Impact of Petroleum Hydrocarbon Contamination on the Indigenous Soil Microbial Community

Ann Microbiol (2015) 65:359–369 DOI 10.1007/s13213-014-0868-1

ORIGINAL ARTICLE

Impact of petroleum hydrocarbon contamination on the indigenous soil microbial community

Simrita Cheema & Meeta Lavania & Banwari Lal

Received: 12 July 2013 /Accepted: 9 March 2014 /Published online: 2 May 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014

Abstract Changes in the microbial community structure in importance, especially since the site had high alkaline and agricultural soils and soils contaminated with hydrocarbons saline characteristics. Soils (arid, alpine and polar) which are were evaluated using the culture-independent method of 16S nutrient and moisture limited are typically often dominated by rRNA gene sequence analysis. The bacterial composition was Actinobacteria that are well adapted to low-resource environ- more diverse in the agricultural soil (AS) samples in terms of ments and do not show major changes in community structure number of species and Shannon diversity index [6.6 vs. 1.94 as a result of hydrocarbon contamination. for the hydrocarbon-contaminated soil (HCS)]. Twelve known bacterial groups were identified in the AS: Keywords Diversity . High alkalinity . Proteobacteria (41 % of bacterial community), Ectothiorhodospiraceae . Polyhydroxyalkanoates . Ectoines Actinobacteria (34 %), Acidobacteria (5 %), Firmicutes (4 %), Chloroflexi (4 %), Bacteroidetes (3 %), Gemmatimonadetes, Planctomycetes, Verrucomicrobia, Introduction Armatimonadetes, Cyanobacteria,TM7andArchaea (the latter 7 each accounting for 1–2%) . In comparison, the clonal Microbes are an essential part of all life forms on Earth. They library from the HCS samples included members from only can live in extreme environments and possess adaptive capa- five groups: Proteobacteria (85 %) and Bacteriodetes, bilities that enable them to adjust to changes in living condi- Actinobacteria, Chloroflexi and Verrucomicrobia (the latter tions. Many microorganisms have been used in industrial- four collectively accounting for 15 %). The family scale processes for the production of antibiotics, preparation Ectothiorhodospiraceae was the most dominant family within of food and beverages, leather industry, production of the Proteobacteria isolated from the HCS. These microbes are biofuels, such as ethanol, among others in which they carry known to synthesize a number of biotechnologically useful out various chemical biotranformations. Microbial communi- products, such as polyhydroxyalkanoates and ectoines, and ties have for billions of generations contributed towards their dominance in the sampled area suggests the possibility of transforming the world around them. Their applications are discovering better adapted novel genes of commercial too widespread to describe in detail, and they represent by far the richest repertoire of molecular and chemical diversity in nature (Kapur and Jain 2004). S. Cheema Centre for Bioresources and Biotechnology, TERI University, Plot The diversity of microorganisms has been explored across No.10, Institutional Area, Vasant Kunj, New Delhi 110070, India a wide range of environments, such as water, soils, the human : : body (Achtman and Wagner 2008), hydrocarbon- S. Cheema M. Lavania B. Lal contaminated soils (Liang et al. 2011), the Arctic and deep Microbial Biotechnology Division, TERI, India Habitat Centre, Lodhi Road, New Delhi 110003, India sea vents (Stoeck et al. 2007). Among the various habitats, soil is considered to house the maximum number of microbes, and B. Lal (*) it has been estimated that 1 g of sediment may contain 1010 Environmental and Industrial Biotechnology, TERI University, bacteria, which is the highest for any environment (Torsvik Darbari Seth Block, India Habitat Center, Lodhi Road, New Delhi 110003, India et al. 1996). However, the cultivation efficiency of soil mi- e-mail: [email protected] crobes is only 0.3 % (Torsvik et al. 1996) due to limitations in 360 Ann Microbiol (2015) 65:359–369 traditional cultivation methods. This implies that most soil inputs of composted cow manure; there has also been no bacteria are difficult to isolate on laboratory media due to tillage and no removal of crop residues during this same time unknown culture conditions, insufficient growth nutrients period. An oil spill occurred in April 2008 which resulted in and the lack of cells in cultivable state. This drawback has one of the agricultural fields being contaminated with hydro- been largely overcome by culture-independent approaches carbons. In our study, soil collected from this site was consid- (Handelsman 2004) which are currently being exploited at a ered as the HCS. Within the same area, we also collected soil very fast pace and have allowed researchers and scientists to samples from uncontaminated AS. gain a better understanding of the number of microbial In both the cases (HCS and AS), about 10-g samples of the communities. surface soil were collected at depths ranging from 0 to 10 after Soil microbial communities residing at sites contaminated the top 3 cm of the soil surface was removed, in the rainy with hydrocarbons are one of the most complex and diverse season of July 2008. Ten samples collected from each site assemblages. Their roles and activities in these soils are the were randomly mixed, and subsamples were used for micro- focus of much interest (Liang et al. 2011). It is expected that bial community analysis. Soil samples were kept in polyeth- such information will result in the identification of many ylene whirl bags on ice and transported to the laboratory. The microorganisms which may represent a potential resource samples were stored at −80 °C until further analysis. All for various biotechnological products, bioenergy production efforts were made to avoid surface contamination. and novel biotransformations (Kalia et al. 2003). In addition, such studies will help to determine bacterial communities with Physiochemical analyses of the soil sample a high tolerance for hydrocarbon contamination. Their physiological response and stronger biodegradation efficiency can Physiochemical analysis of the soil samples was performed as ultimately be exploited for developing economic bioremedia- described by Marinari et al. (2006). Total organic carbon tion technologies. (TOC) was determined by the dichromate oxidation method In the study reported here, we investigated a site contam- (Nelson and Sommers 1982). Soil total lead (Pb), copper, inated with hydrocarbons using a culture-independent ap- cobalt (Co), arsenic, cadmium, selenium and chromium (Cr) proach in order to gain insight into the microbial community were analysed by digesting 0.1 g soil with HCLO4–HNO3– composition. Microbial diversity from a neighbouring uncon- HF (Nautiyal et al. 2010). The digested solution was washed taminated agricultural soil (AS) was also analysed to better into a flask, and deionized water was added to a fixed volume. understand the effects of environmental conditions and soil The digested solutions were analysed with atomic absorption characteristics on microbial diversity. Our results suggest that spectroscopy (model-AA 300; Perkin Elmer, Waltham, MA) the phylogenetic diversity and distribution in the for heavy metals. Soil pH and electric conductivity were hydrocarbon-contaminated soil (HCS) was quite distinct and measured in 1:10 w/v aqueous solution using a pH meter different from that in the unpolluted AS. They also indicates (Thermo Fisher Scientific, Waltham, MA). that microorganisms differ with habitats, are less diverse in contaminated sites and are dominated by populations that are Extraction of total petroleum hydrocarbon from contaminated well adapted to survive under these conditions. soil

