INTRODUCTION:

Mining is the process by which valuable minerals or other geological materials are extracted from the earth. The metals to be extracted are usually bound up in solid minerals. Generally, the ores of these metals exist as metal sulphides and various chemical and physical processes are used to extract the metal of interest. The concentration of the mineral or metal in the rock decides the quality of the ore – high grade ore (with large metal/mineral concentration) or low-grade ore (with less metal/mineral concentration). The quality of the ore directly determines the cost associated with mining of that ore. As the global demand for metals is increasing, the high-grade ores of these metals are depleting at a very high rate and therefore our interest is turning towards low and lean grade ores. If conventional methods are used to mine the low and lean grade ores, it leads to environmental degradation and is very expensive as it requires high energy input¹.

An environmentally and economically accepted method for processing of the low grade lean ores is biomining², which is the process of extracting these valuable minerals using prokaryotes or fungi. Some microorganisms have the ability to oxidise these metals and thereby letting them dissolve in water. This is the main principle behind most biomining operations and it is mostly used for those metals which can be easily recovered when dissolved rather than when directly extracted from the solid rock ores. This process of directly dissolving the metal of interest using microorganisms is called as bioleaching. For example, while extracting copper, copper sulphide is oxidised to copper sulphate by the catalysing reactions of the microorganisms and therefore metal is present in the aqueous phase and remaining solids are castoff.

Alternatively, in some other biomining processes, metals which cannot be dissolved by these microorganisms, uses such microbes which break down the surrounding minerals and therefore leaving rock enriched with the metal of interest. This process is called as biooxidation. For example, in the case of gold mining operations, microorganisms are used to remove pyrite and arsenopyrite from gold ore³.

In principal, there are two types of Bioleaching: Direct and Indirect.

Direct bacterial leaching:

When there is physical contact of the microorganism with the insoluble sulphide surface, it is called as Direct Bioleaching. In this process following reaction takes place:

MS + 2O₂ MSO₄

Where M is a bivalent metal.

Indirect bacterial leaching

When bacteria secrete a lixiviant which in turn chemically oxidises the sulphide mineral. In acid solutions, the lixiviant generated is ferric ion and following reaction takes place:

MeS+ Fe₂(SO₄)₃MeSO₄ + 2FeSO₄ + S^ₒ

Where M is a bivalent metal.

The ferrous ion generated by this process can be re-oxidised to ferric ion by certain bacteria and therefore can participate in the oxidation process again.

Diversity amongst mining bacteria

The bacteria found in these hostile environments are one of the most extraordinary life forms known to us. These microorganisms are chemolithotrophs, i.e., they obtain energy from the oxidation of inorganic substances. Predominantly these metal dissolving microorganisms are extremely acidophilic bacteria (bacteria which thrive at pH<3). Since there are only a limited type of substrate which are available in the mines therefore the microbial diversity in these environments was expected to be poor. Nevertheless, till now mining environment has shown a great deal of microbial diversity with at least 11 putative prokaryotic divisions living at AMD sites.

For many years Thiobacillus ferrooxidans was found to be the only important leaching bacteria. It is an acidophilic bacterium which obtains energy from oxidizing ferrous ions and reducing sulphur compounds. Ferric ions are the product of oxidation of ferrous ions and are a very potent oxidant and can oxidise sulphide minerals. According to direct bioleaching process, the following reactions take place:⁵

A. ferrooxidans

CuS (covellite) + 2O₂ C . uSO₄

A. ferrooxidans

ZnS (sphalerite) + 2O₂ ZnSO₄

According to indirect leaching mechanism theory, the following reactions take place:

Indirect mechanism reaction: 1

A. ferrooxidans

^{4FeSO4 + O2 + 2H2SO4 2Fe2(SO4)3
+ 2H2O}

^{Indirect mechanism reaction:
2}

^{CuFeS2 (Chalcopyrite) +
2Fe2 (SO4)3 CuSO4 +5FeSO4 + 2S˚}

^{FeS2 (Pyrite) + Fe2(SO4)3
3FeSO4 + 2S˚,}

^{UO2 + Fe2(SO4)3 + 2H2SO4
UO2 (SO4)4-3 + 2FeSO4 + 4H+}

Later other important bacteria were found, like Thiobacillus thiooxidans, Leptospirillium ferrooxidans and Thiobacillus organoparus. From detailed investigations it was then later found that Leptospirillium ferrooxidans is found more abundantly than Thiobacillus ferrooxidans. The most robust leaching mechanism is displayed by the bacteria of the genus Sulfolobus. These bacteria flourish in acidic hot springs and volcanic fissures at temperatures that can exceed 60⁰ Celsius. Sulfolobus acidocaldarius and S. brierleyi oxidize sulfur and iron for energy, relying on either carbon dioxide or simple organic compounds for carbon⁶.

