Isolation and molecular characterization of dye tolerant bacteria from dye contaminated soil in Kerala

Table of contents

Table of figures

Table of tables

List of abbreviations

Isolation and molecular characterization of dye tolerant bacteria from the dye contaminated soil in Kerala 1

Abstract

1. Introduction
1.1 Objectives
1.2 Scope of the study

2. Review of literature

3. Hypothesis

4. Materials and Methods
4.1 Study area
4.2 Soil sample collection
4.3 Quantitative analysis of microorganisms present in the collected soil
4.4 Preliminary screening of dye tolerant microorganisms from soil
4.5 Isolation of potential dye decolourizers
4.7 Morphological and biochemical tests
4.8 Optimization of the dye concentration
4.9 Molecular identification and isolation of DNA
4.10 Quantification of DNA
4.11 Agarose gel electrophoresis
4.12 PCR amplification using 16s rRNA
4.13 Data sequencing
4.14 Data analysis
4.15 Cross evaluation of bacterial isolates and MTCC
4.16 Statistical analysis

5. Results and discussion
5.1 Isolation of microorganism from soil
5.2 Preliminary screening of dye tolerant bacteria
5.3 Isolation of potential dye decolourizers
5.4 Pure culture preparation, morphological and biochemical identification
5.5 Optimization of dye concentration
5.5 Bacterial DNA isolation
5.6 PCR amplification using 16s rRNA and sequencing
5.7 Sequence analysis
5.8 Molecular phylogenetic analysis using maximum likelihood method
5.9 Cross evaluation of bacterial isolates and MTCC

6. Conclusions

Acknowledgements

References

ACKNOWLEDGEMENTS

Firstly we thank God Almighty whose blessing were always with us and helped us to complete this project work successfully.

We wish to thank our beloved Manager Rev. Fr. Dr. George Njarakunnel, Respected Principal Dr. Joseph V.J, Vice Principal Fr. Joseph Allencheril, Bursar Shaji Augustine and the Management for providing all the necessary facilities in carrying out the study. We express our sincere thanks to Mr. Binoy A Mulanthra (lab in charge, Department of Biotechnology) for the support. This research work will not be possible with the co-operation of many farmers.

Lastly, we extend our indebt thanks to patents, friends, and well wishers for their love and support.

Prem Jose Vazhacharickal*, Sajeshkumar N.K, Jiby John Mathew and Nimisha Vinod

Table of figures

Figure 1. Classification of dyes according to their chromophore groups (Courtesy: ied.ineris.fr)

Figure 2. Structure of the dyes used in the study. Adapted from commons.wikimedia.org.

Figure 3. Reduction of azo dyes (Courtesy: www.ec.gc.ca)

Figure 4. Mechanism of anaerobic reduction. Adapted from Chacko and Subramanian (2011) [9].

Figure 5. Map of Kerala showing the soil sample collection point. Source: www.researchgate.net/profile/Prem_Vazhacharickal/publication/305143110/figure/fig2/AS:382590403006466%401468228068394/Figure-3-Map-of-Kerala-Showing-the-Various-Sample-Collection-Points.jpg&imgrefurl=https://www.researchgate.net/figure/305143110_fig2_Figure-3-Map-of-Kerala-Showing-the-Various-Sample-Collection-Points&h=680&w=850&tbnid=Un35S8MxWpsznM&tbnh=201&tbnw=251&usg=__hRm-Tbi8iFnKQ3jTX8SeWV_YJb0=&hl=de&docid=gPm71IGVx9x8ZM&itg=1

Figure 6. Details of a) soil sample collected, b) diluted soil samples, c) serial diluted soil samples (10-6 dilution), d), e) and f) decolourization pattern of dyes during 24 hrs incubation of brilliant green, congo red and methylene blue.

Figure 7. Details of a) and b) decolourization pattern of dyes during 24 hrs incubation of congo red and brilliant green , c) pure culture isolate of congo red (CR1), d) pure culture isolate of congo red (CR2), e) pure culture isolate of brilliant green (BG1), f) pure culture isolate of brilliant green (BG2), g) pure culture isolate of brilliant green (BG3).

Figure 8. Samples description and growth on media a) growth of isolates (CR1, CR2, BG1, BG2, BG3) on mannitol agar; C: control, b) indole test, c) methyl red (MR) test, d) voges-proskauer (VP) test, e) citrate utilization test, f) urease test.

Figure 9. CR1 species inoculated on varying congo red dye concentration sequentially, a) 0.05%, b) 0.10%, c) 0.15%, d) 0.20%, e) 0.30%, f) 0.50%.

Figure 10. CR2 species inoculated on varying congo red dye concentration sequentially a) 0.05%, b) 0.10%, c) 0.15%, d) 0.20%, e) 0.30%, f) 0.50%.

Figure 11. CR1 species inoculated on varying congo red dye concentration sequentially a) 0.80%, b) 1.0%, c) 2.0%, d) 3.0%, e) 4.0%.

Figure 12. CR2 species inoculated on varying congo red dye concentration sequentially a) 0.80%, b) 1.0%, c) 2.0%, d) 3.0%, e) 4.0%.

