World J Microbiol Biotechnol (2013) 29:2325–2334 DOI 10.1007/s11274-013-1399-9 ORIGINAL PAPER A meta-analysis of the publicly available bacterial and archaeal sequence diversity in saline soils Bin Ma • Jun Gong Received: 19 April 2013 / Accepted: 3 June 2013 / Published online: 12 June 2013 Ó Springer Science+Business Media Dordrecht 2013 Abstract An integrated view of bacterial and archaeal Halorubrum and Thermofilum were the dominant archaeal diversity in saline soil habitats is essential for understanding genera in saline soils. Rarefaction analysis indicated that less the biological and ecological processes and exploiting than 25 % of bacterial diversity, and approximately 50 % of potential of microbial resources from such environments. archaeal diversity, in saline soil habitats has been sampled. This study examined the collective bacterial and archaeal This analysis of the global bacterial and archaeal diversity in diversity in saline soils using a meta-analysis approach. All saline soil habitats can guide future studies to further available 16S rDNA sequences recovered from saline soils examine the microbial diversity of saline soils. were retrieved from publicly available databases and sub- jected to phylogenetic and statistical analyses. A total of Keywords Archaea Á Bacteria Á Halophilic Á Microbial 9,043 bacterial and 1,039 archaeal sequences, each longer diversity Á Saline soil than 250 bp, were examined. The bacterial sequences were assigned into 5,784 operational taxonomic units (OTUs, based on C97 % sequence identity), representing 24 known Introduction bacterial phyla, with Proteobacteria (44.9 %), Actinobacte- ria (12.3 %), Firmicutes (10.4 %), Acidobacteria (9.0 %), Saline soils are increasingly abundant as a consequence of Bacteroidetes (6.8 %), and Chloroflexi (5.9 %) being pre- irrigation and desertification processes (Rengasamy 2006). dominant. Lysobacter (12.8 %) was the dominant bacterial Increase in salinity greatly affects soil microbial and bio- genus in saline soils, followed by Sphingomonas (4.5 %), chemical properties, and causes shifts in the microbial Halomonas (2.5 %), and Gemmatimonas (2.5 %). Archaeal community structure towards bacteria and archaea. sequences were assigned to 602 OTUs, primarily from the According to a recent meta-analysis, the global microbial phyla Euryarchaeota (88.7 %) and Crenarchaeota (11.3 %). community composition is influenced more by salinity than by extremes of temperature, pH, or other physical and chemical factors (Lozupone and Knight 2007). Accord- ingly, the diversity of halophilic bacteria and archaea in saline soils is expected to be quite different from that in Electronic supplementary material The online version of this non-saline soils. The study of microbial diversity in saline article (doi:10.1007/s11274-013-1399-9) contains supplementary soils is significant for understanding the ecological func- material, which is available to authorized users. tions, saline adaption mechanisms and biotechnical potentials of microorganisms. B. Ma Á J. Gong (&) Laboratory of Environmental Microbiology, Yantai Institute Halophilic and halotolerant bacteria and archaea are of Coastal Zone Research, Chinese Academy of Sciences, essential for the biogeochemical processes in saline soils Yantai, China (Dendooven et al. 2010; Blum et al. 2012), hence for the e-mail: [email protected] utilization of saline soils. Halophilic bacteria and archaea B. Ma Á J. Gong thrive in saline soils because they possess enzymes Network Laboratory for Coastal Microbiology, Yantai, China endowed with unique structural features and catalytic 123 2326 World J Microbiol Biotechnol (2013) 29:2325–2334 power, enabling them to sustain metabolic and physiolog- Methods ical processes under high salt conditions (Munawar and Engel 2012). Specific enzymes from soil halophiles are Sequence data collection perceived to be potentially useful for a variety of appli- cations, including the production of important biomole- Initial sequence sets were obtained on 10 May 2012 from cules (Liszka et al. 2012) and the remediation of pollutants the GenBank (http://www.ncbi.nlm.nih.gov) and RDP in saline conditions (Dastgheib et al. 2011). Soil halophiles (Release 10, http://rdp.cme.msu.edu) databases using the are an excellent source for exploring novel enzymes pos- search terms ‘saline’ OR ‘hypersaline’ AND ‘soil’ AND sessing the inherent ability to function in high salt condi- ‘16S’. Non-16S rRNA sequences from GenBank were tions (Singh et al. 2012). removed by checking the names of the sequences. All 16S Bacteria and archaea have been widely studied with rRNA gene sequences from the two databases were merged respect to their biodiversity in saline soils. Initial studies and duplicate sequences, identified