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Biodecolourization of textile dyes by novel, indigenous Pseudomonas stutzeri MN1 and Acinetobacter baumannii MN3
Accepted Manuscript Title: Biodecolourization of textile dyes by novel, indigenous Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 Author: Sathishkumar Kuppusamy Manivannan Sethurajan Murugan Kadarkarai Rajasekar Aruliah PII: DOI: Reference:
S2213-3437(16)30462-6 http://dx.doi.org/doi:10.1016/j.jece.2016.12.021 JECE 1380
To appear in: Received date: Revised date: Accepted date:
16-8-2016 17-11-2016 17-12-2016
Please cite this article as: Sathishkumar Kuppusamy, Manivannan Sethurajan, Murugan Kadarkarai, Rajasekar Aruliah, Biodecolourization of textile dyes by novel, indigenous Pseudomonas stutzeri L1 and Acinetobacter baumannii L2, Journal of Environmental Chemical Engineering http://dx.doi.org/10.1016/j.jece.2016.12.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Biodecolourization of textile dyes by novel, indigenous Pseudomonas stutzeri L1 and Acinetobacter baumannii L2
Sathishkumar Kuppusamya, Manivannan Sethurajanb*, Murugan Kadarkaraic,d, Rajasekar Aruliaha*
a
Environmental
Molecular
Microbiology
Research
Laboratory,
Department
of
Biotechnology, Thiruvalluvar University, Vellore- 632 115, India b
Universite Paris-Est, Laboratoire Geomateriaux et Environnement (LGE), EA 4508, UPEM,
77454, Marne-la-Vallee, France c
Division of Entomology, Department of Zoology, School of Life Sciences, Bharathiar
University, Coimbatore- 641 046, India d
Thiruvalluvar University, Serkkadu, Vellore-632 115, Tamilnadu, India
* Corresponding Author: Dr. Manivannan Sethurajan, Universite Paris-Est, laboratoire Geomateriaux et Environment (LGE), EA 4508, UPEM, 77454, Marne-la-Vallee, France. #current affiliation,
Biofouling and Biofilm Processing Section, Water and Steam Chemistry
Division, BARC Facilities, Kalpakkam 603102, India, e-mail: [email protected]
Dr. Rajasekar Aruliah, Environmental Molecular Microbiology Research Laboratory, Department of Biotechnology, Thiruvalluvar University, Vellore- 632 115, India. e-mail: [email protected] Tel: +91-8675265635.
Abstract Enormous quantities of toxic dyes containing textile effluents are being discharged in natural water systems and thus contaminate the water quality. Hence it is important to develop an eco-friendly and cost effective technology to treat the dyes contaminated wastewater. In this research, laccase enzyme mediated biodegradation of toxic textile dyes such as congo red and gentian violet was investigated using novel, newly isolated laccase producing Pseudomonas stutzeri L1 and Acinetobacter baumannii L2. Response surface methodology (RSM) with full factorial central composite design (CCD) was applied to optimize pH (pH 6.0 – 10.0), starch (1 - 3 %) and yeast extract (0.1 - 0.3%). The RSM results show that a maximum of 29.41 U/ mL and 41.76 U/ mL laccase activity was achieved by L1 and L2, respectively under optimum conditions (pH 8.0, 2% starch and 0.2% yeast extract). The congo red decolorization studies by L1, L2 and mixed consortia (L1 +L2) showed 84%, 89% and 97% decolourization efficiency, respectively. The gentian violet was decolorized up to 83%, 90% and 95% by L1, L2 and mixed consortia (L1 +L2), respectively. Phytotoxicity assay showed100% germination of Vigna radiata in presence of decolorized synthetic congo red and gentian violet. Crude extract of laccase decolorized congo red and gentian violet up to 70% and 84% respectively within 24 h. The laccase producing L1 and L2 strains can be used to decolorize and detoxify the textile effluents and assist in wastewater treatment. Keywords: Biodegradation, decolourization, response surface methodology, textile effluents, laccase
1. Introduction In recent years, usage of synthetic dyes have been increasing considerably in textile industries. Improper discharge of textile effluents in the environment cause adverse water pollution [1-3]. Improperly discharged textile effluents accumulate in water bodies and affect the physico-chemical characteristics, visual quality, water transparency and thus pollute the aquatic environment [4-7]. Approximately 2, 80,000 tonnes per year of textile dyes/effluent are being discharged worldwide [8]. Congo red and gentian violet are extensively used in the textile, tannery, paper and food industries. Generally these dyes are abundant in the textile industry effluents and extremely toxic to the environment and humans. These dyes are a major concern to environmentalists, since they can cause aesthetic damage to sites and are also carcinogenic. [1,9,10]. Hence the removal of such dyes from the wastewater is necessary. Various physicochemical methods such as membrane separation, photocatalysis, sonication, irradiation, photochemical process, electrochemical oxidation, ion exchange, activated carbon adsorption, coagulation/flocculation, ozonation and Fenton processes have been reported to remove the dyes from the wastewater [1,11,12]. However these approaches have major limitations such as lack of complete removal, high cost and generation of hazardous secondary wastes [13,14]. These drawbacks of physico-chemical methods can be overcome by enzyme mediated biological degradation of toxic dyes from the wastewater [15,16]. Various enzyme mediated biodegradation of wastwater have been reported in the past [17-19]. Especially the use of laccases for the biological degradation of toxic dyes gaining considerable attention in the recent years because of its higher efficiency and accuracy [20,21]. Laccases are group of oxidative enzymes also known as ‘green catalyst’ which catalyze the oxidation of various aromatic compounds by using molecular oxygen as an electron acceptor [22, 23]. Laccases are widely distributed in plants, fungi and bacteria [23]. The use of fungal laccases for the biodegradation of industrial wastes is well documented [24-
