An explosive condition - open cast coal mining soil subsurface biodiversity and the expression of nitrogen cycle genes

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(1)An Explosive Condition: Open Cast Coal Mining Soil Subsurface Biodiversity and the Expression of Nitrogen Cycle Genes. A thesis. Presented by: Nasmille Liceth Mejía Flórez Student code: 200220647. Thesis Director: Jenny Dussán PhD. Centro de Investigationes Microbiologicas (CIMIC) Department of Biological Sciences Science Faculty. Universidad de los Andes Bogotá, Colombia Abr, 2010. 1.

(2) Table of Contents. CONTENT. PAGE. 1. Abstract…………………………….…………………………………………………………………….…………….……….......3 2. Introduction……………………….……………………………………………………………………..…….…………….…….4 3. Materials and methodology ……….…………………………………………..…………………………..……….……..6 3.1. Chapter 1: Coal Mine Pit Diversity 3.1.1. Sample Collection………….…………………..………………………………….………………............6 3.1.2. Sample Processing……………..………………………………………….…………………….……........6 3.1.3. Bacterial Identification………………………………………………………………………..……………..7 3. 2. Chapter 2: ANFO and the Nitrogen Cycle 3.2.1. Plastic and Glass Materials………………………………………………………………………………...8 3.2.2. Treatments and growth mediums with metabolic selection pressures…...………………………………………………………………………………………………………….……8 3.2.3. Morphotype Selection, RNA extraction and Reverse Transcriptase…………………..9 3.2.4. Nitrogen Cycle Genes……………………………………………………………………………………….10 3.2.5. Statistical Analysis………………………………………………………………………………………….…11 4. Results and discusion……………………………………………………….…………………………………….............13 4.1. Chapter 1: Coal Mine Pit Diversity……………………………………………………………………….13 4.2. Chapter 2: ANFO and the Nitrogen Cycle…………………………………………………………….19 6. Conclusions……………………………………………………………………..………………………..………….…..........26 7. References………………………………………………………………........…………………………..………..…........27. 2.

(3) Abstract:. Open coal ANFO-cast pit mines present in the Guajira state are altered daily by anthropogenic activities. Detonation and removal of the earth’s layers for the exposure of coal directly alters microbial ecology. Studying deep subsurface microbial ecology and the factors that influence it is a difficult task but seeing that the environment selects the microorganisms present in a particular place and time, the influence of the ANFO explosive was questioned. Two geographically separate pits were sampled, at three different distances from the surface with different times from the last detonation. Cultivable bacteria abundance, median and standard deviation, values show no statistically significant differences between both pits, while site location did influence plate count. Diversity indexes had the expected tendencies for most sites, except recently detonated locations. At the laboratory ANFO pressures were done to evaluate the influence of the explosive on the cultivated bacteria recovered from both mines. The pressure selected 39% of total cultivable bacteria, 87.5% belonging to recently altered sites and 100%the identified individuals belonging to the Pseudomonas genus. RNA extraction of ANFO treatments as the only nitrogen and carbon sources available demonstrated the expression of narG, napA and nirS genes. Expression of nirS confirms 62.5% of the selected bacteria as denitrifiers. The ANFO explosive may explain the presence and abundance of the Pseudomonas genus in recently detonated sites, altering expected diversity index values. To conclude doubts about the effect of the explosive further studies centered on real time microcosm essays are needed to evaluate differential expression of denitrification genes, along with hydrocarbon degradation genes.. 3.

(4) Introduction. The top layer of the earths’ crust contains the main percentage of living creatures; but, life is not exclusively a surface phenomenon. The vast majority of organisms and microorganisms depend on carbon sources produced by photosynthesis. As we travel deeper inside the subsurface the amount of nutrients and photosynthetically produced organic carbon decreases significantly (11). The terrestrial deep subsurface has been well studied during the past 30 years in different underground scenarios providing scientific evidence of the existence of a microbial biosphere in subsurface horizons (2, 8). Nutrient limitation results in microbial populations and activities in subsurface environments that are commonly 2 to 6 orders of magnitude lower for viable biomass and 4 to 10 orders of magnitude lower for activity than in surface soils (4, 6). Sustainable life in subsurface environments is questioned, especially concerning the different mechanisms used by microorganisms to survive the unfavorable conditions and how those mechanisms need be used for their survival and their own preference over other surface abundant inhabitants.. Microorganisms have been present for many millions of years before the existence of man and other recently surging species. They have developed evolution-powered strategies to survive in other wise inhospitable habitats. Beijerinck said: ‘‘Everything is everywhere, the environment selects’’, referring to the way bacterial communities composition can be affected by biotic and a-biotic factors in a wide range of environments (8). Microbial diversity and ecology function is based on the conditions of the environment, including the available sources of metabolic energy (9).. Open cast coal mining has been present at the Guajira state for more than twenty years, generating environmental disturbances that have altered microbial diversity in a definite manner. Coal extraction pits are later reconstructed and thorough actions are constantly performed for the progressive recovery and future stability of the disturbed territory (4). Inside these coal mines indigenous and contaminant microbes subsist. The factors that influence the abundance of microbes that arrive from different surrounding pit environments include water, wind and other anthropogenic activities such as the entrance of coal transporting WADCO trucks. Only microorganisms capable of using the available nutrients may survive in these pits. The conditions may make any source of energy useful. A compound is used on a daily basis in previously meditated sites inside of the coal bearing pits. The ANFO (ANammonium nitrate, FO-Fuel Oil) explosive is the determined detonator assigned for the exposure of coal and removal of temporarily surfaced spoil material; turning it into a tentative carbon and nitrogen source (19).. 4.

