasons differently 592 inpact the structure of mineral weathering bacterial communities in beech and spruce 593 stands. Soil Biol. Biochem. 43:

28 Ehrlich, H. L Geomicrobiology: its significance for geology. Earth Sci. Rev : Eilers, K. G., S. Debenport, S. Anderson, and N. Fierer Digging deeper to find unique microbial communities: The strong effect of depth on the structure of bacterial and archaeal communities in soil. Soil Biol. Biochem. 50: Faoro, H., A. C. Alves, L. E. Souza, M. U. Rigo, L. M. Cruz, S. M. Al-Janabi, R. A. Monteiro, V. A. Baura, and F. O. Pedrosa Influence of soil characteristics on the diversity of bacteria in the southern Brazilian Atlantic Forest. Appl. Environ. Microbiol. 76: Fierer, N., J. P. Schimel, and P. A. Holden Variations in microbial community composition through two soil depth profiles. Soil Biol. Biochem. 35: Frey, B., S. R. Rieder, I. Brunner, M. Plötze, S. Koetzsch, A. Lapanje, H. Brandl, and G. Furrer Weathering-associated bacteria from the damma glacier forefield: Physiological capabilities and impact on granite dissolution. Appl. Environ. Microbiol. 76: Gleeson, D. B., N. M. Kennedy, N. Clipson, K. Melville, G. M., Gadd, and F. P. McDermott Characterization of bacterial community structure on a weathered pegmatitic granite. Microb. Ecol. 51: Hilley, G. E., and S. Porder A framework for predicting global silicate weathering 613 and CO 2 drawdown rates over geologic time-scales. P. Natl. Acad. Sci. USA : Hutchens, E., E. Valsami-Jones, S. McEldowney, W. Gaze, and J. McLean The 28

29 616 role of heterotrophic bacteria in feldspar dissolution- an experimental approach. Mineral 617 Mag. 67: Lane, D. J S/23S rrna sequencing. In: Nucleic Acid Techniques in Bacterial Systematics (Stackebrandt E. and Goodfellow M., Eds.), John Wiley and Sons, New York, pp Manjanatha, M. G., T. E. Loynachan, and A. G. Atherly Tn5 mutagenesis of Chinese Rhizobium fredii for siderophore overproduction. Soil Biol. Biochem. 24: Mehlich, A Mehlich 3 soil test extractant: A modification of Mehlich 2 extractant. Comm. Soil Sci. Plant Anal. 15: Prakongkep, N., A. Suddhiprakarn, I. Kheoruenromne, M. Smirk, and R. J. Gilkes The geochemistry of Thai paddy soils. Geoderma 144: Puente, M. E., C. Y. Li, and Y. Bashan Rock-degrading endophytic bacteria in cacti. Environ. Exper. Bot. 66: Sambrook, J., and D. Russell Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory Press, NY. 27. Schwyn, B., and J. B. Neilands Universal chemical assay for the detection and determination of siderophores. Anal. Biochem. 160: Sheng, X. F., F. Zhao, L. Y. He, G. Qiu, and L. Chen Isolation and 635 characterization of silicate mineral-solubilizing Bacillus globisporus Q12 from the 636 surfaces of weathered feldspar. Can. J. Microbiol. 54: Uroz, S., C. Calvaruso, M. P. Turpault, J. C. Pierrat, C. Mustin, and P. Frey-Klett. 29

30 Effect of the mycorrhizosphere on the genotypic and metabolic diversity of the 639 bacterial communities involved in mineral weathering in a forest soil. Appl. Environ. 640 Microbiol. 73: Uroz, S., C. Calvaruso, M. P. Turpault, and P. Frey-Klett Mineral weathering by bacteria: ecology, actors and mechanisms. Trends Microbiol. 17: Uroz, S., P. Oger, C. Lepleux, C. Collignon, P. Frey-Klett, and M. P. Turpault. 2011A. Bacterial weathering and its contribution to nutrient cycling in temperate forest ecosystems. Res. Microbiol. 162: Uroz, S., M. P. Turpault, L. Van Scholl, B. Palin, and P. Frey-Klett. 2011B. Long term impact of mineral amendment on the distribution of the mineral weathering associated bacterial communities from the beech Scleroderma citrinum ectomycorrhizosphere. Soil Biol. Biochem. 43: Wang, X. D., N. Yamaguchi, T. Someya, and M. Nasu. 2007A. Rapid and automated enumeration of viable bacteria in compost using a micro-colony auto counting system. J. Microbiol. Meth. 71: Wang, Q., G. M. Garrity, J. M. Tiedje, and J. R. Cole. 2007B. Naïve Bayesian classifier for rapid assignment of rrna sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73: White, A. F., and S. L. Brantley Chemical weathering rates of silicate minerals: 657 an overview. In: White AF, Brantley SL. (Eds.), Chemical Weathering Rates of Silicate 658 Minerals. Reviews in Mineralogy. Mineralogical Society of America, Washington, DC. 659 pp