27]. However, the dye degradation efficiency of fungal laccases is pH dependent and efficient only in mild acidic conditions [25]. Unlike fungal laccases, bacterial laccases are independent of pH and are efficient in wide pH range. Extracellular bacterial laccases were produced from wide range of genera including Bacillus sp, Escherichia sp, Pseudomonas sp and Stenotrophomonas sp [28-30]. The bacterial laccases were reported to decolorize and detoxify methanated distillery effluent and pulp paper mill waste [31]. Laccase mediated synthetic dyes biodegradation and decolourization is gaining considerable interest, as it is eco-friendly. Few reports have been proposed for the laccase mediated biodegradation of synthetic dyes [32-35]. However, laccase biodegradation of synthetic dyes require high capital cost which limits its application in commercial scale [36]. Therefore, it is important to optimize the bioprocess parameters for the maximum production of laccases at relatively cheaper cost. Response surface methodology (RSM) is used to optimize the parameters for enzyme production in order to improve the product yield, reducing the time and process cost. The optimization of medium for the production of laccase by response surface methodology has been reported in different fungal strains [37-39]. Laccase production optimization by RSM has been reported in actinomycetes Streptomyces psammoticus as well [40]. To the best of our knowledge this is first report on the optimization of laccase production from bacteria such as Pseudomonas stutzeri L1 and Acinetobacter baumannii L2. In the present study, laccase producing ability of newly isolated strains Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 was investigated. Optimization of extracellular laccase production was performed using one factor at a time optimization approach and full factorial response surface methodology approach. Decolorization and biodegradation (congo red and gentian violet) of the produced laccases were investigated as well. Phytotoxicity assay of the detoxified dyes was also investigated.
2. Materials and Methods 2.1. Samples collection The crude oil samples were collected from crude oil well head at oil reservoir at Karaikal, Tamilnadu, India (latitude: 10.7694 and longitude: 79.6155).The samples were collected in sterile screw capped vials and transported by using icebox from sites to laboratory. Samples were stored at 4°C until further analysis.
2.2.Isolation and screening of laccase producing bacteria The crude oil samples were used for the isolation of bacteria. The bacterial isolate were isolated by a pour plate technique using nutrient agar plates (Himedia, Mumbai). The plates were incubated at 37°C for 24 hours. Colonies were streaked and restreaked frequently until individual cultures were obtained. Isolated bacterial strains were screened for laccase production using 10 mM guaiacol as a substrate in nutrient agar plates [41]. The individual bacterial consortia were streaked on the plate and plates were incubated at 37°C for 72 hours. Colonies which produce the reddish brown or brown color on guaiacol containing nutrient agar plates were consider as a positive laccase producing bacterial strain [41]. An uninoculated plate was used as a control. The laccase producing bacterial strains were maintained in nutrient agar. 2.3.Identification of bacteria by 16S rDNA Sequence The laccase producing bacterial strains were identified by 16S rRNA gene sequencing. Genomic DNA of laccase producing bacterial strains was extracted using genomic DNA extraction Mini-kit (Himedia, Mumbai) according to manufacturer instructions. The eluted genomic DNA was PCR amplification by 16S rRNA gene sequencing by using universal
primers
specific
for
16S
AGAGTTTGATCCTGGCTCAG-3’
rRNA and
gene
forward
reverse
primer
primer
B27F
U1492R
5’5’-
GGTTACCTTGTTACGACTT-3’. The amplifications were performed using thermal cycler (Eppendorf, USA) and the amplification reaction consisted of an initial denaturation at 95ºC for 3 minutes, followed by 35 cycles of 95°C for 30 seconds, 50°C for 30 seconds and 72°C for 2 minutes, and a final extension step at 72°C for 10 minutes. Sequencing was performed by using Big Dye terminator cycle sequencing kit (Applied BioSystems, USA). Sequencing products were resolved on an Applied Biosystems model 3730XL automated DNA sequencing system (Applied BioSystems, USA). The sequences of bacterial isolates after sequencing were analysed using online NCBI BLAST tool program, http://www.ncbi.nib.gov/blast. Phylogenetic analysis was used for comparative genomics to show evolutionary relationships. The analysis was aligning of sequences using tools like CLUSTAL W version 2.0 and after alignment, phylogenetic tree was constructed using molecular evolutionary genetics analysis (MEGA v 5.05). The evolutionary history/phylogenetic analysis or relationship was inferred using the neighborjoining method [42]. 2.4.Optimization of culture conditions for laccase production 2.4.1. Process optimization – one factor at a time approach The production medium was optimized for various parameters such as agitation (static and shaking), days of incubation (1, 2, 3, 4 and 5 days), pH (5, 6, 7, 8, 9 and 10), carbon sources (lactose, glucose, starch, sucrose, fructose and maltose) and nitrogen sources (ammonium chloride, sodium nitrate, yeast extract and ammonium sulphate). For each experiment 1.6 ×104 CFU/mL cells of Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 were used as inoculum in 100 mL of medium. All the experiments were carried out in triplicate and average of three independent experiments values were taken.
