PROKARYOTIC DIVERSTY OF ACID MINE DRAINAGE PONDS IN ORE ENRICHMENT PLANT

The biodiversity of acidophilic prokaryotes was determined in three AMD ponds (pH 2.7-6.5) in Turkey (Izmir-Halikoy antimony ore enrichment plant) using 16S rRNA cloning and denaturing gradient gel electrophoresis methods. Water samples were taken two times in March 2014 and June 2015. The microbial diversity identified includes species such as Acidiphilium angustum, Acidocella sp., Ferroplasma acidiphilum, Acidithiobacillus ferriphilus, Acidithiobacillus ferrivorans, Acidiphilium rubrum, Thiomonas sp., Acidiphilium multivorum, Acidiphilium cryptum, Ferrovum myxofaciens, Acidocella aluminiidurans with the used techniques. In addition to, it has been determined that biodiversity is variable in the operating mine pools. Aciditihobacillus ferriphilus, Acidiphilium angustum, and Acidiphilium rubrum are new records for Turkey.


INTRODUCTION
Acid mine drainage (AMD) is the largest environmental problem caused by normally associated with mining activities (Garcia-Moyano et al., 2015). The mining wastewater is defined by properties such as low pH, high metal ions (e.g., iron, nickel, copper) and mineral concentrations. Acidophilic microorganisms living in this habitat are very interesting because of their adaptability to extreme pH values, their metabolic diversity, and their ability to be used in biomining applications. Especially, due to their availability in biomining and bioremediation applications, it is important to identify the acidophiles living in AMD. As determined in previous studies, the AMD microbial community changes over time (McGinnes and Johnson, 1993;Edwards et al., 1999;Volant et al., 2014). The variety of microbial community is attached to seasonal changes and environmental conditions in AMD (Auld et al., 2017). Classical microbial ecology methods remain limited in determining microbial diversity. Therefore, culture-independent methods such as 16s rRNA gene cloning, fluorescent in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) are often used to investigate the diversity of microbial community that adapts to this unique environments (Gonzalez-Toril et al., 2003;Nicomrat et al., 2006;Garcia-Moyano et al., 2015). Acidophilic chemolithotrophs such as Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Leptospirillium ferrooxidans have been identified in AMD which extremely low pH and high concentrations of iron, sulfates and other heavy metals (Edwards et al., 1999;Kuang et al., 2012). At the same time, the investigation of microbial community by molecular methods is difficult because of the inhibition of PCR by metals such as Fe and Cu (Nicomrat et al., 2006). For the reason, DGGE and 16s rRNA gene cloning methods are used together to support each other in determining the microbial diversity of AMD. The aim of this research was to determine the acidophilic prokaryotic community of acid mine drainage in ore enrichment plant Halıköy, İzmir (Turkey). Our study area in Halıköy is within the Menderes Massif in western Turkey. The antimony mine was discovered in 1870 and continued to operate until 1918 in Halıköy area. After a long-standing period, production began again in 1974 (Akcay et al., 2006). Our results are the first knowledge about the prokaryotic community of the selected AMD area.

Site description and sample collection
The water samples were collected from the operating antimony mine, ore enrichment plant Halıköy site (38°5'28.09"N, 28°10'09.6"E) in İzmir, Turkey (Fig 1), in two different time (March 2014 andJune 2015). The samples were taken three different points from mine area as drainage water (sample #1 and sample #4), iron oxide pool water (sample #2 and sample #5), and stationary water before iron oxide pool (sample #3 and sample #6) (Fig 2). Water samples were taken in sterile Duran bottles and were filtered from 0.2 um GTTP filter. In situ measurements for pH were made using a WTW Multi350i/SET (WTW, Germany). Metal concentrations of water samples were measured by inductively coupled plasma atomic emission spectrometry (ICP-AES).

DNA isolation and 16S rRNA gene amplification
DNA isolations were performed following the procedure explained by Cifuentes et al. (2000) and Nogales et al. (1999) as modified. We used the primer set 27F (AGAGTTTGATCMTGGCTCAG)-1387R (GGGCGG(AT)GTGTACAAGGC) and 20F (AGAGTTTGATC(AC)TGGCTCAG)-915R (GTGCTCCCCCGCCAATTCCT) for bacteria and archaea, respectively. PCR cycles were as follows: one cycle at 95 °C for five minutes, 30 cycles at 95 °C for 30 seconds, one minute at the corresponding annealing temperature 55 °C and 62 °C (for bacteria and archaea, respectively) and 72 °C for 1.5 minutes, and a final extension step at 72 °C for ten minutes.

16S rRNA gene cloning
The PCR products were cloned using the pGEM-T easy vector system II and colony PCRs were set up with 27F and 1387R; 20F and 907R primer sets of selected colonies, for bacteria and archaea, respectively. Similar profiles were determined by amplified ribosomal DNA restriction analysis (ARDRA) in the 16S rRNA gene libraries. Clones were divided into categories (3 h, 37°C) based on pattern generated by the restriction enzymes MspI and HaeIII (5 units each).