Figure 13. BG1 species inoculated on varying congo red dye concentration sequentially a) 0.05%, b) 0.10%, c) 0.15%, d) 0.20%, e) 0.30%, f) 0.50%.

Figure 14. BG2 species inoculated on varying congo red dye concentration sequentially a) 0.05%, b) 0.10%, c) 0.15% d) 0.20%, e) 0.30%, f) 0.50%.

Figure 15. BG3 species inoculated on varying congo red dye concentration sequentially a) 0.05%, b) 0.10%, c) 0.15%, d) 0.20%, e) 0.30%, f) 0.50%.

Figure 16. DNA isolation a) from bacterial isolates (CR1, CR2, BG1, BG2, BG3), b) 1500bp PCR amplified products of the isolates, c) isolated DNA.

Figure 17. Edited sequences of the isolated bacteriaBioEdit software and Cap contig assembly was done using forward and reverse sequences and finally the Cap contigs having 1057, 1067, 1362 and 1362 bases was generated for CR1, CR2, BG1 and BG2 respectively.

Figure 18. Graphical representation of BLAST of isolate CR1 (1057 sequence).

Figure 19. Table view of BLAST results of isolate CR1 (1057 sequences).

Figure 20. Table view of BLAST results of isolate CR1 (1057 sequences; continued).

Figure 21. Graphical representation of BLAST of CR2 isolates of 1067 sequences.

Figure 22. Table view of BLAST results of isolate CR2 (1067 sequences).

Figure 23. Table view of BLAST results of isolate CR2 (1067 sequences; (continued).

Figure 24. Graphical representation of BLAST of BG1 isolates of 1362 sequences.

Figure 25. Table view of BLAST results of isolate BG1 (1362 sequences).

Figure 26. Table view of BLAST results of isolate BG1 (1362 sequences; continued).

Figure 27. Graphical representation of BLAST of BG2 isolates of 1362 sequences.

Figure 28. Table view of BLAST results of isolate BG2 (1362 sequences).

Figure 29. Table view of BLAST results of isolate BG2 (1362 sequences; continued).

Figure 30. Phylogentic tree by maximum likelihood method.

Figure 31. Comparative study on the growth of bacterial strains (CR1, CR2, BG1, BG1, BG3) in utilizing the azo dyes; congo red (2.00%) and brilliant green (0.10%).

Table of tables

Table 1. Relationship of colour absorbed and complementary colour observed wavelength absorbed (nm) colour absorbed Complementary colour. (Adapted and modified from [5])

Table 2. Table describing various dyes and its applications (adapted and modified from [4, 6]).

Table 3. Characteristics of the dyes used in the study

Table 4. Treatment methods of dyes

Table 5. Description of the sample collection site in Kerala, India.

Table 6. Morphological and biochemical characterization of the isolated bacteria (CR1, CR2, BG1, BG2 and BG3).

Table 7. Biochemical comparison of isolated (CR1, CR2, BG1 and BG2) and microbial type culture collection (MTCC) bacteria.

List of abbreviations

illustration not visible in this excerpt

Isolation and molecular characterization of dye tolerant bacteria from the dye contaminated soil in Kerala

Prem Jose Vazhacharickal1*, Sajeshkumar N.K1, Jiby John Mathew1 and Nimisha Vinod1

1Department of Biotechnology, Mar Augusthinose College, Ramapuram, Kerala, India

Abstract

The dyes are complex chemical compounds that imparts colour to substances. On the urge of urbanisation, the use of synthetic dyes is increasing largely and its untreated effluent release causes serious environmental pollution affecting water bodies by disturbing the aquatic ecosystem, soil, plants, animals, humans. The recalcitrant nature of these dyes limits its treatment using conventional methods wherein biological methods using microorganisms are reported to completely mineralize the dyes lowering the release of degradation products. The present work focuses on the biodegradation of synthetic dyes by isolating bacteria from a dyeing unit. Soil samples from the dye contaminated soils was collected, its degradation potentiality was observed using three major dye of studies congo red, brilliant green and methylene blue within 24 hr incubation. Maximum decoloarized dye (congo red and brilliant green) were chosen, serially diluted to 10-5 and plated to obtain two distinct colonies from decolorized congo red (CR1, CR2) and three distinct colonies from decolorized brilliant green (BG1, BG2, BG3). These isolates were biochemically characterized. Molecular characterization was performed by isolating DNA from five isolates and amplified it using PCR, with the 16s rRNA gene primer. The PCR amplification product having approximately 1500bp were sequenced, edited and searched using BLAST against the known sequences within NCBI databases. The isolates were identified to be CR1 as Pseudomonas (88% identity), CR2 as Aeromonas (89% identical) and BG1/BG2 were confirmed to belong to same genus as Bacillus (99% identical). The phylogenetic tree showed a clear divergence between isolated species. Furthermore, the dye tolerance of isolates were observed by optimization analysis to be as CR1 and CR2 tolerated up to 4% of congo red dye and among BG1, BG2 and BG3; BG1 tolerated up to 0.50% of brilliant green dye. Thus, CR1 and CR2 were observed to be potent azo dye decolourizers. Recombinant DNA Technology can be applied in this field that can make the above future application more reliable.

Keywords: Azo dyes; Aeromonas, Bacillus, BLAST; DNA isolation, PCR; Pseudomonas; 16s rRNA.

