International Research Journal of Public and Environmental Health Vol.3 (11),pp. 244-256, November 2016 Available online at http://www.journalissues.org/IRJPEH/ http://dx.doi.org/10.15739/irjpeh.16.031 Copyright © 2016 Author(s) retain the copyright of this article ISSN 2360-8803

Original Research Article

Comparative study of Dushanzi and Baiyanggou mud volcano microbial communities in Junggar Basin in

Xinjiang, China

Received 9 September, 2016 Revised 13 October, 2016 Accepted 22 October, 2016 Published 11 November, 2016

Zhiyong Huang1,2#, Song Xu2#, Yifan Han2,

Yuan Gao3, Yongli Wang3*

and Chunhui Song1*

1Key Laboratory of Western China’s Mineral Resources of Gansu Province, School of Earth Sciences, University of Lanzhou, Lanzhou, Gansu 73000

2Tianjin Key Laboratory of Industrial Biological Systems and Bioprocessing

Engineering, Tianjin Institute of Industrial Biotechnology, Chinese

Academy of Sciences, Tianjin, 300308 3Key Laboratory of Petroleum Resources Research, Lanzhou

Institute of Geology, Chinese Academy of Sciences, Lanzhou, Gansu 73000

# They made equal contribution to this article.

*Corresponding Author E-mail: wyll6800@lzb.ac.cn;songchh@lzu.edu.cn

Mud volcanoes in Mainland China have not been extensively studied, particularly the microbial community structure. In the present study, we provided the first comparative analysis of the microbial community diversity of Dushanzi (DSZ) and Baiyanggou (BYG) mud volcanoes (MVs), especially for archaea. The universal bacterial and archaeal primers for the 16S rRNA genes were used to identify the microbial community. The results demonstrated that the BYG mud volcano bacterial community diversity is higher than that of the DSZ mud volcano, while the archaeal community diversity of BYG mud volcano is lower than that of the DSZ mud volcano. Phylogenetic analysis indicated that the bacterial and archaeal community structure was significantly different between the DSZ and BYG mud volcanoes. Most of the archaea recovered from DSZ identified in these mud volcanoes are anaerobic or highly thermophilic while recovered from BYG was identified as halobacteria consistent with their particular geographical nature. Moreover, many bacterial and archaeal genera, such as Methanomicrobia, sulfur-oxidizing symbiont bacterium and Alcanivorax sp., have been detected in these two mud volcanoes. The results of the present study contribute greatly to information concerning the species origin and formation mechanisms of mud volcanoes, providing a basis for species accumulation and exploration in the margins of Junggar Basin. Key words: Mud volcano, community structure, Junggar Basin.

INTRODUCTION A mud volcano (MV) is a geological structure formed from the emission of argillaceous material on the Earth’s surface or the sea floor (Etiope et al., 2009; Kopf, 2002; Milkov et al., 2003). Mud volcanoes have attracted the attention of geologists for more than 200 years, and these geological structures are formed through the eruption of mud, fluids and gases (typically methane) from deep-subsurface sediment reservoirs (Milkov et al., 2003). Mud volcanoes play key roles in the carbon budget, biogeochemical cycle, climate regulation, biomass production and maintenance of biodiversity, thereby providing essential ‘goods and services’

for global ecosystems (Jørgensen and Boetius, 2007; Supply et al., 2006). Recent global data have identified approximately 1800 prominent mud volcanoes on land, and more than 20,000 submarine mud volcanoes, primarily concentrated on continental margins and abyssal plains (Judd et al., 2002). These mud volcanoes are primarily located in China, Indonesia, Russia, Trinidad, Barbados, the Caspian Sea, the Black Sea, the Mediterranean Sea, the Barents Sea, the Gulf of Mexico and other places (Judd et al., 2002; Milkov et al., 2003; Schulze et al., 2009; Skinner and Mazzini, 2009).