31 Will, C., A. Thurmer, A. Wollherr, H. Nacke, N. Herold, M. Schrumpf, J. Gutknecht, 661 T. Wubet, F. Buscot, and R. Daniel Horizon-specific bacterial community 662 community composition of german grassland soils, as revealed by pyrosequencing-based analysis of 16S rrna gene. Appl. Environ. Microbiol. 76: Yeomans, J. C., and J. M. Bremmer A rapid and precise method of routine determination of organic carbon in soil. Commun. Soil Sci. Plant. Anal. 19:

32 TABLE 1. Influence of bacteria isolated from the gleyosols and purple-orthic primosols profiles on the element releases from biotite and the ratios of acid and siderophore-producing bacteria Horizon Range of element released ( M)* Ratio of acid-producing bacteria (%) Ratio of siderophore-producing bacteria (%) 692 Fe Si Al K ph < 4 ph = 4-6 ph > 6.0 No Low High Changshu soil profile A ± 4 49 ± 4 9 ± 1 30 ± 3 47 ± 7 23 ± 3 A ± 3 67 ± 7 13 ± 2 18 ± 2 58 ± 6 24 ± 4 Bg ± 2 47 ± 5 37 ± 5 20 ± 3 63 ± ± 2 Bg ± 3 31 ± 4 46 ± 4 51 ± 7 44 ± 8 5 ± 1 Bg ± 2 18 ± 3 53 ± 4 25 ± 3 59 ± 8 16 ± 2 Yanting soil profile P ± 4 35 ± 5 34 ± 3 27 ± 3 42 ± 4 31 ± 3 P ± 5 26 ± 4 29 ± 3 25 ± 3 36 ± 4 39 ± 4 P ± 2 64 ± 7 20 ± 3 10 ± 1 46 ± 9 44 ± 4 P ± 6 34 ± 3 10 ± 1 45 ± 4 33 ± 4 22 ± 3 P ± 3 57 ± 5 17 ± 2 46 ± 4 28 ± 4 26 ± 3 P ± 4 47 ± 4 15 ± 2 38 ± 4 30 ± 7 32 ± 3 * The concentrations of Fe, Si, Al, and K in the liquid modified Bushnell-Haas medium supplemented with biotite 693 mineral during 7 days of incubation in the absence of bacteria were 1.5 ± 0.3, 16.2 ± 0.9, 4.8 ± 0.3, and 21.7 ± µm, respectively

33 Figure captions FIG. 1. The geochemical factors and bacterial counts of the Changshu (A) and Yanting (B) soil profiles. Values are means ± standard error (n = 3). FIG. 2. Average element releases from biotite in the absence and presence of mineral weathering bacteria from different horizons of the Changshu (a) and Yanting (b) soil profiles. Error bars are ± standard error (n = 3). Bars indicated by the same letter are not significantly different (P > 0.05) according to Tukey's test. FIG. 3. Proportion of the least, moderately, and highly effective mineral-weathering bacteria isolated from the Changshu (a) and Yanting (b) profiles. Error bars are ± standard error (n = 3). Bars indicated by the same letter are not significantly different (P > 0.05) according to Tukey's test. The data (bacterial numbers are five) were statistically analyzed. FIG. 4. Proportion of mineral weathering bacterial populations throughout the Changshu soil profile. FIG. 5. Proportion of mineral weathering bacterial populations throughout the Yanting soil profile. FIG. 6. Proportion of least, moderately, and highly effective mineral weathering bacteria throughout the Changshu (a) and Yanting (b) profiles

34 Downloaded from FIG. 1. The geochemical factors and bacterial counts of the Changshu (A) and Yanting (B) on August 19, 2018 by guest 720 soil profiles. Values are means ± standard error (n = 3)

