Microbial Biodiversity Screening for Metal Accumulators from Mineral Ore Rich Site in Andhra Pradesh, India

Problem Statement: Since the site selected for screening was a source of magnetite, the objective of this study was to isolate some groups of magnetotactic bacteria and microbes with potential of metal accumulation. Approach: The culture based approach was used for the study. Pure isolates obtained were characterized on the basis of morphology, biochemical tests, physiology as well as at the 16S rDNA based molecular level. They were screened for their metal accumulating ability. Results: Five pure bacterial isolates were obtained by culture dependent method, isolation being done on a modified Magnetospirillum magnetotacticum specific medium (DSMZ-380). These isolates not only exhibited morphological variation as found from Environmnetal Scanning Electron Microscopy (ESEM), but their cell surface also varied greatly in composition which was confirmed from various staining. Similarly another observed fact was that inspite of the same source of isolation, these strains exhibited a varied temperature and pH tolerance which indicates the diversity in the population. 16S rDNA based analysis shows the molecular identification and real time PCR based pathogenecity analysis proved that they lacked S. aureus or P. aeruginosa like pathogenic gene(s). The isolates were screened for metal accumulating property and all were found to tolerate a wide range of heavy metals and among them certain strains bear high potential of accumulation of metals like copper and chromium. Conclusion: This study reports the isolation and characterization of 5 novel bacterial isolates from the mining area of Andhra Pradesh, India revealing its rich biodiversity. It also points towards the ability of media DSMZ-380 to support growth of metal accumulating non-magnetotactic isolates.


INTRODUCTION
The diversity of microorganisms make them most abundant on the earth, including the extreme environments. Yet the majority of them are unknown due to the non cultivability of about 96% of the microbes under the laboratory condition. The unexplored domain of bacteria can have a great potential in the field of agriculture, foresty, food industry, medicine, recycling of precious metal, environmental detoxification and others. In order to understand the exact role of these microbes in environment and how they can be applied to solve environmental problems, they need to be isolated and characterized.
Microorganisms have important roles in formation of earth crust as well as recycling of matter, thus important in understanding the geochemical cycle. Most of the microbes (>90%) in subsurface or sedimentary environment get attached to particular matters including the dead cells and use them for structural as well as catalytic function [1] . One of the area where microbes are quite important is the metalmicrobe interaction [2] . The mode of interaction with particulate matter like metals may be selective or non selective and also depends upon the external condition. Certain phenomenon exists where the microbial cell surface on getting saturated with metal leads to intracellular uptake of the metal where it gets deposited [3,4] .
Microorganism found in metal rich area showed different types of metal-microbe interaction. Most of the interaction occured either on the cell surface of the microbes (bio-sorption) [5] or they were transported into the cellular compartment for further processing. There can be localized accumulation of metals (bioaccumulation) also. The mechanism of interaction with metals depends on the physiological properties of microbes. Certain trace elements are important for the activation of enzymes like nitrogenase (Mo/Fe or V/Fe, or Fe only), cytochromes (Fe) and cytochrome oxidase (Fe, Cu) [6,7] . All these processes are controlled by genetically determined pathways [8] and sometimes they are fast, nonspecific, and constitutive. The other type of interaction involves the metals/ metalloids as electron acceptors or donors during metabolism.
In the context of bioremediation the microbial interaction with the elements is a means for removal, recovery or detoxification of metal species. This is mainly by the alteration of the behavior of the metal contaminant in the natural or engineered environment. Microorganism also develop a self defense mechanism in their own system against this toxic metal, one of the ways being transport of the metal out of the cell by efflux transport system. The microbial interactions leads to the alteration in the solubility of the metal which cause their mobilization or immobilization and thus helps in removal or detoxification of the contaminant. The alteration can be in terms of oxidation reduction process / involvement of surface charges in interaction with metal / excretion of solvents which helps in solubilization of the metals resulting in their removal or precipitation. One of the potential processes of metal microbe interaction is bioleaching which can be applied to recover valuable metal from a metal rich ore [9] . Bioreduction is an important metabolic process that transforms oxide minerals into reduced mineral phases, such as siderite and magnetite and convert dissolved oxidized metal into precipitate. This controls the fate and transport of multivalent metals like Fe(II) and Mn(II) in groundwater [10] .
Different groups of bacteria like Geobacter, Citrobacter, Acinetobacter, Pseudomonads, Sulfate reducing bacteria are reported to have implications in metal transformation. Another similar group are the magnetotactic bacteria which accumulate metal inside the cell, known as Bacterial Magnetic Particles (BMP) which further can be used for different purpose. These uptake the iron such as Fe 3+ , which moves to cytoplasm, gets converted to ferrous ion (Fe 2+ ) and are deposited as magnetite (Fe 3 O 4 ) or greigite (Fe 3 S 4 ). The metal accumulating bacteria have immense application in the field of biotechnology like enzyme immobilization, nanotechnology, immunology and bioremediation.
This study reports the isolation and characterization of a few microbes, isolated from rock dust collected from a magnetite ore rich area. The objective of the work was to study the culture based microbial diversity of metal accumulating bacteria from that location and to evaluate them as potential candidates of biotechnological importance.