The laccase production medium was used as suggested by Sivakumar et al. [43]. The composition of the production medium is (in g.L-1) starch (20), yeast extract (2), KH2PO4 (1.0), Na2HPO4 (0.05), MgSO4 (0.5), CaCl2 (0.01), FeSO4 (0.01), MnSO4 (0.001), ZnSO4 (0.001), CuSO4 (0.002). The bacterial culture was harvested at every 1 day interval. The culture supernatant was obtained by centrifugation (REMI, India) of overnight cultures such as laccase production medium at 10,000 g at 4ºC for 10 minutes and this supernatant was used to crude extracellular laccase enzyme activity. 2.4.2. Process optimization - Response surface methodology CCD approach Based on the one factor at a time optimization studies, the pH, starch and yeast extract were chosen as the key parameters to be optimized. Minitab v16.0 (United States of America) was used to design the RSM experiments. A full factorial CCD (face centered, unblocked) was used. The process variables such as pH (6 - 10), starch (1 - 3 %) and yeast extract (0.1 - 0.3%) were selected as the factors to be optimized, while the laccase activity (U) was selected as the response variable. The design matrix in non-coded and coded units is presented in Table 1. Temperature (37 °C) and agitation speed (150 rpm) were kept constant. An initial inoculum size containing 1.6 ×104 CFU/mL cells was used in all the experiments. RSM experiments were performed in duplicates and the center point experiments were performed in 6 batches for statistical reproducibility. All the samples were analyzed after 4 days of batch incubations. The statistical analysis, in the form of analysis of variance (ANOVA), of the laccase production efficiencies was performed by the Minitab v16.0 software. A quadratic equation (Equation 1) was derived to determine the optimum conditions based on the responses (laccase activity) after 30 days: Y=β0+β1A+β2B+β3C+β11A2+β22B2+β33C2+β12AB+β13AC+β23BC
2.5.Laccase activity measurement Extracellular laccase activity was measured spectrophotometrically with guaiacol (2 mM) as substrate. The reaction mixture contains 1 mL of crude laccase, 1 mL of 2 mM guaiacol and 3 mL of 0.1 M sodium acetate buffer (pH 5.0). Enzyme blank contains 1 mL of distilled water instead of crude laccase [44]. The reaction mixture was incubated at 30º C for 15 minutes and absorbance was measured at 450nm by using UV spectrophotometer (Elico, Double Beam UV-VIS Spectrophotometer SL 210, India). Enzyme activity was expressed as International Units (IU), where 1 IU is defined as amount of enzyme required to oxidize 1 µM of guaiacol per min. The laccase activity in U/mL is calculated using the extinction coefficient of guaiacol (12,100 M-1 cm-1) at 450 nm by the formula: E.A = (A* V) / (t* e * v), where E.A = enzyme activity (U/mL), A = absorbance at 450nm, V = total volume of reaction mixture (mL), t = incubation time (min) and e = extinction coefficient (M-1cm-1), v = enzyme volume (mL).
