1. Introduction

Microorganisms being the pioneer colonizers of this planet represent the richest repertoire of molecular and chemical diversity in nature. The omnipresence of microorganisms owing to their ubiquitous nature enables them to adapt and exist in virtually every ecological niche on earth, including natural as well as man-made ecosystems. However, studies indicate that the nature and identity of only a tiny fraction of the available microbial diversity is known ^[31]. Current evidence suggests that of the 300,000 to one million species of prokaryotes existing on earth only 3,100 bacteria have been described in the Bergey’s Manual of Systematic Bacteriology ^[15]. Around 5,000 species of prokaryotes have been reportedly identified, which represent only about 1 to 10% of the existing bacterial species.

Current research needs to focus on screening the microbial diversity in man-made or adaptive environments, which will be useful for specific biotechnological applications, such as bioremediation. There has been a tremendous increase in the number and types of various industries, with all industrial operations generating wastes in one form or the other. Liquid waste, either treated or untreated, is generally described as ‘effluent’. Nature of the microbial diversity of industrial effluents, in particular, the effluent from the electroplating industry remains largely uncharacterized. There is, therefore, a need for screening the microflora from man-made or adaptive environments such as the electroplating effluent which are likely to have variety of toxic heavy metals.

Electroplating sector contributes a major part in deteriorating the local environment due to the persistent accumulation of heavy metals in the environment which are toxic, non-biodegradable and accumulate in the food chains through ‘bio-magnification’ ^[14]. Microbial communities residing in electroplating effluents encounter and interact with various types of heavy metals of which, some are also precious metals ^[18]. Metals exert an inhibitory action on microorganisms by blocking essential functional groups, displacing essential metal ions or modifying the active conformations of proteins and nucleic acids ^{[9, 12]}. In spite of this, microbes have learnt to survive in such environments. Microbial survival in heavy metal polluted environment is facilitated by acquiring intrinsic biochemical and physiological properties by genetic changes triggered by necessity and also ‘by random occurrence followed by selection. Thus, long term exposure to heavy metals leads to the selection of a microbial community which then adapts to the polluted environment.

Manefield et al. reported the detrimental effect of heavymetalson the microbial populations and reasoned only such microbial flora will survive that can tolerate the heavy metal concentrations ^[18]. Microbial diversity studies in heavy metal polluted watersby Satchanska et al. have reported a reduction in diversity of the indigenous water micro flora and alteration of the microbial community structure of the region ^[29]. Ezzouhriet al. reported that pollution of water by heavy metals promotes the selection of resistant species and decreases the microbial diversity ^[7].

There are few reports on the microbial diversity of industrial effluents. Some among them include microflora of effluents of pesticide, pharmaceutical and textile industry. Rani et al. reported the presence of species belonging to the genera Alcaligenes, Bacillus, Pseudomonas, Brevundimonas, Citrobacter, Pandoraea and Stenotrophomonas from the effluent of a pesticide industry. They also reported the presence of species ofAgrobacterium, Brevibacterium, Micrococcus, Microbacterium, Paracoccus and Rhodococcus in the effluent of a pharmaceutical industry ^[23]. Faryal and Hameed described the presence of fungi including species of Rhizopus, Aspergillus, Penicillium, Candida, Drechslera and Rhodotorula in the effluents of a textile industry ^[8].

Malekzadeh et al. reported the presence of a Pseudomonas strain, designated MGF-48 that he isolated from the effluent of a metal melting factory in Tehran. The organism was shown to be efficient in the bioaccumulation of uranium, lead, copper and other metal ions from polluted effluents ^[17].

Parameshwari et al. reported the isolation of metal-tolerant fungi including Aspergillus niger, Phanerochaete chrysosporium and Trichoderma viride from municipal sewage contaminated soil ^[21].

Jamaluddin et al. obtained five metal tolerant bacterial isolates from the wastewaters of a Malaysian gold mine. They were identified to be belonging to the genera Bacillus and Achromobacter. ^[13].