1. Introduction

Dyes are compounds used to colour substances. It is used widely in textile, leather, plastic, cosmetics and food industries. The use of dyes dates back to late Bronze Age, the first of which to be used was indigo. Indigo was a plant extract named Indigofera tinctoria by formation and possess a characteristic blue colour of itself [1]. Natural dyes were obtained from extracts of fruits, leaves, roots etc from plants used for dyeing clothes long back. Since the dyeing procedure took more time using natural dye, an alternative dyeing method was implemented, the use of synthetic dyes began in 1856 synthesised with the first dye being mauve dye (aniline), a brilliant fuchsia colour [2]. From then to the present date synthetic dyes finds its applications by many industries making itself an indispensible part of human life.

A chromophore is an unsaturated group of atoms attached to the coloured compounds [3]. These compounds are the colour induced group present in a dye without which a substance could remain colourless (Figure 1). The intensity in the colour absorption is brought about by another group of compounds associated with chromophore in a dye structure called Auxochromes. The unshared pair of electrons in the auxochrome group interacts with the π electrons within the aromatic ring [3].

Common auxochromes are:-

1. -NH2

2. -OH

3. -NR2

4. -CO2H

5. -SO3H

A substance is coloured due to its absorption property. The absorption of UV/V radiations are due to the electronic transitions occurring within the molecular orbital’s of the organic molecule in coloured substance.

When light strikes on dye molecules the π electrons within the unsaturated atoms in chromophoric group gets delocalised between its ground stage and excited stage by absorbing a light of wavelength in the visible range thus producing colour in the dye molecule [3,4].

Some of the white light will be absorbed by the substance and may reflect (or transmit) the remaining light. In this case, substance is coloured but the colour transmitted is different from that of the absorbed colour. The colour of the transmitted light is called the complementary colour [1] (Table 1).

Dyes are classified according to their chromophoric group as azo, anthraquinone etc and according to their solubility in aqueous solution as Acid dyes (anionic), Basic dyes (cationic), and disperse dyes (non-ionic) [4].

The different class of dyes finds its major use in cotton, hair, plastic, gasoline, waxes, ink, polyester, cellulose acetate, soaps and detergents, food and drugs, paints, oil, leather and paper industries. Among dyes, the most commonly used is the azo dyes as they are easily soluble in water. Azo dyes are compounds consisting of azo groups (-N=N-) group within it (Table 2 and 3; Figure 2).

The toxic side of dyes are the end products say for; in the case of azo dyes are aromatic amines such as benzidine, 3, 3’-dimethoxybenzidine and p-amino benzene. The destruction process mainly occurs due to the redox reaction, the products being aromatic amines [1] (Figure 3).

Many dye substances have been classified as human carcinogens by World Health Organisation (WHO) and International Agency for Research on Cancer (IARC, 1982). Benzidine degradation product of azo dyes is classified as Class-1 known human carcinogen by IARC and 3, 3’-dimethoxybenzidine and p-amino benzene as group 2B carcinogens thus limiting its usage [7, 8].

Textile effluents released from industries contain a combination of polluting substances like organic, non-biodegradable, poisoning compounds. These when released untreated directly into the water bodies poses a threat to the aquatic life. Majority of dyes are not completely degraded and they interfere in the aquatic ecosystem and causing mutagenic effects to other organisms including Humans. The usage of many dyes is studied to be carcinogenic in nature. Thus, the FDA (Food and Drug Administration) declares only certain dyes to be used [9]. Dye poisoning is a major concern as it affects the skin, respiratory tract and neural systems. Many dyes (crystal violet, tartrazine), used in the food industry are reported harmful for usage due to the health problems caused. Analysing the toxological aspects of the dye usage, dye decolourization treatment method is a need of the hour. Various treatment procedures may be devised for the safe treatment of dye substances [1, 10, 12, 13] (Table 4).

Among these physical and chemical are less appreciated concerning the cost and the time consumed. Thereby, biological methods are more widely used being easier, less costly and degradation process being in an environmental friendly manner.

Biodegradation is defined as the breakdown of complex structures of chemical compounds into simpler structures using microorganisms and it consists of two process namely, growth and co metabolism where growth factor microorganisms utilize organic pollutants as their carbon source resulting in complete mineralization of pollutants and in co metabolism growth substrate mediates the degradation of organic pollutants wherein growth substrate is used as energy source [12].

The bio reduction of azo dyes consists of mainly three methods:-

I. Anaerobic treatment process
II. Aerobic treatment process
III. Aerobic-Anaerobic treatment process

In anaerobic process, the reduction of dye is by azo link breakage enzymatically with FMNH2 () and FADH2 ( as co factors. The end products are mainly carbon dioxide (CO2) and methane (CH4) along with toxic release of aromatic amines. Methanogenic organisms are usually involved in this process. It is not involved in the energy generation process [13, 14, 15].

In aerobic degradation process, involves the oxidation of the dye by free radical mechanism avoiding formation of toxic aromatic amines production. Mainly oxidoreductases enzymes are involved [13, 16].

In aerobic-anaerobic treatment process, the amines formed during the anaerobic treatment are mineralized in the aerobic condition. Hydroxylation of aromatic compounds resulted after degradation is later exposed to aerobic environment in which oxygen is introduced into bonds for further bond breakage. A cell growth temperature of 35-45˚C and at pH 7-7.5. [13, 15].

