3.1 Characterization of the rock samples
As shown in Table 1, different patterns in available element content were found among different groups (Table 1). A significantly lower content of available Ca was found in group LT than in groups MT and F. All the rock samples had a relative alkaline pH (7.79~8.40). Based on the phase analysis of XRD, rock collected was found to be slate, and similar mineral constituents were found among these rock samples. The possible major mineral phases included 2:1 type clay minerals, kaolinite, biotite, and quartz (Fig S2). The proportion of gravel (> 2 mm) was significantly higher in group LT than in groups MT and F, suggesting a lower alternation degree of rock samples of group LT (Supplementary Table S1).
Table 1
Contents of available elements and organic matter, pH, and cell number of slate rocks collected from less (LT group) and more altered rock samples (MT group) from triggering area, and rock samples from flowing area of debris flow (F group). The values are arithmetic means ± standard error (n = 3). Different letters represent significant differences within a row (i.e. among groups LT, MT, and F) according to Tukey's Honestly Significant Difference (HSD) post-hoc test (P < 0.05).
|
Rock samples
|
LT
|
MT
|
F
|
Available element content (mg kg− 1)
|
Na
|
9.4 ± 0.4b
|
9.0 ± 0.5b
|
11.4 ± 1.0a
|
Fe
|
112 ± 5a
|
92 ± 3b
|
129 ± 25a
|
Mg
|
394 ± 26b
|
597 ± 40a
|
377 ± 6b
|
Al
|
524 ± 41a
|
244 ± 8b
|
54 ± 11c
|
K
|
60.5 ± 2.7b
|
76.9 ± 5.2a
|
41.3 ± 3.9c
|
Si
|
414 ± 23a
|
130 ± 7c
|
169 ± 10b
|
Mn
|
30.7 ± 0.8a
|
13.6 ± 0.9c
|
27.0 ± 0.6b
|
Ca
|
784 ± 49b
|
2369 ± 586a
|
2295 ± 143a
|
Organic matter (g kg− 1)
|
3.19 ± 0.15a
|
0.75 ± 0.05c
|
2.67 ± 0.21b
|
pH
|
7.87 ± 0.08c
|
8.33 ± 0.07a
|
8.17 ± 0.05b
|
Bacterial count (× 105 cfu g− 1)
|
15.2 ± 1.8a
|
5.1 ± 1.3b
|
6.4 ± 1.4b
|
3.2 Comparison of α-diversity and composition of bacterial communities among different rock samples.
Totally, 123,379 high-quality sequences (11,662 − 15,258 for each sample) were obtained, with 275 genera belonging to 29 phyla, 92 classes, 134 orders, and 200 families. Firmicutes (accounting for about 66 % of the total sequences), Proteobacteria (15 %), and Actinobacteria (11%) were the three most dominant phyla.
Overall bacterial α-diversity indices exhibited significant differences (P < 0.05 in all cases, Tukey's test) (Fig. 1a). Collectively, the values of all the indices were highest in group LT, followed by groups F and then MT, suggesting that the less alerted slate harbored a higherα-diversity of bacterial communities
UPGMA was used to compare the relatedness of bacterial communities based on the distribution of OTUs. The nine samples under investigation were divided into three groups, with group MT closely relating with those of group F (Fig. 1c). These results are supported by the results of two-dimensional PCoA using Bray-Curtis similarity distances (Fig. 1b), unweighted unifrac distance (Fig. S3a), and weighted unifrac distance (Fig. S3b). The results were further confirmed by ADONIS and ANOSIM (Table S2).
At the phylum level, the relative abundances of Acidobacteria and Proteobacteria were highest in group LT, followed by groups F and then MT. The highest relative abundances of Chloroflexi and Firmicutes were found in group MT, followed by groups F and then LT. The relative abundance of dominant taxa at class level also varied among rock samples from different sampling sites (Fig. 1c).The highest relative abundance of Bacilli was found in group MT, followed by groups F and then LT (Fig. 1d).
