Bacterial diversity in south coast of the Caspian Sea: Culture-dependent and culture-independent survey

[Research]

Bacterial diversity in south coast of the Caspian Sea: Culture-dependent and culture-independent survey

Makhdoumi A.

Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran

E-mail: a.makhdomi@um.ac.ir                                                              (Received: May 08. 2018 Accepted: Aug. 16. 2018)

Bacterial diversity in the south coast of the Caspian Sea was studied by analyzing 16S rDNA clone library and cultivation technique. Analysis of inserts of 30 clones revealed a total of 13 OTUs. All of these sequences were related to Proteobacteria including Betaproteobacteria (60%), Gammaproteobacteria (22%) and Alphaproteobacteria (18%). The majority of these sequences (40%) branched with member of Limnobacter. Within the cultivation effort, phylotypes related to Gammaproteobacteria (60%), Firmicutes (27%), Actinobacteria (9%) and Bacteroidetes (4%) were retrieved. Members of the Bacillus (14%) and Rheinheimera (18%) were the most common isolates. The secretions of eight hydrolytic enzymes and antibiotic compounds, as well as resistance to heavy metals were studied in these marine strains. Among them 46, 45, 38, 27, 19, 19, 7 and 4% of bacterial isolates were able to produce protease, pectinase, xylanase, amylase, cellulase, lipase, urease and DNase, respectively. Two strains which are phylogenetically related to Streptomyces and Stenotrophomonas produced antimicrobial compounds and could inhibit the growth of Candida albicans and Bacillus subtilis,respectively. A total of 2, 1, 2, 3 and 2 strains could survive in the presence of lead (1500 ppm), cadmium (1000 ppm), zinc (2000 ppm), copper (2000 ppm), and Chromium (2000 ppm), respectively. The investigation showed that this marine environment harbors a high bacterial diversity which is a potential source of hydrolytic enzymes and other valuable biotechnological activities.

INTRODUCTION

Marine environments, the largest ecosystem on earth, harbor high microbial species (high evenness). This is because of the conditions in marine systems, which are neither selective nor inhibitory to specific groups of microorganisms (in contrast to the environments like hypersaline lakes harbor high species richness). The majority of metabolites isolated from marine microorganisms, as well as decreasing rate of discovery of novel compounds from soilled to interest in studying the marine microbial population (Lane & Moore 2011). There are various published reports about the biotechnological applications of marine microorganisms for novel bioactive compounds (Bhatnagar & Kim 2010; Imhoff et al. 2011) or biocatalysts (Zhang & Kim 2010, Trincone 2011). Ecological studies also emphasized on the potential applications of marine microorganisms in metal detoxification, nutrient cycling and greenhouse gas reduction (Zaky et al. 2014; Salazar & Sunagawa 2017). Hence, awareness of microbial variation in aqueous environments will provide great source for biotechnological application.

The Caspian Sea is the largest inland body of water in the planet with a surface area of about 373,000 km² (Dumont 1998). It is bordered by five countries including Azerbaijan, Iran, Kazakhstan, Russia and Turkmenistan. The water surface lies 28 m below sea level. It reaches its maximum depth of 1025 m in the south while the northern half averages only about 5 m depth. About 130 rivers of varying size drain freshwater into the sea thereby decreasing its salinity to the maximum amount of 1.3 (% w/v) (one-third of the salinity of seawater) (Leroy et al. 2007). This landlocked system suffers from anthropogenic pollutants discharged from municipals and industrial wastes (Zonn 2005) which can influence the microbial population in coastal regions. Recently-published reports considered its sediments and brackish water microbial diversity by next generation sequencing (Mahmoudi et al. 2015, Mehrshad et al. 2016). However, there is no report about bacterial diversity in the coastal region of the sea. The following research focuses on bacterial diversity in the south coast of the Caspian Sea (Iranian part) and aims toa)describe its bacterial community using both cultivation and culture-independent approaches, b) survey on some biotechnological activities including: hydrolytic enzymes production, antimicrobial activity and resistance to heavy metals in the indigenous bacterial strains.

MATERIALS AND METHODS

Site description, samples collection and analysis

The Caspian Sea water was sampled from 0.5 and 30 m depth, and about 200 m away from the sea shore of four cities including Behshahr (36°41′40ʹʹ N, 53°32ʹ33ʹ′ E), Babolsar (36°42′43′′ N, 52°38′44′′ E), Nowshahr (36°66′04′′ N, 51°48′12′′ E) and Tonekabon (36°48′31′′ N, 51°52′55′′ E) in Mazandaran Province, north of Iran in October 2013. The salinity and pH of the samples was determined in situ with SevenMulti dual meter pH/conductivity (Mettler). The anions and cations present in the samples were analyzed using titration and atomic absorption methods, respectively (Saad et al. 1998). Samples were pooled for culture dependent and culture-independent analyzes.