Total petroleum hydrocarbon (TPH) was extracted in an equal Materials and methods ratio of hexane and dichloromethane (1:1, each 100 ml) from 10 g of HCS and AS using the Soxhlet method (Soxtherm; Sampling site Gerardt GmbH, Königswinter, Germany). Extracts were dried at room temperature by evaporating the solvents under a The heavy oil belt of Gujarat, India is 30 km long, with a gentle nitrogen stream in the fume hood. After evaporation surface area of about 45 km2. The majority shareholder is the the amount of residual TPH recovered was determined gravi- Oil & Natural Gas Corporation Ltd., and several exploratory metrically (Mishra et al. 2001). oil wells with an extensive pipeline network are located at various sites in this oil belt. The village of Kalol, located Analysis of TPH fractions by gas chromatography 27 km east of Ahmadabad city (73°15′N/ 72°28′60E) also falls within this belt. It is characterized by a mean annual Following gravimetric quantification, the residual TPH was rainfall of 742 mm and maximum and minimum temperatures fractionated into alkane, aromatic, asphaltene and nitrogen– ranging between 45 °C and 4 °C, respectively. The farmers sulphur–oxygen fractions in a silica gel column (Mishra et al. grow mainly Pennisetum glaucum (L), Ricinus communis and 2001). The TPH sample (200 mg) was dissolved in a mixture Brassica juncea in an inter-cropping cycle. For the past 14 of hexane, loaded onto a silica gel column and eluted with years, the crops have been under organic management with solvents of different polarity. The alkane and aromatic Ann Microbiol (2015) 65:359–369 361 fractions were eluted with 100 ml of hexane and dichloro- Sequencing methane, respectively. Aliphatic and aromatic fractions were analysed by gas chromatography (GC-FID; 6890 series N; The template used for the sequencing of inserts was processed Agilent Technologies, Santa Clara, CA) using a flame ioniza- by heat lysis of the overnight-grown clones at 96 °C for tion detector (FID) fitted with a DB5 column (length 30 m, 10 min in a thermocycler (Eppendorf, Hamburg). About 96 inner diameter 0.25 mm- film thickness 0.25 μm). The oper- clones were randomly picked, and the 16S rDNA insert was ating conditions of the GC system were: an oven temperature amplified with the primer set M13F (5′ GTA AAA CGA CGG programmed to increase from 55 °C for 1 min to 290 °C at CCA G-3′) and M13R (5′-AGG AAA CAG CTATGA C-3′).