Extensive research has been going-on on these mining bacteria in the fields of metallurgy and biotechnology. New high throughput sequencing techniques and molecular methods such as 16S rRNA gene sequencing and DNA-DNA hybridization etc. are being used to isolate and identify new strains. The results reveal that the microbial leaching communities consist of a plethora of microorganisms and the processes used by all these microorganisms during bioleaching are unique⁷. As in the case of studying the site of a red coloured acidic geothermal pond, ‘Chinoike Jigoku’ in Japan, several methods were applied to investigate the microorganisms present there which can survive in highly acidic environment and have metal reducing ability. Acidophiles like Acidithiobacillus sp.⁸, Sulfolobus sp., and Alicyclobacillus sp. were characterised and one new strain of Sulfolobus sp. was isolated⁹. In other study in China on coal mine drainage, several strains were isolated and majority of them belonged to Acidithiobacillus sp.¹⁰ and a few of them belonged to Pseudomonas sp. and Legionella sp.¹¹.Moreover, locally in India itself, a number of strains have been isolated and characterized, for example in one study of Malanjkhand mines, several strains of Proteobacteria, Actinobacteria and Chloroflexi were isolated and characterized¹².

New strains are being isolated locally and are being genetically manipulated to increase the rate of bioleaching and biooxidation and consequently there is a rapid increase in the data related to mining bacteria. Therefore, there is a necessity of an integrated database which can serve as repository for the mining bacteria and hence we created an interactive and informative web database in which the information related to these bacteria can be deposited as well as extracted. There are numerous approaches to identify potential drug target, for example, uncharacterized essential genes, virulence genes, species– specific gene and membrane transporter¹³. Comparative genomics gives a novel way to deal with perceived new drug focuses among prior known targets in based on their related biological function in pathogen and host. In the arranged work subtractive genomic approach is utilized, where subtraction dataset looking at two genomes i.e. pathogen and host. This approach is productively utilized as a other bacteria, for example Pseudomonas aeruginosa^14,Helicobacter pylori¹⁵ etc. The possibility of complete Proteome sequence report of Mycoplasma Pneumonia from NCBI- FTP site helps to complete the BLAST against human proteome database¹⁶.

There are some infections viruses which live just for a few of minutes outside the host while the spore-forming microorganisms may live in a dormant state for over 10 years. A few infection spread through the inhalation of virus infected air while other infections are transferred through the ingestion of infected water or food¹⁷. TADB2.0 (http://bioinfo-mml.sjtu.edu.cn/TADB2/) is a amend database that gives extensive data about bacterial type II toxin– antitoxin (TA) loci. It also helps to consider the genomic context of predicted TA loci for putative virulence factors, antimicrobial resistance determinants to the definitive public databases. A renew type II TA-particular resource which is relied to encourage proficient examination of extensive quantities of these systems, recognition of patterns reciprocal to cell focuses of differing toxins, and an enhanced comprehension of biological roles and significance¹⁸. Mining bacterial genomes for bacteriocins is a require for because of the substantial structure and sequence diversity, and little sizes, of these antimicrobial peptides. The scoring arrangement was fine-tuned utilizing expert information on information obtained from screening every single bacterial genome currently accessible at the NCBI. BAGEL2 is openly accessible at http://bagel2. molgenrug.nl. Rapid approach in computational meta-genomics, sequencing methods and refining contig lengths, will permit bacteriocin mining in meta-genomic information with BAGEL2¹⁹.

In this database we have focused on microbiome which are related to mines and listed them out (mining bacteria, morphology, biochemical identification, culture media, molecular study, applications, and references). Since there are many databases existing on various information about microbiome eg. pathogenic, non-pathogenic, airborne, waterborne, RNA databases. Here we have constructed database which is strongly focused on mine related microbiome (Pathogenic and non-pathogenic and their consequences). This will help the researcher to get the detail information on mine related microbiome²⁰.

