Characterization of Fe(III)-Reducing Enrichment Cultures and Isolation of Enterobacter sp. Nan-1 from the Deep-Sea Sediment, South China Sea

WANG Dunfan, JIN Chong, JIN Aimin, and LOU Zhanghua

Characterization of Fe(III)-Reducing Enrichment Cultures and Isolation ofsp. Nan-1 from the Deep-Sea Sediment, South China Sea

WANG Dunfan1), 2), JIN Chong3),*, JIN Aimin1), and LOU Zhanghua1)

1),,,316021,2),,,572000,3),310007,

To characterize the Fe(III)-reducing bacteria, enrichment cultures were initiated by inoculating deep-sea sediment from the South China Sea (SCS) into the media with hydrous ferric oxide (HFO) as the sole electron acceptor. As indicated by Meta 16S rDNA Amplicon Sequencing, the microorganisms related to Fe(III)-reduction in the enrichment cultures were mainlyand. A new facultative Fe(III)-reducing bacterium was obtained and identified assp. Nan-1 based on its 16S rRNA gene sequence and physiological characterizations.sp. Nan-1 was not only a mesophilic bacterium capable of reducing HFO with a wide range of salinity (4, 34, 40, 50 and60gL−1) efficiently, but also a piezotolerant bacterium that can proceed Fe(III)-reduction sustainedly at hydrostatic pressures between 0.1 and 50MPa using glucose and pyruvate as carbon source. Furthermore, the geochemical characteristics of deep-sea sediment indicated that the microbial metabolism and iron reduction both remain active in the well-developed Fe(III)-reducing zone where the strain Nan-1 was obtained. To our knowledge,sp. Nan-1 could serve as a new applicative Fe(III)-reducing bacterium for future investigation on the iron biogeochemical cycle and diagenetic process of organic matter in the deep-sea environment.

deep-sea sediment; Fe(III)-reducing bacteria; Meta 16S rDNA Amplicon Sequencing; high hydrostatic pressures;sp. Nan-1

1 Introduction

Dissimilatory iron reduction (DIR) has long been known to play an important role in biogeochemical processes involved in multiple element cycles in marine environments (Lovley, 2006). It has demonstrated that the oxidation of organic matter, circulation of trace elements, and even magnetization of minerals are linked to DIR (Wu., 2013). The organisms capable of coupling oxidation of organic matter to iron reduction have been descri- bed as Fe(III)-reducing bacteria. These organisms could transfer extra electrons from energy production to solid Fe(III)-bearing minerals outside of bacterial cell while con- verting the insoluble Fe(III) to soluble Fe(II) (Amstaetter., 2012).

Lovley. (1987) discovered the first Fe(III)-redu- cing bacterium Geobacter metalli-reducens GS-15, which could simultaneously decompose benzene, toluene and other aromatic compounds. Roh. (2006) isolated a Fe(III)- reducing bacteria, strainsp. PV-4, which is capable of reducing metals in temperatures from 0 to 37℃,and proposed that it played a vital role in biogeochemical activities in marine sedimentary environment. Although many Fe(III)-reducing bacteria have been isolated and their contributions to the iron cycle in marine environment have been investigated, previous studies have mainly focused onandgenes. It’s still poorly understood about such bacteria living in the deep-sea environment due to the presence of complex sedimentary mi- crobial communities and tedious recovery process of pure cultures for laboratory experimentation (D’Hondt., 2004; Li., 2015).

It was suspected that iron biogeochemical cycle is pre- valent in deep-sea sediments due to the enrichment of Fe- oxide which is readily available for anaerobic respiration as electron acceptor (Beckler., 2016). However, the lack of pure cultures and isolation of Fe(III)-reducing bacteria from deep-sea environment results in a poor laboratory model application for investigators to further examine the biotic iron reduction mechanism. To better understand the iron biogeochemistry cycle in deep-sea environ- ment, here we have investigated the geochemical characteristics of deep-sea sediment and the biological community structure of Fe(III)-reducing enrichment cultures. A new Fe(III)-reducing bacterium,sp. Nan-1, was obtained from the enrichment cultures after multiple screening. The physiological characterizations of strain Nan-1 were also identified under different conditions.

2 Materials and Methods

2.1 Site Description and Sampling

Deep-sea sediment was collected from the SCS (18˚09.57΄N and 116˚40.52΄E) with a water depth of 3915 m during the cruise ofin from January to March 2017. Sediment column (260cm) were divided into segments and placed in sterile plastic bags at 4℃ for further process after gravity core was recovered.