5 °C/min over a period of 20 min; total run time of 68 min; The PCR mix consisted of 1× PCR buffer, 2.5 mM MgCl2, injector port temperature of 250 °C; detector temperature of 0.1 μM of each primer (M13F/M13R), 0.2 mM dNTPs 300 °C. Helium was used as the carrier gas at a flow rate of (Fermentas) and 1.25 U Taq Hot Start (Promega). The PCR 2 ml/min with a split ratio of 1/50. Individual compounds cycling conditions consisted of an initial denaturation step at present in the alkane and aromatic fractions were determined 94 °C for 3 min followed by 30 cycles of denaturation at 94 °C by comparing retention times with those of authentic stan- for 30 s, annealing at 60 °C for 30 s and elongation at 72 °C for dards (Sigma Chemicals, St. Louis, MO) as described earlier 90 s, followed by a final extension step of 3 min at 72 °C. The (Lal and Khanna 1996). 96 PCR reactions were purified using the Qiagen MinElute 96 UF PCR Purification kit (Qiagen, Venlo, the Netherlands). The amplified insert after purification was cycle sequenced Total community DNA extraction using the BigDye® Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA). The forward and Total microbial community DNA was extracted from soil reverse sequencing reactions were done in separate reactions samples, following the removal of stones and plant roots, using the M13F and M13R primers, respectively. A 10-μL using the PowerMax® Soil DNA Isolation kit (Mo Bio reaction mixture was prepared consisting of 1 μL purified Laboratories, Solana Beach, CA) according to the manufac- PCR product, 2 μL BigDye Ready Reaction Mix, 1 μLof turer’s protocol. The DNAwas quantified using the NanoDrop BigDye sequencing buffer and 1 μL of M13F / M13R Lite Spectrophotometer (Thermo Fisher Scientific). (25 μM) primers. De-ionized water (5 μL) was added to a final volume of 10 μL. The cycling conditions for the ampli- 16S rRNA gene library construction fication reaction included an initial denaturation step at 96 °C for 1 min followed by 25 cycles of denaturation at 96 °C for The community DNA extracted from the HCS was amplified 20 s, annealing at 56 °C for 20 s and elongation at 60 °C for for the 16S rRNA gene using the universal bacterial primers 4 min with a final extension step at 60 °C for 1 min. The 27 F (5′-AGA GTT TGATCC TGG CTC AG-3′) and 1492R sequencing PCR reactions were purified using the Qiagen- [5′-ACG G(CT)T ACC TTG TTA CGA CTT-3′). A PCR DyeEx 96 kit. The elutes were dried using the DNA reaction mixture (50 μL) was prepared using the extracted SpeedVac-DNA 110 vacuum system for 30 min at medium community DNA (50 ng/μL) and PCR reagents [1× PCR speed (Thermo Fisher, Waltham, MA). The sequenced prod- buffer, 2.5 mM MgCl2, 0.5 μM of each primer, 0.2 mM uct was re-suspended in 20 μL Hi-Di Formamide (PE Applied dNTPs (Fermentas, Vilnius, Lithuania), 1.25 U Taq Hot Biosystems, Foster City, CA) and transferred to sequencing Start (Promega, Madison, WI)]. The PCR cycling conditions vials for sequence analysis (ABI prism 310 genetic analyser; consisted of an initial denaturation step at 94 °C for 2 min PE Applied Biosystems). The sequenced DNA was resolved followed by 30 cycles of denaturation at 94 °C for 30 s, on ABI PRISMTM 310 genetic analyser (PE Applied annealing at 56 °C for 30 s and elongation at 72 °C for Biosystems). The DNA samples were sequenced with the long 1 min , with a final extension of 2 min at 72 °C. The amplified capillaries (5–61 cm×50 μm). Electrophoresis was performed 16S rDNA product (about 1,500 bp) was confirmed by load- in 1× electrophoresis buffer with EDTA and performance- ing about 5 μL of the PCR product onto a 1 % agarose gel. optimized polymer (POP6). The various parameters that were The PCR product was purified with using Microcon 100 PCR set for the electrophoresis in the genetic analyser were tem- centrifugal filter device (Millipore, Billerica, MA) and was perature (50 °C), current (4 μA), voltage (12 kV) and argon ligated into pGEM-T Easy vector (Promega) as per the man- ion laser power (9.8 mW). ufacturer’s protocol. The ligated 16S rRNA was transformed into Escherichia coli JM109 high-efficiency competent cells Phylogenetic analysis which were plated on LB plates supplemented with ampicillin (100 μg/mL), IPTG (100 mM) and X-Gal (34 μg/mL) and The 16S rRNA sequences were checked for purity with the incubated overnight. About 150 clones were picked, arrayed Check–Chimera program (http://rdp.cme.msu.edu/). into 96-well plates and stored at −80 °C as glycerol stocks. Sequences in certain cases were manually edited by using 362 Ann Microbiol (2015) 65:359–369 the