This database fills the void in scientific community as no such database which caters need of researchers. This database gives one stop solution to all details of microorganisms. The data of microorganisms are scattered in the galaxy of vast published material. We have dug deep into published material and burned night oil to consolidate all microbial, biochemical and molecular details of an industrially useful bacteria which is found in mines throughout the globe and created this database. This database provides detailed featuring on mines related microbiome (mining bacteria, morphology, biochemical identification, culture media, molecular study, applications, and references). Substantially we have included that added information about mining bacteria, morphology, biochemical identification, culture media, molecular study, applications, and references. As well as, we have extended and identified different species of the bacteria²¹.

MATERIALS AND METHODS:

Data Source and Content:

The isolate developed well on HMC media. Numerous sorts of metal resistance bacteria have been isolated. The majority of them have a place with genera Bacillus, Pseudomonas, Comamonas, Alcaligenes, and Streptomyces^{22, 23}. Upon reviewing research articles related to bacteria involved in bioleaching and biomining, a database of the bacteria using MS excel sheet was created. In the database, every bacterium has its basic information of morphological characteristics, biochemical identification, culture media, molecular study, type of bacteria, country they were isolated in and their applications in mining industry as their attributes in the database. The information was derived from reviewing and studying several research and review articles.

Database Construction and Design:

A cumulative and informative data repository in collaboration with a universally accessible and user-friendly interface was developed. Two interfaces were created- the main homepage and an admin page. The main homepage will be available to all the users and the admin page can only be accessed by the website administrator. The purpose of this interface is to provide users with a platform where they can view or update existing entries and add new entries to the database (by providing legitimate references), pending approval of the administrator. The user can ‘search’ the database by using any information related to any attribute as a query, making the database accessible in any possible way. The front-end script is developed using HTML, CSS and JavaScript and the back-end script is developed using MySQL and PHP.

For the back end, the database prepared on the excel sheet was converted to .csv format and imported on phpMyAdmin. phpMyAdmin is a free and open source administration tool for administration of MySQL tools and hosting web services. In the main database, the attributes were defined by morphological characteristics, biochemical identification, culture media, molecular study, type of bacteria, country they were isolated in and their applications in mining industry. Each bacterial entry defined a new row. Additionally, one more attribute was made, Status, which described if that row entry of bacteria is approved by the administrator or not. Since the database can be updated with new entries, it serves as a mechanism for displaying only those entries which are approved by the administrator. The status can be 1, 0 or -1 which denote the status as approved, pending or disapproved, respectively.

In order to make the data accessible by searching for any attribute, the front end was developed using MySQL search queries. For any attribute if some information was sent as an input, it would run a query and search the database for that particular attribute which matches with query and display results. To obtain data regarding biochemical tests, drop-down input format type was used and for countries checkbox input format was used. The front end and back end were connected using JavaScript functions and PHP so that the webpage is more dynamic.

Bacterial 16S rRNA sequences were obtained from the database and in comparison the frequency of the amino acids coded by the RNA sequences. The 16S rRNA sequences were information mined from BFDB i.e. Biofilm Forming Bacteria Database (http://www.info.vit.ac.in/bfbd/home.html) which is control by VIT University. The SERB funded the formation of this BFDB site to have a global database on Biofilm forming bacteria. This site is an easy to understand and interactive stage to understand the contribution of individual bacteria from planktonic form to surface adsorbed conversion²⁴. BFBD is world's first Biofilm forming bacterial database which is serving scientists to abundantly investigate metabolic interaction, phylogenetic grouping and competition among members of these communities in a more extensive range²⁵.A C⁺⁺ program was made to determine the frequency of the codons that codes for the respective amino acids²⁶.ATMs were very much contaminated with pathogenic bacteria forms particularly on numeric keys. Even the user interface was infected with high amount of pathogenic bacteria. Subsequently, the investigation finds out that ATM interfaces may act as potential source for the transmission of diseases²⁷.

RESULTS AND DISCUSSION:

Data Source and Content:

A database of bacteria was created using MS excel sheet upon reviewing research articles related to bacteria involved in bioleaching and biomining. The database is created for majority of the bacteria that belong to the genera Bacillus, Pseudomonas, Comamonas, Alcaligenes and Streptomyces^22,23. In the database, every bacterium has its basic information about morphological characteristics, biochemical identification, culture media, molecular study, type of bacteria, country they were isolated in and their applications in mining industry as their attributes in the database. The information was derived from reviewing and studying several research and review articles. Database was successfully created and hosted by VIT which can be utilized by link https://minemicronet.000webhostapp.com/index.php

Utility of the database:

A freely accessible, informative interface is formed. The database is designed in such a way that the user can access the database via all the attributes of the bacteria, i.e., the user can input any information he knows related to attributes, and the bacterium(a) corresponding with those attributes is shown.

