Variations in the Bacterial Community Compositions at Different Sites in The

Microbiological Research 196 (2017) 26–33

Contents lists available at ScienceDirect

Microbiological Research

j ournal homepage: www.elsevier.com/locate/micres

Variations in the bacterial community compositions at different sites

in the tomb of Emperor Yang of the Sui Dynasty

a,∗ b c d d,∗∗

Zhi Huang , Fei Zhao , Yonghui Li , Jianwei Zhang , Youzhi Feng

Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing

210095, China

Nanjing Institute for Comprehensive utilization of Wild Plants, China CO-OP, Nanjing 210042, China

Key Laboratory of Urban and Architectural Heritage Conservation of Ministry of Education, School of Architecture, Southeast University, Nanjing 210096, China

State Key Laboratory Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China

a r t i c l e i n f o a b s t r a c t

Article history: To fully understand the bacterial processes in tomb environments, it is necessary to investigate the details

Received 20 August 2016

of the bacterial communities present under such oligotrophic conditions. Here, high-throughput sequenc-

Received in revised form

ing based on partial 16S rRNA gene sequences was used to fully evaluate the bacterial communities at

29 November 2016

different sites in the tomb of Emperor Yang of the Sui Dynasty. We also aimed to identify the soil factors

Accepted 7 December 2016

that were signiﬁcant related to bacterial diversity and community composition. The results showed the

Available online 16 December 2016

presence of a broad taxonomic diversity that included nine major phyla. Actinobacteria, Firmicutes and

Proteobacteria dominated the bacterial proﬁles in all tomb soil samples. However, signiﬁcant differences

Keywords:

between deposited soils (DS) and covering soils (CSA, CSB and CSC) were revealed by chemistry-based

Bacterial community

Tomb principal component analysis (PCA), the number of OTUs, and the Chao 1 and Shannon indexes. At the

Actinobacteria family level, hierarchically clustered heatmap and LefSe analyses showed differences in the bacterial

Emperor Yang of the Sui Dynasty community compositions at different sampling sites. Notably, CSA contained signiﬁcant populations of

High-throughput sequencing Nocardioidaceae, Pseudonocardiaceae and Streptomycetaceae, which are often reported to be associated

with biodeterioration in cave environments. Further, the most abundant group (>10%) in all soil samples

was Streptococcaceae, whose abundance decreased from 34.66% to 13.43% with increasing soil depth.

The results of redundancy analysis (RDA) and the Monte Carlo permutation test indicated that soil pH and

Cu and Mn levels were signiﬁcantly related to the bacterial communities in this tomb. This research offers

new insight into bacterial communities in cave environments and also provides important information

for the protection of this historically important tomb.

1. Introduction these extreme environments. The aphotic and oligotrophic envi-

ronment restricts the development of microbial communities and

Subterranean cave environments, including catacombs and only allows for the survival of species that are adapted to such con-

tombs, are dark, with nearly constant temperature and high rel- ditions. Recently, some studies regarding microbial communities

ative humidity (Groth and Saiz-Jimenez, 1999). Due to the lack in caves have been reported (Stomeo et al., 2008; Krakova et al.,

of sunlight, primary production via photosynthesis is absolutely 2015), primarily because microorganisms affect cultural heritage,

impossible. The limited nutrients and oxygen come from the such as rock art paintings (Portillo et al., 2009). However, a com-

surface via sinkholes, underground hydrology and drip waters prehensive and detailed understanding of microbial diversity and

(Barton and Jurado, 2007), but the supplies of these resources community composition in subterranean caves is still lacking.

are unpredictable. Microorganisms, particularly bacteria, inhabit Previous surveys have shown that subterranean caves con-

tain highly diverse populations of bacteria. In particular, it was

emphasized that Actinobacteria was the dominant group in the

studied caves (Groth et al., 1999; Saarela et al., 2004). Krakova

∗

Corresponding author at: No. 1 Weigang, Nanjing, Jiangsu, 210095, China.

et al. (2015) investigated the white bioﬁlm colonization in cata-

∗∗

Corresponding author at: No. 71 East Beijing Road, Nanjing, Jiangsu, 210008,

combs of St. Callixtus in Rome, showing that Actinobacteria was the

China.

most abundant phylum in both samples. Similar studies involving

E-mail addresses: [email protected] (Z. Huang), [email protected] (Y. Feng).

http://dx.doi.org/10.1016/j.micres.2016.12.004

Z. Huang et al. / Microbiological Research 196 (2017) 26–33 27

white and brown bioﬁlms in caves and catacombs demonstrated

the signiﬁcant presence of Actinobacteria (Stomeo et al., 2008;

Diaz-Herraiz et al., 2013; Vasanthakumar et al., 2013). In addition

to Actinobacteria, Firmicutes and Proteobacteria have also been

routinely found in subterranean caves (Wu et al., 2015; Krakova

et al., 2015; Vasanthakumar et al., 2013). Although the bacterial

diversity and community structure in caves were surveyed in all of

those studies, to our knowledge, little is known about the relation-

ships between environmental factors and the distributions of the

bacterial communities in the tomb environments.

Previous studies have shown that bacteria in caves are versatile

and also participate in capturing CO2, ﬁxing N2, biodeterioration,

and bioprecipitation (Laiz et al., 2009; Desai et al., 2013; Krakova

et al., 2015; Sanchez-Moral et al., 2004). To better understand

the roles and relative abundances of these functional bacteria in

the cave environment, it is necessary to investigate the details

of the bacterial communities occurring under such oligotrophic

conditions. However, previous surveys generally used DNA/RNA-

based clone libraries and denaturing gradient gel electrophoresis

(DGGE) to study the bacterial community in cave environments

(Schabereiter-Gurtner et al., 2002; Krakova et al., 2015). Compared

Fig. 1. A simple sketch map indicates the sampling locations in the tomb of Emperor

with these conventional techniques, high-throughput sequencing Yang of Sui. Deposited soil in the tomb is indicated by the green color at the bottom,

(e.g., Illumina MiSeq sequencing) can produce more sequence infor- and the covered soils are divided into three sections, CSA, CSB and CSC. The black

dots mean the sampling site. (For interpretation of the references to colour in this

mation (Song et al., 2013) and provide further insight into the rare

ﬁgure legend, the reader is referred to the web version of this article.)

bacteria in these communities.