(5) The use of the detonator inside the pits may be a factor that influences the presence of a certain type of bacteria, affecting naturally present subsurface microbial ecology. Some organic sources such as petroleum or other oily fuels were thought to be not biodegradable in the subsurface, but they are in fact very important substrates for subsurface microorganisms (11). Most importantly is ammonium nitrate, which was used before international constrain as a fertilizer, and inside a pit is a good source of nitrogen for a large variety of organisms. The purpose of this study was to analyze the biodiversity differences found in two geologically separate and age different coal mine pits using laboratory approaches and molecular analysis for the presence and expression of nitrogen cycle genes in ANFOpressure selected bacteria.. 5.

(6) Materials and Methodology. CHAPTER 1: COAL MINE PIT BIODIVERSITY. 1.. Sample Collection:. The samples were collected February 2009 from two coal mine pits, Patilla (P) and Tabaco (T). Both pits are property of Carbones del Cerrejón and are located at La Guajira state, northeast Colombia (11° 5′ 22″ N, 72° 40′ 31″ W). Three sites at each coal pit were sampled, the first designated place for recollection was at sites that had been recently exploded with ANFO (Recent-R), the second was a place exploded several years ago (with no recent coal extracting activities; Quiet-Q), and finally the third was a place adjacent to the coal mine (Adjacent-A). From each site ten samples of approximately 200 grams were taken with a sterile ~30cm plastic cylinder, homogenized in a sterile plastic bag and a total of 200grams was stored and transported carefully in a cold (~0°C) styrofoam box at room temperature from the pit to the laboratory in Bogotá (CIMIC), Universidad de los Andes. Each sample was taken with a new pair of gloves, tightly sealed and properly managed. One sample (20 grams), taken from a recently disturbed site at Patilla was mixed during 20seconds with 20ml of sterile distilled water in a 50ml sterile (RNAse and DNase free) falcon tube . The sample was left to settle for 10 seconds and the supernatant was passed to a new plastic 15ml falcon tube. The tube was transported in the Styrofoam box as well. Another sample, from Patilla recently exploded, was taken in a new 15ml plastic falcon tube and transported in liquid nitrogen (~-80°C) to the laboratory in Bogotá.. 2.. Sample processing:. Dilutions. Serial dilutions were made for each one of the samples and they were plated for isolation of cultivable microorganisms present. Fifty grams of material were taken from each bag and placed in 50ml of sterile distilled water. The flacks were agitated during twelve hours at 220rpm. After that time each flask was left to settle for 10 minutes and well homogenized dilutions (10-2, 10-3, 10-4) were made and plated on Plate Count Agar (PCA, Scharlau) and Cetrimide Agar Base (CBA, Difco Laboratories) incubated at 30° C during 48 hours. Each sample had three replicates of the plated dilutions.. 6.

(7) Colony counts, differentiation, individual isolation and conservation of each morphotype. Each plate was counted for the number of different morphologies (morphotype) found and the total number of colonies present for each one (Colony forming units per gram, CFU/gram). Each morphotype was isolated individually in the same medium from where they were taken, incubated at 30°C for 24 to 48 hours. Macroscopically different morphological characteristics of each colony were registered and named. The name registration for each morphotype was based on the pit from where the sample was taken (P or T), site from where the sample was collected (A, Q or R), and the number of the morphotype. Each pure individual colony was conserved in 10% glycerol at -80°C.. 3.. Bacterial identification:. Isolate DNA extraction. All the mayor isolates from the plate count were isolated and then identified. The procedure began with individual over night cultures in Luria Bertani (LB) medium for 14 hours at 30°C and 220 rpm. After the ON, 25ul of each culture were transferred to 1.5ml eppendorf tubes. For other microorganisms, a colony of the isolates was taken from PCA or CBA mediums and pressed on the bottom of a 1.5ml eppendorf, with a sterile wooden stick. The tubes were maintained in boiling water during 15 minutes and were then centrifuged at 13000 rpm for a minute. 0.9ul of the supernatant were used as the DNA template (19).. V3 to V5 hypervariable region (16S rDNA) PCR. The 25ul volume reactions PCR amplifications were performed using an iCylcer thermocycler (BioRad). Negative controls were done using ultra-pure water instead of DNA. PCR assays for primer sets targeting the16S rRNA gene included 0.9ul or 5ul template DNA, 2U of Taq Polymerase (Bioline), 1X PCR buffer, 2.5mM MgCl2, 0.2mM of each dNTP and 0.3uM of each primers. PCR protocols for 16S rRNA gene (35cycle) were performed as previously reported (11) with annealing temperatures between 66 and 61° C. A 622 bp fragment was expected and was confirmed by taking 5ul aliquots of PCR product and loading them to 1% agarose gels. Gels were stained with ethidium bromide and images were captured with a GelDoc imaging system (BioRad)(19). The sequences of the genes used for identifying the bacteria V3-V5 hypervariable region are 352f (5’ GGT TAC CTT GTT ACG ACT T 3’) and 975r (5’ AGA GTT TGA TCC TGG CTC AG 3’).. Nucleic Acid sequencing and analysis. 7.