Sarkar et al. isolated and characterized novel bacteria from a mineral-ore site in Andhra Pradesh, India revealing its rich biodiversity. The cultures were identified as Pseudomonas fluorescens, Janibacter anopheles, Bacillus licheniformis and Acinetobacter baumannii. The culture isolateswere screened for their potential for metal accumulation and all of them were found to tolerate a wide range of heavy metals ^[28].

In recent years, microbes have been drawing tremendous attention due to their extraordinary ability to metabolize waste materials and thereby improving water quality. Taking the above facts into consideration, the nature of microbial flora including bacteria and fungi were studied. This study presents one of the few studies aimed at characterizing the microbial diversity prevalent in the effluents of a gold electroplating unit.

2. Materials and Methods

Effluent samples were obtained from a gold electroplating industry located at Kandivali, Mumbai (India). The sample was collected in sterile glass bottles and stored at 4°C for further use.

Metal content of the effluent sample was analyzed at Sophisticated Analytical Instrument Facility (SAIF), at Indian Institute of Technology (IIT) Bombay, India, using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES). Also, concentration of the metals Copper, Cadmium, Lead, Silver and Gold was determined by the same technique.

[Report details: Ref. – ICP-39; Date – 21/07/2010].

Prior to isolation of bacteria, pH, temperature, odor and clarity of the effluent sample was noted. Isolation of chemo heterotrophic bacteria was done by streak plate method and pour plate method. Pour plate method was done using sterile Nutrient Agar and Glucose Yeast Extract Agar medium adjusted to pH 7.4. Decimal dilutions of the effluent samples were plated in triplicates and the plates were incubated at 30°C for 48 hours.

Also, enriched samples were used for isolation of species that may be present in low numbers. Aerobic spore bearing bacilli were isolated by pasteurizing the effluent samples by holding at 80º C in boiling water bath for 10 minutes.

Features of surface colonies including shape, margin, elevation and color were recorded using electronic colony counter and viewer. Gram-stained smears of the isolates were observed recording the morphology, cell arrangement, Gram reaction and other features such as the presence of endospores.

The biochemical characterization of the isolates was done using various biochemical media. The media used for biochemical identification are shown in Table 3. Sugar fermentation was studied using peptone water-base with inverted Durham’s tube and pH indicator. Suitable reagents were used with the biochemical media to read the results. The results were fed into and analyzed using online software “ABIS (Advanced Bacterial Identification Software) ONLINE” and the cultures were identified.

(https://www.tgw1916.net/software.html).

The bacterial isolates were subcultured on fresh Nutrient Agar medium slants and submitted to National Centre for Cell Science (NCCS), Pune, India for molecular identification based on their 16S rRNA gene sequences. The partial nucleotide sequences were obtained. The data was then subjected to BLAST analysis and the identity was established on the basis of closest neighbor showing the maximum % of identity. If no close match to an existing 16S rRNA gene sequence is found, then the test sequence represents a new species. The sequence data of novel isolates obtained were submitted at NCBI, GenBank to acquire unique accession numbers subsequent to which new strain designations were given to them by the authors.

Fungal species were isolated using sterile Sabouraud’s Dextrose Agar and Glucose Yeast Extract Agar medium adjusted to pH 5.4 using suitable decimal dilutions of the effluent samples in triplicates by pour-plate method and incubated at 25°C for 48 -72 hours. Chloramphenicol was added to the media to prevent bacterial contamination.

Growth of the fungal isolates was assessed by measuring diameter of zone of growth (in mm) on Sabouraud’s Dextrose Agar, 4% Malt Extract Agar, Malt Extract Peptone Agar, 2% Yeast Malt Glucose Agar and Potato Dextrose Agar plates by central spot inoculation of the spore suspension prepared in 0.05% Tween 80 solution (density, 0.01Å unit) for a period of five days.

All the fungal isolates were submitted on fresh Sabouraud’s Dextrose Agar slants to National Centre for Cell Science (NCCS), Pune, India for molecular identification based on 18S rRNA gene sequences. The partial nucleotide sequences were obtained. The sequences were then subjected to BLAST analysis and the identity of the cultures was established on the basis of closest neighbor showing the maximum % of identity. The sequence data of novel isolates obtained were submitted at NCBI, GenBank to acquire unique accession numbers subsequent to which new strain designations were given to them by the authors.