Site of isolation and cultivation conditions:
The source was the rock dust from magnetic ore rich area in Andhra Pradesh, India. This site was selected because it was expected to be rich in microbes which would bear the property of metal tolerance. Since the site was an iron ore rich area, the primary aim was to screen for magnetotactic bacteria, therefore the isolation medium selected was Magnetospirullum magnetotacticum specific DSMZ-380 medium. The medium composition was slightly modified (K 2 HPO 4 0.5 gL −1 , NH 4 Cl 0.1 gL −1 , CH 3 COONa 1.0 gL −1 , yeast extract 0.1 gL −1 ). Later on, the pure isolates obtained were grown in the enriched medium Luria Bertani broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 1.5% agar, pH 7-7.5). Since no changes were observed in their morphological features when grown in the enriched medium, hence LB was used for further characterization and maintenance. Unless mentioned otherwise, the cultivation conditions for liquid culture involves overnight incubation at 37 o C with shaking at 150 rpm.

Morphological characterization:
The preliminary morphological characteristics were determined by bright field microscopy (Zeiss Axiostar Plus microscope) post simple staining with crystal violet dye. The details of the cell surface were found through general staining procedures like Gram staining, acidfast staining, endospore, capsule and flagellar staining. The cell shape and surface features were explored in more detail by Environmental Scanning Electron Microscopy (FEI QUANTA 200 MARK 2 at 15kV).

Molecular identification:
The molecular identification was on the basis of sequence analysis of 16S rDNA which occur as a conserved molecule in the bacterial domain. The initial step involved genomic DNA isolation by modified alkaline lysis method followed by PCR amplification using universal 16S rDNA primers. The detailed procedural steps, the primer specification and PCR conditions were followed as reported by RayChaudhuri et al. [11] .The PCR product was cloned into pGEMT-Easy vector and transformed into E. coli DH5α host by TSS method as reported by Chung et al. [12] .Transformed cells containing the desired insert were selected by gel retardation assay and were send for sequencing. The 16S rDNA sequences obtained were subjected to BLAST and were submitted to GenBank. The phylogenetic analysis was done by Neighbour Joining method.
Biochemical characterization: Presence of certain extracellular enzymes like protease, lipase, DNase, oxidase, catalase and lecithinase were confirmed following the biochemical characterization techniques as reported by Nandy et al. [13] . Specific ready made media (HiMedia laboratory) were used for detection of enzymes like DNase, lipase and lecithinase. Protease was detected on milk medium plates where the enzyme if present degraded the milk protein casein producing a clean zone around the colony. Catalase and oxidase tests are based on the basic property of the enzyme and were done on LB plates containing single colony of pure isolates.
Physiological Charcterization: Since pH & temperature are limiting factors governing the microbial growth, determination of the optimum conditions for these parameters are necessary. For temperature profile, the culture (1%) was grown in LB broth and was incubated for 12 h at 20 o C, 30 o C, 37 o C and 40 o C at 150rpm shaking condition. The Optical Density measured in UV-visible spectrophotometer (Beckman UV Vis spectrophotometer) at 660nM gave the growth efficiency. To find the pH range, LB broths of different pH were prepared using NaOH and HCl for acidic and alkaline range respectively. 1% inoculum was provided to medium of each pH range and the bacterial culture was incubated for 12 h at the optimum temperature found for each isolate. The presence or absence of growth was defined in terms of Optical Density at 660nm (UV Vis spectrophotometer).