The geochemical characteristics of the sediment were inferred from the layered pore-water. Sediment pore-water was extracted using Rhizons sampler with 0.1μm pore size polymer filter (Seeberg-Elverfeldt., 2005) and frozen in brown glass bottle for further various analyses. Dissolved oxygen (DO) was measured using amperome- tric Clark-type microelectrode (Unisense, Denmark) calibrated by N2-purged (as a zero) and air-saturated seawater (D’Hondt., 2009). Pore-water profiles of NO3−and NH4+were obtained by the photometrical (Schnetger and Lehners, 2014) and fluorescence methods (Holmes., 1999), respectively. Sulfate was quantified using an ion chromatograph (ICS900, Dionex) equipped with a 4mm×25mm AS19 analytical column, a 4mm AERS500 suppres- sor and an AS-DV auto-sampler. 20mmolL−1potassium hydroxide was used as the eluent with a flow rate of 0.6mLmin−1.

The sediment was freeze-dried and analyzed for total organic carbon (TOC) using elemental analyzer, before which approximately 500mg of sediment powder was first digested in 5mL of 20% HCl for 24h to remove any inorganic carbon. As for total organic carbon isotope (δ13C- TOC), the CO2gas was extracted from 100mg of sample powder treated with 100% orthophosphoric acid at 75℃ for 45min and measured using a continuous flow isotope- ratio mass spectrometry (Delta V Advantage, Thermo Fisher Scientific Inc., USA) coupled with GasBench II device with a precision of 0.2‰ for carbon isotope analysis. The results were reported in standard δ notation relative to the Vienna-PeeDee Belemnite (V-PDB) standard.

2.2 Enrichment Conditions

The enrichment culture was set up as 10% (w/v) sediment slurry in the Fe(III)-reducing medium (Fe(III)=10 mmolL−1, pH=7.0) by using 100mL serum bottles which contained the following ingredients (gL−1): peptone 5.0, yeast extract 1.0, NaCl 34.0, L-cysteine 0.5. HFO was pre- pared according to the method of Glasauer (Glasauer., 2003) by rapidly adding 1molL−1NaOH to 2molL−1FeCl3· 6H2O until the pH reached 7.0, meanwhile, stirring vigorously to avoid local high pH. The precipitation of iron slurry was washed five times with 18.2Ω water to remove any trace salts. The serum bottles were purged with nitrogen gas for 30min and capped with butyl rubber stoppers for the required anaerobic condition. Culture broth was diluted 10 times with sterile water and inoculated into the fresh medium repeatedly to enrich the Fe(III)- reducing microbes after maintained at 4℃ and 25℃ for a week respectively. Final enrichment culture was used for composition analysis of microbial community.

2.3 Composition Analysis of Microbial Community

Composition of Fe(III)-reducing enrichment cultures were characterized by Meta 16S rDNA Amplicon Sequen- cing. The V4–V5 region of 16S rDNA was amplified by performing a PCR using fusion primer (515F: 5’-GTGCC AGCMGCCGCGG-3’, 907R: 5’-CCGTCAATTCMTTT RAGT-3’) with dual index and adapters. After too short fragments were removed by AMPure beads, all qualified fragments were used to construct a library and then for PE300 sequencing. The raw sequencing data were filtered to obtain clean reads (Fadrosh., 2014), then paired- end reads with overlap were merged to tags by FLASH (Magoc and Salzberg, 2011). Tags were clustered into OTUs with a 97% threshold by scripts of software USEARCH (Edgar, 2013). OTUs representative sequences were ta- xonomically classified by using Ribosomal Database Pro- ject (RDP) Classifier (v.2.2) trained on the Greengenes databass, using 0.8 confidence value as cutoff. Different species screening was analyzed based on OTUs and taxonomic ranks.