The biodegradation of dyes is due to the enzymes secreted by the microorganisms (often being extracellular). The enzymes involved in the dye decolourization are [17]:-

1. Flavin Reductases (EC 1.5.1.30)
2. Lignin Peroxidises (EC 1.11.1.14)
3. Aryl alcohol Oxidases (EC 1.1.3.7)
4. Laccases (EC 1.10.3.2)
5. Azo Reductases (EC 1.7.1.6)
6. NADH-DCIP Reducatses (Reduced Nicotinamide Adenine Dinucleotide-2,6 Dichloroindophenol; EC 1.6.99.3)
7. Tyrosinases (EC 1.14.18.1)

The predominant enzymes are azoreductases, laccases, lignin peroxidises, and manganese peroxidises, hydroxylases.

1.1 Objectives

The objectives of this research work are to identify the microorganisms that are tolerant to the dye and characterization using molecular method. From these organisms the ability of it to degrade the dye can also be identified by growing the organism in nutrient agar with the supplementation of various dyes. Polymerase chain reaction (PCR) amplification of deoxyribonucleic acid)DNA of the organism is done using the primer made from 16s rRNA (16 svedberg ribosomal ribonucleic acid) to identify the organism.

1) Isolation of dye tolerant bacteria from dye contaminated soil sample
2) Molecular characterisation of isolated strains using 16s rRNA sequence

1.2 Scope of the study

The current research work aims to isolate and characterize various dye tolerant bacteria from dye contaminated soil in Kerala which could be further explored to effective bioremediation in industrial dye contaminated soils.

2. Review of literature

Dyes are used to colour any substances. Suggested that dyes are used to resist the laundering and sunshine effect with their complex aromatic structures. Hasan (2008) [18] suggested that dyes find its major applications in textile, paper, plastic, hair, fur, leather, wax, food stuff and cosmetic industries. The annual production of dyes in the world is estimated to be more than 7 x 105 tonnes suggested by the reviews [19, 20] of which the annual dye production in India nears up to 80,000 tonnes making it the second largest exporter of dyes after china [21,22,35]. The major consumers of dyes are the textile industry. Azo dyes are one of the most profoundly used among dyes [21, 23, 24, 25, 26, 27]. Anthroquinone ranks the second largely used dye after azo dyes [19].

Sulfonated group and azo bonds provide a recalcitrant property to azo dyes featuring its xenobiotic effect [23]. The industrial effluents waste water contains many dye varieties like acidic dyes, basic dyes, dispersants, oxidants and detergents [28]. The degradative products of azo dyes, a xenobiotic compound are reported as mutagenic and carcinogenic due to their structural characterisation consisting of aromatic rings [4, 32, 35] .These degradation end products are primarily aromatic amines. Thus being harmful to humans [9] as their use has reported internal injuries. Many harmful effects of azo dyes have been studied [29, 30].

During dyeing process only a smaller proportion of dye is used in the dyeing process of dyes rest are disposed into water [2, 4, 31, 32]. The untreated waste effluent contains aromatic amines and phenols [33, 34]. A dye concentration above 1mg/L is harmful [4, 19].

The direct exposure of dyes especially azo dyes from textile industries causes toxicity flora and fauna in the ecosystem thereby inversely affecting environment. It affects mainly the aquatic life by blocking the light availability to aquatic flora during photosynthesis process [35]. A review of studies from [19] reports the toxicological studies 98% of dyestuffs with a dye concentration more than 1mg/L and 59% of dyestuffs with a dye concentration more than 100 mg/L is lethal to fishes also, a dye concentration lesser than 2000 mg/L is responsible for the bioaccumulation in water bodies. Fishes have shown more sensitivity to basic dyes released untreated into water bodies [4].The toxicity of untreated dye effluent is reported to human beings too as they injure various the internal organs [4, 22, 36].

In aquatic environment they increase total organic carbon (TOC), biological oxygen demand (BOD), and chemical oxygen demand (COD) levels that are the pollution indicator influencing the aquatic growth [2, 11, 39]. Dye concentration of (10-50 mg/ L) influences the water transparency, solubility of gases in water and aesthetic value [28].

As mentioned by the reviews, the dye pollution influences aquatic ecosystem, soil fertility affecting plants, and other resources in nature summing up the ecosystem in whole [9].

Rather than just dyes the released untreated effluent waste water from industries also showed the presence of heavy metals like chromium (Cr), cadmium (Cd), copper (Cu), lead (Pb), nickel (Ni), zinc (Zn), cobalt (Co), magnesium (Mg), iron (Fe) and arsenic (As) has been reported in mainly studies [37].

Among dyes only a quarter of them of the total synthetic dyes produced annually are biodegradable in nature. Conventional wastewater treatment processes do not degrade the substituent rings of dye structure being recalcitrant in nature. The treatment protocol consists of physical, chemical and biological methods [1, 4, 18, 19, 86]. Absorption, coagulation – flocculation, oxidation, electrochemical are the various physical methods used being expensive. Chemical methods are also devised one of which is hydrogen peroxide [38, 39].