Culture media and growth conditions

Marine bacteria were isolated under aerobic conditions on different growth media: The marine agar (Merck); SW medium contained (g L^-1): NaCl 9.1, MgSO₄.7H₂O 0.75, MgCl₂.6H₂O 0.17, KCl 0.13, CaCl₂.2H₂O 0.22, (NH₄)₂SO₄ 0.86, FeSO₄.7H₂O 0.009, peptone 10.0, yeast extract 2.0, and agar 15.0; pH 8.2; sea water salt medium consisted of filtered Caspian Sea water supplemented by (g L^-1): peptone 1.0, yeast extract 0.2 and agar 15.0; pH 8.2. All samples were spread plated to culture media. The plates were incubated aerobically at 20 °C for 8 weeks. After successive cultivation, pure isolates were obtained.

DNA extraction, amplification of 16S rRNA genes and gene library construction

Genomic DNA was extracted using a Genomic-DNA extraction kit (Thermo Scientific, Lithuania), according to the manufacturer’s instructions.

A modified method of Benlloch et al. (1996) including freezing and thawing step was applied for environmental DNA extraction. The 16S rRNA genes were amplified using Bacteria-specific froeward, 5’-AGAGTTTGATCATGGCTCAG-3’, and reverse, 5’- GGTTACCTTGTTACGACTT-3’, primers (Lane et al. 1985). Following  PCR conditions were employed: 94 °C for 2 min, followed by 30 cycles of 94 °C for 60 s, 55 °C for 60 s and 72 °C for 60 s, with final 7 min extension at 72 °C. In amplifications involving environmental DNA, mentioned PCR condition varied with different annealing temperatures (48-60 °C). The PCR products of expected size (1500 bp) were gel purified (DNA extraction kit, Roche, Germany), pooled and ligated into pGEM-T cloning vector (Promega, USA) and employed to transform E.coli DH5α cells according to the manufacturer’s instructions.

Sequencing and sequence analysis

The sequencing was conducted on ABI 3730XL DNA sequencer at Macrogen (Seoul, South Korea). The 16S rRNA genes of isolates were sequenced directly. The nucleotide sequences of the cloned products were determined from plasmid preparations (Miniprep plasmid extraction kit, Bioneer, South Korea). Putative chimeric sequences were recognized using Bellerophon server [8]. Relevant sequences were extracted from GenBank (www.ncbi.nlm.nih.org) using BLASTN and through EzTaxon server (Chun et al. 2007). The sequences were considered to belong to an operational taxonomic unit (OTU) if they shared ≥97 % sequence identity. The alignments were generated using MUSCLE web server (http://www.ebi.ac.uk/T- ools/msa/muscle/). Phylogenetic analysis was performed using the software package MEGA version 6 (Tamura et al. 2013).

Screening for extracellular hydrolytic activities

Qualitatively detection of extracellular amylase, cellulase, DNase, lipase, pectinase, protease, urease and xylanase production was performed in solid suitable SW medium. After 5 days incubation at 20 °C standard methods were applied for screening approach as described by Rohban et al. (2009) and Ramesh et al. (2014)

Screening for heavy metals (Cd, Cr, Cu, Pb and Zn) resistance

MIC (Minimum inhibitory concentration) determination was conducted in SW medium following the method described by Khammar et al. (2015).

Screening for antimicrobial activity

For preliminarily antimicrobial activity study, marine strains were streaked onto TSA (Merck) and incubated at 20 °C for 5 days. Test strains including Staphy­lococcus aureus (PTCC1431), Bacillus subtilis (PTCC1023), Pseudomonas aeruginosa (PTCC1074), Escherichia coli (PTCC 1330) and Candida albicans (PTCC 5027)were streaked perpendicular to the marine strains and incubated overnight. Inhibitory activity was indicated by inhibited growth of pathogenic strains as compared to the negative control. Selected marine strains (based on the zone of inhibition) were inoculated into 100 mL of SW broth medium and incubated at 20 °C in a rotary shaker at 150 rpm for 5 days. The broth culture was extracted using equal volume of ethyl acetate and then solvent was removed by evaporation.

The antibacterial effects of crude extract were compared with tetracycline (30 μg disc^-1) and cycloheximide (100 μg disc^-1) as positive control against bacterial and fungal test strains, respectively.

RESULTS

Diversity of bacterial isolates and sequences of environmental 16S rRNA genes

The physicochemical properties of water samples collected are presented in Table 1. We identified Na⁺ and Cl^- as major ions in the samples followed in abundance by SO₄^2- and Mg²⁺. The temperature of samples was about 19 °C.

Table 1. Chemical characterization of the Caspian Sea water.