The tomb of Emperor Yang of the Sui Dynasty (CE 569–618)

was discovered accidentally during late 2013. In the view of the site, and all samples were taken back to the laboratory immedi-

◦

important historical value, it is necessary to protect this valuable ately. Soil subsamples were stored at 4 C for biological analysis

◦

−

cultural heritage. Although the conservation of monuments is a or at 80 C for molecular analysis, and the remaining soils were

multifaceted aspect, such as the effect of lights, number of visitor air-dried in the laboratory over several weeks for further analysis.

and humidity (Bourges et al., 2014; Russell and MacLean, 2008), Soil pH was measured with a pH meter (PHS-3CT, Shanghai)

bacterial communities in the caves also have diverse impacts. Bac- using a soil extract solution made at a ratio of 1:2.5 soil:distilled

teria usually cause deterioration (De Leo et al., 2012) and are even water (w/v). Soil total organic carbon (TOC) and total nitrogen (TN)

pathogenic and opportunistic in cave environments (Valme et al., were determined according to previously reported methods (Lu,

2010). Therefore, monitoring the microbial community and envi- 1999). In brief, soil available potassium (K) was extracted using 2 M

ronmental parameters in the cave, in some degree, will help us to HNO3 and analyzed by ﬂame photometry (FP640, INASA, China).

understand the biological processes in such environments. How- Soil available phosphorus (P) was extracted using 0.5 M NaHCO3

ever, little is known regarding the differences among bacterial and analyzed using the molybdenum blue method (Bao, 2005). Soil

communities at the different sites in the tomb of Emperor Yang available Fe, Mn, Zn, Al and Cu were extracted using 0.05 M DTPA

of the Sui Dynasty, as well as regarding the responses of these (diethylenetriaminepentaacetic acid) and analyzed using an induc-

bacterial communities to environmental variation. Therefore, the tively coupled plasma optical emission spectrometry (ICP-OES,

ﬁrst objective of this study was to identify the bacterial communi- Optima 8000). The chemistry-based principal component analysis

ties in the tomb. Second, we aimed to identify which factors drive (PCA) was conducted using Canoco software for Windows (version

bacterial richness, diversity and community composition. Finally, 4.5).

the main bacterial communities that might be associated with cave

biogeochemical cycles in this tomb were discussed. 2.2. DNA isolation, PCR conditions, multiplexing and sequencing

Community DNA was extracted from 500 mg of each soil sam-

2. Materials and methods ple using the FastDNA 24 instrument (MP Biomedicals) according

to the manufacturer’s instructions. DNA concentration was deter-

2.1. Soil sampling and physicochemical analysis mined using the NanoDrop-2000 (Thermo Scientiﬁc, USA). DNA

samples were ampliﬁed using the bacterial primers 519F (5 -CAG

The tomb was discovered in Yangzhou, which was naturally CMG CCG CGG TAA NWC-3 )/907R (5 -CCG TCA ATT CMT TTR AGT

closed for thousands of years and accidentally discovered at the T-3 ) according to the literature (Caporaso et al., 2012; Shehab et al.,

end of 2013. A stone epitaph found in the western side of the brick- 2013). For Illumina sequencing, a 5-bp barcode sequence was added

lined tomb was inscribed with the title “Tomb Epitaph of the late to the 5 end of each primer. The PCRs were carried out in a total vol-

Emperor Yang of Sui”, indicating that the tomb was that of Emperor ume of 50 ␮l, containing 5 ␮l of 10× buffer with 15 mM MgCl2, 4 ␮l

Yang. The footprint of the tomb is 4.98 × 5.88 m in dimension. The of dNTPs (2.5 mM), 1 ␮l of each primer (5 ␮M), 0.5 ␮l of Taq DNA

top of the tomb had been damaged by buildings subsequently built polymerase (5 U) and 50 ng of DNA. The following cycling param-

◦

on top of it, and the surface soil was moved away. During the burial, eters were used: initial denaturation at 95 C for 5 min, followed

◦ ◦

approximately 10 cm of soil was deposited in the tomb. In June by 30 cycles of denaturation at 95 C for 45 s, annealing at 58 C for

◦ ◦

2014, soil samples were collected from various sites in this tomb. 45 s, extension at 72 C for 1 min and a ﬁnal extension at 72 C for

The deposited soils (DS) in the bottom of the tomb were collected. 10 min. Triplicate reaction mixtures for each soil sample were then

In addition, covering soils (CSA, CSB and CSC) that were on top of mixed. The PCR products were puriﬁed using a PCR Clean Up Kit

the tomb gate were also collected every 30 cm. A sketch map is (Amresco). The amplicons from each triplicate sample were sub-

shown in Fig. 1. Three replicate soil samples were collected at each jected to Illumina MiSeq sequencing. All the sequences used in this

28 Z. Huang et al. / Microbiological Research 196 (2017) 26–33

study are available from the DDBJ Sequence Read Archive under a b

b b accession number DRA005284. 0.07 0.26 0.31 0.12

± ± ± ±

(mg/kg)

2.3. Data analysis

Zn 0.51 1.07 0.61 0.40

The high-throughput sequencing data were analyzed using the a

b a a

QIIME data analysis package (Caporaso et al., 2010). Brieﬂy, the 3.8 9.3 14.6 7.4

sequences with the same barcode were assigned to the same sam- ± ± ± ±

(mg/kg)

ple, and then the barcode and primer sequences were removed.