Bio treatment offers a cheaper and environmentally friendlier alternative for colour removal in textile effluents [2, 4, 28, 32, 47]. It is suggested as a cost efficient alternative to chemical process [28]. The Biodegradation of dyes involves two processes namely, Adsorption by changing the chromophoric group (Decolourisation) and Breaking down dye molecule through biological process (Degradation) [32]. Biodegradation of dyes could be in aerobic, anaerobic or with a combination of the former and latter process occurs [24]. Pearce et al. (2003) [16] suggested that the dye decolourisation mechanism is the bio adsorption of dye onto the cell biomass. Anaerobic dye degradation involves the chromophoric bond cleavage releasing the substituent with no adenosine triphosphate (ATP) production while aerobic dye decolourisation is the transfer of electrons among the substrate and enzyme through oxidation process along with ATP production [16, 42]. The major disadvantage of the either methods is that in anaerobic treatment method dye molecules produced aromatic amines upon reduction while many synthetic dyes show oxidative biodegradation resistance as they are manufacture in a way remaining stable to light and chemical oxidations [86]. The sequential anaerobic-aerobic method overcomes the above mentioned limitations. Combined anaerobic-aerobic method results in complete mineralization of molecules as no biodegradable compounds remained during the anaerobic mechanism are degraded in the aerobic process followed [4, 15, 41].

The reduced dye such as azo dyes are utilised by the microorganisms as their energy source by reducing or mineralising the complex dye molecule to their simplest monomeric form [42, 43]. Bioremediation involves the participation of microorganisms in the degradation of complex molecular structures of xenobiotics like dyes. From the literatures reviewed a wide range of dye decolourising organisms’ microorganisms of different taxonomic many have been studied.

The bacterial dye decolourisation has been reported by many literatures [1, 11, 15, 16, 45, 46, 47, 87]. Shyamala et al. (2014) [48] studied the decolourisation activity on methyl orange of Bacillus sp. Strain TVU-M4 , a halophilic bacterial, that showed 88.24 % within 32 h of incubation and Aftab et al. (2011) [23] has reported the decolourisation activity on azo red 2G commonly used as a food colour of Bacillus megaterium that showed 64.89%. Studies on intestinal bacteria are gaining its importance over year. Organisms like Enterococcus faecium, has been studied by Susan et al. (2010) [50] and reported tolerant to many azo dyes proving its decolourisation ability [51, 52, 53].

Zimmermann et al. (1982) [54] have reported the azoreducatse activity of Pseudomonas KF46 species in orange II dye. Shah et al. (2014) [55] the commonly found milk bacteria Lactobacillus decolorized reactive orange 16 dye within 24 h to 95% at a concentration of 1mg/L. Srinivasan et al. (2014) [44] isolated mangrove sediment habitat bacteria Paenibacillus sp degrading azo dyes Nitomill Brill crimson, nito green B and methyl red along with its characterisation. Joshi and Mhatre (2015) [88] have reported decolourisation efficiency of Enterococcus spp.to Malachite green dye by 100% and 92% to Carbol fuchsin. Babu et al. (2015) [89] have studied another marine bacterium capable of degrading Congo red dye Dietzia sp. (DTS26) by 94.5 %.

The decolourising activity of algal species has also been reported [1, 11, 15, 47, 56, 57]. Daneshvar et al. (2005) [58] have studied the 92% decolourisation of highly coloured malachite green , a triphenylmethane dye so called Brilliant green, by Cosmarium sp. and algal biomass to 85% [59] under appropriate temperature conditions [47].The decolourisation of widely used food colorants like by a green algae by azoreductase enzyme have been studied [57]. The decolourisation of actinomycetes has also been reported [93].

According to the literatures fungal strains studied have shown maximum decolourisation to a number of synthetic dyes. Lignin degrading fungi are involved in this biodegradation since they degrade lignin, a complex structural polymer, [67, 93] and are extensively used in the biodegradation of azo, triphenylmethane and anthraquinone. Lavanya et al. (2014) [15] have reported the dye decolourisation activity in Phanerochaete chrysosporium in immobilised stage with calcium alginate in batch colour of Direct Violet 51, Reactive Black 5, Ponceau Xylidine and Bismarck Brown R azo dyes. The decolourisation activity of Mucor mucedo to Congo red dye has been reported [90].

A research on Pleurotus ostreatus decolourising activity on synthetic dyes like Synozol red [63], food colours [64] have been reported at efficient decolourisation rate. Pleurotus ostreatus MTCC 142 decolourisation pattern on crystal violet dye has being studied [65], which is widely used as food colour causes allergies. Many yeasts varieties [1, 2, 15].

On a comparative analysis white rot fungi have been studied to be the most efficient dye decolourisers with its extracellular enzymes like lacasses and peroxidises [37,47,61,62,66,67,82,83]. Murugesan et al. (2007) [68] have reported the white rot fungi Ganoderma lucidum KMK2 decolourisation activity on remazol brilliant blue R dye with its laccase enzyme. Daedalea flavida, a white rot fungus, has been studied for a good decolourisation rate of azo dyes in the absences of Carbon source [90].