2.6.Decolorization of synthetic dyes The identified laccase producing bacterium Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 were tested for their ability to decolorize congo red and gentian violet (100ppm- 500ppm). For decolorization experiment hundred milliliters of the optimized laccase production medium was taken in 250 mL conical flasks inoculated with individual strains L1 and L2 with the different concentration 100 ppm to 500 ppm of each dye. The inoculated flasks were incubated at 37ºC for 5 days. The culture was harvested at every 1
day interval. Culture supernatant of decolorization experiment flask was obtained by centrifugation (10,000 g at 4ºC for 10 min) from the overnight cultures [33]. This supernatant was used to measure the decolorization of synthetic dyes by UV spectrophotometer.
Crude laccase (culture filtrate) was also used to study the decolorization of congo red and gentian violet. Crude laccase (41.76 U/mL) was incubated with 100 ppm of each dyes and incubated at 37ºC for 24 h. The percentage of decolorization was calculated with reference to the control samples that were not treated with the enzyme or culture. The supernatant was also treated with the inhibitor 1 mM EDTA and tested for decolorization to check whether decolorization is due to enzyme or other metabolites. The percentage of dye decolorization was calculated by the following formula: (
)×100
2.7.Phytotoxicity assay The Phytotoxicity tests were conducted to evaluate the toxicity of the untreated and treated dye on Vigna radiata. The Phytotoxicity tests were conducted using the protocol suggested by Ayed et al. [45]. Ten seeds of Vigna radiata were sowed into a plastic pot. These pots were sprayed with a solution of 5mL containing congo red (100ppm) and gentian violet (100ppm). L1, L2 and mixed consortia treated congo red and gentian violet containing solution were added to the pot. 5 mL of sterile distilled water was used as the control. Germination, length of the root and shoot were monitored during the entire course of the experiments. The experiments were carried out at room temperature (27ºC).
3. Results 3.1.Isolation, screening and identification of laccase producing bacteria The laccase producing bacteria were isolated from crude oil sample and the results were shown in Fig. 1. Total bacterial count was 41× 104 CFU/mL and five different bacterial colonies Pseudomonas stutzeri L1, Acinetobacter baumannii L2, Pseudomonas aeroginosa L3, Chelatococcus caeni L4 and Achromobacter xylosoxidans L5 were observed. The L1, L2, L3, L4 and L5 individual bacterial colonies were screened for laccase and the results were shown in Fig 1. In the present study L1 and L2 strains were further investigated for their ability to oxidize the laccase substrate and identified as potential laccase producing bacteria. The isolated strains L1 and L2 were characterized for optimization studies to enhance the production of laccase. The parameters including incubation days, agitation, pH, carbon sources and nitrogen sources were carried out to examine the production of laccase by individual isolates L1 and L2. The bacterial isolates were amplified and sequenced by 16S rDNA gene sequencing. The amplified DNA sequences were analyzed using NCBI database and L1, L2, L3, L4 and L5 strains were found as Pseudomonas stutzeri, Acinetobacter baumannii, Pseudomonas aeruginosa, Chelatococcus caeni and Achromobacter xylosoxidans respectively. The sequence similarity and phylogenetic tree were constructed and shown in Fig. 2. The nucleotides sequences data have been deposited in GenBank under the sequence accession numbers KU708859, KU708860, KU708862, KU708863 and KU708865. 3.2.Bioprocess parameters optimization for the production of laccase. The optimum conditions for agitation, pH, carbon source and nitrogen source for the production of laccase were studied. The maximum laccase production by the L1 1.2 U/mL
and L2 1.9 U/mL was higher in shaking condition when compared to static condition L1 0.44 U/mL and L2 0.69 U/mL were shown in Fig 3. The optimization of pH in the production medium was investigated by L1 and L2 the results were shown in Fig 4. The pH 5,6,7,8,9 and10 shows in L1 strain laccase activity 0.57 U/mL, 0.74 U/mL, 0.68 U/mL, 3.69 U/mL, 0.66 U/mL and1.2 U/mL respectively. The L2 strain shows laccase activity 1.07 U/mL, 0.97 U/mL, 1.57 U/mL, 3.39 U/mL, 1.31 U/mL and1.65 U/mL for pH 5, 6, 7, 8, 9 and10 respectively. The optimization of carbon sources for bacterial growth and maximum laccase production were studied. Six different carbon sources such as lactose, glucose, starch, sucrose, fructose and maltose were tested at a range of 2% (w/v) for laccase production by L1 and L2. L1 strain shows laccase activity 7.33 U/mL, 7.6 U/mL, 9.41 U/mL, 1.28 U/mL, 1.58 U/mL and 7.31 U/mL respectively. L2 Strain shows laccase activity 6.79 U/mL, 9.11 U/mL, 11.67 U/mL, 1.59 U/mL, 10.75 U/mL and 7.57 U/mL respectively. Among these carbon sources tested, starch provided a maximum laccase activity which was about 9.41 U/mL in L1 and 11.67 U/mL were observed in L2 shown in Fig 4. The optimization of nitrogen sources for bacterial growth and laccase production were studied. Four different nitrogen sources such as ammonium chloride, sodium nitrate, yeast extract and ammonium sulphate were tested at 0.2% (w/v) for laccase production by L1 and L2. L1 strain shows laccase activity 12.03 U/mL, 16.7 U/mL, 29.41 U/mL and 11.28 U/mL. L2 strain shows 16.79 U/mL, 19.11 U/mL, 41.76 U/mL and 18.95 U/mL respectively. Among these nitrogen sources investigated, yeast extract provided a maximum laccase activity or production of 29.41 U/mL in L1 and 41.76 U/mL were observed in L2. Surface plots obtained from the response surface methodology – full factorial design were shown in Fig 5. It can be observed from Fig 5, that the interaction between starch and the yeast extract was significant.