Mn 16.1 36.2 42.4 47.9

Denoised sequences with one mismatched base in the barcode,

overlapped shorter than 10 bp, containing ambiguous characters,

a b b

or with a sequence length shorter than 50 bp were eliminated. The 0.39 1.19 0.59 0.43

chimeric sequences were also removed from aligned sequences. To ± ± ± ±

(mg/kg)

prevent bias due to different sequencing depth, all samples were

Cu 5.75 3.27 3.43 3.27

subsequently rareﬁed to 16000 bacterial sequences per sample. The

valid reads were then clustered into operational taxonomic units

b b

(OTUs) based on 97% sequence similarity. The diversity indexes b 2.1 1.7 3.9

Chao 1 and Shannon were estimated to indicate the community 2.5

± ± ±

(mg/kg)

richness. The Pearson’s correlations between soil factors and the

Al 20.6 9.8 13.8 13.7

relative abundances of bacterial communities, and the least sig-

niﬁcant difference (LSD) pairwise multiple comparison analysis a

a ab were analyzed using Statistical Analysis System (SAS, 9.1). Linear b 9.9 11.6 6.2

2.1

discriminant analysis (LDA) effect size (LefSe), a novel method to ± ± ± ±

support the high-dimensional class comparisons in metagenomics (mg/kg)

Fe 52.0 71.8 78.9 64.8

analysis (Zettler et al., 2013), was performed using the online

LefSe Program (http://huttenhower.sph.harvard.edu/galaxy/root/ a b

index). Signiﬁcantly discriminant taxon nodes are colored, and b

branch areas are shaded according to the highest-ranked variety for 17.9 3.6 34.0 23.9

± ± ± ±

that taxon. Redundancy analysis (RDA) was performed to arrange (mg/kg)

soil bacterial community structure based on soil properties using

Si 253.9 139.1 112.1 112.7

Canoco for windows (version 4.5). The environmental variables

were evaluated using a partial Monte Carlo permutation test (499 0.05).

a ab b

permutations) with an unrestricted permutation to investigate the (p

2.94 2.72 0.81 2.59

statistical signiﬁcance (Salles et al., 2004). ± ± ± ±

(mg/kg)

3. Results P 24.38 19.98 15.91 17.63 signiﬁcantly

3.1. Physical and chemical characterization a a a differ

b 24.8 23.2 29.1

2.4

± ± ±

The soil physical and chemical characteristics of the soil samples ±

letters

are shown in Table 1. In general, deposited soils (DS) were signiﬁ- (mg/kg)

K 79.3 135.5 136.0 124.9

cantly different from covering soils (CSA, CSB and CSC). The pH and

the levels of P, Si, Al and Cu were markedly higher in the deposited different

a a a a

tomb.

soil than in the covering soils. In comparison, the levels of K, Fe and by

0.07 0.09 0.10 0.02 the Mn were signiﬁcantly lower in the deposited soil. However, TOC,

± ± ± ±

TN and Zn showed no differences between deposited soil and cov- from

C/N 1.04 0.95 1.01 1.02

ering soils. Chemistry-based principal component analysis (PCA) followed

showed that deposited soils were grouped together and were sep- a a a

arate from the covering soils (Fig. 2), indicating that the chemistry Values

collected

of deposited soils is more similar to other deposited soils than to 0.17 0.12 0.47 0.01

SD).

± ± ± ±

that of covering soils. ± (g/kg)

samples TN 4.04 4.03 3.69 3.74

3.2. Diversity of bacterial communities (mean soil

a a a a

the

In this study, Illumina MiSeq sequencing was employed to char- of

0.16 0.14 0.00 0.40

± ± ±

(g/kg) replicates

acterize the bacterial communities in soil samples from the tomb.

There were 16658–32326 valid reads obtained from each sample. TOC 3.88 3.83 4.02 3.75 three

Using a 97% sequence similarity cutoff, the number of bacterial of

OTUs ranged from 1442 to 2891 (Table 2). The Chao 1 and Shannon c a b b characteristics

index values ranged from 3964.1 to 9823.1 and from 5.73 to 8.13, means 0.03 0.05 0.16 0.20

respectively. Although the number of sequence reads was relatively ± ± ± ±

the

small, the number of OTUs and the Chao 1 and Shannon index val- chemical

pH 6.84 5.69 5.40 4.98 are

ues of the deposited soil samples were signiﬁcantly higher than and

those of other soil samples. 1

values

Microorganisms assigned to unclassiﬁed bacteria were present DS CSA CSB CSC Table The

Physical in each soil sample, with relative abundance levels of 6.4–14.9%

Z. Huang et al. / Microbiological Research 196 (2017) 26–33 29

Fig. 2. The ﬁrst two principal coordinate axes (PC1 and PC2) for PCA and the distri-

Fig. 4. Hierarchically clustered heatmap of bacterial distribution of different com-

butions of soil samples in response to these axes. Soil chemical factors are indicated

munities from the four soil samples at the family level. Row represents the relative

by lines with arrows.

abundance of each bacterial group, and column indicates different soil samples. The

relative abundance for each bacterial family were depicted by color intensity with

the legend indicated at the top of the ﬁgure.

(Fig. 3). The rarefaction analysis (data not shown) indicated that

the analyzed sequences covered the entire diversity of bacterial

populations in the investigated tomb. A total of 9 major bacterial

abundance of each phylum. For example, beta-Proteobacteria was

phyla were identiﬁed in the tomb soils: Acidobacteria, Actinobac-

relatively abundant in DS (LSD, p < 0.05), whereas Actinobacteria

teria, Bacteroidetes, Chloroﬂexi, Firmicutes, Gemmatimonadetes,

and Firmicutes were more abundant in CSA (LSD, p < 0.05) and CSC

Nitrospirae, Planctomycetes and Proteobacteria. Sequences afﬁli-

(LSD, p < 0.05), respectively.

ated with Actinobacteria, Firmicutes and Proteobacteria dominated

To further compare the differences in bacterial communities

the soil samples, ranging from 8.4% to 34.4%, from 16.6% to 42.8%,

at different sites in the tomb, the 10 most abundant bacterial

and from 30.7% to 36.0%, respectively. Among these bacterial

families in each soil sample are shown in Fig. 4. A simple clus-

phyla, Proteobacteria were the most abundant group in all soil

ter analysis, based on the OTU patterns, revealed the differences

samples, mainly consisting of classes alpha-, beta- and gamma-

among the tomb soils. The soil samples were divided into two clus-

Proteobacteria. Certain other phyla (e.g., Verrucomicrobia) were

ters. One cluster was DS and the other was covering soils (CSA,

also detected from the tomb soils but with low abundances (<0.1%)