The release of special enzyme by the microorganisms is responsible for the decolourisation process in dyes [40]. The common feature of these biocatalysts is that they are active redox molecules providing them a broad spectrum of action in substrates [17] that mediate the electron transfer enzymes and dye substrate [4,10,17,69,70]. The enzymes are mostly extracellular released. Pearce et al. (2003) [16] the redox potential is necessary for a decolourisation reaction for the redox equilibrium within substrate molecule and redox mediators involved.

The major enzymes involved in the decolourisation process are azoreductase, peroxidises, laccases etc. Most of the enzymes are extracellular secreted by microorganisms. Azoreductase enzymes are generally secreted by bacterial species [9, 11, 40, 69, 71] that show specificity to Nicotinamide adenine dinucleotide (NADH) or Flavin adenine dinucleotide (FADH) as cofactors [40, 73]. This enzyme being a membrane bound is inhibited in the aerobic condition since oxygen, the natural electron acceptor, oxidises the reduced redox mediator and not the dye substrate molecule during the reduction mechanism [16, 42]. It was reported evident by Russ ET al.in 2000[69] the action of cytosolic flavin-dependent azoreductase activity in recombinant Sphingomonas sp. strain BN6 [70] suggesting that the enzyme activity was fair well in vitro condition rather than in vivo. Azoreductase activity has also been detected in yeast (Saccharomyces cerivisae) in reduction of azo dyes with the help of Fre1p gene [1].

A number of fungal enzymes have been reported [11, 15, 27, 31, 40, 67, 74]. Fungal enzymes have been studied to be the most potential dye decolourisers as per the literatures to a wide range of synthetic dyes used. According to literatures among enzymes, laccases have shown the highest potential decolourisation. Trametes species due to their laccase activity [74, 75, 76, 77] are potent decolourisers of azo, anthroquinone etc dyes. Rodriguez et al. (1999) [78] have studied decolourisation activity on reactive blue acid blue etc in Trametes hispida.

3. Hypothesis

The current research work is based on the following hypothesis

1) Dye contaminated soil samples posse’s diverse bacteria capable of utilizing various dyes.
2) These bacterial isolates differ in their dye degrading capabilities.
3) The isolated bacteria shares common ancestry as a part of better environmental adaptation and evolution.

4. Materials and Methods

4.1 Study area

Kerala state covers an area of 38,863 km2 with a population density of 859 per km2 and spread across 14 districts. The climate is characterized by tropical wet and dry with average annual rainfall amounts to 2,817 ± 406 mm and mean annual temperature is 26.8°C (averages from 1871-2005; Krishnakumar et al ., 2009). Maximum rainfall occurs from June to September mainly due to South West Monsoon and temperatures are highest in May and November.

4.2 Soil sample collection

Soil samples were collected from a region which was treated textile dye dump up area for several years. The soil samples were collected up 10 cm depth using standard soil collection protocols.

4.3 Quantitative analysis of microorganisms present in the collected soil

About 10 g of soil sample was diluted with 100 ml of distilled water. The soil sample was serially diluted to a dilution rate of 10-6 dilution. 25 ml nutrient agar plate was prepared and 0.1ml sample was spread plated (10-5 dilution). The plates were kept for 24 hrs incubation at 37˚C in the bacteriological incubator. Colonies were counted on reaching the incubation time by the following equation:-

Total number of bacterial colony forming units in the sample

(CFU/ml) = No. of colonies/ Volume of sample added* Dilution factor.

4.4 Preliminary screening of dye tolerant microorganisms from soil

The soil used for the enrichment and isolation of dye tolerant bacteria was obtained. Dyes such as Congo red, brilliant green and methylene blue were chosen. 0.02 gm of each dye was weighed such that the concentration of the dye is 800 ppm by which not many organisms could survive and added to autoclaved nutrient broth media in the Erlenmeyer flasks. To the dye containing nutrient broth, 1ml of 10-5 from the above mentioned diluted soil sample was added and kept for incubation for 24 hr at 37˚C. The decolourization was observed.

4.5 Isolation of potential dye decolourizers

The tubes that showed maximum decolourization were chosen and 1ml of the sample was serially diluted to 10-5 from it. 0.1 ml from the sample serially diluted to 10-4 was spread plated and kept for 24 hr incubation at 37˚C in the bacteriological incubator. Single colonies with distinct colony morphology were observed and each single colony was streaked onto nutrient agar plates to obtain pure cultures of isolates.

4.6 Pure culture preparation

Bacterial pure culture was prepared by streak plate method. One loop full of enrichment culture from the flasks was streaked on nutrient agar plates. The growth of the bacterial colonies was measured after 24-48 hr incubation at 28°C. Morphologically dissimilar colonies were randomly selected and sub cultured onto nutrient medium and maintained at 4°Cfor bacterial characterization.

4.7 Morphological and biochemical tests

Identification of the isolates were performed according to their morphological, cultural and biochemical characteristics by following Bergey’s Manual of Systematic Bacteriology [94]. All the isolates were subjected to Gram staining and specific biochemical tests.

4.7.1 Colony morphology

This was done to determine the morphology of selected strains on the basis of shape, size and colour.