3. Results and Discussion

Microbial communities residing in electroplating effluents encounter and interact with various types of heavy metals of which some of them are also precious metals and only such microbial flora are likely to survive which can tolerate the elevated heavy metal concentrations. Effluent samples obtained from a gold electroplating unit were used for studying the bacterial and fungal diversity. pH of the sample was checked by using pH paper and was found to be 7.5. Temperature was found to be 32°C. The sample was clear and had an ammoniacal odor.

Metal content of the effluent sample was analyzed both qualitatively and quantitatively using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) to detect the presence of heavy metals. The effluent was found to have twenty one elements of which seventeen were found to be heavy metals (Table 1). The metal content analysis of the effluent sample showed the presence of a cocktail of metals including Cu, Zn, Mn, Fe, Ca Cr, K, Na, Ni, Pb, Al, Ag, Mg, Cd, As, Ti and Au. Concentration of five metals including three heavy metals (Copper, Lead, and Cadmium) and two precious metals (Silver and Gold) were quantified. Their concentrations are shown in Table 1. The synergetic toxicity of a variety of heavy metals present in the effluent was considered a good reason for recovering many metal-tolerant microbial species and possibly multi metal resistant (MMR) species.

Both traditional as well as molecular-based approaches were used for the identification and characterization of culturable bacterial and fungal species. A unique set of chemo-heterotrophic organisms comprising of nine bacteria and eleven fungi were isolated.

About the bacterial isolates: The streak plate method did not show the presence of many colony types because of the low standard plate count of the sample. Pour plate method gave isolated colonies as undiluted and decimal dilutions of the sample were used for plating. Features of well isolated surface colonies were studied. The size, shape, surface, margin, elevation and pigmentation of the colonies were recorded (Table 2). Gram-stained smears of the isolates were observed for recording the morphology, cell arrangement, Gram reaction and other features such as the presence of endospores (Table 2).

Table 1. Results of ICP-AES Analysis of the Effluent Sample

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Table 2. Colony Characteristics of the Bacterial Isolates (Culture Medium: Nutrient Agar; Incubation Time: 48 hours; Incubation Temperature: 30°C)

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The biochemical characterization of the isolates was done using various biochemical media. The media used for biochemical identification and the results of the biochemical tests are shown in Table 3.

The biochemical test results were analyzed using online software “ABIS (Advanced Bacterial Identification Software) ONLINE” and the cultures were identified.

Table 3. Biochemical Characteristics of Culture Isolates

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Table 4. Partial 16S rRNA Sequence Based Identification of Bacterial Isolates

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The isolates were submitted to National Centre for Cell Science (NCCS) Pune, India for 16S rRNA sequencing. The partial nucleotide sequences obtained were further subjected to BLAST analysis and the identity was established on the basis of closest neighbor. On the basis of morphological features, biochemical and molecular identification, the bacterial isolates were found to belong to five genera including Staphylococcus, Achromobacter, Klebsiella, Macrococcus, Pseudomonas and Bacillus. (Table 4).

Among the nine isolates, four were found to be new bacterial species. The sequence data of novel isolates obtained were submitted at NCBI, GenBank to acquire unique accession numbers subsequent to which new strain designations were given to them by the authors.

The isolate BGW1 was identified as a novel uncultured species belonging to the Staphylococcus genus (GenBank Accession no. KC009762) and most closely the resembled Staphylococcus gallinarum strain. A high chromate resistant strain of Staphylococcus gallinarum has been reported earlier from soil contaminated with tannery effluents ^[1].