2.4 Isolation and Identification of the Strains

Pure culture of Fe(III)-reducing bacteria was obtained by spread-plating the enrichment cultures onto agar medium in the anaerobic chamber (BACTRONEZ-2, USA). Individual colonies were re-screened at least 5 times using anaerobic culturing techniques (Mille and Wolin, 1974) prior to assessing the culture purity by Repetitive Extragenic Palindromic (REP)-PCR (Debruijn, 1992). Once the isolated strain met the required purity, the final test was performed for its ability of iron reduction. The identification of the isolated strain was carried out by 16S rRNA gene sequence analysis. The 16S rRNA genes were amplified using a pair of primers: 27F: 5’-AGAGTTTG ATCMTGGCTCAG-3’; 1492R: 5’-TACGGYTACCTTG TTACGACTT-3’. The PCR conditions were as follows: an initial denaturation step for 5min at 95℃, followed by 35 cycles of 30s at 95℃, 30s at 55℃, 60s at 72℃ and then final extension for 10min at 72℃. The sequence of PCR products was performed by the BGI (Wuhan, China). Phylogenetic tree was constructed by the neighbor-joining methods using the Mega program. Similar topologies of the trees with different algorithms were confirmed. Robustness of the branch clustering was assessed by bootstrap values on the basis of 2000 replications.

2.5 Physiological Characterization of Isolated Strain

To describe the effects of different NaCl concentrations (0–60gL−1), temperatures (15–40℃) and hydrostatic pre- ssures (0.1–50MPa) on biomass accumulation and Fe(III) reduction of the isolated strain, a serial of batch operations was performed under the given environmental conditions in 100mL serum bottles (working volume 50mL). using pyruvate (20mmolL−1) as electron donor. The biomass accumulation and Fe(III)-reduction were determined by time course sampling at 6h intervals during the axenic incubation. All tests were performed in triplicate to assure data reproducibility.

Morphological examination was carried out by transmission electron microscopy (TEM) (JEM-2100F, Japan). Cell counting was performed by fluorescence microscopy method described previously (Hobbie., 1997). Fe(II) was determined by measuring the absorbance at 562nm on the spectrophotometer by the ferrozine method (Stookey,1970) after 0.5molL−1HCl extraction with anaerobic water for sample dilution. Briefly, 0.5mL cell suspension of the isolated strain were taken using sterile syringes and immediately transferred to a screw top plastic tube containing 4.5mL HCl (0.5molL−1). After 24h of extraction at 25℃ in the Coy anaerobic glove box, 0.1mL aliquots of supernatant filtered with 0.22μm filter membrane were used testing in final volume of 5mL.

3 Results and Discussion

3.1 Geochemical Features of the Sedimentary Environment

Pore-water profiles of the deep-sea sediment showed that both DO and NO3−declined exponentially with depth, while DO penetration depth is apparently shallower than that of NO3−(Fig.1A). The nitrate penetration depth (30cm) was approximately twice as deep as the DO penetration depth (16cm). In the whole depth profiles, no distinct variation of SO42−concentration was observed. The concentration value (25.13mmolL−1) at the base of core is close to that in the overlying seawater (28.05mmolL−1). In contrast, the ammonium concentration was low at the surface but accumulated to a significant value of 60μmolL−1at the bottom of sediment column. Iron reducing peaks appeared below thecomplete removal depth of oxygen and nitrate in the sediment column, consisting with the theoretical biogeochemical zonation model for the marine se- diment (Jahnke., 1982; Salwan and Murray, 1983). Nevertheless, the iron reduction zone exhibited a wide depth scale ranging from 25 to 230cm with average Fe(II) concentration of 2.33μmolL−1. Previous study claimed that the depth scale over which the remineralization processes occur varies among different sedimentary environments due to the factors suchas the contents of organic matters and sediment flux (Froelich., 1979). Our studied site is located in the SCS where is characterized by a high organic matter content and sedimentary rate (Chen and Wang, 1999), which leaded to the abundant matters available for DIR. As a result, iron reducing zonation was well-de- veloped and exhibited a wide band of penetration on geo- chemical profiles. Hence, we declared that the iron re- mineralization process was predominant in studied site.