4.7.2 Gram staining

A clean grease free slide was taken and a smear of the bacterial culture was made on it with a sterile loop. The smear was air-dried and then heat fixed. Then it was subjected to the following staining reagents:

(i) Flooded with Crystal violet for 1 min. followed by washing with running distilled water.
(ii) Again, flooded with Gram’s Iodine for 1 min. followed by washing with running distilled water.
(iii) Then the slide was flooded with Gram’s Decolourizer for 30 sec.
(iv) After that the slide was counter stained with Safranin for 30 sec, followed by washing with running distilled water.
(v) The slide was air dried and cell morphology was checked under microscope.

4.7.3 Motility test

This test is done to check the motility of the microorganism. The isolates were taken in a cavity slide from a 24 hr inoculated broth and observe under the microscope.

4.7.4 Mannitol salt agar test

This experiment is generally performed to determine whether the bacteria are capable of fermenting mannitol sugar or not. Whenever organisms ferment mannitol agar, the pH of media becomes acidic due to production of acids. The fermentation of the media from red to yellow which shows positive test result.

4.7.5 Catalase test

The catalase test was performed to detect the presence of catalase enzyme by inoculating a loopful of culture into a drop of 3% of hydrogen peroxide solution taken in a glass slide. Positive test was indicated by formation of effervescence or appearance of bubbles, due to the breaking down of hydrogen peroxide (H2O2) to oxygen (O2) and water (H2O).

4.7.6 IMViC test

(i) Indole Test: Indole test is done in sulfide-indole-motility medium (SIM). A loopful of experimental organism is inoculated into SIM medium. Result is read after adding Kovac’s reagent.
(ii) Methyl Red Test: MR test is done in Methyl-Red-Voges-Proskauer broth (MR-VP). A loopful of experimental microorganism is inoculated into the medium. Result is checked by adding methyl red.
(iii) Voges-Proskauer Test: VP test is done in MR-VP broth. A loopful of experimental microorganism is inoculated into the medium. Result is checked by adding Barrit’s A and Barrit’s B reagent.
(iv) Citrate Test: It is done in Simmon Citrate media. A loop of organism is streaked in zigzag manner in slant of media. Colour change is observed after 24 hr incubation.

4.7.7 Urease test

Using sterile technique, inoculate a loopful of experimental organism into urea broth tube. Incubate the tube for 24 to 48 hrs at 37°C and then observe the colour change.

4.8 Optimization of the dye concentration

The bacteria chosen were analysed for the optimisation using the varying dye concentration as 0.05%; 0.10%; 0.15%; 0.20%, 0.30% and 0.50%. The organisms were streaked onto the nutrient agar plates containing the above mentioned dye concentration and the growth pattern were observed. Enhancement of the dye concentrations (0.80%, 1.00%, 2.00%, 3.00% and 4.00%) were done when the initial dye concentrations were found to be satisfactory for the corresponding isolates.

4.9 Molecular identification and isolation of DNA

The DNA from bacteria was isolated using a modified Tris-EDTA (TE) buffer method with the addition of Proteinase K [95, 96]. Two ml of the overnight bacterial culture pellets were suspended in TE buffer with the addition of 10% sodiumdodecylsulfate (SDS) and Proteinase K. Phenol-chloroform mixture was added; mixed well and incubated at 35°C for 5 min. The tubes were centrifuged at 10,000 rpm for 10 min at 4°C. The highly viscous jelly like supernatant was collected using cut tips and transferred to a fresh tube. The phenol-chloroform treatment was repeated for one more time. 100 µl of 5 M sodium acetate was added and the DNA was precipitated using ice-cold isopropanol for overnight at 4°C. The tubes were further centrifuged at 5,000 rpm and the pellet washed with 2-3 drops of 70% alcohol, air dried and redissolved in 200 µl TE buffer and stored at -20°C till further analysis [95, 96].

4.10 Quantification of DNA

The purity of the isolated DNA was checked spectrophometrically in a UV-Vis spectrophotometer by checking the 260/280 values (Thompson and Dvorak, 1989; Muller et al., 2003). Concentration of DNA (ng/µl) were calculated using the formula

DNA (ng/µl) = OD @ A260 x 50 x 100 x 0.1

Where OD @ A260 is the optical density at absorbance 260 nm

50 is the calculation factor

100 is the dilution factor

0.1 is the total volume of DNA

4.11 Agarose gel electrophoresis

Amplicons were separated by agarose gel. For this 50 mL of 1% agarose was prepared by melting it in boiling water bath and 1µL of Ethidium bromide (10mg/ml) was added when temperature dropped to 45°C. The molten agarose was poured after assembling the gel-casting tray with comb at one end (near cathode) and allowed to set. 1 x tris acetate-Ethylenediaminetetracetic acid (TAE) buffer was poured in the tank to sub-merge the gel. 15 µL of the sample (mixed with gel loading buffer) was loaded in the wells and electrophoresis was carried out at 60 V for 60-90 min. Thereafter, gel was removed and examined over UV transilluminator for observing the DNA bands. The stained DNA bands were observed and the fluorescent profile was photographed by gel documentation system electrophoresis.