Isolates BGW2 and BGW8 were identified as species of genus Achromobacter. Isolate BGW2 was also found to be an uncultured Achromobacter species (GenBank Accession No. JX966369) and isolate BGW8 was identified as a strain of Achromobacter xylosoxidans [Table 4]. Classen et al. have reported the frequent occurrence of Achromobacter xylosoxidans in aqueous environments ^[5]. Jamaluddin et al. have reported the detection of an Achromobacter species in the wastewater samples obtained from a gold mine environment in Malaysia ^[13]. Interestingly, two species of the genus Achromobacter namely, Achromobacter xylosoxidans subsp. denitrificans and Achromobacter piechaudii, were also reported from the gold mine environments of Australia by Santini et al. ^[27]. Further studies can be done to examine the adaptation of these organisms to inhabit Gold-rich environments from an ecological point of view.

Isolate BGW3, grew as a highly viscous colony with a pearl like surface and was identified as a strain of Klebsiella pneumoniae. A viscous flowing mass of exopolysachharide was produced by this culture. Gum production was found to be enhanced on medium supplemented with Glycerol. This culture hence holds promise as a potential candidate for biogum production.

Isolate BGW4 was identified as a new species of Macrococcus genus (GenBank Accession No.JX944685) This genus Macrococcus at present includes only seven species which were typically isolated from animal origin and the present work identifies yet another species of Macrococcus from an effluent sample.

Isolates BGW5, BGW6 and BGW9 were found to belong to the genus Bacillus. Isolate BGW5 was identified as a strain of Bacillus thuringiensis. B. thuringiensis is a soil-dwelling organism naturally occurring in aquatic environments, insect-rich environments, leaf surfaces and animal feces. Oves et al. have reported the presence of Bacillus thuringiensis in soil contaminated with industrial effluents. They have found the organism to show efficient biosorption for heavy metals including cadmium, chromium, copper, lead and nickel ^[20]. BGW6 was identified as a novel Bacillus species (GenBank Accession No.KC009763). Isolate BGW9 was identified as a strain of Bacillus licheniformis. It showed colony morphology that resembles the appearance a flower with five to six petals on Glucose Yeast Extract Agar plate. A metal-accumulating Bacillus licheniformis strain has been reported by Sarkar et al. from a mineral-ore site and was found to tolerate a wide range of heavy metals ^[28].

Isolate BGW7 was identified as Pseudomonas aeruginosa. This bacterium is ubiquitous in most heavy metal polluted environments and is capable of accumulating several heavy metal ions suggesting their excellent potential for bioremediation. Pseudomonas aeruginosa has also been reported to degrade synthetic-detergents and possess high degree of heavy metal tolerance and multiple antibiotic resistances ^{[10, 17, 19, 22, 29]}. Thus, among the nine cultures isolated and identified, four were found to be novel and hitherto unreported species of bacteria including the isolates BGW1 (Staphylococcus sp. ss-1), BGW2 (Achromobacter sp. ss-2), BGW4 (Macrococcus sp. ss-4) and BGW6 (Bacillus sp. ss-6).

About the fungal isolates: Eleven fungal cultures were isolated from the effluent sample and their growth characteristics were studied on different plate media [Table 5]. Spore suspensions (density, 0.01Å unit) of the fungal cultures were prepared in 0.05% Tween 80 solution and spot inoculated in the center of Sabouraud’s Dextrose Agar, 4% Malt Extract Agar, Malt Extract Peptone Agar, and 2% Yeast Malt Glucose Agar and Potato Dextrose Agar plates. The relative growth rates were assessed by measuring the diameter of the zone of growth (in mm) over a period of five days at ambient temperatures. Most isolates (BPF3, G1F4, G2F5, G3F6, G4F7, B1F8, B2F9 and B3F10) grew best on Sabouraud’s Dextrose Agar medium (Table 5).

Table 5. Relative Growth of the fungal isolates on plate media

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Table 6. Partial 18S rRNA Sequence based Identification of Fungal Isolates

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The isolates were identified based on their growth characteristics and their 18S rRNA gene sequences. The partial 18 S rRNA sequences of the fungal isolates are shown in Table 6. A total of eleven fungal species were obtained. Of the eleven fungal cultures, seven belonged to the genus ‘Aspergillus’, two of the isolates were species of genus Penicilliumand the remaining two were found to be Cladosporium sphaerospermum and Cochlioboluslunatus based on the 18S rRNA sequence match analysis. Ten of the eleven fungi were found to be new strains and their sequence data was submitted to NCBI, Gen Bank.