Furthermore, some peaks on behalf of low TOC contents with low δ13C values were observed within the iron reduction zone, which nearly correspond to the high Fe(II) concentration intervals in depth profile (Fig.1B). This is because Fe(III)-reducing bacteria are heterotrophic microbe capable of coupling the organic matter oxidation to the iron reduction, which lead to the depletion of organic matter with the accumulation of Fe(II). The smaller negative peaks of δ13C for TOC provided a clear evidence to suggest that the organic matter as electron donor for DIR was produced by the autotrophic microbes. Fe(III)-reducing bacteria could harvest energy for growth during the DIR process, which may facilitate microbial symbiosis and boom. This result confirms that autotrophic microbes supply a portion of the biomass and organic matter for heterotrophy in the marine sediment (Lovley, 2001). A slight malposition may well be caused by the Fe(II) diffusion upward and bioturbation. Stapleton. (2005) in- vestigated the geochemical characteristics and isolated many Fe(III)-reducing bacteria related tofrom distinct marine sediment at the depth from 119m to 2530m. Their results claimed that there were great geochemical differences between the sediments from the deep ocean and shallow station. Our study focused on deeper site with the water depth of 3915m and apparently showed that active microbial metabolism and prevailing iron biogeochemical cycle were significant in the deep-sea environment.

Fig.1 Pore-water geochemistry (A) and sediment geochemistry (B) in the core from the SCS.

3.2 Microbial Community Composition of Enrichment Cultures

After multiple enrichment, the composition of Fe(III)- reducing enrichment cultures was investigated by Meta 16S rDNA Amplicon Sequencing. 39041 and 37564 clean reads were obtained from each temperature-dependent sample, whose utilization ratio was 36.59% and 75.69% respectively (Table 1). The high quality paired-end reads were combined to tags based on overlaps. Tags without pri- mers were 37653 and 36235 in per sample, and the aver-age length was 377bp (Table 2). All tags were clustered into OTUs assigned to,andspecies.

Meta 16S rDNA Amplicon Sequencing is a useful technique to reveal community structures of microbial enri- chment cultures from different environments. Althoughandexist in both samples at 25℃ and 4℃, their abundance is distinctly different (Fig.2). The predominant microbe in the enrichment culture at 4℃ refers to, which substantially supports the claim that some members of the genusadapted to the marine environment and participated in iron biogeochemical cycle actively at cold temperature (Stapleton., 2005). Biogeochemical metabolism is widespread in marine sediment. The indigenous microorganisms are well adapted to their environment and remain participating in the remineralization processes actively at a wide range of temperature (Kostka., 1999).andspecies are both found in enrichment cultures at 25℃, which might be attributed to the fact that the metabolite of organic acids and H2potentially provide excellent substrates for each other as electron donors in the iron reduction reaction, forming the structure of mutualistic symbiosis (Lovley, 1987).

Table 1 Reads filtered out with default parameters

Table 2 Tags combined with the high quality paired-end reads and OTUs numbers

Fig.2 The taxonomic composition in samples of Genus- level.

3.3 Isolation and Identification of the Pure Bacterial Strain

A strain named Nan-1 was obtained from the enrichment cultures at 25℃. The individual colonies of Nan-1 were milky white, opaque and round. TEM observation showed that the strain was rod about 2.1×0.9μm (Fig.3) in dimension. Strain Nan-1 was closely related tosubsp. DSM 16691 showing a similarity of 99% based on the 16S rRNA sequence date (1500bp) deposited in Gen Bank with the accession of MK311344. Analysis of the phylogeny places strain Nan-1 within the genuswhich has been previously cultured but does not reduce iron and isn’t likely to be Fe(III) reducers (Fig.4).

Fig.3 TEM of strain Nan-1 (2.1μm×0.9μm) grown in medium aerobically at 25℃.

As shown in Fig.5, the tendency of Fe(II) accumulation and cells growth exhibited two different phases during the Fe(III)-reducing activity. From 6h to 30h, the cells grew logarithmically and Fe(II) accumulated effectively. But from 30h to 48h, the Fe(II) accumulation reduced with the slow cell growth because of the inhibiting effects of the Fe(II)-containing minerals on cells (Royer., 2004). Fe(II) accumulation corresponded to the cell growth almost in the whole process, which indicated that DIR could yield energy to support microbes growth (Nickel., 2008). No Fe(II) accumulation was detected in abiotic control group, implying that the Fe(III)-reduction process was caused by bacteria. The strain Nan-1 was also able to use glucose and pyruvate as electron donors for Fe(III)- reduction (Table 3). The maximum cell number ((6.413±0.001)×107mL−1) and the highest Fe(III)-reducing activity (4.69±0.35mmolL−1) were detected in the culture solution when using pyruvate as the electron donor for Fe(III)-reduction. In contrast, Nan-1 remained viable but did not grow in the culture solution with acetate, formate, lactate, and propionate as the sole electron donor. The strain Nan-1 caused the release of soluble Fe(II) by coupling the oxidation of organic matter to the Fe(III)-reduction. This result offers convincing evidence for the idea that Fe(III)-reduction are intimately linked to the carbon fate in deep-sea sediments (Roh., 2002). As shown in Table 4, plenty of Fe(III)-reducing organisms have been isolated from variable environments, while members of thespp. were rarely obtained. The differences about original source, electron donors and acceptor betweensp. Nan-1 and previously reported Fe (III)-reduction bacteria are stark.