Int. Res. J. Public Environ. Health 245 Numerous studies have focused on the eruptions, variable geometry, tectonic settings, activities and products, oil and gas accumulations, greenhouse climate contributions, formation mechanisms and petroleum prospecting potential of mud volcanoes (Dimitrov, 2002; Gao et al., 2012; Milkov et al., 2003). In China, numerous mud volcanoes have developed in the southern Junggar Basin, particularly in Baiyanggou, Sailiketi farm in Wusu City and the Dushanzi oilfield (Wang, 2000). However, currently, only a limited number of studies have addressed the community characterization of land mud volcano systems, particularly for Xinjiang mud volcanoes. Previous experimental prove these areas have developed large-scale mud volcano groups, providing convenient access for Chinese mud volcano research and microbial communities mud volcano (Ma et al., 2009). Many mud volcanoes have developed in the southern Junggar Basin in Northwest China, and recent studies in mud volcanoes worldwide have indicated that the Dushanzi anticline, comprising a single yellowish or gray-green hill, is one of numerous folds structurally formed through the ongoing northward thrusting of the Tianshan Mountains (Sun et al., 2004). A normal fault along the crest of the anticline provides pathways for hydrocarbon seepages and eruptions from mud volcanoes. A large mud volcano crater with a diameter of 115 m is located at the top of the Dushanzi Hill, just at the apex of the anticline south of Dushanzi Town, and numerous small vents in various sizes are active around this crater. Mud volcanoes with typical structures and geomorphology characteristics provide uncommon environmental conditions for unique microbial resources. The DSZ and BYG mud volcanoes were formed on the Earth’s surface north of the Tianshan Mountains in the Xinjiang Tianshan piedmont depression band at the shaft portion of the anticline fold. Mud volcanoes are a significant marker for the modern crustal movement and new tectonic activity.

However, the microbial community composition from DSZ and BYG is lacking, in particular, the composition of archaeal community. Here, we characterized the microbial community compositions associated with the geochemical characterization of DSZ and BYG mud volcanoes in Xinjiang, China. The results of the genetic analysis of Dushanzi and Baiyanggou mud volcanoes revealed these two different adaptive microbial characterizations. To address these aims and determine whether these sequences represent indigenous microorganisms of their geothermal mud volcanoes, bacterial and archaeal 16S rRNA gene clone libraries were constructed from samples of these two mud volcanoes obtained from Junggar Basin in China. MATERIALS AND METHODS Field description This field study was conducted on the northern piedmont of Dushanzi (DSZ) (44°18.3'N, 84°50.8'E) and Baiyanggou

(BYG) (44° 11'N, 84° 23'E) mud volcanoes in Tianshan Mountains where these formations constitute the southern margin of Junggar Basin (Figure 1). This area has an arid inland continental climate with a high evaporation rate, abundant sunshine, large diurnal fluctuations in temperature, and sharp seasonal changes, with hot summers (as high as 45 °C) and cold winters (as low as -35 °C). Most of the region has sparse plant cover, excluding some oasis-like areas typically located along rivers or streams commonly located at the outlets of the mountains. Junggar Basin, one of the largest Mesozoic-Cenozoic sedimentary basins in Northwest China, is known for coal, asphalt, crude oil and natural gas deposits. (Saltsburg and Flytzani-Stephanopoulos, 2003; Wan et al., 2013). Sample collection and DNA extraction Mud volcano samples of approximately 10 kg were collected in May 2015, wrapped in a Ziploc bag containing ice cubes, immediately transported to the laboratory with ice, and stored at – 80 °C until further use in subsequent studies. The Power Max DNA isolation kit (MO BIO Laboratories, Solana Beach, CA, USA) was used to extract DNA from 0.25 g mud samples using sterile tweezers and a spoon, according to the manufacturer’s instructions. The concentration of extracted DNA was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Mud volcano geological information Mud volcanoes temperature is measured directly with a thermometer inserting 20 cm depth. The mud volcanoes gas component and carbon isotopic composition were collected and analyzed in Geology and Petroleum Resources Research Center, Lanzhou Institute of Geophysics (Gao et al., 2012). The total water-soluble salt is determinate according to the national standard method (weighing method) (NY/T 1121.16-2006). Bacterial and archaeal clone library construction We will amplify a region of the 16S rRNA using the primers using the primer 27F 5-AGAGTTTGATCMTGGCTCAG-3 combined with a modified version of 1492R primer 5- RGYTACCTTGTTACGACTT-3 (Song et al., 2015), and 21F and 926R primers were used for the amplification of archaeal 16S rRNA sequences (Liu, Marsh, Cheng, and Forney, 1997). Each 50-μl amplification reaction contained 25 μl of 2×mix (Taq DNA Polymerase, PCR Buffer, dNTP), 20 μl of double distilled water (ddH2O), 2 μl of each primer (5ng/μl), and 1 μl of DNA template (3ng/μl). The PCR conditions included initial denaturation at 95°C for 3 min, followed by 30 cycles of 95°C for 1 min, annealing at 55°C for 30 s and elongation at 72°C for 1 min, with a final extension step at 72°C for 10 min (Song et al., 2015). Approximately 5 μl of PCR products were electrophoresed