Isolates RF1 and RF2 were identified as species belonging to the genus Penicillium. Both the isolates turned the growth medium red due to secretion of colored metabolite. Isolate RF1 was identified as a strain ofPenicillium marneffei (Teleomorph: Talaromyces marneffei). It showed a distinct thermal dimorphic nature with a mycelial morphology at 25°C (Figure 1) and yeast-like morphology at 37°C (Figure 2) ^[32]. Isolate RF2 was identified as a strain of Penicillium pinophilum. A metabolite from Penicillium pinophilum, 3-O-methylfunicone (OMF), has been reported to have anti-proliferative effects and was effective in selective inhibition of breast cancer stem cells and inducing apoptosis ^[4].

Figure 1.

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Figure 2.

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Isolate BPF3 was identified as Curvularia lunata (teleomorph: Cochliobolus lunatus). The growth of the culture caused darkening of the medium. The medium turned brown in color. The darkening has been attributed to melanin synthesis by Rizner and Wheeler ^[24]. The pigment was used for textile dyeing. The pigment imparted a skin color to woolen and silk fabrics ^[30]. Cochliobolus lunatus is also reported to synthesize mammalian-like steroid hormones and steroid-binding proteins. 17β-Hydroxy steroid dehydrogenase (17β-HSD) from this fungus has been described as the first known fungal pluripotent HSD ^[25]. Curvularia lunatahas been used for producing silver nanoparticles that was shown to have larvicidal activities ^[26].

Isolates G1F4, G2F5, G3F6, G4F7,B1F8, B2F9 and B3F10 all belonged to the genus Aspergillus. Isolates G1F4, G2F5, G3F6, and G4F7 produced colonies in various shades of green color owing to the green colored spore masses.

Isolate G1F4 and G2F5 were identified as strains of Aspergillus tamarii producing abundant conidial heads in dull yellowish-green shade which became metallic-bronze in color at maturity. Aspergillus tamarii has been reported earlier as a potential tannase producer that finds application in the food and beverage processing ^[6].

Isolates G3F6 and G4F7 were identified as strains of Aspergillussydowii and Aspergillus flavus respectively [Table 6]. Asperigillus sydowii produced blue-green to grayish-turquoise colored colonies. The extracellular enzyme ‘β-glucosidase’ purified from Aspergillus sydowii has reportedly shown great potential for several biotechnological applications such as the production of bio-ethanol from agricultural biomass and for improving aromatic character of wines and fruit juices ^[16]. Aspergillus flavusproduced powdery masses of yellow-green spores. Aspergillus flavus is a well-documented fungus for its aflatoxinproducing properties.

Isolates B1F8, B2F9 and B3F10 gave black colored colonies. They were identified as species of genus Aspergillus. Two of them, Isolates B1F8 and B3F10 were identified as strains of Aspergillus niger and B2F9 isolate was identified as a strain of Aspergillus awamori. The latter grew in broth cultures as star-like pellets. Aspergillus awamori has been reported to produce an extracellular lipase that could reduce 92% fat and oil content in effluents laden with oil and indicated scope for potential applications in bioremediation ^[3].

Isolate WBF11 showed a wrinkled black appearance on plate cultures and was identified as a strain of Cladosporium sphaerospermum. Cladosporium species are common in indoor and outdoor air and occasionally from plants and humans. Cladosporium sphaerospermum has been described as one of the most common air-borne organisms. The fungus is known to produce Gibberellins plant growth-promoting metabolites ^[11].