CSB and CSC). The bacterial communities in deposited soils were

(Fig. 3). As shown in Fig. 3, the soil samples all essentially contained

distinguished from those in covering soils based on higher abun-

the same bacterial taxa, but they showed differences in the relative

dances of Nocardioidaceae, Methylophilaceae, Comamonadaceae,

Syntrophobacteraceae and Xanthomonadaceae, and lower relative

Table 2 abundance of Micrococcaceae and Moraxellaceae. Bacterial com-

Observed bacterial diversity estimates based on 97% OTU cluster. munities in CSA, which showed relatively high levels of sequences

belonging to Micrococcaceae, Pseudonocardiaceae, Streptomyc-

OTU Chao 1 Shannon

etaceae and Nocardioidaceae, were also different from those in CSB

a a a

DS 2891 ± 53 9823.1 ± 95.6 8.13 ± 0.17

b b b and CSC. In contrast, the sequences related to Burkholderiaceae and

CSA 1600 ± 443 5120.8 ± 1406.0 6.17 ± 0.94

b b b

CSB 1587 ± 581 4649.8 ± 2466.8 6.07 ± 1.21 Pseudomonadaceae were relatively abundant in CSC. In addition,

b b b

± ± ±

CSC 1442 523 3964.1 1440.9 5.73 1.18 Streptococcaceae was the only taxon that was relatively abundant

(>10%) throughout the soil samples, ranging from 13.43% to 34.66%.

The values are the means of three replicates (mean ± SD). Values followed by differ-

ent letters differ signiﬁcantly (p < 0.05).

3.3. LefSe analysis of bacterial community structures in different

soils

LefSe was performed to obtain further insights into the differen-

tiation and internal interactions of the identiﬁed bacterial afﬁliation

in soil samples collected from the tomb, with the taxonomic tree

generated as shown in Fig. 5. For clarity, the cladogram shows taxa

with LDA values higher than 2.0. Notably, Actinobacteria was the

dominant group in CSA, and at the family level, the abundances

of Nocardioidaceae, Pseudonocardiaceae and Streptomycetaceae

were signiﬁcantly higher in CSA than in other soils. Streptococ-

caceae was signiﬁcantly present in CSC. The results also showed the

occurrence of a variety of low-abundance bacterial communities

in DS, including families of Nitrososphaeraceae, Kouleothrixaceae,

Pirellulaceae, Rhizobiaceae, Methylophilaceae and Entotheonel-

laceae. In addition, members of Frankiaceae and Acetobacteraceae

Fig. 3. Bacterial community structure in the different tomb soils. Stacked bar graph

were markedly present in CSB.

represent the relative abundance of the major phyla.

30 Z. Huang et al. / Microbiological Research 196 (2017) 26–33

4. Discussion

In this study, the bacterial diversity and community composi-

tion in the tomb of Emperor Yang were obtained using Illumina

MiSeq sequencing. The results showed that the number of obtained

bacterial OTUs and the Chao 1 and Shannon diversity indexes

were signiﬁcantly greater in DS than in covering soils (Table 2).

These differences are probably caused by soil and tomb environ-

ments. The hierarchically clustered heatmap and LefSe analysis also

revealed differences in the bacterial community compositions at

different sites. Pearson’s correlation analysis indicated that soil pH

and Cu were positively related to OTU number, Chao 1 and Shannon

diversity and that Mn was negatively related to these parameters

(Table 3). It is widely accepted that pH has a signiﬁcant effect on

bacterial diversity in a range of terrestrial and aquatic environ-

ments (Fierer and Jackson, 2006; Li et al., 2014a; Kuang et al., 2012).

Portillo and Gonzalez (2010) also reported that pH could inﬂuence

the bacterial communities in a cave environment. However, soil

Fig. 5. Taxonomic tree generated using the LefSe online software highlighting the

biomarkers that statistically differentiated the tomb soils. pH can be considered a master variable that integrates a variety of

other soil characteristics, so we cannot determine whether pH has a

direct inﬂuence on bacterial communities (Li et al., 2014b). In addi-

tion to soil pH, Cu and Mn have also been reported to signiﬁcantly

correlate with bacterial communities, which was consistent with

Huang et al. (2013) and Zhang et al. (2015). The Monte Carlo per-

mutation test of RDA also emphasized that pH, Cu and Mn were the

main factors determining the bacterial community composition in

the tomb soils (Table S2 in Suppelmentary material). Although we

observed strong correlations between soil properties and bacterial

community compositions in this study, explaining the factors that

shape the bacterial diversity and community composition remains

difﬁcult.

Using culture-independent methods, our study has shown that

Actinobacteria, Firmicutes and Proteobacteria were the three most

abundant groups in the tomb proﬁle (Fig. 3). Similar results have

also been reported for other tombs or caves, such as the Cave

of Altamira, the Roman catacombs and Jinjia Cave (Portillo et al.,

2008, 2009; Krakova et al., 2015; Wu et al., 2015). These phyla are

described as versatile particularly for playing important geochem-

ical roles in the subterranean environment (Tebo et al., 2005).

Previous studies had noted that Actinobacteria is a ubiqui-

tous group in natural caves and in catacombs (Groth et al., 1999;

Fig. 6. RDA ordination diagram of environmental factors in related to bacterial phy-

Sanchez-Moral et al., 2005; Krakova et al., 2015). Similar studies

logenetic groups at the family level. Environmental parameters are indicated by lines

involving white and brown bioﬁlms in caves and catacombs were

with arrows, and phylogenetic groups are represented by triangles.

carried out and showed the signiﬁcant presence of Actinobacte-

ria (Stomeo et al., 2008; Portillo et al., 2009; Vasanthakumar et al.,

2013). In the present study, a high percentage of Actinobacteria

3.4. Impact of soil properties on the relative abundances of

was also detected in all soil samples. Actinobacteria in general

dominant taxa

have been known for their ability to adapt to resource-limited con-

ditions (Hartmann et al., 2009) and to solubilize phosphate and

In this study, RDA was used to describe the correlations between

calcium carbonate (Ortiz et al., 2013). There is no doubt that the

the edaphic factors and the relative abundances of bacterial

bacteria in this group can decay stones and minerals (Abdulla et al.,

communities (Fig. 6). The data showed that soil factors were sig-

2008; Abdulla, 2009). Notably, Actinobacteria can be easily cultured

niﬁcantly related to the bacterial communities at the family level.