DGGE
Amplification of the 16S rRNA gene was carried out with specific primers for DGGE analysis. Primer set including 344F-GC (CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCGACGGGGC GCAGCAGGCGCGA) and 907R (CCGTCAATTCCTTTGAGTTT) was used for the archaeal gene amplification while for the bacterial gene, the forward primer, 341F-GC (CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCGCCTACGG GAGGCAGCAG) in combination with 907R were used (Muyzer et al., 1993). DGGE PCR condition for bacteria: one cycle at 94 °C for five minutes, one minute at 65 °C and three minutes at 72 °C and nine cycles at 94 °C for one minute, one minute at the annealing temperature was decrease 64-55 °C and 72 °C for three minutes, and one cycle at 94 °C for five minutes, one minute at 55 °C and three minutes at 72 °C and a final step 94 °C for five minutes, one minute at 55 °C and ten minutes at 72 °C. DGGE PCR condition for archaea: one cycle at 94 °C for five minutes, 29 cycles at 94 °C for three seconds, 56 °C for 45 seconds, 72 °C for two minutes, and one cycle at 94 °C for 30 seconds, 56 °C for 45 seconds, 72 °C for seven minutes. The PCR products were purified with the Wizard SV Gel and PCR Clean-Up System (Promega, Italy). Denaturing gradient gel electrophoresis (DGGE) was performed with the DCode Universal Mutation Detection System (Bio-Rad Laboratories, Inc.). PCR product was loaded on 1 mm thick 8% (w/v) polyacrylamide (37.5: 1 acrylamide: bisacrylamide) gels containing a 45-60% linear denaturing gradient. Gels were run in 1X TAE buffer at 60°C and 90 V for 18 h. Gels were stained in 1X TAE buffer containing ethidium bromide solution (1 μg/ ml) and photographed under UV transillumination. Each band in different positions was cut with a sterile lancet from the polyacrylamide gel and was stored at 37 °C in solvent buffer (ammonium acetate 5M, magnesium acetate 10 mM, EDTA (pH 8.0) 1mM, SDS 0.1%) during overnight and DNA fragments were isolated. The DNA fragments were used reamplification with same primer pairs without GC clamps and were sequenced.

Accession numbers and construction phylogenetic tree of nucleotide sequences
Multiple gene alignments were applied using MUSCLE software. Phylogenetic trees were made using MEGA version 7 and the neighbor-joining method (Saitou and Nei, 1987). The 16S rRNA gene sequences and results of DGGE analyses were uploaded into the GenBank Database.

Characteristics of site and samples
The sampling site is antimony mine site being operated. The AMD samples were characterized by acidic pH values ranging from 2.7 to 6.5 and high concentrations of dissolved metals (Table 1). The sample points show the typical orange and red colors of dissolved ferric iron as shown in Figure 1. It was determined that the samples had high iron, zinc, lead and manganese ratios. Treatment and neutralization studies of outlet water caused increase the pH value and decrease iron concentration between two sampling times (sample #5). This pH change also affected the prokaryotic diversity at the sample site (Table 1).

DGGE
DGGE analyses were performed with each different sample to determine the level of microbial diversity in the mine area. (Fig 4). The samples collected from the same sample points determined to have different profiles. Especially, due to pH change, it was found that bacterial diversity quite different sample #5 and sample #2. Blast analyses of DGGE bands sequences are given in Table 3. Archaeal diversity was determined only in sample #1 and sample #3. The sequence of bands YT_D1, YT_D2, YT_D3, YT_D4, and YT_D5 showed similarity with uncultured archaeon clones, as a show that in Table 3. Differences were observed in the DGGE profiles of taken water samples at different times from the same sampling points. It was determined that the sample #4 have more bacterial diversity from sample #1. According to sequence analysis results, there are bands matched with Ferrovum myxofaciens, Acidithiobacillus ferrooxidans, Acidithiobacillus ferrivorans, Acidithiobacillus ferriphilus and uncultured Acidithiobacillus sp. in sample #1. In the case of water sample #4, it was determined that the majority of the species are Acidocella (Table 3). Although samples #2 and #5 were taken from the same spot, it was thought that the change in pH at sample #5 caused the formation of different profiles. Bands at sample #5 were showed similarity with Acidocella aluminiidurans, uncultured Acidocella sp. and uncultured Acidiphilium sp. In sample #3, bacterial diversity is less than in sample #6. The band of sample #6, it was determined to match with Thiomonas sp. (Table 3).

Accession numbers and construction phylogenetic tree of nucleotide sequences
16S rRNA gene sequences were deposited in GenBank under accession numbers MH057089-H057162. In order to determine the phylogenetic group, the phylogenetic tree was constructed with sequences obtained by 16 rRNA clone library and DGGE analyses (Fig 5, 6).

Figure 5
Phylogenetic tree based on 16S rRNA gene sequences of 16S rRNA clone libraries.  As seen in Figure 7, the variety of prokaryotic diversity was observed by used molecular techniques in AMD ponds.  It was determined later that this strain separated from other acidithiobacilli (Falagan and Johnson, 2016). The clones and DGGE bands sequences showed to match the most Acidiphilium genus. The mesophilic and obligately acidophilic bacteria Acidiphilium angustum grow in the pH range of 2.0-5.9. The clone YT_K8 matched with Acidiphilium angustum (99% similarity) was obtained from water sample #2 (pH 2.9). Auld and coworkers have isolated Acidiphilium rubrum from an AMD site in Copper Cliff, Ontario (Auld et al.,  2013). The isolate was isolated from AMD water at pH 2.5, similar to the water sample (pH 2.7) in which identified YT_K16.

CONCLUSION
Future studies will focus on the roles of these species on the biogeochemical cycles of the region where microbial diversity is determined. The AMD is also likely to contain new species. To better understand community dynamics in acid formation, more studies are needed to identify predominant species in AMD environments.