(8) The PCR products were purified using Wizard® SV Gel and PCR Clean-Up System kit (Promega) according to the manufacturer’s instructions. Purified products were sequenced using the BigDye® Terminator v3.1 Cycle Sequencing Kit according to the manufacturer’s instructions using 325f primer and 975r primer.. Sequences were assembled and manually edited using BioEdit v7.0.9. Sequences for the V3-V5 hypervariable region of the 16S rRNA gene were submitted to the Ribosomal Database Project II Classifier (27) which uses a naïve Bayesian rRNA classifier algorithm, and greengenes website NAST program (7). All isolates sequences were aligned using the D’Align software.. CHAPTER 2: NUCLEIC ACID EXTRACCION AND THE NITROGEN CYCLE. 4.. Plastic and glass materials:. All samples were stored and handled with RNase free plastic- and glassware. Plastic ware was washed with RNase free 10% SDS, soaked with DEPC treated water (0.1% diethyl pyrocarbonate, SIGMA), and then double autoclaved. All glassware was previously treated by baking at 200°C for at least four hours. All the solutions used were prepared with DEPC treated water (stirred overnight with 0.1% DEPC at room temperature and double autoclaved) in pretreated glassware.. 5.. Treatments and growth mediums with metabolic selection pressures:. ANFO Agar and Liquid Pressure. The next step, after the isolation each one of the chosen morphotypes, was the selection of the microorganisms that could develop with ANFO as an important component of their growth medium. Total pressure time for all the thirty-five bacteria was 4 months. Each microorganism was placed in a solid and liquid medium pressure with ANFO. For the solid ANFO agar pressure the procedure was made as done in previous work (19) with some modifications. ANFO Agar was formulated by mixing 1.5% of Agar (Agar Agar, Scharlau), 20% of the density of ANFO (0,8g/ml) and a medium free of nitrogen and carbon sources spiked with CaCO3 (MFNC; 0.5% KH2PO4, 0.25% MgSO4 7H2O, 0.25% Na2MnO4, 1% FesO4 7H2O 0.1% MnSO4 4H2O, 0.001% CaCO3 and 0.016% AN) at 30° C during only seven days. After those seven days each morphotype was passed to 40% ANFO Agar and finally 80% ANFO for seven days each at 30°C. After each pressure in ANFO Agar an approximate estimation of the biomass growth content of. 8.

(9) each microorganism was registered. Liquid pressures and registrations were made for 4 consecutive weeks.. For ANFO liquid pressure was formulated with 20% of the density of ANFO (0,8g/ml) (2) and medium free of nitrogen and carbon sources spiked without CaCO3 (MFNC; 0.5% KH2PO4, 0.25% MgSO4 7H2O, 0.25% Na2MnO4, 1% FesO4 7H2O 0.1% MnSO4 4H2O, 0.001% CaCO3 and 0.016% AN) without agar. Each morphotype was added to 5ml of the liquid medium during seven days at 30°C for 5 consecutive weeks (five 300uL passes).. “Replica plating” growth on MFNC Agar + Diesel. Each one of the isolates taken from 80% ANFO liquid pressure were placed in order on 20ml of Agar (Agar Purificado, Scharlau) and medium free of nitrogen and carbon sources with no CaCO3 (MFNC; 0.5% KH2PO4, 0.25% MgSO4 7H2O, 0.25% Na2MnO4, 1% FesO4 7H2O 0.1% MnSO4 4H2O, 0.001% CaCl2 and 0.016% AN). The carbon source was supplemented by placing a little lid with 1ml of Diesel in the middle of the Petri dish (19) and using a sterile paper on the inside of the top lid to prevent the condensation and spilling of Diesel on the medium. In each dish 8 isolates were placed. The treatments were incubated for 9 days at 30° C for 5 consecutive weeks using sterile wooden stick passes. Five days into the treatment the amount of Diesel had lowered significantly (highly volatile) so 500ml of Diesel were added to the lid. Photographs of day1 and day7 were compared to determine which organisms had grown significantly with Diesel as their only carbon source.. 6.. Morphotype selection, RNA extraction and Reverse Transcriptase:. The thirty-five bacteria were submitted to the replica plating and thirteen were interesting prospects for molecular analysis after the pressures. After an essay using all thirteen bacteria with 4 pairs of gene amplification primer couples eight bacteria had acceptable results (view Nitrogen Cycle Genes>Genes, Primers Selection and PCR for information on specific protocols). The final eight bacteria were submitted to the final pressures before bacteria RNA extraction. The treatments consisted of 47mL of liquid MFNC and 3mL of Diesel fuel with the bacteria (108- 109) for seven days with replicas. In case of poor quantity and quality of the RNA extractions 25mL of one replica was centrifuged at 8000rpm for 30min and stored at -80°C. RNA bacteria RNA extraction from 7 day samples was made using RiboPure™-Bacteria Kit (AMBION) manual protocol (1). Low efficiency was observed for the morphotype PRIV at day seven caused by the formation of a biofilm and RNA extraction for the bacteria was made for samples that had three days of treatment.. 9.