3.3.Decolorization of synthetic dyes Two different synthetic dyes such as congo red and gentian violet were used for dye decolorization study and the results were shown in Fig. 6 and Fig. 7. The synthetic dye congo red were decolorized by L1 up to 84% and L2 showed 89% decolorization as well as treated with both L1 and L2 mixed consortia for congo red decolorization showed 97% on 5th day (Fig. 6). The gentian violet were decolorized by L1 up to 83% and L2 showed 90% decolorization as well as treated with both L1 and L2 mixed consortia for gentian violet decolorization showed 95% on 5th day (Fig. 7). Also the culture supernatant of L2 showed 70% of congo red and 84% of gentian violet decolorization within 24 h (Fig 8). The culture supernatant was treated with laccase enzyme inhibitor such as EDTA (10 mM), there was no change in color which indicating that the decolorization of congo red and gentian violet was due to laccase enzyme not by any other metabolites. 3.4.Phytotoxicity assay The phytotoxicity for untreated and treated dye effluent on Vigna radiata was evaluated and the results were shown in Fig 9. The untreated congo red dye effluent showed 30% germination, L1 treated effluent showed 70% germination, L2 treated effluent showed 90% germination and L1 and L2 mixed consortia degraded dye effluent showed 100% germination. The untreated gentian violet dye effluent showed 40% germination, L1 treated effluent showed 80% germination, L2 treated effluent showed 90% germination and L1 and L2 mixed consortia degraded dye effluent showed 100% germination. The shoot and root are grown better in the degraded dye effluent than the untreated dye effluent. 4. Discussion Laccase enzyme has wide range of applications such as pulp delignification, [46] bioremediation, [47] ethanol production, [48] biosensors, [49, 50] wine clarification, [51]
detergent manufacturing, transformation of antibiotics and steroids [52] and herbicide degradation [53]. In the present study indigenous laccase producing bacteria were isolated in the crude oil sample collected from oil reservoir, Karaikal, Tamilnadu, India. Crude oil is naturally unrefined petroleum product which contains mixture of hydrocarbons. Bacteria are adapted to this condition are able to degrade the hydrocarbons and toxic pollutants. Laccase received much attention from researchers in the last decade due to its ability to oxidize both phenolic and non-phenolic lignin related compounds and also environmental pollutants. Most of the laccases are derived from fungi (especially white rot fungi). However, the major disadvantage of fungal laccases is that they are acidic in nature and could not be used for the treatment of textile effluents (due to the alkaline nature of the textile effluents). Bacterial laccases with wide pH range can work better than the fungal laccases. Bacterial laccases are highly active and more stable at high temperature, pH and chloride concentrations [54-57]. Laccases from S. psammoticus and S. ipomoea showed high activity at the alkaline pH 7.0 to 8.0 found in wastewater, and tolerance to high concentrations of sodium chloride [58, 59].