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Figure 1: Sketch map (A) and pictures (B) of the study area showing the Dushanzi (a) and Baiyanggou (b) mud volcanoes.

on a 1.0% agarose gel. The PCR products were cloned into the pUC19-T vector and transformed into Escherichia coli DH5 alpha. For each sample site, 96-well plates of glycerol stocks were prepared using each primer set and sequenced

using M13+(-47) and M13-(-48) through colony PCR. The PCR products were digested using Hha I and Rsa I (37 °C, 4 h). The digestion products were purified through 2% agarose gel electrophoresis after ethidium bromide

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Table 1. The16S rRNA library community diversity statistical analysis for DSZ and BYG mud volcanoes

Category Sample ID Clones No. OTU No. Chao I Shannon-Weaver index Simpson index Coverage C

Bacteria BYG 351 22 26 2.947 0.94 99.7% DSZ 135 21 22 2.638 0.91 95.6%

Archaea BYG 83 5 5 1.052 0.52 97.6% DSZ 38 14 16 2.406 0.89 86.8%

staining. After analyzing the gel restriction map, a randomly representative from each restriction strip was sequenced and selected. A total of 650 sequences from the mud DNA clones were sent to Huada for commercial gene sequencing. Phylogenetic tree and data analysis To determine whether the communities were similar between the DSZ and BYG mud volcanoes, pair-wise sequence comparisons were conducted. Statistical analysis of the library was performed using MOTHUR v1.25.0 (Schloss et al., 2009). Chimeric sequences were eliminated from the analysis prior to consensus sequence construction. The sequences from bacterial and archaeal libraries were pooled for DOTUR analysis to perform operational taxonomic unit (OUT) determinations, respectively (Schloss and Handelsman, 2005). The sequences were assigned to OTUs using the nearest-neighbor algorithm in DOTUR at 3% identity differences. This algorithm adds a query sequence to an existing OTU whenever that sequence is within the specified percent identity level of an existing OTU; otherwise, a new OTU is generated.

Good’s nonparametric coverage was estimated using the equation [1-(n/N)] x 100, where “N” is the total number of clones evaluated and “n” is the number of singleton OTUs (Good, 1953). The phylogenetic trees were constructed through both neighbor-joining and maximum likelihood analyses using the MEGA V5.0 software package. For the phylogenetic tree analysis, the reference sequences were obtained from the National Center for Biotechnology Information database (NCBI) (http://www.ncbi.nlm.nih.gov) (Johnson, Scholes, and Whittington, 2008).

All 16S rRNA gene sequences determined in the present study have been deposited in GenBank. The accession numbers for the archaeal and bacterial sequences are KT933233 to KT933250 and KT943980 to KT944020 for the two mud samples, respectively. RESULTS Geochemistry of mud volcanoes DSZ spout alkane gas (C1-C4) volume content was 94.78%. In gas collected from DSZ, the methane content was up to 90.10%, CO2 content was 3.22%, and N2 content was 1.92 % by volume (Gao et al., 2012). The gas collected from BYG,