35 723 a Contents of element released ( M) b Contents of element released ( M) A1 a A2 a a Bg1 ab Bg2 ab Bg3 Control (no bacteria) a a ab ab c a b a a c c a a d b d c e c Fe Si Al K Element released P1 a P2 b P3 c P4 d P5 d d P6 Control (no bacteria) a a a b b b b a b b b b b b c c c c c e c d Fe Si Al K Element released FIG. 2. Average element releases from biotite in the absence and presence of mineral weathering bacteria from different horizons of the Changshu (a) and Yanting (b) soil profiles. Error bars are ± standard error (n = 3). Bars indicated by the same letter are not significantly different (P > 0.05) according to Tukey's test. 35

36 730 a Least effective bacteria Moderately effective bacteria Highly effective bacteria b a Proportion (%) b Proportion (%) 80 c c c d e e ef ef e 20 g 0 A1 A2 Bg1 Bg2 Bg3 Soil horizon 100 a Least effective bacteria 90 Moderately effective bacteria a a 80 Highly effective bacteria ab 70 c 60 d e e 30 f f f f fg h 20 h i 10 0 P1 P2 P3 P4 P5 P6 Soil horizon FIG. 3. Proportion of the least, moderately, and highly effective mineral-weathering bacteria isolated from the Changshu (a) and Yanting (b) profiles. Error bars are ± standard error (n = 3). 735 Bars indicated by the same letter are not significantly different (P > 0.05) according to Tukey's test. The data (bacterial numbers are five) were statistically analyzed. 36

37 Downloaded from FIG. 4. Proportion of mineral weathering bacterial populations throughout the Changshu soil profile. on August 19, 2018 by guest

38 Downloaded from FIG. 5. Proportion of mineral weathering bacterial populations throughout the Yanting soil profile. on August 19, 2018 by guest

39 757 a b FIG. 6. Proportion of the least, moderately, and highly effective mineral weathering bacteria isolated from the Changshu (a) and Yanting (b) soil profiles. 39

40 ERRATUM Depth-Related Changes in Community Structure of Culturable Mineral Weathering Bacteria and in Weathering Patterns Caused by Them along Two Contrasting Soil Profiles Jing Huang, Xia-Fang Sheng, Jun Xi, Lin-Yan He, Zhi Huang, Qi Wang, Zhen-Dong Zhang Key Laboratory of Agricultural and Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agricultural University, Nanjing, People s Republic of China Volume 80, no. 1, p , Page 30, column 1, line 44: 2-mm-size should read 2- m-size. Page 30, column 2, line 26: mm should read m. Page 30, column 2, line 28: 200-mm should read 200- m. Page 30, column 2, line 37: 5-mm should read 5- m. Page 30, column 2, lines 52 to 55: The PCR system contained 2 ml of purified DNA extract, 0.2 mm each primer, 12.5 ml premix Ex Taq (containing DNA polymerase, buffer, and deoxynucleoside triphosphate mixture) (TaKaRa), and sterile deionized water to a final volume of 50 ml should read The PCR system contained 2 l of purified DNA extract, 0.2 M each primer, 12.5 l premix Ex Taq (containing DNA polymerase, buffer, and deoxynucleoside triphosphate mixture) (TaKaRa), and sterile deionized water to a final volume of 50 l. Page 31, column 2, lines 37 and 38: mm should read M. Page 31, column 2, lines 53 and 54: mm should read M. Page 34, column 1, lines 14 and 15: 20 mm Fe, 40 mm Si, 20 mm Al, and 100 mm K should read 20 M Fe, 40 M Si, 20 M Al, and 100 M K. Page 34, column 1, line 16, and column 2, line 1: 20 to 40 mm Fe, 40 to 80 mm Si, 20 to 40 mm Al, and 100 to 300 mm K should read 20 to 40 M Fe, 40 to 80 M Si, 20 to 40 M Al, and 100 to 300 M K. Page 34, column 2, lines 3 and 4: 40 mm Fe, 80 mm Si, 40mM Al, and 300 mm K should read 40 M Fe, 80 M Si, 40 M Al, and 300 M K. Copyright 2014, American Society for Microbiology. All Rights Reserved. doi: /aem aem.asm.org Applied and Environmental Microbiology p February 2014 Volume 80 Number 4