based on accession employed traditional culture-dependent methods and numbers, were removed. Sequences with vector nucleo- resulted in the discovery of a large number of new bacterial tides or chimeras were checked by Mallard (http://www.cf. and archaeal taxa (Lefebvre et al. 2006). The primary ac.uk/biosi/research/biosoft/). The 16S rRNA gene sequen- methods in current use are DNA-based molecular biology ces of Escherichia coli (accession number: U00096) and approaches such as cloning and sequencing of 16S rRNA Methanothermobacter thermoautotrophicus (accession genes, denaturing gradient gel electrophoresis (DGGE), number: AE000666) were selected as reference sequences terminal restriction fragment length polymorphism (T- for bacteria and archaea, respectively. Sequences shorter RFLP) and quantitative PCR (Lefebvre et al. 2006; Cui than 250 bp were removed from the dataset in order to et al. 2009; Blum et al. 2012). Cloning and sequencing of avoid uncertainties when comparing and classifying short 16S rRNA genes has been generally favored over other sequences that have little or no sequence overlap. The methods because it allows the detection both of known and remaining sequences comprised the redacted composite unknown microbes present in saline soils. dataset used in this study. Our knowledge of the diversity of bacteria and archaea in saline and hypersaline soils is, however, still limited and Phylogenetic analysis fragmented. In most cases only a few soil samples of a single specific saline system, or a small number of cloned Sequences were sorted by length and aligned with Kalign sequences or DGGE bands, have been investigated and (Lassmann and Sonnhammer 2005). The sequences were analyzed. Some studies have focused only one particular classified into taxonomic ranks using the RDP Classifier microbial group such as Archaea (Valenzuela-Encinas with default settings (Cole et al. 2005). Treemaps based on et al. 2009; Youssef et al. 2012) or Actinobacteria (Singh the output classifications from the RDP Classier were et al. 2012). Numerous sequences recovered from saline constructed using the treemap packages in R. Based on the soils are available from the GenBank, but many of these classifications, the dataset was divided into the following are largely overlooked because they are not published in groups: Archaea, Bacteria, Proteobacteria, Actinobacteria, the literature. Recently developed high-throughput Firmicutes, Acidobacteria, Bacteroidetes, Chloroflexi, and sequencing technologies produces large datasets of the collected ‘‘minor phyla’’ of bacteria that comprised sequence reads. However, unlike the longer sequences sequences not assigned to any of the aforementioned phyla. generated with Sanger sequencing, the length of these reads Distances matrices of aligned sequences were computed is too short to classify the sequences to genus level (Nelson within ARB using Jukes-Cantor correction (Cohen and et al. 2011). Currently, there is no consensus on the size or Townsend 2009). Individual distance matrices were ana- nature of the microbial diversity generally found in saline lyzed using Mothur (Schloss et al. 2009) to cluster OTUs, soils. generate rarefaction curves, and determine the nonpara- In this study, we performed a meta-analysis of all pub- metric ACE and Chao1 richness estimates. The genetic licly available 16S rRNA gene sequences identified from distance B0.03 was used to define species-level OTUs. The saline and hypersaline soils in order to provide an updated distance cut-offs were set at 0.05, 0.10, 0.15, and 0.2, overview of the bacterial and archaeal diversity in saline which were assumed for taxonomic ranks genus, family, soil environments. Estimates of the current coverage of the class/order and phylum, respectively. All the estimated halophilic bacterial and archaeal diversity already identi- asymptotes of the rarefaction curves were determined fied in saline soils were made, and particular gaps in the through R package monomol (https://github.com/binma/ knowledge and understanding of the microbial populations monomol). The coverage percentages were calculated as present in saline soil habitats were identified. described by Nelson et al. (2011). 123 World J Microbiol Biotechnol (2013) 29:2325–2334 2327 Fig. 1 Treemap of observed bacterial and archaeal taxa shown in their hierarchical order. Treemap showing taxonomic ranking of all taxa for all retrieved sequences. The Firmicutes Acidobacteria size of each box is proportional to the number of sequences assigned to that taxon with respect to the entire dataset. The Proteobacteria placement of boxes is arbitrary Euryarchaeota with respect to boxes within the Bacteria Archaea same taxonomic rank and does not correspond to any form of Bacteroidetes Chloroflexi