under nutrient-rich conditions (Portillo et al., 2009). If the environ-

For example, signiﬁcant correlations were found between Nocar-

mental conditions or nutrients are variable, the explosive growth of

dioidaceae and pH (r = 0.70, p < 0.05), Si (r = 0.58, p < 0.05) and Mn

this group may become a potential risk for the cave environments

(r = −0.74). The relative abundance of Streptococcaceae was cor-

(Groth et al., 1999).

related positively with Mn (r = 0.77, p < 0.01) and negatively with

At the family level, our study revealed that the most frequently

pH (r = −0.79, p < 0.01), Si (r = −0.61, p < 0.05) and Cu (r = −0.60,

occurring Actinobacteria were Micrococcaceae, Nocardioidaceae,

p < 0.05). Details of the correlations between soil factors and bacte-

Pseudonocardiaceae and Streptomycetaceae (Fig. 3). These groups

rial communities are shown in Table S1 in Supplementary material.

have often been found in subterranean environments (Stomeo

The Monte Carlo permutation test was used to examine whether

et al., 2008; Krakova et al., 2015). In this study, all four of these

environmental variables signiﬁcantly affected the distribution of

subgroups of Actinobacteria were signiﬁcantly more abundant in

the bacterial community. The results showed that pH, Mn and

CSA and CSB (Figs. 4 and 5). Streptomyces are the most important

Cu signiﬁcantly correlated with the bacterial community structure

representatives of Streptomycetaceae and have been reported to

(Table S2 in Supplementary material), which was in accordance

be the ﬁrst colonizers in tombs and caves (Ciferri, 1999). Strep-

with the data in Table 3.

tomyces are involved in the weathering of mural paintings in

Z. Huang et al. / Microbiological Research 196 (2017) 26–33 31

Table 3

Statistical analysis of correlation coefﬁcients between bacterial diversity indexes and soil characteristics.

Parameter pH TOC TN C/N K P Si Fe Al Cu Mn Zn

b a a b a

OTU 0.80 0.13 −0.01 0.13 −0.54 0.62 0.68 −0.42 0.62 0.85 −0.68 0.07

b a b b a

Chao 1 0.83 0.20 −0.03 0.20 −0.50 0.68 0.72 −0.43 0.63 0.87 −0.69 0.12

b b a

Shannon 0.72 0.07 0.04 0.04 −0.49 0.52 0.58 −0.33 0.53 0.78 −0.61 0.11

Correlation is signiﬁcant at the 0.05 level.

Correlation is signiﬁcant at the 0.01 level.

such environments and also cause color changes (Abdel-Haliem 90% of the species in this family belonged to Streptococcus. Some

et al., 2013). Members of Micrococcaceae, Nocardioidaceae and species of genus Streptococcus, such as S. thermophilus, are ben-

Pseudonocardiaceae have also been demonstrated to be involved in eﬁcial for humans, but the majority of them are detrimental to

biodeterioration and in the important geochemical cycles in cave human health and are frequently present in pathogenic animals

environments (Gorbushina et al., 2004; Diaz-Herraiz et al., 2013; or fecal materials (Zhu et al., 2013; Sinton et al., 2007). Previous

Pan et al., 2015; Pinar˜ et al., 2014). studies have not reported such abundant Streptococcaceae in tomb

Proteobacteria were the dominant group in the studied tomb environments, so it is concluded that this tomb was probably con-

soils, which was consistent with previous cave studies (Barton taminated by animal or human feces at an early stage.

et al., 2004; Krakova et al., 2015). In our study, this phylum was

mainly represented by the beta and gamma subdivisions. In beta-

5. Conclusion

Proteobacteria, Burkholderiaceae and Comamonadaceae were the

dominant groups. As the type genus of family Burkholderiaceae,

In this study, the bacterial community compositions of soils at

members of Burkholderia occupy diverse ecological niches. Several

different sampling sites in the tomb of Emperor Yang of the Sui

Burkholderia species have been reported to weather diverse min-

Dynasty were investigated using a high-throughput sequencing

erals (Lepleux et al., 2012; Calvaruso et al., 2013; Wu et al., 2008),

method. The results showed that a broad bacterial diversity was

and several are capable of nitrogen ﬁxation (Coenye, 2013). Coma-

present in tomb soils, and the bacterial communities were signif-

monadaceae are chemoorganotrophic or chemolithotrophic. These

icantly different among the soils that were collected at different

organisms can use only very few carbohydrates but use a wide vari-

sites. Actinobacteria, Firmicutes and Proteobacteria were demon-

ety of organic acids. Some species of this family had been described

strated to be the dominant bacterial groups in the tomb. Cu, Mn,

as oxidizing H2 and CO (Willems et al., 1991) and as ﬁxing nitrogen

and pH were the important environmental factors that were sig-

(Jenni et al., 1989). In gamma-Proteobacteria, Moraxellaceae, Pseu-

niﬁcantly related to bacterial communities. Microorganisms in the

domonadaceae and Xanthomonadaceae were the dominant groups

tomb were versatile and have been demonstrated to play many

in the tomb soils. The family Moraxellaceae includes species that

important roles in biological processes. Because of their potential

colonize mucosal membranes or skin of humans and animals and

functional complementarity, it is expected that a diverse bacterial

that can occasionally cause a variety of infections, as well as appar-

community is more beneﬁcial for microorganisms to adapt to such

ently harmless species occurring in the environment, including

an oligotrophic environment. On the other hand, bacteria that are

soil and water. Two important genera of this family, Acinetobac-

potentially associated with biodeterioration and pathogenicity in

ter and Moraxella, have been reported to play important roles in

the tomb should be properly recognized.

human and veterinary medicine (Teixeira and Merquior, 2014). In

this tomb, the members of this family were present at signiﬁcant

Acknowledgments

levels, especially in CSA, CSB and CSB. The potential risk of members

of this family should be assessed because of their pathogenic-

The authors thank Dr. Zhiying Guo (State Key Laboratory Soil and

ity. The family Pseudomonadaceae has always been described as

Sustainable Agriculture, Institute of Soil Science, Chinese Academy

involved in multiple ecological roles, and it is known for the ability

of Sciences) for assisting analyzing the data of high-throughput

to degrade a wide range of organic substrates (Ikner et al., 2007;

sequencing. The authors also thank the editor and reviewers for

Lin et al., 2016). Members of Pseudomonas can produce malic acid

giving many suggestions to improve this manuscript. This work

in tombs and can further cause the brown spots (Vyas and Gulati,

was supported by National Nature Science Foundation of China

2009; Vasanthakumar et al., 2013). Xanthomonadaceae is also a

(41101229, 41201251 and 51478103) and the Natural Science

widespread family of bacteria. Its current taxonomy lists 22 gen-

Foundation of Jiangsu Province (BK20161424).