Thus, among the eleven fungal cultures isolated and identified, seven of them belonged to the genus Aspergillus, one to genus Penicillium, and the rest one each to Talaromyces, Curvularia and Cladosporium genera. We see that Fungi Imperfecta members comprise a big group followed by Ascomycetes group. Ten of them were found to be new strains of fungi including Talaromyces marneffei GEF-1 (RF1), Penicillium pinophilum GEF-2 (RF2), Curvularia lunata strain GEF-3 (BPF3), Aspergillus tamarii strain GEF-4 (G1F4), (Aspergillus tamariiGEF-5 (G2F5), Aspergillus sydowii GEF-6 (G3F6), (Aspergillus flavus GEF-7 (G4F7), Aspergillus niger strain GEF-8 (B1F8), Aspergillus awamori GEF-9 (B2F9) and Cladosporium sphaerospermum strain GEF-11 (WBF11).

Microbial diversity studies in heavy metal polluted waters report a reduction in diversity of the indigenous water micro flora and alteration of the microbial community structure of the region ^[33]. Additionally, pollution of water by heavy metals is known to promote the selection of resistant species and decrease the indigenous microbial diversity ^[7]. Characterization of the indigenous micro flora with better methods for culturing the otherwise viable but non-culturable organisms may add-on to the repertoire of the microbial collections by providing better strains or new culture isolates with better features for specific biotechnological applications.

4. Conclusion

The findings of the present work indicate a unique biodiversity prevalent in man-made ecosystems as seen from the types of new, hitherto unreported species of both bacteria and fungi in the effluent of the Gold electroplating units. The sample was found to have low counts of bacteria and fungi as pour plate method had to be used for isolating the indigenous flora. Nine bacterial and eleven fungal isolates were obtained upon culturing the effluent sample on suitable media. They could survive in effluent having seventeen heavy metal ions. The culture isolates were characterized for their growth attributes and bacterial isolates were also characterized for their biochemical attributes. Partial 16S rRNA and 18S rRNA sequencing was done for bacterial and fungal isolates respectively. Identification of the isolates was done using all the information.

The bacterial isolates obtained were found to belong to five genera including Staphylococcus, Achromobacter, Klebsiella, Macrococcus, Pseudomonas and Bacillus. Among the nine bacterial isolates, four were found to be new bacterial species including Staphylococcussp. ss-1, Achromobacter sp. ss-2, Macrococcus sp. ss-4 and Bacillus sp. ss-6 as per the identification given by GenBank (NCBI) entries.

Of the eleven fungal isolates, seven were to belong to the genus ‘Aspergillus’, suggesting the abundant diversity of species of the genus Aspergillus. The species include Aspergillus sydowii, Aspergillus flavus, Aspergillus awamori,two strains of Aspergillus tamarii and two strains of Aspergillus niger. Two of the isolates were species of genus Penicillium including Penicilliummarneffei and Penicillium pinophilum. The two remaining isolates include Cladosporium sphaerospermum and Cochliobolus lunatus. Ten of the eleven fungi were found to be new strains and their sequence data was submitted to NCBI, GenBank and new strain designations were assigned to them and unique accession numbers were also acquired.

Long term exposure to metals leads to the selection of a microbial community which survives in polluted environments. It is also to be noted that the overview of the diversity picture emerging from the results indicate a rather narrow spectrum of microbial flora and low counts. The synergetic toxicity of a variety of toxic metals present in the electroplating effluent results in selective pressure and reflects the unique adaptability of certain microbes to survive therein. The discovery of new species is not only exciting but also suggests the possibility for finding more novel species with better methods for culturing the otherwise viable but non-culturable organisms. Also, the study highlights the potential for finding new isolates in heavy metal contaminated environments encoding genes for multiple metal resistance. Indigenous microorganisms isolated from the electroplating effluents may be studied to examine their potential to produce enzymes, and bioactive compounds for application in agricultural, pharmaceutical, industrial, environmental and medical sciences.

All the cultures isolated in this work are deposited at Microbial Culture Collection (MCC), National Centre for Cell Science, Dept. of Biotechnology, Pashan, Pune, India. MCC is now recognized as an International Depository Authority for microbial culture collection.

Acknowledgements

The authors thank NCCS, Pune for the support in the molecular characterization of the bacterial and fungal isolates and IIT, Bombay for the help extended inthe metal content analysis of the effluent sample.

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