BioEdit software version 7.0.5.3. Sequences were determined. In the AS, all metals were below the detection subjected to a BLAST search with the NCBI database and limits; in the HCS, Pb (1,477 mg/kg), Co (997 mg/kg) and Cr Classifier tool of the Ribosomal database project II. Multiple (3,869 mg/kg) were present at significant levels (Table 1). The sequence alignments were performed using CLUSTAL W, AS and HCS also differed TOC (Table 1), with the AS having ver. 1.8 (Thompson et al. 1994). A phylogenetic tree was a higher TOC (4.32 %) than the HCS (0.8 %). This difference constructed with the evolutionary distances using the was likely due to no organic manure being applied to the HCS neighbour-joining method (Saitou and Nei 1987). Tree topol- since the oil spill in 2008 which made the field unfeasible for ogies were evaluated by performing bootstrap analysis of agricultural purposes. The AS showed negligible (residual 1,000 data sets with the PHYLIP version 3.61 packages 60 mg) TPH per kilogram soil and was not characterized by (Felsenstein 1989). The tree was generated with the any hydrocarbon signatures indicative of an uncontaminated TREEVIEW program (Page 1996). healthy soil. In comparison, the crude oil concentration of the HCS was approximately 5,100 mg TPH/kg soil. The HCS Nucleotide sequence accession numbers included hydrocarbons belonging to all saturated alkanes (C14–C36) and aromatics (anthracene, fluoranthene, pyrene, The 16S rRNA sequences of representative isolates from this benzo[a] anthracene, chrysene, benzo[b] fluoranthene, study were submitted to the NCBI Genbank Database with benzo[k] fluoranthene, dibenzo[a,h] anthraene), as shown in accession numbers JN217142 through to JN217218 (HCS) Fig. 1. and KC820818 through to KC820893 (AS). Diversity analysis Statistical analysis Shannon’s diversity index varied remarkably across the two Sample diversity richness based on 16S rRNA sequences was environments, being 6.6 for the AS, which indicates an ex- calculated using the Shannon–Weaver index with the help of tremely diverse and dynamic community structure (Köberl Mothur (Schloss et al. 2009), which is a comprehensive et al. 2011; Steven et al. 2013), and 1.94 for the HCS. High software package for analysing community sequence data. concentrations of TPH have been shown to have contrasting The rarefaction curve was also calculated in Mothur by plot- effects on microbial diversity (Sutton et al. 2013). In our study ting the number of operational taxonomic units (OTUs) ob- there was clearly a decrease in diversity in the HCS, most served against the number of sequences sampled (Schloss likely caused by the high selection pressures imposed by the et al. 2009). high level of TPH contamination (5,100 mg/kg of soil). All groups of Proteobacteria respond positively to the influx of hydrocarbon contaminants, and members of the class Results and discussion Gammaproteobacteria are commonly predominant when sup- plemental nutrients (C:N:P) are provided. The concomitant In the last three decades metagenomics as a tool for investi- presence of heavy metals, such as Cr, Pb and Co, has also been gating microbial communities has gained momentum since it provides access to the large gene pool of unculturable bacteria Table 1 Physico-chemical properties of soil sampled from the site con- and thereby to microorganisms which are missed due to the taminated with hydrocarbons and an agricultural site at Kalol, Ahmedabad inherent limitations of conventional culturing methods (Handelsman 2004). To date, a number of bacterial commu- Properties Hydrocarbon- Agricultural soil nities have been examined using this approach and hydrocar- contaminated soil bon contaminated sites have also been studied (Liang et al. Texture Dark-brown, Copper-brown, 2011). In our study bacterial diversity was investigated in two granular, loamy granular, loamy different soil environments, a HCS and a neighbouring AS, by pH 8.5 7.8 determining the phylotype richness and the distribution and Total organic carbon 0.8 % 4.32 % similarity among the 16S rDNA clones investigated. Heavy metals (mg/kg) Pb 1477 ND (<5) Characteristics of the sampling sites Co 997 ND (<5) As ND (<10) ND (<10) Soils from the Kalol site (Gujarat) were loamy with a surface Cd ND (<5) ND (<5) temperature of 28 °C. The pH of the HCS was alkaline (pH Cr 3869 ND (<5) 8.5) with a high salt content (6.4 %), and that of the AS was Se ND (<5) ND (<5) also alkaline (pH 7.8) with a low salt content (1.3 %). The occurrence of heavy metal contamination was also ND, Not detected Ann Microbiol (2015) 65:359–369 363