Laccase which was at first derived from the rhizosphere of rice (Azospirillum lipoferum) [60]. Previously laccase producing bacteria are isolated from seawater, [61] river sludge/top-soil containing organic litter, [62] soil contaminated with dyes and lignocellulosic wastes [63, 64] and soil samples containing sawdust and dairy effluents [33]. The pH of the culture medium strongly affects many enzymatic reactions and influences the secretion of enzyme in the medium [65]. The optimum pH for production of laccase by white rot fungi was found to be 4.0 to 6.0 [66]. But in the present study, pH 8 was found to be the optimum pH for the growth and extracellular laccase production by
L1 and L2 Fig. 4. The maximum laccase production was observed in pH 8 for L1 3.39 U/mL and L2 3.69 U/mL. Hence, the laccase produced by the isolates L1 and L2 are suitable to treat alkaline industrial effluents/wastewater as well. Nitrogen sources are essential for the appropriate growth and metabolism of microorganisms and enzyme production. The use of costeffective nitrogen sources is important for the production of laccase as these can significantly reduce the cost [67,68]. Among the tested nitrogen source, both the isolates L1 29.41 U/mL and L2 41.76 U/mL gave highest yield, with respect to yeast extract Fig. 4. Yeast extract is the commonly used as a nitrogen source for cell growth and to enhance hydrogen photo-production by Rhodobacter sphaeroides. [69]. Synthetic dyes commonly present in industrial (such as textiles, printing, paper, plastics and leather) effluents [70]. Colored industrial effluent contains different of synthetic dyes including azo dyes which are mostly toxic, mutagenic and carcinogenic in nature. Congo red is a benzidine based azo dye which is used in various industries such as textile, paper and pulp, and leather industries [71]. Synthetic dyes are very complicated to remove because of their complex aromatic structure. In the present study the L1 and L2 has the ability to decolorize the both dyes up to 84% to 90%. It indicates that the strain L2 has the higher decolorization capacity and also mixed consortia. The dye decolorization efficiency of the isolates (pure and mixed consortia) was decreased while increasing the dye concentration which could be attributed by toxicity of the dye. The higher concentration of the dye was found to be lethal to bacterial cells in many instances [72-73]. Increase in dye concentration, decreased the decolorization rate and biodegradation of congo red by Bacillus sp [72]. Enterobacter sp growth was inhibited by higher concentrations (50 and 100 mg L-1) of reactive red 195 [73].
On one hand, 29.41 U/mL and 41.76 U/mL laccase activity was observed in the pure cultures of isolates L1 and L2, respectively. 23 ×104 CFU/mL of individual bacterial cultures L1 and L2 were used for the decolorization study. On the other hand, the mixed consortia had higher laccase activity (58.22 U/mL). Mixed consortia contain 23 ×104 CFU/mL of individual bacterial cultures of L1 and L2. Higher dye decolourizing efficiency of the mixed consortia (among the tested pure and mixed cultures) could be influenced by higher laccase activity of the mixed consortia. Most of the organic dyes are harmful and affect aquatic life [74]. The removal of dyes from industrial effluents prior being discharged into the water bodies is most important in view of health and environmental protection [75]. Gentian violet dye is a carcinogen at several different organ sites [76]. Phytotoxicity assay reveals that treated dyes with the strain L1, L2 and mixed consortia have up to 100% germination efficiency (Fig. 8). A majority of industrial processes to treat dye contaminated wastewater are ineffective and too expensive [77]. Therefore, the development of laccase enzyme mediated decolorization or degradation is an attractive solution due to their potential capability in degrading dyes of different chemical structure as well its eco-friendly biodegradation of toxic pollutants. 5. Conclusion In this study two potent laccase producing bacteria Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 were isolated from crude oil sample. These strains show the laccase activity in guaiacol containing nutrient agar plates which was confirmed by oxidation of the laccase substrate. Biomolecular identification by 16S rRNA gene sequencing confirms that the laccase producing bacteria were Pseudomonas stutzeri and Acinetobacter baumannii. The optimization of various parameters shows the maximum laccase activity were observed by L1 on 4th day (1.2 U/mL), pH 8 (3.39 U/mL), starch 2%
(w/v) (9.41 U/mL), yeast extract 0.2% (w/v) (29.41 U/mL). While at the same conditions, L2 showed maximum laccase activity on 4th day (1.9 U/mL), pH 8 (3.69 U/mL), starch 2% (w/v) (11.67 U/mL), yeast extract 0.2% (w/v) (41.76 U/mL). The synthetic dye congo red were decolorized by L1 showed 84% and L2 showed 89% decolorization as well as treated with both L1 and L2 mixed consortia showed 97% on 5th day. Gentian violet were decolorized by L1showed 83% and L2 showed 90% decolorization as well as treated with both L1 and L2 mixed consortia for gentian violet decolorization showed 95% on 5th day. The culture supernatant of L2 (41.76 U/mL) showed 70% of congo red and 84% of gentian violet decolorization were observed within 24 hours. It can be concluded that these laccase producing Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 strains can be used to decolorize and detoxify the textile effluents and assist in wastewater treatment. It can be used for bioremediation of textile effluent as well.