After eight weeks of incubation, viable counts obtained on different media were comparable and ranged from 2.5 - 4 × 10⁶ CFU mL^-1. A total of 124 isolates were obtained and subsets of 30 different strains were analyzed. Bacterial isolates clustered into 17 OTUs (Table 2) and belonged to Bacteroidetes, Firmicutes, Actinobacteria and Gammaproteobacteriaphyla (Fig. 1). They were phylogenetically related to the following genera: Aeromonas (3.5 % of bacterial isolates obtained), Bacillus (14%), Cyclobacterium (3.5%), Halomonas (3.5%), Jeotgalicoccus (3.5%), Kocuria (3.5%), Marinobacter (7%), Micrococcus (3.5%), Paenibacillus (7%), Pseudoalteromonas (7%), Pseudomonas (7%), Psychrobacter (3.5%), Rheinheimera (18%), Sporosarcina (3.5%), Staphylococcus (3.5%), Stenotrophomonas (3.5%) and Streptomyces (3.5%). A total of 30 bacterial clones were selected randomly and sequenced. Chimeric sequences (3 sequences) were removed, while others were assigned to OTUs sequences and used for phylogenetic analysis (Table 2). Environmental sequences of bacteria, which formed 13 OTUs, belonged to Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria (Fig. 1). None of the detected groups by cultivation were identified in culture-independent approach. The majority of sequences (40%) branched with member of Limnobacter. Other phylotypes were related to Citreimonas (4.5% of sequences analyzed), Acinetobacter (4.5%), Acidovorax (4.5%), Spongiibacter (4.5%), Perlucidibaca (4.5%), Seohaeicola (4.5%), Youngimonas (4.5%) andSulfitobacter (4.5%). A remarkable part of recovered sequences (20%) were unrelated to any previously reported sequences.

Table 2. Comparison of isolates and clones 16S rRNA sequences obtained from the Caspian Sea with those available in EzTaxon (Chun et al. 2007).

Enzymes production by marine isolates

Survey on enzymes production was tested qualitatively among strains isolated from the Caspian Sea (Fig. 2). A total of 12, 12, 10, 7, 5, 5, 2 and 1 bacterial isolates were able to produce protease, pectinase, xylanase, amylase, cellulose, lipase, urease and DNase, respectively.

Fig. 1. Neighbor-joining phylogenetic reconstruction of 16S rRNA of bacterial sequences recovered from the Caspian Sea. Gray: isolate sequences; bold black: environmental sequences. The sequence of the halophilic archaeon Halopenitus malekzadehii CC65^T (KC0180183) was used as an outgroup. Bootstrap values (%) are based on 1000 replicates. Only values higher than 70% are shown. Bar, equals 0.02 substitutions per nucleotide position.

Combined hydrolytic activity was also detected in many marine strains. Two strains (belonged to Pseudomonas and Streptomyces genera) presented 6 hydrolytic activities. Protease, pectinase and xylanase were the most common

hydrolytic enzymes among strains by 46, 38 and 33% abundance, while amylase (25%), cellulase (20%) and lipase (20%), had an intermediate diversity. The rare rate of urease (6%) and DNase (3%) were observed.

Screening of heavy metal tolerant in marine bacteria

Marine isolates were screened to identify most potential metal resistant bacteria. All of the bacterial strains could survive at: cadmium (Cd) (100 ppm), chromium (Cr) (100 ppm), copper (Cu) (100 ppm), lead (Pb) (100 ppm) and zinc (Zn) (250 ppm). Strain ZP37 (Pseudomonas) could survive in 1000 ppm cadmium. Strains ZC15 and KP10 which is phylogenetically related to Jeotgalicoccus and Psychrobacter could tolerate up to 2000 ppm chromium. High level of copper resistance (2000 ppm) were observed in strains MN24 (Micrococcus), KP10 (Psychrobacter) and FZC6 (Bacillus). Strains Mn23 (Kocuria) and KC19 (Sporosarcina) could tolerate 2000 ppm zinc. Finally two strains including KP10 (Psychrobacter) and ZP35 (Stenotrophomonas) could grow in the presence of 1500 ppm of lead (Fig. 3).

Fig. 2. Phylotype diversity for enzyme-producing bacteria in the Caspian Sea. The bar chart compares 16S rRNA sequence diversities of strains produced various hydrolytic enzymes. Abbreviations; Bac: Bacillus; Hal: Halomonas; Koc: Kocuria; Mar: Marinobacter; Mic: Micrococcus; Pae: Paenibacillus; Pse: Pseudomonas; Rhe: Rheinheimera; Ste: Stenotrophomonas; Str: Streptomyces.

Fig. 3. Phylotype diversity for heavy metal resistance bacteria in the Caspian Sea. The bar chart compares 16S rRNA sequence diversities of strains resistant to various amount of metals. Abbreviations; Bac: Bacillus; Hal: Halomonas; Koc: Kocuria; Mar: Marinobacter; Mic: Micrococcus; Pae: Paenibacillus; Pse: Pseudomonas; Rhe: Rheinheimera; Ste: Stenotrophomonas; Str: Streptomyces.

Antimicrobial activity of marine isolates

Only two strains, ADR10 and ZP35 which are phylogenetically related to Streptomyces and Stenotrophomonas, represented antimicrobial activity against test strains according to the cross-streak method. The growth inhibition was also observed by well diffusion assay in the cultures of C. albicans and B. subtilis by strains ADR10 and ZP3, respectively. The high

antifungal activity was observed by a crude extract of strain ADR10 culture (22 mm inhibition zone compared to 11 mm zone of cycloheximide [100 µg mL^-1]) (Fig. 4). However ZP35 crude extract represented low antibacterial activity (8mm inhibition zone compared to 20 mm zone by tetracycline [30 µg mL^-1]).

Fig. 4. Antifungal effect of strain ADR10. Culture extract (right), cycloheximide (left).