Fig. 1 Gas chromatographs of hydrocarbon components present in contaminated soil. a Saturated alkanes (C14–C36). Number indicates chain length. b Aromatic hydrocarbons

observed to decrease microbial diversity due to their deleteri- 77 (HCS) and 76 (AS) 16S rDNA clones were successfully ous effects on susceptible microorganisms (Sheik et al. 2012). sequenced and ultimately deposited in the GenBank database The combined effect of heavy metal and hydrocarbon con- under accession numbers JN217142–JN217218 and tamination likely caused a reduction in microbial diversity in KC820818–KC82089, respectively. Sequences were subject- the HCS, which is also highlighted by the rarefaction curves ed to BLAST searches as well as to the Ribosomal Database (Fig. 2). A comparison between the rarefaction curves from Project II Classifier tool for determining the phylogenetic both sites shows that, at the phylum level, the number of affiliations of each of the clones. The microbial community OTUs observed decreased and reached a plateau at 20 % of the HCS site was characterized mainly by a single phy- distance for the HCS, suggesting that most of the diversity at lum—Proteobacteria (88 %). This phylum was also predom- the phylum level had been detected. In contrast, for the AS, inate in the AS sample, where 41 % of OTUs belonged to the number of phylotypes seemed to be increasing, indicating Proteobacteria (Fig. 3a). However, within this phylum differ- the possibility that many more unique phylotypes or species ent trends were observed for the two soils. The AS site could be found. included Delta Proteobacteria (3 %), which was not observed in the HCS. Almost all sequences (85 %) in the HCS were Bacterial community structure affiliated to Gamma Proteobacteria (Fig. 3b) whereas in the AS, Alpha Proteobacteria formed the dominant group (33 %). Among the two libraries, a total of 350 transformants were The prevalence of Gamma Proteobacteria at such contam- obtained of which 192 clones were analysed further. A total of inated sites (HCS) has also been observed in previous studies 364 Ann Microbiol (2015) 65:359–369

Fig. 2 Rarefaction curves of the bacterial 16S rRNA clonal library of the agricultural soil (AS; a)and hydrocarbon-contaminated soil (HCS; b) at the species (3 % difference, filled diamond), genus (5 % difference, open square), family/class (10 % difference, X) and phylum (20 % difference, shaded triangle) levels. OTUs Operational taxonomic units

(e.g. Stoffels et al. 1998). These bacteria have the capabilities of utilizing such contaminants for growth and therefore survive easily. Heavy metals are usually present at sites contaminated with hydrocarbons, and only those microbes which have the ability to tolerate both can survive. Specific adaptive mechanisms enable hydrocarbon-tolerant bacteria to survive and grow in the presence of these toxic hydrocarbons, and these generally involve either modification of the membrane and/or cell surface properties, changes in the overall energy status and activation and/or induction of active transport sys- tems for extruding hydrocarbons (Lăzăroaie 2009). However, heavy metal contamination has been observed to lower microbial biomass and activity, which in turn affects the decom- position of soil organic matter and its accumulation in soil (Shi et al. 2002) and explains why such sites typically show less diverse and more selected microbial communities. Our comparison of microbial diversity in our samples re- vealed that the family Ectothiorhodospiraceae made up the largest group of the HCS library. Ectothiorhodospiraceae are halophilic and haloalkaliphilic, purple sulfur, phototrophic bacteria belonging to Gamma Proteobacteria which prefer to Fig. 3 Distribution of cloned 16S rRNA sequences at the phylum level grow anaerobically under light conditions using reduced sul- from the AS (a) and the HCS (b) phur compounds as electron donors (Tourova et al. 2007). Ann Microbiol (2015) 65:359–369 365