the methane content was up to 91.15%. The total water-soluble salt of DSZ and BYG was 2.995g/kg and 13.77g/kg, respectively. The measured temperature of DSZ and BYG was 35°C and 27°C. Samples overview A total of 607 available clones were obtained from the mud volcano samples: 173 from Dushanzi (DSZ) and 434 from Baiyanggou (BYG). These sequences were grouped into OTUs at a 97% identity value. A total of 43 bacterial OTUs were obtained from the mud volcano samples: 21 OTUs from the Dushanzi (DSZ) mud volcano and 22 OTUs from the Baiyanggou (BYG) mud volcano. A total of 19 archaeal OTUs were obtained from the mud volcano samples: 14 OTUs from the Dushanzi (DSZ) mud volcano and 5 OTUs from the Baiyanggou (BYG) mud volcano (Table 1). Normally, the comparison of the bacterial and archaeal community diversity in these two mud volcano samples revealed that the bacterial community richness was much higher than the archaeal community richness.

Moreover, each library had a moderate number of clones, rarefaction analysis (Hammer, Harper, and Ryan, 2001) suggested that these clones represented the majority of bacterial and archaeal diversity in these habitats, suggesting that the sequence information obtained in the present study could basically represent mud volcanoes in microbial community composition (Supplementary Figure 1). The bacterial Good’s nonparametric coverage of DSZ and BYG mud volcanoes was approximately 95.6% and 99.7%, respectively. The archaeal Good’s nonparametric coverage of DSZ and BYG mud volcanoes was approximately 86.8% and 97.6%, respectively. According to the microbial data, the bacterial richness (Chao I index) and diversity (Shannon-Weaver index) for the DSZ and BYG mud volcanoes were higher compared with the archaeal data. The bacterial community diversity of the BYG mud volcano was higher than that of the DSZ mud volcano, while the archaeal community diversity of the DSZ mud volcano was higher than that of BYG mud volcano (Table 1).

Generally speaking, the phylogenetic tree analysis showed that the bacterial and archaeal community structure was significantly different between the DSZ and BYG mud volcanoes (Figure 2). Proteobacteria 16S rRNA gene clones, particularly for Gammaproteobacteria, dominated the DSZ mud volcano bacterial clone library, suggesting that Gammaproteobacteria the dominate

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Figure 2: Bacterial and archaeal community structure comparison between the Dushanzi (DSZ) and Baiyanggou (BYG) mud volcanoes. Radar Chart, the X-axis represents the Genus, the Y-axis represents the logarithmic scale.

bacteria in the populations detected this location. At the phylum level, the DSZ archaeal sequences belong to Euryarchaeota, Thaumarchaeota, Crenarchaeota and Unclassified Archaea, with abundances of 46%, 3%, 28% and 23%, respectively (Figure 3), while BYG archaeal sequences belong to Euryarchaeota and Thaumarchaeota. The archaeal community in the Dushanzi mud volcano primarily comprised Methanomicrobia (23%) ，

Thermoplasmata (3%)，Thermococci (5%), Nitrososphaeria

(3%) and Thermoprotei (6%) (Figure 3). Similarly，the Baiyanggou mud volcano also harbored Methanomicrobia (6%) and Halobacteria (69%),indicating a vital role for aceticlastic and methylotrophic methanogens in methane production in these two mud volcanoes. The major bacterial phylotypes in DSZ and BYG mud volcanoes were Gammaproteobacteria (62% and 42%) and Bacteroidetes (2% and 32%), respectively (Figure 4). These results suggest the potential isolation and utilization of extremophilic microbial resources from these two mud volcanoes．

As shown in Figure 2, the same genera between these two

mud volcanoes samples were Alcanivorax sp., sulfur-oxidizing symbiont bacterium and Methanomicrobia, old types of archaea typically present in the mud at the bottom of the seabed or "smoky area "(rich in iron and sulfur minerals), which use CO2 as a carbon source for growth and methane production (Haymon and Kastner, 1981).

DSZ mud volcano phylogenetic tree analysis

Four phyla identified in the DSZ mud volcano bacterial phylogenetic tree analysis included Gammaproteobacteria (62%), Alphaproteobacteria (11%), Deltaproteobacteria (25%), and Bacteroidetes (2%). A total of 18 genera were identified as belonging to the four phyla according to the Ribosome Database Project using the RDP classifier database and phylogenetic tree construction (Figure 3).