era, among them the two plant-pathogenic genera Xanthomonas

and Xylella, as well as the related genus Stenotrophomonas, iso-

lates of which are increasingly recognized as an important cause Appendix A. Supplementary data

of severe disease of crops and ornamentals (Mhedbi-Hajri et al.,

2011). The genus Dyella, another representative of this family, is Supplementary data associated with this article can be found, in

capable of mineral weathering (Uroz et al., 2011; Zhao et al., 2013), the online version, at http://dx.doi.org/10.1016/j.micres.2016.12.

degradation of pollutants (Li et al., 2012) and oxidizing thiosulfate 004.

(Anandham et al., 2011).

Firmicutes was the third most dominant phylum in the studied

References

tomb. Notably, more than 80% sequences of this phylum in each soil

sample belonged to the family Streptococcaceae. Our data showed

Abdel-Haliem, M.E.F., Sakr, A.A., Ali, M.F., Ghaly, M.F., Sohlenkamp, C., 2013.

that the abundances of Streptococcaceae decreased with the soil Characterization of Strptomyces isolates causing colour changes of mural

paintings in ancient Egyptian tombs. Microbiol. Res. 168 (7), 428–437.

depth, dropping from 34.66% to 13.43%. The observed members of

Abdulla, H., May, E., Bahgat, M., Dewedar, A., 2008. Characterisation of

Streptococcaceae belonged to four genera, Lactococcus, Lactovum,

actinomycetes isolated from ancient stone and their potential for

Pilibacter and Streptococcus. Based on the valid species in these four deterioration. Pol. J. Microbiol. 57 (3), 213–220.

Abdulla, H., 2009. Bioweathering and biotransformation of granitic rock minerals

genera (http://www.bacterio.net/-allnames.html), approximately

by actinomycetes. Microb. Ecol. 58 (4), 753–761.

32 Z. Huang et al. / Microbiological Research 196 (2017) 26–33

Anandham, R., Kwon, S.W., Gandhi, P.I., Kim, S.J., Weon, H.Y., Kim, Y.S., Sa, T.M., Lin, Z., Zhen, Z., Wu, Z., Yang, J., Zhong, L., Hu, H., Luo, C., Bai, J., Li, Y., Zhang, D.,

Kim, Y.K., Jee, H.J., 2011. Dyella thiooxydans sp. nov., a facultatively 2016. The impact of the soil microbial community and enzyme activity of two

chemolithotrophic, thiosulfate-oxidizing bacterium isolated from rhizophere earthworm species during the bioremediation of

soil of sunﬂower (Helianthus annuus L.). Int. J. Syst. Evol. Microbiol. 61 (2), pentachlorophenol-contaminated soils. J. Hazard. Mater. 301, 35–45.

392–398. Lu, R.S., 1999. Methods of Soil Agricultural Chemical Analysis. Chinese Agricultural

Bao, S.D., 2005. Agricultural Chemical Analysis of Soil. China Agriculture Press, Science and Technology Publishing House, Beijing.

Beijing. Mhedbi-Hajri, N., Jacques, M.A., Koebnik, R., 2011. Adhesion mechanisms of

Barton, H.A., Jurado, V., 2007. What’s up down there? Microbial diversity in caves. plant-pathogenic Xanthomonadaceae. Adv. Exp. Med. Biol. 715, 71–89.

Microbe 2, 132–138. Ortiz, M., Neilson, J.W., Nelson, W.M., Legatzki, A., Byrne, A., Yu, Y., Wing, R.A.,

Barton, H.A., Taylor, R.M., Pace, N.R., 2004. Molecular phylogenetic analysis of a Soderlund, C.A., Pryor, B.M., Pierson III, L.S., Maier, R.M., 2013. Proﬁling

bacterial community in an oligotrophic cave environment. Geomicrobiol. J. 21 bacterial diversity and taxonomic composition on speleothem surfaces in

(21), 11–20. Kartchner Caverns, AZ. Microbiol. Ecol. 65 (2), 371–383.

Bourges, F., Genthon, P., Genty, D., Lorblanchet, M., Mauduit, E., D’Hulst, D., 2014. Pan, X., Ge, Q., Pan, J., 2015. Damage to ancient mural paintings and petroglyphs

Conservation of prehistoric caves and stability of their inner climate: lessons caused by Pseudonocardia sp. —a review. Acta Microbiol. Sinica 55 (7), 813–818

from Chauvet and other French caves. Sci. Total Environ. 493, 79–91. (in Chinese).

Calvaruso, C., Turpault, M.P., Frey-Klett, P., Uroz, S., Pierret, M.C., Tosheva, Z., Kies, Pinar,˜ G., Kraková, L., Pangallo, D., Piombino-Mascali, D., Maixner, F., Zink, A.,

A., 2013. Increase of apatite dissolution rate by Scots pine roots associated or Sterﬂinger, K., 2014. Halophilic bacteria are colonizing the exhibition areas of

not with Burkholderia glathei PML1(12)Rp in open-system ﬂow microcosms. the Capuchin Catacombs in Palermo, Italy. Extremophiles 18 (4), 677–691.

Geochim. Cosmochim. Acta 106 (4), 287–306. Portillo, M.C., Gonzalez, J.M., 2010. Differential effects of distinct bacterial bioﬁlms

Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, in a cave environment. Curr. Microbiol. 60 (6), 435–438.

E.K., Fierer, N., Pena, A.G., Goodrich, J.K., Gordon, J.I., Huttley, G.A., Kelley, S.T., Portillo, M.C., Gonzalez, J.M., Saiz-Jimenez, C., 2008. Metabolically active microbial

Knights, D., Koenig, J.E., Lozupone, C.A., McDonald, D., Muegge, B.D., Pirrung, communities of yellow and grey colonizations on the walls of Altamira Cave,

M., Reeder, J., Sevinsky, J.R., Turnbaugh, P.J., Walters, W.A., Widmann, J., Spain. J. Appl. Microbiol. 104 (3), 681–691.