(10) 7.. Nitrogen Cycle Genes. Genes, Primers Selection and PCR. Six genes from the nitrogen cycle were selected for further analysis (nirS, nirK, napA, narG, nosZ and nifH). A total of ten degenerate primer pairs were selected from previous studies for the amplification of the genes (table1). The bacteria that would continue in the final analysis of nitrogen cycle gene expression were selected by a final molecular PCR analysis in which all thirteen bacteria were submitted to PCR of nirS, nir K and nozZ (results not shown). The eight bacteria with the best results were selected based on amplification efficiency (band presence and resolution).. For genes nirS primer pair cd3aF-R3cd(20), nirK primer pair F1aCu-R3Cu (13, 26) and nosZ’ s primer pairs a) nosZF-nos1773R, b) nosZF-nos1622R; c) nos1527F-nos1773R (14, 21, 24, 26) PCR mixes were constituted by 1x PCR Buffer, 1.5mM of MgCl2, 0.2mM of dNTP’s and 1.25U of Taq Polymerase (Bioline) in a final volume of 25uL. Thermocycler protocol started with 2 minutes at 94°C, 35 cycles of 30sec at 94°C followed by 1min at 51°C and 1min at 72°C, and a final cycle at 72°C for 10min. Although previous work suggested a final concentration of 1mM of each degenerate primer, a final concentration of 0.8uM worked well. Product bands were seen in an ethidium bromide stained 1% agarose gel.. For gene napA with primer pairs V16 and V17, followed by nested PCR with V66 and V67 (10) 1X Buffer, 3mM MgCl2, 0.2mM dNTPs, and 3.75U of Taq Polymerase (Bioline) were used for the first PCR. For nested PCR the final concentration of MgCl2 was changed to 1.5mM. Both had 100uL as the final volume. The final primer concentration was changed to 20uM. Product bands were seen in an ethidium bromide (0.5 mg/mL) stained 1.5% agarose gels.. NarG protein’s nucleotide sequence was targeted using primer pair 1960m2f-2050m2r (17) with 1x PCR buffer, 1.5mM MgCl2, 0.2mM dNTP’s, 1U Taq Polymerase (Bioline) and 5uM as the adjusted final concentration of degenerate primers and a final volume of 50uL. The enzyme was added after the first denaturation step. Thermal cycling was carried out by an initial denaturation step of 5 min at 95°C, followed by a touch down PCR (BioRad Thermocycler).This consisted of a denaturation step of 30 s at 94°C, a primer annealing step of 30s at 60°C, and an elongation step of 45 s at 72°C; cycling was completed by a final elongation step of 72°C for 6 min. During the first eight cycles, the temperature was decreased by 0.5°C for each cycle, starting at 59°C, until it reached 55°C. The additional 30 cycles were performed at an annealing temperature of 55°C (22) The presence and size of the amplification products were determined by agarose (1%) gel electrophoresis of the reaction product and ethidium bromide staining.. 10.

(11) Finally, for nifH the first PCR was performed with the primer pair forA-forB. Reaction mix consisted in 1x buffer, 1.5mM MgCl2, and 0.2mM dNTP’s, 0.8uM of each primer, 1.25U of Taq Polymerase (Bioline) and a final volume of 25uL. Nested PCR and a final reaction volume of 100uL with the same concentrations of each one of the reaction components, except the units of Taq Polymerase (Bioline) passed to be 0.8 U. The cycling conditions used for both reactions were as follows: denaturation for 11 s at 94°C and for 15 s at 92°C, annealing for 8 s at 48°C and for 30 s 50°C, and extension for 10 s at 74°C and for 10 s at 72°C. A final 10-min extension step at 72°C was performed after the 40 cycling steps. The nested reaction was performed for 35 cycles(28). Product bands were seen in an ethidium bromide stained 2% agarose gel. Gels were stained with ethidium bromide and images, as well as for the other genes mentioned above, were captured with a GelDoc imaging system (BioRad).. DNA was extracted from each one of the eight selected bacteria following the extraction protocol described previously (Isolate DNA Extraction) for the evaluation of the presence of the desired band sizes. 5ul of the extracted DNA from each one of the selected bacteria were added to each individual gene reaction. For cDNA-PCR reactions 5ul of the resulting retro-transcriptase RNA reaction was added (50-500ng). Retro-transcription was done using Random Primers (Invitrogen) and QIAGEN LongRange 2 Step RT-PCR (23) with two DNase treatments (PCR of 16S rRNA gene was evaluated to test absence of DNA in the samples).. 8.. Statistical Analysis and Bioinformatic tools.. Data analysis from bacteria abundance and graph construction were elaborated using the SPSS 16.0 program. Alignment of gene fragments for 16s rRNA gene, nirS, nosZ, napA, narG and nifH for identification of the fragments were done using blastn and tblastx, available in the NCBI homepage (http://www.ncbi.nlm.nih.gov/). Nitrogen cycle gene sequences were assembled and manually edited using BioEdit v7.0.9 and aligned using D’Align Website using default options (25). Phylogenetic trees were calculated using the program ClustalW (http://srs.ebi.ac.uk) and constructed with the iTOL program (16).. 11.

(12) Table1. Degenerate primers used during this study. Gene target, primer name, specific sequence, annealing position inside the gene, expected fragment size and original reference from where all primers where taken are informed in the table. Gene Primer name Sequence (5'-3') nirS. nirK. nosZ. Position. Fragment (bp). cd3aF. GT(C/G) AAC GT(C/G) AAG GA(A/G) AC(C/G) GG. R3cd. GA(C/G) TTC GG(A/G) TG(C/G) GTC TTG A. F1aCu. ATC ATG GT(C/G) CTG CCG CG. R3Cu. GCC TCG ATC AG(A/G) TTG TGG TT. 1021–1040. (26). nosZF. CG(C/T) TGT TC (A/C) TCG ACA GCC AG. 1169–1188. (14). nos1773R. AAC GA(A/C/G) CAG (T/C)TG ATC GA(T/C) AT. 1396–1415. 246. (21). nos1622R. CGC (G/A)A(C/G) GGC AA(G/C) AAG GT(G/C) CG. 1603–1622. 453. (26). nos1527F. CTG TTC ITC GAC AG(T/C) CAG. 1171–1188. 225. (24). napA V16. GC(A/T/C/G)CC(A/T/C/G)TG(C/T)(A/C)G(G/A/T/C)TT(C/T)TG(C/T)GG. 916–935. Reference (20). 1322–1341. 425. (26). 568–584. 472. (13). 136-156. V17. (G/A)TG(C/T)TG(G/A)TT(G/A)AA(G/A/T/C)CCCAT(G/A/T/C)GTCCA. 1131-1155. V66. TA(C/T)TT(C/T)(C/T)T(G/A/T/C)I(G/C)(G/A/T/C)AA(G/A)ATIATGTA(C/T)GG. 256-282. V67. (G/A/T))AT(G/A/T/C)GG(G/A)TGCAT(C/T)TC(G/A/T/C)GCCAT(G/A)TT. 663-687. 1040 (10). narG 1960m2f. 385. TA(C/T) GT (G/C) GGG CAG GA (A/G) AAA CTG (17). nifH. 2050m2r. CGTAGA AGA AGC TGG TGC TGT T. 110. univ for A. GCI (A/T) TITA(C/T) GG (GA/T/C) AA (A/G) GG (A/T/C/G) GG. 19-38. 464. univ for B. GGITGTGA(C/T) CC (G/A/T/C) AA (A/C/G) GC (G/A/T/C) GA. 112-131. 371. univ rev. GC (A/G) TAIA(C/G/T) (G/A/T/C) GCCATCAT(C/T) TC. 463-482. (28). 12.