They are usually found in soils with high alkalinity and salin- represented by just one clone each in our study. More species ity. Singh et al. (2010) earlier described the occurrence of these could have been found with greater sampling sizes, as indi- haloalkalophilic bacteria in saline habitats of Gujarat and the cated by the rarefaction curve. The optimum medium, inor- biotechnological applications of their enzymes (such as prote- ganic salt content, temperature and growth factors present in ases), which are active at alkaline pH and high salt concentra- the AS promoted the sustainability of such a rich microbial tions. Some of the members of this family are also known for community. The widespread and ubiquitous distribution of accumulating two useful products: polyhydroxyalkanoates phyla may indicate their important role in plant–soil (PHAs) and ectoines (Galinski and Herzog 1990;Zhang interactions. et al. 2004). Consequently, the HCS could represent a reservoir The AS soil was also characterized by additional phyloge- of a group of useful bacteria that can be exploited for the netic groups, although these were low in numbers—Archaea production of these valuable products. The survivability of (2 %), Gemmatimonadetes (1 %), Acidobacteria (5 %), these organisms in such an environment may be due to better Firmicutes (4 %), Planctomycetes (1 %), Armatimonadetes metabolic adaptabilities which have evolved over time. It is (1 %) and Cyanobacteria (1 %). Acidobacteria,althoughan particularly noteworthy that these genes are involved in the extremely prevalent bacteria in soil, was underrepresented at processing of PHAs and ectoines as it suggests the probability both sites. In case of the HCS, their absence can be attributed that these genes may possess properties that are unique and to the higher alkalinity of the soil (Jones et al. 2009). At least which have evolved for a better expression of enzymes than 15 taxonomic orders were present with a diversity index of their counterparts belonging to other environments. The genus 6.67. Reports of such a high diversity index from agricultural Methylonatrum formed the second largest subgroup within soils with high nutritional levels are not known. The combined Gamma Proteobacteria. These bacteria also thrive in data for the AS library represents a phylogenetically broad alkaliphic and hypersaline ecosystems, and its members have spectrum of microorganisms. Members of Alpha been previously detected from sediments of hypersaline chlo- Proteobacteria were the most dominant group, and members ride–sulfate lakes in Altai, Russia (Sorokin et al. 2007). These of this group are frequently found in soils where they are bacteria can degrade the greenhouse gas methane and other known to form symbiotic relationships with plants organic pollutants (Sorokin et al. 2007). (Herschkovitz et al. 2005). Crop rotation involving Papaver T HCS and AS shared some similarities in terms of the glaucum (L), Ricinus communis and Brassica juncea is prac- presence of Bacteroidetes (8 vs. 3 %, respectively), ticed at the AS site, and these plants form associations with Actinobacteria (1 vs. 34 %), Chloroflexi (1 vs. 4 %), nitrogen-fixing bacteria such as Azotobacter chroococcum. Verrucomicrobia (1 % each) and TM7 (1 vs. 2 %) but also Therefore, this type of farming system with the inclusion of showed significant differences. The major decline in the dom- these plants in the rotation amplifies the presence of the root inance of Actinobacteria in AS and the shift to Proteobacteria nodule bacteria, and with their degradation there is excess of dominance in HCS has also been observed in other studies nitrogen availability in soil which stimulates microbial (Sheik et al. 2012). This shift is likely due to Proteobacteria activity. possessing metal-tolerant genes and, therefore, they can easily survive in contaminated soils. High concentrations of metals Phylogenetic analysis harm cells by displacing the enzyme metal ions, competing with structurally related non-metals in cell reactions and The phylogenetic analysis of the 16S rRNA sequences from the blocking functional groups in the cell bio-molecules (Hetzer HCS site placed the members of the phyla Gamma et al. 2006). Microbial survival in soils polluted with heavy Proteobacteria together (Fig. 4a), although there were some metals depends on the intrinsic biochemical properties and sequences which were placed within the Gamma physiological and/or genetic adaptation mechanisms, includ- Proteobacteria but as a clear distinct group (Fig. 4a). These ing morphological, of the specific microbial species, as well as consisted of the sequences from phyla Alpha Proteobacteria, environmental modifications of metal speciation (Abou- Verrucomicrobia,TM7andBacteroidetes. Most of the sequences Shanab et al. 2007). The few Actinobacteria observed in the from Bacteroidetes, however, formed a separate group. One HCS may be either those species which carry such resistance OTU each belonging to Actinobacteria and Chloroflexi formed genes and have been earlier reported to be involved in metal distinct separate branches in the tree. The Gamma Proteobacteria cycling (Kothe et al. 2010) or their presence was possibly mainly represented by Ectothiorhodospiraceae showed a high affected by higher than normal soil moisture (Goodfellow and genetic diversity as clearly reflected by the low bootstrap Williams 1983). values (Fig. 4a). In addition to the Ectothiorhodospiraceae, Verrucomicrobia is a major phylogenetic group but is rep- the Gamma Proteobacteria included the subgroup resented by very few cultured isolates (Hedlund et al. 1997) Methylonatrum which were mostly closely associated with These bacteria account for 1–10 % of the bacterial 16S rRNA each other, although some clones were also associated within in soils (Buckley and Schmidt 2003) and therefore were also the Ectothiorhodospiraceae cluster. 366 Ann Microbiol (2015) 65:359–369 Ann Microbiol (2015) 65:359–369 367