Acknowledgement This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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Figures Fig. 1. Screening of laccase production on nutrient agar containing 10 mM guaiacol Fig. 2. Phylogenic tree of 16S rRNA gene sequence of Pseudomonas stutzeri L1, Acinetobacter baumannii L2, Pseudomonas aeroginosa L3, Chelatococcus caeni L4 and Achromobacter xylosoxidans L5 and taxonomy Fig. 3. Time course study to determine maximum laccase activity by Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 in shaking and static condition (A) L1 Shaking (B) L2 Shaking (C) L1 Static (D) L2 Static Fig. 4. Effect of pH, carbon and nitrogen sources to determine the maximum laccase activity by Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 (A) L1 (B) L2, SU-sucrose, FR-fructose, ST-starch, LA-lactose, MA-maltose, AC- ammonium chloride, SN-sodium nitrate, YE-yeast extract, AS-ammonium sulphate Fig. 5. 3-D surface plots for the optimization of laccase production by Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 (a) starch versus yeast extract, (b) pH versus yeast extract and (c) pH versus starch. Fig. 6. Decolorization of congo red by laccase producing bacterial strain (a) Pseudomonas stutzeri L1 (b) Acinetobacter baumannii L2 (c) Mixed consortia L1 and L2 Fig. 7. . Decolorization of gentian violet by laccase producing bacterial strain (a) Pseudomonas stutzeri L1 (b) Acinetobacter baumannii L2 (c) Mixed consortia L1 and L2 Fig. 8. Decolorization of (A) congo red and (B) gentian violet by crude laccase Fig. 9. Phytotoxicity study on vigna radiata CR-UT- congo red untreated, CR-L1- congo red L1 treated, CR-L2- congo red L2 treated, CR-M- congo red mixed consortium treated, GVUT-gentian violet untreated, GV-L1- gentian violet L1 treated, GV-L2-gentian violet L2 treated, GV-M-gentian violet mixed consortium treated
L1
L2
Fig. 1. Screening of laccase production on nutrient agar containing 10 mM guaiacol
Fig. 2. Phylogenic tree of 16S rRNA gene sequence of Pseudomonas stutzeri L1, Acinetobacter baumannii L2, Pseudomonas aeroginosa L3, Chelatococcus caeni L4 and Achromobacter xylosoxidans L5 and taxonomy
Fig. 3. Time course study to determine maximum laccase activity by Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 in shaking and static condition (A) L1 Shaking (B) L2 Shaking (C) L1 Static (D) L2 Static
Fig. 4. Effect of pH, carbon and nitrogen sources to determine the maximum laccase activity by Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 (A) L1 (B) L2, SU-sucrose, FR-fructose, ST-starch, LA-lactose, MA-maltose, AC- ammonium chloride, SN-sodium nitrate, YE-yeast extract, AS-ammonium sulphate
Fig. 5. 3-D surface plots for the optimization of laccase production by Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 (a) starch versus yeast extract, (b) pH versus yeast extract and (c) pH versus starch.
Fig. 6. Decolorization of congo red by laccase producing bacterial strain (a) Pseudomonas stutzeri L1 (b) Acinetobacter baumannii L2 (c) Mixed consortia L1 and L2
Fig. 7. Decolorization of gentian violet by laccase producing bacterial strain (a) Pseudomonas stutzeri L1 (b) Acinetobacter baumannii L2 (c) Mixed consortia L1 and L2
Fig. 8. Decolorization of (A) congo red and (B) gentian violet by crude laccase
Fig. 9. Phytotoxicity study on vigna radiata CR-UT- congo red untreated, CR-L1- congo red L1 treated, CR-L2- congo red L2 treated, CR-M- congo red mixed consortium treated, GVUT-gentian violet untreated, GV-L1- gentian violet L1 treated, GV-L2-gentian violet L2 treated, GV-M-gentian violet mixed consortium treated
List of Tables Table 1: Laccase production parameters optimization and experimental runs based on 23 – central composite design.
Table 1: Laccase production parameters optimization and experimental runs based on 23 – central composite design.
Std Order
pH
Starch (%, wt/vol)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
6 10 6 10 6 10 6 10 6 10 8 8 8 8 8 8 8 8 8 8
1 1 3 3 1 1 3 3 2 2 1 3 2 2 2 2 2 2 2 2
Yeast extract (%, wt/vol) 0.1 0.1 0.1 0.1 0.3 0.3 0.3 0.3 0.2 0.2 0.2 0.2 0.1 0.3 0.2 0.2 0.2 0.2 0.2 0.2
S2213-3437(16)30462-6 http://dx.doi.org/doi:10.1016/j.jece.2016.12.021 JECE 1380
To appear in: Received date: Revised date: Accepted date:
16-8-2016 17-11-2016 17-12-2016
Please cite this article as: Sathishkumar Kuppusamy, Manivannan Sethurajan, Murugan Kadarkarai, Rajasekar Aruliah, Biodecolourization of textile dyes by novel, indigenous Pseudomonas stutzeri L1 and Acinetobacter baumannii L2, Journal of Environmental Chemical Engineering http://dx.doi.org/10.1016/j.jece.2016.12.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Biodecolourization of textile dyes by novel, indigenous Pseudomonas stutzeri L1 and Acinetobacter baumannii L2
Sathishkumar Kuppusamya, Manivannan Sethurajanb*, Murugan Kadarkaraic,d, Rajasekar Aruliaha*
a
Environmental
Molecular
Microbiology
Research
Laboratory,
Department
of
Biotechnology, Thiruvalluvar University, Vellore- 632 115, India b
Universite Paris-Est, Laboratoire Geomateriaux et Environnement (LGE), EA 4508, UPEM,
77454, Marne-la-Vallee, France c
Division of Entomology, Department of Zoology, School of Life Sciences, Bharathiar
University, Coimbatore- 641 046, India d
Thiruvalluvar University, Serkkadu, Vellore-632 115, Tamilnadu, India
* Corresponding Author: Dr. Manivannan Sethurajan, Universite Paris-Est, laboratoire Geomateriaux et Environment (LGE), EA 4508, UPEM, 77454, Marne-la-Vallee, France. #current affiliation,
Biofouling and Biofilm Processing Section, Water and Steam Chemistry
Division, BARC Facilities, Kalpakkam 603102, India, e-mail: [email protected]
Dr. Rajasekar Aruliah, Environmental Molecular Microbiology Research Laboratory, Department of Biotechnology, Thiruvalluvar University, Vellore- 632 115, India. e-mail: [email protected] Tel: +91-8675265635.