Yatsunenko, T., Zaneveld, J., Knight, R., 2010. QIIME allows analysis of Portillo, M.C., Saiz-Jimenez, C., Gonzalez, J.M., 2009. Molecular characterization of

high-throughput community sequencing data. Nat. Methods 7 (5), 335–336. total and metabolically active bacterial communities of white colonizations in

Caporaso, J.G., Lauber, C.L., Walters, W.A., Berg-Lyons, D., Huntley, J., Fierer, N., the Altamira Cave, Spain. Res. Microbiol. 160 (1), 41–47.

Owens, S.M., Betley, J., Fraser, L., Bauer, M., Gormley, N., Gilbert, J.A., Smith, G., Russell, M.J., MacLean, V.L., 2008. Management issues in a Tasmanian tourist cave:

Knight, R., 2012. Ultra-high-throughput microbial community analysis on the potential microclimatic impacts of cave modiﬁcations. J. Environ. Manage. 87

Illumina HiSeq and MiSeq platforms. ISME J. 6 (8), 1621–1624. (3), 474–483.

Ciferri, O., 1999. Microbial degradation of paintings. Appl. Environ. Microbiol. 65 Saarela, M., Alakomi, H.-L., Suihko, M.-J., Maunuksela, L., Paaska, L.,

(3), 879–885. Mattila-Sandholm, T., 2004. Heterotrophic microorganisms in air and bioﬁlm

Coenye, T., 2013. The family burkholderiaceae. In: The Prokaryotes. Springer, samples from Roman catacombs, with special emphasis on actinobacteria and

Berlin Heidelberg. fungi. Int. Biodeterior. Biodegrad. 54 (1), 27–37.

De Leo, T., Iero, A., Zammit, G., Urzì, C.E., 2012. Int. J. Speleol. 41 (2), 125–136. Salles, J.F., Van Veen, J.A., Van Elsas, J.D., 2004. Multivariate analyses of

Desai, M.S., Assig, K., Dattagupta, S., 2013. Nitrogen ﬁxation in distinct microbial Burkholderia species in soil: effect of crop and land use history. Appl. Environ.

niches within a chemoautotrophy-driven cave ecosystem. ISME J. 7 (12), Microbiol. 70 (7), 4012–4020.

2411–2423. Sanchez-Moral, S., Luque, L., Canaveras,˜ J.C., Laiz, L., Jurado, V., Hermosin, B.,

Diaz-Herraiz, M., Jurado, V., Cuezva, S., Laiz, L., Pallecchi, P., Tiano, P., Saiz-Jimenez, C., 2004. Bioinduced barium precipitation in St. callixtus and

Sanchez-Moral, S., Saiz-Jimenez, C., 2013. The actinobacterial colonization of domitilla catacombs. Ann. Microbiol. 54 (1), 1–12.

Etruscan paintings. Sci. Rep. 3 (3), 1440. Sanchez-Moral, S., Luque, L., Cuezva, S., Soler, V., Benavente, D., Laiz, L., Gonzalez,

Fierer, N., Jackson, R.B., 2006. The diversity and biogeography of soil bacterial J.M., Saiz-Jimenez, C., 2005. Deterioration of building materials in Roman

communities. Proc. Natl. Acad. Sci U. S. A. 103 (3), 626–631. Catacombs: the inﬂuence of vistors. Sci. Total. Environ. 349 (1–3), 260–276.

Gorbushina, A.A., Heyrman, J., Dornieden, T., Gonzalez-Delvalle, M., Krumbein, Schabereiter-Gurtner, C., Saiz-Jimenez, C., Pinar,˜ G., Lubitz, W., Rölleke, S., 2002.

W.E., Laiz, L., Petersen, K., Saiz-Jimenez, C., Swings, J., 2004. Bacterial and Phylogenetic 16S rRNA analysis reveals the presence of complex and partly

fungal diversity and biodeterioration problems in mural painting unknown bacterial communities in Tito Bustillo cave, Spain, and on its

environments of St. Martins church (Greene-Kreiensen, Germany). Int. Palaeolithic paintings. Environ. Microbiol. 4 (7), 392–400.

Biodeterior. Biodegrad. 53 (1), 13–24. Shehab, N., Li, D., Amy, G.L., Logan, B.E., Saikaly, P.E., 2013. Characterization of

Groth, I., Saiz-Jimenez, C., 1999. Actinomycetes in hypogean environments. bacterial and archaeal communities in air-cathode microbial fuel cells open

Geomicrobiol. J. 16 (1), 1–8. circuit and sealed-off reactors. Appl. Microbiol. Biotechnol. 97 (22), 9885–9895.

Groth, I., Vettermann, R., Schuetze, B., Schumann, P., Saiz-Jimenez, C., 1999. Sinton, L.W., Braithwaite, R.R., Hall, C.H., Mackenzie, M.L., 2007. Survival of

Actinomycetes in karstic caves of northern Spain (Altamira and tito bustillo). J. indicator and pathogenic bacteria in bovine feces on pasture. Appl. Environ.

Microbiol. Methods 36 (1-2), 115–122. Microbiol. 73 (24), 7917–7925.

Hartmann, M., Lee, S., Hallam, S.J., Mohn, W.W., 2009. Bacterial, archaeal and Song, Z.-Q., Wang, F.-P., Zhi, X.-Y., Chen, J.-Q., Zhou, E.-M., Liang, F., Xiao, X., Tang,

eukaryal community structures throughout soil horizons of harvested and S.-K., Jiang, H.-C., Zhang, C.L., Dong, H., Li, W.-J., 2013. Bacterial and archaeal

naturally disturbed forest stands. Environ. Microbiol. 11 (12), 3045–3062. diversities in Yunnan and Tibetan hot springs. China Environ. Microbiol. 15 (4),

Huang, J., Sheng, X., He, L., Huang, Z., Wang, Q., Zhang, Z., 2013. Characterization of 1160–1175.

depth-related changes in bacterial community compositions and functions of a Stomeo, F., Portillo, M.C., Gonzalez, J.M., Laiz, L., Saiz-Jimenez, C., 2008.

paddy soil proﬁle. FEMS Microbiol. Lett. 347 (1), 33–42. Pseudonocardia in white colonizations in two caves with Paleolithic paintings.