Four phyla identified in the DSZ mud volcano archaeal phylogenetic tree analysis included Euryarchaeota (46%), Thaumarchaeota (3%), Crenarchaeota (28%), and Unclassified Archaea (23%) (Figure 5). A total of 5 genera were identified as belonging to the four phyla based on the

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Figure 3: The composition of bacteria (A) and archaea (B) 16S rRNA gene clone library phylotypes in the DSZ MV based on RDP classifier results.

Ribosome Database Project using RDP classifier database and phylogenetic tree construction. Interestingly, the unclassified archaea might contain an unknown new phylum (Figure 6). Among the archaea clones, 23% of the clones were related to Methanomicrobia, including the most predominant phylotype observed. The second most dominant phylotype, unclassified archaea (Figure 6), was

most closely related to an environmental clone sequence from deeply buried coral carbonates and sediments (23% of the total library) (Hoshino et al., 2011). BYG mud volcano phylogenetic tree analysis The samples in this region of the BYG mud volcano were

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Figure 4: The composition of bacteria (A) and archaea (B) 16S rRNA gene clone library phylotypes in the BYG MV based on RDP classifier results.

dominated by Gammaproteobacteria (42%), but had considerable contributions from Bacteroidetes (32%), Verrucomicrobia (10%), Acidobacteria (6%), Planctomycetes (3%), Deinococcus-Thermus (1%) and Unclassified Bacteria (5%) (Figure 5). A total of 16 genera were identified as belonging to the four phyla based on the

Ribosome Database Project using the RDP classifier database and phylogenetic tree construction with unclassified bacteria (Figure 7).

Halobacteria (69%) was the most predominant and distantly related archaeal phylotype (Figure 7B), which was only detected in the BYG samples (Figure 7). The GenBank

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Figure 5: Phylogenetic relationships of the bacteria 16S rRNA gene sequences recovered from the DSZ

MV. DB: DSZ bacteria, DA: DSZ archaea, BB: BYG bacteria, BA: BYG archaea. (x): clones number.

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Figure 6: Phylogenetic relationships of the archaea 16S rRNA gene sequences recovered from the DSZ MV. database analysis showed that most of the sequences in the BYG archaeal phylogenetic library showed a higher degree of similarity (>97%) based on the BLAST analysis, and most of these microorganisms were halobacteria, producing pigments with such features as Halalkalicoccus paucihalophilu, Halohasta litchfieldiae, Halorhabdus tiamatea and Haloarchaeon clone, belonging to a class of primarily photosynthetic bacteria. Archaeal clone library sequencing through GenBank BLAST analysis revealed two main phyla in

BYG mud volcano samples, where BA66 (BA23) and BA40 represent a class of advantageous microbes with a high degree of similarity to the reported genus Halobacteria and Methanomicrobia: the latter is a class of methanogenic archaea, widely distributed in the sea "white smoke zone", and the former is a class of halophilic microorganisms primarily located in the area of inland saltwater (Yassine, Tagomori, Henein, and Bryzik, 1996). BA13 represents a class of fewer microbes that might be a new genus of the

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Figure 7: Phylogenetic relationships of the bacteria 16S rRNA gene sequences recovered from the BYG MV

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Figure 8: Phylogenetic relationships of the archaea 16S rRNA gene sequences recovered from the BYG MV.

phylum Thaumarchaeota, which is widely distributed in the area of seabed smoke archaea, and these thermophilic anaerobes use sulfide as an electron donor to obtain energy (Figure 8)(Brochier-Armanet et al., 2008). DISCUSSION The aim of the present study was to analyze the microbial composition of DSZ and BYG mud volcanoes to discuss the species origin and formation mechanisms of mud volcanoes, and to provide the basis for species accumulation and exploration in the margins of Junggar Basin. To our knowledge, this study is the first to report and describe the composition of the microbial assemblages of the archaeal populations (based on 16S rRNA-derived libraries) extant in these two mud volcanoes in Junggar Basin of Mainland China (Gao et al., 2012; Jian-hui, 2010; Jian Hui and Zhang, 2011).