4.12 PCR amplification using 16s rRNA

The polymerase enzymes, adaptors and primers were purchased from Genie life science technology, Bangalore, India. The PCR reaction was performed with a Biorad MJPt100 thermocycler (Bio-Rad Laboratories, Bangalore, India). Gene fragments specific for the highly variable region of the bacterial 16S rRNA forward and reverse prime. The PCR amplification was performed with an amplification profile of 94°C for 30 sec, anneling at 50°C for 1 min, extension at 72°C for 1 min, repeated for 20 cycles, and then cooling at 10°C for 30 min. The PCR products were stored at -20°C in a deepfreezer. Electrophoresis of the samples was carried out on agarose gels, by loading 10 µl of each DNA samples at 50 V for 3 hrs tracking dye has properly moved across the gel. The gels were later viewed on a gel docking station and photographed.

4.13 Data sequencing

The PCR products were cleaned up using GenEluteTM PCR Clean-Up Kit (Sigma-Aldrich). Purified PCR products were sequenced by dideoxy chain termination method [99] using AB3730XL capillary sequencer for the bacterial samples.

4.14 Data analysis

Taxonomical identification of the bacteria collected from different locations was carried out using molecular techniques. The sequences were aligned using the ClustalW algorithm [97] in Bioedit 7.0 (DNA Sequence Analysis Software 224 package). Molecular phylogeny and phylogenetic trees were constructed by the maximum likelihood (ML), neighbour joining (NJ) and unweighted pair group method with arithmetic mean (UPGMA) analysis with the software MEGA version 7 (Tamura et al., 2007), and using 16s r-RNA sequence of strain 1 and strain 2 bacteria with 1,000 times bootstrapping. Pair wise genetic distances between the strains (1 and 2) were calculated based on Kimura 2 parameter model and was used to calculate estimates of nucleotide diversity [98], singleton variation, parsimony informative sites, and haplotype diversity. Statistical significance of strain 1 and 2 within the inferred trees was evaluated using the bootstrap of 1,000 replications.

4.15 Cross evaluation of bacterial isolates and MTCC

The biochemical tests of the isolated bacteria were compared with the microbial type culture collection and gene bank (MTCC), Chandigarh parent culture for the respective species. All the isolated bacteria were cross examined with their respective MTCC strains to prove the correctness of the molecular and phylogenetic analysis.

4.16 Statistical analysis

The survey results were analyzed and descriptive statistics were done using SPSS 12.0 (SPSS Inc., an IBM Company, Chicago, USA) and graphs were generated using Sigma Plot 7 (Systat Software Inc., Chicago, USA).

illustration not visible in this excerpt

Figure 1. Classification of dyes according to their chromophore groups (Courtesy: ied.ineris.fr)

illustration not visible in this excerpt

Figure 2. Structure of the dyes used in the study. Adapted from commons.wikimedia.org.

illustration not visible in this excerpt

Figure 3. Reduction of azo dyes (Courtesy: www.ec.gc.ca)

illustration not visible in this excerpt

Figure 4. Mechanism of anaerobic reduction. Adapted from Chacko and Subramanian (2011) [9].

illustration not visible in this excerpt

Figure 5. Map of Kerala showing the soil sample collection point. Source: www.researchgate.net/profile/Prem_Vazhacharickal/publication/305143110/figure/fig2/AS:382590403006466%401468228068394/Figure-3-Map-of-Kerala-Showing-the-Various-Sample-Collection-Points.jpg&imgrefurl=https://www.researchgate.net/figure/305143110_fig2_Figure-3-Map-of-Kerala-Showing-the-Various-Sample-Collection-Points&h=680&w=850&tbnid=Un35S8MxWpsznM&tbnh=201&tbnw=251&usg=__hRm-Tbi8iFnKQ3jTX8SeWV_YJb0=&hl=de&docid=gPm71IGVx9x8ZM&itg=1

illustration not visible in this excerpt

Figure 6. Details of a) soil sample collected, b) diluted soil samples, c) serial diluted soil samples (10-6 dilution), d), e) and f) decolourization pattern of dyes during 24 hrs incubation of brilliant green, congo red and methylene blue.

illustration not visible in this excerpt

Figure 7. Details of a) and b) decolourization pattern of dyes during 24 hrs incubation of congo red and brilliant green , c) pure culture isolate of congo red (CR1), d) pure culture isolate of congo red (CR2), e) pure culture isolate of brilliant green (BG1), f) pure culture isolate of brilliant green (BG2), g) pure culture isolate of brilliant green (BG3).

illustration not visible in this excerpt

Figure 8. Samples description and growth on media a) growth of isolates (CR1, CR2, BG1, BG2, BG3) on mannitol agar; C: control, b) indole test, c) methyl red (MR) test, d) voges-proskauer (VP) test, e) citrate utilization test, f) urease test.

illustration not visible in this excerpt

Figure 9. CR1 species inoculated on varying congo red dye concentration sequentially, a) 0.05%, b) 0.10%, c) 0.15%, d) 0.20%, e) 0.30%, f) 0.50%.

illustration not visible in this excerpt

Figure 10. CR2 species inoculated on varying congo red dye concentration sequentially a) 0.05%, b) 0.10%, c) 0.15%, d) 0.20%, e) 0.30%, f) 0.50%.

illustration not visible in this excerpt

Figure 11. CR1 species inoculated on varying congo red dye concentration sequentially a) 0.80%, b) 1.0%, c) 2.0%, d) 3.0%, e) 4.0%.

[...]