(13) Results and discussion:. CHAPTER 1: COAL MINE PIT BIODIVERSITY. Two coal mine pits, Tabaco and Patilla, belonging to the open coal mining operation Carbones del Cerrejón at the Guajira state were each sampled at three previously established sites. Both pits were collected at a recently exploded site, in contact with the explosive ANFO for 24 hours before the detonation. The second site was altered about 15 to 20 years ago, for coal extraction purposes, but had no recent changes. The latter was not affected directly by mining, but rather by precipitation that constantly moves particles from the surface to the inside of the pit. The last site sampled in the pit was from places adjacent to the coal mine, surface soil with indirect mining alterations. The three sites were designated according to previous work (19) and may be a guide to the effect that mining activities have on microbial communities inside an open cast pit (12).. Microbial cultivable community diversity was determined by observing the total number of species, in this case the morphotypes (species richness) and relative abundances of the morphotypes (species evenness) at the three sites (9). Samples were processed and plated on PCA medium and total colony forming units (CFU) per gram of soil, number of distinct morphotypes and individual morphotype counts were registered at the end of 72 hours of incubation at 30°C and 24 hours at room temperature. Central tendency of data (median) and variety (standard deviation) were calculated in relation to the abundance of each morphotype for both pits and each site separately (Fig 1) (18).. 13.

(14) A. B Figure1. Box Plot graphs representing plate counts (CFU/gram) for each open coal mine pit Patilla (P) and Tabaco (T) and for each sampled site Adjacent (A), Quiet (Q) and Recent (R). N: number of morphotypes stipulated at each pit/site.. 14.

(15) Results show (Fig1A) that although samples were taken from two geographically separate pits, abundance statistical values are very similar for both pits. This fact is demonstrated by the similarity of the box plots that concerns the mean values for each individual pit (P: Patilla 4.980; T: Tabaco 4.565), the standard deviation (P: 0.991; T: 1.131), and the total number of morphotypes observed (P: 17; T: 16). Both values are very close to the total mean and standard deviations values of both pits together (mean 4.779; standard deviation 1.065). Variance analysis using ANOVA statistical analysis to determine the spatial heterogeneity show non significant differences between pits with values of P= 0.270 (>P≤0.05) and Rsquared=0.039.. Species richness is a concept that defines a sites biodiversity by defining the number of phenotypically /genotypically different organisms can be recognized. The richness of a particular environment is determined by a large number of biotic and a-biotic factors (9). Some of the species that arrive at a particular place are not necessarily potential inhabitants of the site. Inside the quarry only organisms capable of establishing themselves are considered an important part of that distinct sites original biodiversity. Richness values are therefore an estimate of the immediate number of distinct species/morphotypes in a particular place and time that, especially in extreme environments, are largely influenced by their surrounding environment’s biodiversity. Results shown in Fig1B for species richness in each site are very close to what is expected when A-site and Q-sites in both pits are compared; however, when comparing Q-site and R-site’s results the difference is not clear. In R-site a low number of morphotypes and a low number of species abundance were expected, but results manifest a different scenario when comparing both pits simultaneously. A greater number of morphotypes was determined, and some of these morphotypes are in high abundance. Both pits needed to be analyzed individually to understand species number and abundance.. Fig 2 exhibits the data found for each individual pit. Abundance values, and variance, are closer to what is expected. For richness, A-site has the largest number of morphotypes, followed by Q-site and R-site (with an exception in Tabaco R-site). At the colony count level values Tabaco Q-site and Patilla R-site change the tendency that is expected. Tabaco’s Q-site abundance values are very close to what is seen in the A-site. Because Tabaco pit is a much younger mining operation, compared with the years of excavation of Patilla, then Tabaco Q-site is much closer to the surface. This fact may also be influencing the species richness in Tabacos’ R-site. For Patilla’s R-site, although there are only 3 distinguishable morphotypes, recounts where high and close to what was observed in the same pit’s A-site, passing over Q-site values. This difference is of great importance and will be discussed later.. As seen in previous work, the level of microbial diversity is much lower in the subsurface than in surface soils (19, 29) The same effect is seen for total amount of bacteria and the number of morphotype counts in the three sites for both pits (A: 16; Q: 9; R: 8; see Fig 1B). Mean values for the three sites show the differences in colony count between the places are statistically significant with P=0.01 (< P≤0.05) and. 15.

(16) Rsquared= 0.267 without taking into account the pit from where the samples were taken. Previous results show there is a statistically significant difference between plate count values in the three sites and most importantly, the site from where samples are taken explains and influences abundance values. In other words, some of the change in species abundance is explained by the site (R>0.25)(3).. Although mean values confirm differences between the different sites sampled, standard deviation values show there is a great amount of variance for abundance values in sites Q and R. Variance in Q-site and R-site may be due to precipitation, erosion and wind currents, that transport microorganisms from other places to the inside of the pit. The effects of environmental abiotic sources of bacterial movement have larger consequences for Q-site than R-site because of their position inside the pits. Q-sites are generally located towards the edges of the pit, where truck tracks are located, used to transport of coal. Q-sites are therefore established closer to the surface and are most affected by precipitation and wind currents than R-sites. The latter are found in the lowest sections of the coal pit, where coal activity is recent and functioning. Large variance in abundance (poor evenness) for this site can also be due to water precipitation transport of microorganisms to the lowest parts of the coal mine, concentrating bacteria number and species.. Figure2. Box Plot graphs that show Plate counts (CFU/gram) for each site sampled at both open coal mine pits. Patilla (P), Tabaco (T), Adjacent (A), Quiet (Q) and Recent (R). N: number of morphotypes stipulated at each pit/site. Data is graphed based on the mean of three replicas.. 16.