At genus level, 190, 125, and 172 genera were obtained from LT, MT, and F, respectively. The distribution of major bacterial genera (accounting for more than 0.25 % of the total sequences) were presented in Table S3. Especially the ammonia oxidizing-related genus Nitrospira appeared significantly more abundant in group LT than in groups MT and F. Furthermore, Rhodobacter, Hydrogenophaga, and Limnobacter were only observed in group F (Table S3).
Regarding OTU level, the exclusive and shared species-level OTUs among rock samples were inspected. The results showed that 281 out of 3,114 total OTUs (9.5%) were shared among groups LT, MT, and F (Fig. 1e, left panel), accounting for 84.7 % of all the sequences, indicating that these shared OTUs are mostly more abundant species. While OTUs unique to each group were mostly species with low abundance (Fig. 1e, right panel).
3.3 Correlations between rock properties and bacterial community composition
Rock properities (including contents of Ca, Mg, Si, and Mn) significantly correlated with the RDA model (P < 0.05) were selected to analyze their respective influences on bacterial community composition (Fig. 2). RDA explained nearly 99 % of the total variation in the rock bacterial community structure, with the first two axes explaining 93.13% of the variation (Fig. 2a). Rock bacterial communities formed similar clusters on the RDA plot (Fig. 2a), in agreement with PCoA analysis (Fig. 1b). Canonical variation partitioning revealed that the available Ca content was the major contributor of bacterial community variation, explaining 36.6% of the variation (Fig. 2b). The bacterial diversity indices were correlated negatively with available Ca and Mg contents (r = -0.694~-0.907) and positively with Si and Mn contents (r = 0.760~0.973) of rock samples (Table S4). In addition, the diversity indices correlated positively with OM content (r = 0.795~0.913) and negatively correlated with pH (r = -0.932~-0.913) of rock samples.
3.4 Predicted microbial metabolic profiles
Using PICRUSt, 28 of 43 level 2 KEGG Orthology groups (KOs) were represented by the data set, and 23 genetic families showed significant differences (P < 0.05) among groups LT, MT, and F (Fig. S4). As for the mineral weathering-related functional potential, the relative abundances of genetic families associated with organic acid production and flagellar assembly were highest in group LT, followed by groups F and then MT (Fig. 3). The highest relative abundance of CA was found in LT group. However, the relative abundance of genetic families involved in siderophore biosynthesis in groups MT and F was significantly higher than that of group LT. Collectively, these results indicate that the bacterial communities may influence the rock weathering through a variety of mechanisms in the rock environments.
3.5 Rock weathering assays
On the rock surfaces, 105-106 bacterial counts per gram of rock were obtained. Furthermore, cultivable bacterial counts in group LT were significantly higher than those in groups MT and F (Table 1). Totally, 248 bacterial strains were obtained, of which 90, 81 and 77 strains from LT, MT and F groups, respectively. Dissolved Fe, Si, Al, and Ca from slate was used as an overall indicator of rock weathering. It was found that all the strains can enhance slate weathering compared with the uninoculated control (Table 2, TableS6). As shown in Fig. 4, the ratios of active Fe, Al, and Si solubilizers were significantly higher in group LT than in groups MT and F. The proportion of active Fe solubilizer was significantly higher in group F than group MT (Fig. 4b). No significant difference in the ratio of active Ca was found among different rock samples (Fig. 4f).
Table 2
Influence of bacteria isolated from slate rock samples on the element releases from slate. The values are arithmetic means ± standard error (n = 3). Different superscript letters represent significant differences within a row (i.e. among groups LT, MT, and F) according to Tukey's Honestly Significant Difference (HSD) post-hoc test (P < 0.05).