Growth profile:
The overnight grown culture in LB medium acted as the parent source. 1% inoculation from the parent culture was added to 100mL of sterile LB medium and incubated at 37 o C under 150rpm shaking. At regular interval of 1 h, 3mL of culture was aliquoted and the optical density was measured at 660nM (Beckman UV-Vis spectrophotometer) which represented the extent of growth.
Effect of light/dark on growth: Not only the pH and temperature but also presence of light play a rate limiting step in bacterial growth. Each isolate was inoculated in LB medium and incubated for the entire growth phase in either darkness or in presence of light (13Watt incandescent lamp from Phillips). In the third set each culture was allowed to grow under alternative cycle of light and darkness. In each set after the entire growth cycle, the extent of growth was measured by checking the Optical Density at 660 nM (Beckman Coulter DV -530, UV Vis spectrophotometer).

Substrate profile:
The isolation medium DSMZ-380 contained sodium acetate as the main carbon source which was replaced by substrates like methanol, ethanol, D.L lactate at the similar concentration. After inoculating 1% culture, incubation was done in rotary shaker (150 rpm) for overnight at 37 o C. Bacterial growth was measured spectrophotometrically in terms of Optical Density at 660 nM.
Esterase assay: Esterase assay was done by spectrophotometric method reported by Lin and Ko [14] with certain modifications. To 930 µl of Tris HCl buffer pH 9, 20µl of 10 mM pNPP was added. 50µl of enzyme solution was added to the reaction mixture, mixed well and incubated at 50 o C for 20 min. The reaction was stopped by adding 330µl of acetone alcohol mixture at ratio of 1:1 and absorbance was measured at 410 nM. One unit of enzyme activity was expressed as 1µM of PNPP released / mL / min. Antibiotic assay: Susceptibility towards different antibiotics was checked using the antibiotic discs provided by HiMedia Pvt Ltd. The susceptibility was checked on a log phase culture. The antibiotic groups selected, the culture conditions, and the assessment procedure were followed as per Nandy et al. [13] Metal sensitivity test: Each metal was added as supplement in the LB medium and to it 1% inoculum was given from a log phase culture. The growth conditions were maintained at 37 o C with constant shaking of 150rpm. The tolerance was determined in terms of growth as visible from the appearance of turbidity. The concentration of metal was incremented in the medium and on the cessation of growth the MIC was determined. Multiple mechanisms could be involved in this metal tolerance, one of them being intracellular uptake. Metal accumulation was determined by Energy Dispersive X Ray Fluroscence which gives the quantification of the net metal accumulation as compared to untreated cells. The EDXRF analysis was done for two metals Cu and Cr that occur as common contaminants in the environment. Preparation of cells for EDXRF analysis, the procedural details were followed as per Adarsh et al. [15] and Chowdhury et al. [16] Pathogeneicity detection: The isolates were checked for the presence of pathogenic gene of P. aeruginosa and S. aureus by Taqman Detection Kit of Applied Biosystem. This detection was done by real time PCR to check the amplification of desired gene as per the procedure mentioned in the kit.   Fig 1). The staining details as put forth in Table 1 indicates that these isolates are of different identity in spite of the same source of isolation. These specific staining procedures revealed the surface details of the isolates, two of them were found to bear flagella while one was found to have a capsule. These structural features also indicated the ability of that particular isolate to use them for survival in extreme environment. Many more typical structural features like presence of pilli like structure, conjugation tubes (Fig 1 i, iii) were detected with the application of Environmental Scanning Electron Microscopy.