Fig.4 Phylogenetic relationships of the strain Nan-1.

3.4 Effect of Salt Concentration, Temperature and Hydrostatic Pressure on Fe(III)-Reduction

The salt concentration is essential for microbial growth and Fe(III)-reduction process (Pollock., 2007). The strain Nan-1 was examined for Fe(III)-reduction under different NaCl concentrations (0, 4, 34, 40, 50 and 60gL−1). The result showed that it could grow and reduce Fe(III) at a wide range of NaCl concentration from 0 to 60gL−1(Fig.6). Optimum growth occurred at a NaCl concentration of 34gL−1with the cells number of (10.480±0.051)×107mL−1and Fe(II) accumulation of (3.1996±0.331)mmolL−1. This result was consistent with the salinity of the deep ocean water overlying the site wheresp. Nan-1 was isolated. Although many Fe(III)-reducing bacteria have been characterized for their capability of Fe(III)-reduction, their tolerance to salt is different (Bongoua-Devisme., 2012). Nan-1 was capable of adapting to a wide range of salinity and even reducing Fe(III) under fresh-water culture condition. This unique property madesp. Nan-1 a significant model Fe(III)-reducing bacterium from marine environment.

Table 3 The characterization of different electron donor of Nan-1

Notes: Fe(II), mmolL−1; Cells, ×107mL−1; ND, not determined.

Table 4 The comparison of Fe(Ⅲ)-reducing bacteria obtained from different environments

Fig.5 The curve of Fe(III) reduction and cell growth of the strain Nan-1 (n=3, Error bars=s.e.m.).

Fig.6 Effect of salt concentration on Fe(III) reduction and cell growth of the strain Nan-1 (n=3, Error bars=s.e.m.).

The effects of incubation temperature on Fe(III)-re- duction and microbial growth were also examined. Available Fe(III)-reduction and cell growth temperature for strain Nan-1 ranged from 4℃ to 50℃. The maximum Fe(III)-reduction degree ((4.3330±0.149)mmolL−1Fe(II) was generated) and bacterial growth ((13.400±0.133)× 107mL−1) were observed at 37℃ (Fig.7).sp. Nan-1 could reduce Fe(III) at the temperature between 4℃ and 50℃ with the optimum at 37℃, suggesting that it was a mesophilic bacterium. Temperature variations were very common in the ocean environment from cold seeps to hydrothermal vents (Slobodkin., 2001; Roh., 2006). The adaptability to different temperatures indicates thatsp. Nan-1 may play a vital role in carbon and iron biogeochemical cycle process of marine environment (Burdige, 1993).

The strain Nan-1 was isolated from the deep-sea sediment at a water depth of 3915m, where the hydrostatic pressure is 39MPa equivalently. Different hydrostatic pressure effects were examined to assess the rate of Fe(III)- reduction and cell growth after 48h of incubation. Bacterial iron reduction proceeded continuously at the six experimental pressures (0.1, 5, 20, 35, 40, 50MPa). However, the extent of Fe(III)-reduction consistently decreased with the pressure (Fig.8). More specifically, 3.39, 3.09, 2.24, 1.11, 0.71 and 0.25mmolL−1of Fe(II) concentration were produced at 0.1, 5, 20, 35, 40 and 50MPa pressures respectively. And the cell growth was quite associated with the Fe(II) accumulation at each of changes in pressure. The average rate of Fe(II) accumulation at the six pressures were 70.8, 64.3, 46.7, 23.2, 14.7 and 5.3μmolL−1h−1, respectively. By extrapolating the linear regression of the Fe(II) concentration versus time, the Fe(II) accumulation rate dropped to zero by 53MPa (Fig.9). This may due to the shutoff of enzymatic iron reduction under this pressure level. It accounts for the previous report which claimed that Fe(III)-reduction done by bacteria is an enzymatic process (Roh., 2002). Several piezotolerant Fe(III)-re- ducing bacteria have been isolated from the deep-sea se- diments at water depths ranging from 1000 to 5000m (Xu., 2003; Toffin., 2004), but the feature of pressure adaptability was very different. Our results suggest thatsp. Nan-1 is a piezotolerant bacterium, which adapted to the deep-sea environment and remained active.