(17) Shannon and Simpson’s diversity indexes were calculated separately for each mine site (Fig3). Simpson’s index expresses the possibility of finding an organism of the same species during random sampling at each location. Calculated values for each site show the expected tendencies in each mine. The values are greater as a deeper stage of the subsurface is reached. The probabilities of finding two microorganisms of the same species in A-sites are low, and they increase in R-sites. Q- and R-site values at Tabaco are very similar due to similar richness and abundance values, but the value continues to be greater in the latter.. When comparing with Shannon’s index values, proportional to the total bacterial diversity found at each site, Patilla’s results display the expected results: less diversity in the deepest site of sampling. Richness in Patilla’s R-site had greater importance than the abundance mean and std. deviation values found at this site, and showed the expected tendency. Results were also as expected for A- and Q-sites at Tabaco pit, with exception of R-site. This last site had low abundance of species, but richness value was high, compared to Q-site. This result is due to the morphological differences between the bacteria that were cultivated from the site. Phenotypical differences in color shape and consistency made it easy to differentiate five species from that location. For further analysis of these results, a molecular identification of the different morphotypes was implemented.. Figure3. Histogram divided by pit and sample sites of two diversity indexes (Shannon and Simpson). Pits: Patilla (P), Tabaco (T); Sites: Adjacent (A), Quiet (Q) and Recent (R).. 17.

(18) Sequencing and identification using sequence comparison to GenBank database for the amplified V3-V5 hypervariable region of the rRNA 16S gene (Table2). 51% of the cultivated bacteria correspond to the genus Pseudomonas, 18% correspond to the genus Stenotrophomonas and 15% to the genus Bacillus. Although the number of bacteria that were not identified corresponds to 6%, the amount of different genera was less than expected for the total 6 sites.. Interesting results surge with the presence and abundance of Pseudomonas in both R-sites. The expected diversity in R-sites, according to results seen in A- and Q- sites, was null, but the species charge of Pseudomonas-identified morphotypes change what was expected. The influence of the genus is tremendous, to the point which if it were not taken into account the numbers would fit perfectly to what was expected. But the presence of the genus seems to be allowed in the “inhospitable” environment.. Table2. Morphotype identification and species count for each pit site. P pit (Patilla pit), T pit (Tabaco pit), A-site (Adjacent site), Q-site (Quiet site), R-site (Recien site). Species Count A-site. Q-site. R-site. Specie (Genus). P pit. T pit. P pit. T pit. P pit. T pit. Pseudomonas. 1. 4. 3. 3. 2. 4. Stenotrophomonas. 4. -. 1. -. 1. -. Bacillus. 2. 2. -. 1. -. -. Microbacterium. 1. -. -. -. -. -. Paenibacillus. -. 1. -. -. -. -. Serratia. 1. -. -. -. -. -. Not Identified. -. -. 1. -. -. 1. TOTAL. 9. 7. 5. 4. 3. 5. A phylogram tree was constructed for the sequence comparison of the thirty-five bacteria (Fig4). The results show a clear differentiation between the different genera recovered from the coal mine pits. Short distances are also registered for some individuals from the same specie. The possibility of having replica species in the sampling of Tabaco R-site was also examined. Since most Pseudomonas-identified morphotypes from the site corresponded to the phenotypically variable specie Pseudomonas stutzeri the possibility of two or more identical bacteria was studied. This particular specie changes in appearance with time(15), for which the number of morphotypes was registered after 24h incubation at 20°C. All phenotypically different bacteria were individually separated as morphotypes but, being identified in the same specie could also explain R-site richness due to abundance of the same microorganism in the site.. 18.

(19) Figure4. iTOL phylogenetic tree display using ClustalW phylogram tree from D’Align results for the alignment of V3-V5 hypervariable region of the rRNA 16S gene for the thirty-three bacteria isolates recovered from both coal mine pits. Specific site from where species were recovered is written before the specie.. Divergence- based methods consider a community more diverse if the individuals in it are highly divergent from each other, or are phylogenetically distinct from organisms found elsewhere (species within a sample are equally related to each other). When taking about species divergence in a determined environment related to the differences between genomes, the comparison of specific amplified and sequenced genes becomes vital. Genome relations superior to 70% using DNA-DNA hybridization (DDH) may consider being from the same species. In 16S rRNA gene information a 97% of sequence identity threshold is generally used(18). Considering the information given eight distinct species can be designated (two species in the Pseudomonas genus). Species divergence diversity using this form of analysis condenses all data and makes it clear that, even though some previously calculated diversity indexes say the contraire, R-sites poor species divergence can be accounted and more appreciated than the value set in Tabaco R-site (0.644).. 19.