Rock
sample
|
Concn. range (µM) of element released a
|
Ratio of acid (or alkali)-producing
bacteria (%) by pH
|
Fe
|
Si
|
Al
|
Ca
|
< 4
|
4–7
|
> 7
|
LT
|
3.7–105
|
104–211
|
5.2–82
|
372–2667
|
2.5 ± 0.3c
|
15 ± 2.1a
|
82 ± 1.2a
|
MT
|
3.3–44
|
103–144
|
3.7–12
|
526–2437
|
12 ± 1.9b
|
3.3 ± 0.3c
|
84 ± 2.4a
|
F
|
3.4–47
|
104–150
|
4.0–46
|
707–3572
|
26 ± 2.6a
|
9.0 ± 1.2b
|
65 ± 5.6b
|
a The concentrations of Fe, Si, Al, and Ca in the liquid Bushnell-Haas medium supplemented with slate mineral during 7 days of incubation in the absence of bacteria were 2.6 ± 0.13, 97 ± 4.6, 3.0 ± 0.4 and 339 ± 12 µM, respectively. |
Based on the distribution of the concentrations of Fe, Si, Al, and Ca in cultural medium in the presence or absence of bacterial strains (Table S6), these strains could be grouped into the following three categories: strains with poor rock-weathering potential (< 5 µM Fe, < 8 µM Al, < 110 µM Si, and < 800 µM Ca in the culture), moderate rock-weathering potential (5–10 µM Fe, 8–10 µM Al, 110–120 µM Si, and 800–1000 µM Ca), and high rock-weathering potential (> 10 µM Fe, > 1 µM Al, > 120 µM Si, and > 1000 µM Ca). As shown in Fig. 4, the proportion of highly active Fe, Al, and Si solubilizers was highest in group F, followed by groups LT and then MT. The proportion of highly active Ca solubilizer in groups LT and F was significantly higher than that in group MT, and no significant difference was found between groups E and F (Fig. 4f). Collectively, the rock weathering potential of slate-weathering bacteria form groups LT and F was higher than mineral-weathering bacteria from group MT.
The pH in the culture medium ranged from 3.21 to 8.76 in the presence of different bacterial strains (Table 2, Table S6). Most of the bacterial strains (about 78 %) showed high alkaline pH values. The ratio of highly (pH < 4) active acid-producing strains was highest in group F, followed by groups MT and LT. In addition, the proportion of the alkaline-producing (pH > 7) strains in groups LT and MT was higher than group F.
3.6 Linking of culturable bacterial taxonomy with their rock weathering potential
As shown in Fig. 5, the rock-weathering bacterial strains were affiliated with 14 genera (8, 7 and 8 genera were obtained from groups LT, MT and F, respectively). Bacterial strains assigned to Pseudarthrobacter (n = 129), Arthrobacter (n = 11), and Bacillus (n = 59) were common in the rock samples. Strains belonging to Microbacterium (n = 2) and Sphingomonas (n = 4) were detected in both LT and MT groups, and Pseudomonas (n = 18) was found in both MT and F groups. Furthermore, each group had its unique genera (Fig. 5a)
The relative activity of solubilizing Fe, Al, Si, and Ca from slate were compared among different genera (with a minimum of 3 strains for each genus). As shown in Fig. 5b, representatives of Pantoea and Pseudomonas exhibited considerably higher activities promoting the release of Fe, Al, Si, and Ca from slate, compared with other genera. The medium pH of Pantoea (4.41 ± 1.96) and Pseudomonas (4.95 ± 1.94) strains was significantly lower than that of other genera. In addition, pH showed negative correlations with Fe, Al, and Ca released from slate (r = -0.824 ~ -0.876).
When inter-comparing the taxonomic data obtained from the cultivation-dependent and -independent methods, 4 of the 25 most abundant bacterial genera identified through 16S rRNA gene Miseq sequencing (19 % of total sequences) were also identified by the cultivation-dependent method (Table S3). Most of the cultured genera with rock-weathering potential were also detected by the culture-independent method, except for Pesudarthrobacter, Micrococcus, Terrabacter, Ensifer, Pantoea, and Enterobacter (Fig. 5a). Generally, the cultivable rock-weathering bacterial genera took up a small proportion of the bacterial community detected by cultivable-independent method.