In recent years, there have been several research focused on the microbiology of the mud volcanoes in Xinjiang (Gao et al., 2012; Jian-hui, 2010; Jian Hui and Zhang, 2011). The bacterial diversity from mud volcano in Xinjiang by culture-independent approach revealed the rich microbial

communities in these mud volcano and the important research significance for the exploration in the margins of Junggar Basin, yet failed to identify the bacteria associated with desulfurization species (Ma et al., 2009). However, Desulfobacteraceae was detected in high abundance in both the Dushanzi and Baiyanggou mud volcanoes, and this strictly anaerobic chemoorganotrophic or inorganic autotrophic archaea can use H2 and sulfate as energy sources and CO2 as a carbon source. The common electronic acceptors for this microbe are sulfates (typically sulfide, thiosulfate or sulfur), but this organism can also use simple organic compounds or carbon as electron acceptors, such as long chain fatty acids, ethanol, or polar aromatic or aliphatic compounds (Kuever, Rainey, and Widdel, 2005). This was firstly detected in the mainland China, but has been repeatedly found in the international arena mud volcanoes.

The technology single-strand conformation polymorphism (SSCP) and a target 16S rDNA gene on V3 region were used to analyze mud volcano microbial community and structure. And the results showed Pseudomonas is a dominant microorganism in Xinjiang mud volcano, it distributes widely in mud volcano and was affected little by ecological and climatic conditions, which is consistent with our results

Int. Res. J. Public Environ. Health 255 Pseudomonas was also recovered from these mud volcanos (Lu, Li, and Zhang, 2011). However, they failed to describe the archaeal community and structure which could response more realistic characterization of their unique geographical nature. Moreover, others pointed out these mud volcano areas belong to natural gas and oil mineral-rich region with such extreme environments widespread in high salinity, low nutrient (Gao et al., 2012). In our study, Halobacteria was detected in high abundance in the BYG mud volcano, and these bacteria are typically present in strict anaerobic saline environment, which is coincided with the corresponding measured with high salinity in BYG mud volcano (L. Jian-hui, 2010). Candidatus Methanoplasma termitum, recovered from the DSZ mud volcano, primarily exist in the high-temperature environments (75℃~78℃) of hot springs on land (Itoh, Suzuki, Sanchez, and Nakase, 2003). These archaea are likely derived from the crust magmatic or volcanic eruption since the surface temperature of the mud volcano is not particularly high. In addition, an Unclassified Archaea was identified in the DSZ mud volcano, which might be a new archaeal phylum.

An increasing number of studies have attempted to address the fate of methane originating from these undersea mud volcanoes, and determine the potential impact of this gas on the global carbon cycle and the role of benthic marine microbial communities. Sites characterized by high methane discharges and high salinities have not previously been described in terrestrial settings. Methanogenic archaea are widely distributed in nature as prevalent and natural anaerobic reactors in soils, sediments and other artificial environments and for senate methane and organic matter degradation. The results of the present study revealed that Dushanzi and Baiyanggou contained the highest abundance of Methanosaeta, belonging to obligate acid nutrition methanogenic archaea, which only use acetic acid as a carbon source for growth and methane production. Methanolobus in the two samples was also detected at a high abundance, and this archaea uses methanol and methylamine to produce methane refer. These findings indicate that there are three different types of nutrition methanogenic archaea in DSZ and BYG mud volcanoes, including efforts describing potential bacterial and archaeal partnership(s) promoting the anaerobic oxidation of methane. The methane, regardless of origin, could act as an energy and carbon source for sustaining microbial life through Methanomicrobia metabolism in archaea that inhabit extremely cold or hot, salty mud volcanoes. The community composition reflects the sampling locations with high-temperature and high-sulfur landscapes. The clones related to anaerobic sulfur oxidizing populations (i.e., sulfur-oxidizing symbiont bacterium) were detected in these two samples. The occurrence of metabolically active anaerobic methane- and sulfur-oxidizing populations suggests a highly favorable environment for the anaerobic oxidation of methane in mud volcano sediments.

Thus, the mud volcano samples contained rich microbial resources, although many species remain unknown.

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