(20) CHAPTER 2: ANFO AND THE NITROGEN CYCLE. Subsurface environmental selection. Species divergence related to the metabolic capabilities that share or distinguish species from each other(18). If any certain specie is considered from the same phylogenetic branch as others in the same sample, there is a possibility of divergence inside the same branch that cannot be measured using molecular clock genes such as ribosomal genes. The divergence is created by an environmental condition, which triggers the selection and expression of certain genes, if present. Chromosomal information, plasmids, transposons and other forms of horizontal gene transfer alter microorganism function in a determined environment. In this case, lack of an obtainable metabolic energy source may activate certain protein routes to compromise with the pressure. ¿But, can the ANFO explosive play an important role in the determination of microbial subsurface diversity and function?. Metabolic pressures using the ANFO explosive as the only available nitrogen and carbon sources for the thirty-five cultivated bacteria was designed to test their ability all through the treatments (approximately 4 months). Attractive results were encountered for the number of morphotypes (species) selected and original pit recovery site. The first day 57% of the bacteria submitted to pressure corresponded to A-site individuals, while only 43% belonged to both Q- and R-site. After the first round of selection 36.3% bacteria were from A-site samples, 18.1% from the Q-sites and 40.9% from the R-site (22 total individuals selected). Finally, when all carbon sources were eliminated only 13 individuals survived the pressure, 0% from A-sites, 12.5% from Q-sites and 87.5% from R-sites. ANFO pressures have an evident effect on the individuals recovered from the coal mine pits. Selection of 39% of the total individuals was evidence of the explosives effect.. Selection was not homogeneous or site independent. While the number of initially abundant A-site organisms decreased rapidly, R-site individuals continued almost intact from beginning to the end. The explosive’s pressure seemed to disturb surface original microorganisms that are normally acquainted with a broad amount of nitrogen and carbon sources, extinguishing all of the A-site members at the end of the treatment. To our huge interest, the effect also influenced Q-site bacteria and had almost no consequences for R-site bacteria. Somehow the idea of the explosive being used recently in R-sites may establish a pressure over other bacteria that could be contaminating the site. This means that some days before the exposure of new underground sites with detonation selection may already be. 20.

(21) happening and, after the disruption is made, residue traces of the explosive may continue pressuring and selecting the bacteria at that site. Results are comparable in both pits, the same outcome is observed for both although they are kilometers apart and the depth of recollection of Q-site and R-sites were not the same.. Retaking previous results, in the R-site a low number of morphotypes and a low number of species abundance were expected but not seen. On the contraire, a greater number of morphotypes and high abundance values were observed. ANFO pressure analysis may play an important role in these results because it may not only confirm the maintenance of R-site original bacteria throughout the ANFO treatment, it also strengthens the possibility of a selection pressure inside the coal mine pit that brings abundance to bacteria that are capable of using it. The next step in our analysis was molecular, searching for genomic and transcriptional evidence of the explosive’s use.. PCR and cDNA PCR. The final eight selected bacteria were screened for the presence and expression of genes important in nitrogen cycling. DNA extraction and PCR amplification of 6 genes using 8 primer pairs is shown in table 3. RNA extraction from seven day pressure selected bacteria (three-day treatment for morphotype PRIV), followed by reverse transcription and cDNA-PCR of five genes using five primer pairs (best resulting from Table3, Fig5). Results of PCR amplifications for the eight bacteria are shown in table4. Table5 shows the genera and, in some cases, the species to which each morphotype corresponds to.. Figure5. 1% agarose gel with the best results from initial random primer screening in the 13 selected bacteria after ANFO treatment. 100bp weight marker (1), Primer pair nosZb: PRIV (2), TRI (3), TAI (4), Primer pair nosZ1: TAI (5), Primer pair nosZ3: TAI (6), TRIV (7), PRIV (8), TRI (9), Primer pair nirS: TRI (10), TRIV (11), PRIV (12), Primer pair nosZ3 optimized annealing temperature: TRI (13), TRIV (14), PRIV (15).. 21.

(22) Presence of all genes, reflected as a band of the expected size, was seen in at least one of the eight morphotypes used. The degenerate primers used are visually effective in the amplification of the evaluated genes. Since degenerate primers were used, it was common to find more that one band for the same gene. Sequencing of the questioned bands was necessary to confirm identity of the bands. No detectable bands were seen for the expression of nirK and nosZ at the seventh day of treatment. Results for enzyme expression amplification and gene presence in the bacteria genome showed comparable results in nirS gene. For genes narG and napA bands that did not present themselves in the initial screening, appeared expressed. Results may be due to poor amplification efficiency when amplification was done using the whole genome, while expression results reflect only the information that is expressed, making it easier to find target cDNA.. 22.

(23) Table3. PCR amplification of 6 genes using 8 primer pairs with extracted DNA from each individual morphotype. Presence (+) is established as the presence of the band of the expected size. Negative (-) as the absence of the expected band. Since primers are degenerate, in some cases where the expected band was not present, other nonspecific bands could be detected (*). Presence of the Expected Band (present + or absent -) Gene nirS nirK nosZ. Primer pair (fwd-rvse) Expected Band Size (bp) cd3aF-R3cd 425 F1aCu-R3Cu 472 nosZF-nos1773R 246 nosZF-nos1622R 453 nos1527F-1773R 225 napA V66-V67 (Nested) 385 narG 1960m2f-2050m2f 110 nifH univ forB-uni rev (Nested) 371 *Bands of a different size than what was expected are present.. PQII -* -* + + + -*. PRIII -* -* + + -. PRIV + + + -* + + + -*. TRI + + -* -* + +. TRII + + + + + + +. TRIII + + + -* + + -. TRIV + + + -* + + + -*. TRV -* -* + + + -*. Table4. PCR amplification of 5 genes of interest (using 6 primer pairs) after a seven day essay (3-day for PRIV) RNA extraction and retro-transcriptase cDNA production. Expected ban size presence (+) or absence (-) was registered. No non-specific bands were detected. Bands for nirS and narG were confirmed using specific band sequencing. Presence of the Expected Band (present + or absent -) Gene nirS nirK nosZ napA narG. Primer pair (fwd-rvse) cd3aF-R3cd F1aCu-R3Cu nosZF-nos1773R nos1527F-1773R V66-V67 (Nested) 1960m2f-2050m2f. Expected Band Size (bp) 425 472 246 225 385 110. PQII +. PRIII + +. PRIV + + +. TRI + + +. TRII + + +. TRIII + + +. TRIV + + +. TRV +. 23.