Fig.7 Effect of incubation temperature on Fe(III) reduction and cell growth of the strain Nan-1 (n=3, Error bars= s.e.m.).

Fig.8 Fe(III) reduction and cell growth rate of the strain Nan-1 under different hydrostatic pressures (n=3, Error bars=s.e.m.).

Fig.9 The correlation between the Fe(III) reduction rate and the hydrostatic pressure.

Liu. (2016) have reported thesp. L6, a Fe(III)-reducing bacteria, isolated from the coastal sludge in Bohai sediment at the depth of approximately 15m below the seafloor. L6 could reduce ferric iron at the NaCl concentration of 4gL−1using acetate as the sole electron donor, which is different from strain Nan-1. The effect of hydrostatic pressure on L6 is still unknown, but strain Nan-1 obtained from the deep-sea sediment could grow well at hydrostatic pressures ranging from 0.1 to 53MPa with a preference for seawater salinity. It could also reduceFe(III) at temperatures ranging from 4℃ to 50℃ with theoptimum rate at approximately 37℃. Such specific features in physiology and phylogeny make strain Nan-1 distinct from L6. We therefore proposed that strain Nan-1 was an alternative mode of Fe(III)-reducing bacterium that had never been optimized from the deep-sea environment for studying the iron biogeochemical process.

More importantly, one of striking characteristics of strain Nan-1 was that its metabolic activities appear to create an acidic microenvironment (pH values not show here), which distinguishes it from the known strain for biomineraliz- tion that prefer alkaline conditions (Bell., 1987; Roh., 2001). Strain Nan-1 could utilize glucose during iron respiration in which making a microenvironment with CO2and low Eh value is considerable. Because this cha- racteristic facilitates the formation of siderite (low Eh and pH) thermodynamically (Zachara., 2002; Roh., 2003), Fe(III)-reducing bacteria is suspected to be important for forming a ‘biological carbon pump’ through which CO2is consumed in surface water and then transported to the deep sea as sinking particulate organic carbon (Tortell., 1999). The biomineralization of HFO to siderite by such kind of Fe(III)-reducing bacteria may be potentially functional for ‘biological carbon pump’ and magnetic properties of oceanic sediments (Ellwood., 1988). In this respect, future studies are needed to understand the mechanism of electron transfer and biomineralization about genus. Our work just provided an alternative pathway to have an insight into the iron biogeoche- mical process in deep-sea environment.

4 Conclusions

It has already been recognized that bacteria capable of using Fe(III) as a terminal electron acceptor may modify Fe chemical species through DIR metabolism and thereby play a vital role in regulating the iron biogeochemistry cycle process in the deep-sea environment. As part of the study on the microbial diversity of Fe(III)-reducing bacteria, deep-sea sediment sample was used as the inoculum for enrichment cultures in media containing HFO as the sole electron acceptor. A novel Fe(III)-reducing bacteriumsp. Nan-1 was recovered and determined for Fe(III)-reduction effects under different conditions. Nan-1 is a facultative anaerobe that grows by coupling organic matter oxidation to the reduction of HFO with an optimum salinity of 34gL−1. The cells are rod shaped about 2.1μm×0.9μm. It’s also a piezotolerant bacterium that could reduce ferric iron effectively at a wild range of hydrostatic pressures from 0.1 to 50MPa. To our knowledge,sp. Nan-1, recovered from the deep-sea sedi- ment of SCS, represents a new microbe among the Fe(III)- reducing bacteria. An alternative strain to provide an in- sight into the iron biogeochemical cycle and early dia- genetic process associated with organic matter in the marine environment is proposed.

Acknowledgements

The authors acknowledge all the participants for helpful discussions and assistance to this research work in the laboratory. We also acknowledge the financial support by the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDB06020000), the Zhejiang Geological Prospecting Bureau Science Projects (No. 2017 13), and the Geological Fund of Zhejiang Province (No. 20150012).

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. E-mail: jin-chong@foxmail.com

April 2, 2019;

May 24, 2019;

October 23, 2019

(Edited by Chen Wenwen)