Fig. 4 Phylogenetic tree based on 16S rRNA sequences of clones obtained from the HCS (a) and AS (b). Bootstrap values (based on 1,000 replication) are given on each node. Reference entries from public databases are given by accession numbers, and entries from this work are given as the clone number

The phylogenetic distribution of the cloned 16S rDNA sequences from the AS did not show a consistent branching order of the bacterial divisions which was representative of the HCS (Fig. 4b), although the topologies of two major divisions, Actinobacteria and Alpha Proteobacteria,werecoher- ent. However, even within these divisions few sequences were phylogenetically interspersed into other groups. The most notable ones were a group of sequences belonging to Actinobacteria (TERI-AS10, 13, 17, 19, 21, 20, 22, 24, 42, 57, 69) which clustered separately and were not associated with the representative sequences obtained from the BLAST results. The sequences are probably very unique and diverse and thus remained independent. The phylogeny of Acidobacteria (TERI-AS55, 63, 66, 79), Firmicutes (TERI- AS16, 82, 84), Gamma Proteobacteria (TERI-AS2, 23, 52, 80), Chloroflexi (TERI-AS44, 47, 56), Verrucomicrobia (TERI-AS59), Gemmatimonadetes (TERI-AS75), Planctomycetes (TERI-AS31) and TM7 (TERI-AS72) also did not agree, which reaffirms that sequences from this site are highly mobile and varied. Sequences from Delta Proteobacteria (TER-AS36, 45), Bacteroidetes (TERI-AS77, 78), Armatimonadetes (TERI-AS65), Cyanobacteria (TERI- AS68) and Archaea (TERI-AS73) were phylogenetically di- vided into distinct branches. Even though this study is based on 16S rRNA gene sequences and is limited in determining the functional profiles of only the isolated microbes, the results obtained can be useful for constructing specific DNA primers and probes to target bacterial groups of interest. Diversity profiles, especially of agricultural soils, can help in improving soil quality for better productivity and management. It would also enable the design and development of better bioremediation strategies which would be targeted specifically towards these inherent microorganisms and therefore would be more effective. The study of stressed sites can facilitate the identification of microbial genera which have developed genes with superior biocatalytic properties which possibly would be of higher biotechnological importance than existing ones. In the context of the HCS site it would be especially beneficial for identify- ing novel PHAs and/or ectoines with a better adaptability to extreme conditions of alkalinity and salinity. The possibility of detecting those microorganisms which might have applications in the field of pharmaceuticals, bioremediation or other industrial biosynthetic processes, and previously thought to be non-existent, not only speeds up the screening process but also provides information on whether a sampling site is worth targeting for further exploration or not. Fig. 4 (continued) 368 Ann Microbiol (2015) 65:359–369

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