Any user can easily access the database and can view it. Whilst updating old entries or uploading new entries, the user needs to log in and then fill in the form. The details will be updated/uploaded after the approval of administrator so as to avoid incorrect entries. If there is experimental data to support the claim of the user while adding/ updating entries, the administrator approves it and the database is updated. Therefore, this is an ever-expanding database which can be accessed from any part of the world.

It is an interactive database from which users can retrieve as well as deposit data regarding mining bacteria. It will serve as a cumulated informative platform for users where they can get information regarding various mining bacteria at one place and can refer it for further research. The database is created in such a way that the user can access it in such a way that he can input any information he knows about any of the attributes and the bacteria corresponding to that hits up and is shown on screen. Similarly, the user can search the bacteria by its name and all the information which is known about the attributes is shown. For example, if a user wants to know all the gram-positive bacteria involved in copper mining, he can enter this information in the corresponding attributes and the matching bacteria is sorted and shown. This way, the user can easily identify the domains in which a certain bacterium needs to be studied more and vice versa²⁸.

Figure 3: Search the database by any attribute

Figure 4: Admin page for approval or declination of requests to update database.

Moreover, since it’s an interactive platform, any researcher from any part of the world can access it and input their data regarding an existing bacterium in the database or create a new entry for new bacteria they have isolated from the mines. Therefore, this ever-expanding database can be updated from entries all over the world and will have latest entries related to the mining bacteria.

Through this database, the user can identify the bacteria involved in biomining operations industrially, identify and study the genes and proteins involved in metal leaching and therefore can create superior strains which can make biomining operations faster and industrially viable. Through the experiments and research, a large amount of data related to genes and proteins and microbiological growth conditions is being generated and we can incorporate all this data at one place so that the researchers all over the world are updated with newest experiments²⁹. This database will act as a repository for all the data related to bacteria involved in biomining and therefore is an indispensable tool for researchers and industrialists all over the world³⁰.

FUTURE SCOPE AND EXPANSION:

This database is and will act as a repository for all the mining bacteria and can later be expanded to other classes of microorganisms as well, like yeasts and fungi, involved in biomining. As the research in the field of biomining and mining bacteria advances, the isolation of new strains from mines and development of new strains of microorganisms using gene manipulation, the researchers will be able to deposit the details regarding them and hence it will be an auto updated database with the details of the newest mining microbe found. In future, as the advancement of available technology continues, additional data related to these microorganism like microscopic images and SEM images can be added. Molecular data related to the microorganism and the primer information can be put in. Therefore, as the database expands in number of organisms, it will also expand in the number of attributes, that is, the data related to each and every microorganism related to genes and proteins, their microbiological growth conditions, and their applications in different areas of biomining will also grow.

Industrially it will help as a separate section about industrial applications of the bacteria related to bio-beneficiation, E- waste management etc. will be added in order to aid the research of big mining industries will be added. A large number of microbes are associated with the solubilisation of minerals and metals as this approach offers various favourable circumstances over conventional methods³¹. Therefore, this database will act as cumulated repository of and for all the researchers around the globe.

While plants have been an extraordinary of beneficial bioactive compounds, consideration has as recently turned to endophytes which are microorganisms (bacteria and fungi). Those are living in the intercellular spaces of plant tissues denied of triggering any noticeable detriment. They are perceived as potential sources of novel secondary metabolites with potential application in pharmaceutical, farming and industry³².

CONCLUSION:

A freely accessible, informative and cumulated database, from which the data related to any kind of mining microorganism can be extracted and any new data related to existing or new strains can be uploaded so that a uniform informational repository is formed. The database is designed in such a way that the user can access the database via all the attributes of the bacteria, i.e., the user can input any information he knows related to attributes, and the bacterium(a) corresponding to those attributes are shown. Any user can easily access the database and can view it.

ACKNOWLEDGMENTS:

The authors are grateful to VIT for hostage site of database for mining bacteria and CTS department of VIT University. We would also like to acknowledge VIT lab facility and Gargi Vyas, student of VIT University for helping us in the development of the interface.

CONFLICT OF INTEREST:

The authors declare that there are no conflicts of interest regarding the publication of this manuscript.

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Received on 31.05.2018 Modified on 12.07.2018

Research J. Pharm. and Tech. 2019; 12(3): 1122-1128.

DOI: 10.5958/0974-360X.2019.00185.9