Molecular characterization:
The 16S rDNA based molecular phylogeny indicated that all the five isolates obtained were novel and belong to different genus. Moreover the partial sequence based analysis can confirm the identity to the genus level. Table 2 shows Diplococci Gm-ve ----   Fig 2( Biochemical characterization: All the isolates were found to be catalase positive ( Table 3). The enzyme has been reported to play a role in the defensive mechanism and would be important in the phenomenon of metal microbe interaction. Isolates like SAP 2 and SAP 5 were found to secrete extracellular lipase. The quantitative assay for lipase indicated that SAP 5 secretes about 9 units of enzyme whereas 7 units of lipase were secreted by SAP 2. The enzyme can be of commercial importance when applied in formulation of pharmaceutics and detergents.
Physiological characterization: All the isolates were found to tolerate temperature from 20 o C to 60 o C and the optimum temperature for all lies between 37 o C-40 o C ( Table 3). The pH profile of the isolates demonstrated an important finding where isolates SAP 5 and SAP 6 were found to tolerate a wider range of pH. The ability to grow at a lower pH (pH 3) would be important for the application in area contaminated with acid mine drainage. The light dark profile showed that the growth efficiency was affected differently by light/dark conditions (Table 4). In case of SAP 2, SAP 3 and SAP 4, growth was not much affected by alterations in these conditions where as for SAP 5, growth was accelerated in dark as well as with alternate dark and light phase. In case of SAP 6, light Table 3: Table showing    respectively. This indicates significant variation in case of SAP3, SAP5 and SAP6 at 95% confidence level. Different alternative carbon sources were replaced in place of sodium acetate in DSMZ 380 and the utilization efficiency of the isolates were compared. Isolates SAP 5 and SAP 6 were found to utilize these different substrates more efficiently in comparison to others ( Table 5). The different substrate utilization indicates the different metabolic pathway of the isolates, for example the pathway of starch utilization would be different from the sugars which would enter the TCA cycle.

Growth phase:
The growth pattern of the isolates was found to vary drastically. Isolate SAP 5 and SAP 6 demonstrated a very short lag phase followed by a long logarithmic phase as compared to SAP 2 and SAP 3 (Fig 2). These details were important for any application where the microbial growth can influence the probable functioning. For example the extent of biodegradation of any contaminant depends on the time length of interaction with the microbe along with the metabolic cycle of the microbe where the contaminant can act as a carbon source.
Antibiotic sensitivity: Table 6 indicates the sensitivity profile of the isolates towards various groups of antibiotics. The resistance and tolerance towards different antibiotics gives an idea of the nature of the isolate and the sensitivity profile provides the correlation with the tolerance towards metals. Since the metal and antibiotic resistance genes are reported to be localized in the same plasmid, the effect of one stress factor can be related to the induction of the other. This explains the co-occurrence of metal and antibiotic resistance together.
Heavy metal tolerance: Sine the objective was to look for metal accumulating bacteria, all the isolates were checked for tolerance for a spectrum of heavy metals and the Minimum Inhibitory Concentration (MIC) gives an idea about the concentration of a metal that a bacterium can tolerate. As depicted in Table 7, all the isolates shows tolerance towards different heavy metals and among them isolates SAP 5 and SAP 6 exhibited maximum tolerance towards Fe and Pb. The tolerance      SAP 2  3  3  1  3  2  3  3  1  SAP 3  3  3  2  2  2  3  3  3  SAP 4  1  1  1  1  1  3  3  2  SAP 5  1  4  1  4  2  6  6  8  SAP 6  10  8  1  5  2  6  11  12   Table 8: Table showing   towards Fe was expected since the site of origin being a mineral ore rich site (magnetite ore). The next objective was to check for the efficiency of intracellular accumulation. Cr and Cu metals were selected since they are the most common contaminants from industrial exhausts. SAP 5 and SAP 6 showed relatively higher accumulation of Cu (Table 8) which can be of importance for Cu recovery as well as detoxification.

CONCLUSION
Thus this study reveals the presence of 5 novel isolates from the mineral ore rich site in Andhra Pradesh, India. The heavy metal tolerance property of the isolates could be further exploited for means of bioremediation as well as processes like bioleaching for the economical recovery of important metals like Fe and Cu. The detailed characteristics of the isolates in terms of their temperature, pH tolerance, their growth profile would together facilitate in designing the application process.