(24) Table5. Morphotype identification of the final 8 selected individuals. Percentage identity and score are also informed (16S rRNA gen database and workbench, greengenes). Morphotype PQII PRIII PRIV TRI TRII TRIII TRIV TRV. Specie Identification Identity (%) Score Not identified Pseudomonas sp. 100 451 Pseudomonas stutzeri 98.82 487 Pseudomonas stutzeri 96.27 510 Pseudomonas stutzeri 98.24 485 Pseudomonas stutzeri 99.22 503 Pseudomonas sp. 94.81 520 Not identified -. In each treatment the initial nitrogen source is ammonium or nitrate. In a past study, using coal mine original bacteria as well (19), biochemical analysis using colorimetric methods showed that the initial amount of ammonium increased, production of large amounts of nitrite and nitrate decrease at the end of seven days of treatment. Results suggested denitrification as the mechanisms used by the bacteria when submitted to the metabolic pressure. In this study the expression of three genes important in the denitrification cycle genes was detected after seven days of treatment for the selected bacteria.. In using studies which target metabolic function, in some cases all the organisms of a species or genus possess the same metabolic function (20). This effect is also seen in this study for Pseudomonas stutzeri specifically. Although all identified bacteria correspond to the genus Pseudomonas, morphotypes PRIII did not express nirS, establishing differences inside the genus. P. stutzeri is largely known as model denitrificator because of a large gene cassette that includes the whole denitrification machinery. Finding P. stutzeri in the deep subsurface of both pits could seem strange, but when encountered with ANFO as the explosive of preference, the relation is not as strange.. Denitrification is an essential and natural process for nitrogen cycling, returning fixed nitrogen, nitrates (NO3), into the atmosphere via gaseous nitrogen (i.e. NO, N2O, or N2) (9). Some bacteria are nitrate respiring (Pseudomonas genus included), using nitrate as their alternative electron acceptor. NarG (membrane bound nitrate reductase) and NapA (periplasmic nitrate reductase) were confirmed as expressed in some bacteria. These genes pass nitrate to nitrite, but are not definite role players to determine denitrifiers, these bacteria may be initiating the denitrification cycle but further analysis must be made for confirmation.. The way to differentiate nitrate respiring bacteria from real denitrificants is through the expression of nitrite reductases (NirS and NirK) (5). Nitrite reductase catalyzes the reduction of nitrite to nitric oxide. The reaction is central to denitrification and is catalyses by two different types of nitrite reductases (Nir), either a cytochrome cd1 enzyme encoded by nirS or a Cu-containing enzyme encoded by nirK (26).. 24.

(25) NirS is more widely distributed, while nirK is only found in 30% of the denitrifying bacteria species. Although both genes were detected as present in some of the evaluated, only the nirS gene was expressed (100% identity between morphotypes). This result confirms the bacteria that express the gene as denitrifiers, corroborating suspicion of denitrifiers in the previous study (19).. Since there is a reduction in the amount of nitrate, for the generation of nitrite and other metabolic functions, bacteria selected by the explosive are processing the available nitrogen source. Abundance of these bacteria belonging to the Pseudomonas genus, who distort the expected diversity index results, is then pressure selected to inhabit zones with recent exposure to the detonator. Thanks to the presence of the explosive these bacteria inhabit R-sites, influencing subsurface microbial ecology and contribute to nitrogen cycling.. Establishing the linkage between ecosystem function and ecosystem biodiversity is a substantial scientific challenge. Nowhere is this challenge greater than in soils (9). Surface soils are complex systems where infinite factors play roles in function and community. For subsurface soils and deep subsurface ecosystems the challenge is no different, but important factors such as distance from the surface and presence/use of specific explosives are factors that have been affecting, to some grade, subsurface microbial ecology.. Further individual biochemical and molecular studies that evaluate the real time effect of the explosive (ammonium nitrate) are necessary to determine its processing and the specific intermediates in the nitrogen cycle. Since abundance values in R-sites are low, microcosm essays would be the best way to make an approximation to what is seen on the field. Fuel oil (Diesel) investigation is also recommended by targeting specific genes and detecting expression all the way through the essays.. 25.

(26) Conclusions:. 1.. Although sampled pits are differentiated by geographical location and the year in which anthropogenic activity began, abundance values show no statistically significant differences between values obtained in each. Values are between 3 x 10. 2. and 2 x 10 6 UFC/gram were. registered in both pits (p≤0.05).. 2.. In both pits, statistically significant differences were found in plate count values for the three sites sampled (p≤0.05).. 3.. Change in species abundance inside the pit is influenced by the site where the samples were taken (R=0.267).. 4.. Simpson and Shannon’s diversity indexes reflected the expected results for each pit site in both pits, except for Tabaco’s R-site. Poor evenness in abundance values and high richness in R-sites may be due to water precipitation that concentrates microorganisms in the lowest parts of the pit (number and species).. 5.. All phenotypically different bacteria were individually separated as morphotypes, but being identified in the same specie could also explain R-site richness due to abundance of the same phenotypically variable microorganism in the studied site.. 6.. The expression of NirS (100% identity between morphotypes) at the seventh day of treatment with the ANFO explosive as their only available carbon an nitrogen sources available confirm 5 of the total 8 selected bacteria as denitrifiers (4 Pseudomonas stutzeri and 1 Pseudomonas sp).. 7.. The presence of the explosive may influence the presence of the Pseudomonas-identified morphotypes to inhabit R-sites. Their presence changes expected diversity index results, to higher values, and contributes to nitrogen cycling.. 26.

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