Ikner, L.A., Toomey, R.S., Nolan, G., Neilson, J.W., Pryor, B.M., Maier, R.M., 2007. Int. Biodeterior. Biodegrad. 62 (4), 483–486.

Culturable microbial diversity and the impact of tourism in Kartchner Caverns. Tebo, B.M., Johnson, H.A., Mccarthy, J.K., Templeton, A.S., 2005. Geomicrobiology of

Arizona. Microbiol. Ecol. 53 (1), 30–42. manganese (II) oxidation. Trends Microbiol. 13 (9), 421–428.

Jenni, B., Isch, C., Aragno, M., 1989. Nitrogen ﬁxation by new strains of Pseudimonas Teixeira, L.M., Merquior, V.L.C., 2014. The family moraxellaceae. In: The

pseudoﬂava and related bacteria. J. Gen. Microbiol. 135 (2), 461–467. Prokaryotes.

Krakova, L., De Leo, F., Bruno, L., Pangallo, D., Urzì, C., 2015. Complex bacterial Uroz, S., Turpault, M.P., Van Scholl, L., Palin, B., Frey-Klett, P., 2011. Long term

diversity in the white bioﬁlms of the Catacombs of St. Callixtus in Rome impact of mineral amendment on the distribution of the mineral weathering

evidenced by different investigation strategies. Environ. Microbiol. 17 (5), associated bacterial communities from the beech Scleroderma citrinum

1738–1752. ectomycorrhizosphere. Soil Biol. Biochem. 43 (11), 2275–2282.

Kuang, J.L., Huang, L.N., Chen, L.X., Hua, Z.S., Li, S.J., Hu, M., Li, J.T., Shu, W.S., 2012. Valme, J., Leonila, L., Veronica, R.N., Patrick, B., 2010. Pathogenic and opportunistic

Contemporary environmental variation determines microbial diversity microorganisms in caves. Int. J. Speleol. 39 (1), 15–24.

patterns in acid mine drainage. ISME J. 7 (5), 1038–1050. Vasanthakumar, A., DeAraujo, A., Mazurek, J., Schilling, M., Mitchell, R., 2013.

Laiz, L., Miller, A.Z., Jurado, V., Akatova, E., Sanchez-Moral, S., Gonzalez, J.M., Microbiological survey for analysis of the brown spots on the wall of the tomb

Dionísio, A., Macedo, M.F., Saiz-Jimenez, C., 2009. Isolation of ﬁve Rubrobacter of King Tutankhamun. Int. Biodeterior. Biodegrad. 79 (4), 56–63.

strains from biodeteriorated monuments. Naturwissenschaften 96 (1), 71–79. Vyas, P., Gulati, A., 2009. Organic acid production in vitro and plant growth

Lepleux, C., Turpault, M.P., Oger, P., Frey-Klett, P., Uroz, S., 2012. Correlation of the promotion in maize under controlled environment by phosphate-solubilizing

abundance of betaproteobacteria on mineral surfaces with mineral weathering ﬂuorescent Pseudomonas. BMC Microbiol. 9 (1), 174.

in foresee soil. Appl. Environ. Microbiol. 78 (19), 7114–7119. Willems, A., De Let, J., Gillis, M., Kersters, K., 1991. Comamonadaceae, a new family

Li, A., Qu, Y.Y., Pi, W.Q., Zhou, J.T., Gai, Z.H., Xu, P., 2012. Metabolic characterization encompassing the acidovorans rRNA complex, including Variovorax paradoxus

and genes for the conversion of biphenyl in Dyella ginsengisoli LA-4. Biotechnol. gen. nov., comb. nov., for Alcaligenes paradoxus (Davis 1969). Int. J. Syst.

Bioeng. 109 (2), 609–613. Bacterol. 41 (3), 445–450.

Li, H., Ye, D., Wang, X., Settles, M.L., Wang, J., Hao, Z., Zhou, L., Dong, P., Jiang, Y., Ma, Wu, L., Jacobson, A.D., Hausner, M., 2008. Characterization of elemental release

◦

Z., 2014a. Soil bacterial communities of different natural forest types in during micro-granite interactions at T = 28 C. Geochim. Cosmochim. Acta 72

Northeast China. Plant Soil 383 (1), 203–216. (4), 1076–1095.

Li, J., Sun, W., Wang, S., Sun, Z., Lin, S., Peng, X., 2014b. Bacterial diversity, Wu, Y., Tan, L., Liu, W., Wang, B., Wang, J., Cai, Y., Lin, X., 2015. Proﬁling bacterial

distribution and insight into their role in S and Fe biogeochemical cycling diversity in a limestone cave of the western Loess Plateau of China. Front.

during black shale weathering. Environ. Microbiol. 16, 3533–3547. Microbiol. 6, 244.

Z. Huang et al. / Microbiological Research 196 (2017) 26–33 33

Zettler, E.R., Mincer, T.J., Amaral-Zettler, L.A., 2013. Life in the Plastisphere: Zhao, F., Guo, X.Q., Wang, P., He, L.Y., Huang, Z., Sheng, X.F., 2013. Dyella

microbial communities on plastic marine debris. Environ. Sci. Technol. 47 (13), jiangningensis sp. nov., a ␥-proteobacterium isolated from the surface of

7137–7146. potassium-bearing rock. Int. J. Syst. Evol. Microbiol. 63 (9), 3154–3157.

Zhang, J., Yang, Y., Zhao, L., Li, Y., Xie, S., Liu, Y., 2015. Distribution of sediment Zhu, W., Wu, C., Sun, X., Zhang, A., Zhu, J., Hua, Y., Chen, H., Jin, M., 2013.

bacterial and archaeal communities in plateau freshwater lakes. Appl. Characterization of Streptococcus suis serotype 2 isolates from China. Vet.

Microbiol. Biotechnol. 99 (7), 3291–3302. Microbiol. 166 (3–4), 527–534.