Abstract Enormous quantities of toxic dyes containing textile effluents are being discharged in natural water systems and thus contaminate the water quality. Hence it is important to develop an eco-friendly and cost effective technology to treat the dyes contaminated wastewater. In this research, laccase enzyme mediated biodegradation of toxic textile dyes such as congo red and gentian violet was investigated using novel, newly isolated laccase producing Pseudomonas stutzeri L1 and Acinetobacter baumannii L2. Response surface methodology (RSM) with full factorial central composite design (CCD) was applied to optimize pH (pH 6.0 – 10.0), starch (1 - 3 %) and yeast extract (0.1 - 0.3%). The RSM results show that a maximum of 29.41 U/ mL and 41.76 U/ mL laccase activity was achieved by L1 and L2, respectively under optimum conditions (pH 8.0, 2% starch and 0.2% yeast extract). The congo red decolorization studies by L1, L2 and mixed consortia (L1 +L2) showed 84%, 89% and 97% decolourization efficiency, respectively. The gentian violet was decolorized up to 83%, 90% and 95% by L1, L2 and mixed consortia (L1 +L2), respectively. Phytotoxicity assay showed100% germination of Vigna radiata in presence of decolorized synthetic congo red and gentian violet. Crude extract of laccase decolorized congo red and gentian violet up to 70% and 84% respectively within 24 h. The laccase producing L1 and L2 strains can be used to decolorize and detoxify the textile effluents and assist in wastewater treatment. Keywords: Biodegradation, decolourization, response surface methodology, textile effluents, laccase
1. Introduction In recent years, usage of synthetic dyes have been increasing considerably in textile industries. Improper discharge of textile effluents in the environment cause adverse water pollution [1-3]. Improperly discharged textile effluents accumulate in water bodies and affect the physico-chemical characteristics, visual quality, water transparency and thus pollute the aquatic environment [4-7]. Approximately 2, 80,000 tonnes per year of textile dyes/effluent are being discharged worldwide [8]. Congo red and gentian violet are extensively used in the textile, tannery, paper and food industries. Generally these dyes are abundant in the textile industry effluents and extremely toxic to the environment and humans. These dyes are a major concern to environmentalists, since they can cause aesthetic damage to sites and are also carcinogenic. [1,9,10]. Hence the removal of such dyes from the wastewater is necessary. Various physicochemical methods such as membrane separation, photocatalysis, sonication, irradiation, photochemical process, electrochemical oxidation, ion exchange, activated carbon adsorption, coagulation/flocculation, ozonation and Fenton processes have been reported to remove the dyes from the wastewater [1,11,12]. However these approaches have major limitations such as lack of complete removal, high cost and generation of hazardous secondary wastes [13,14]. These drawbacks of physico-chemical methods can be overcome by enzyme mediated biological degradation of toxic dyes from the wastewater [15,16]. Various enzyme mediated biodegradation of wastwater have been reported in the past [17-19]. Especially the use of laccases for the biological degradation of toxic dyes gaining considerable attention in the recent years because of its higher efficiency and accuracy [20,21]. Laccases are group of oxidative enzymes also known as ‘green catalyst’ which catalyze the oxidation of various aromatic compounds by using molecular oxygen as an electron acceptor [22, 23]. Laccases are widely distributed in plants, fungi and bacteria [23]. The use of fungal laccases for the biodegradation of industrial wastes is well documented [24-
27]. However, the dye degradation efficiency of fungal laccases is pH dependent and efficient only in mild acidic conditions [25]. Unlike fungal laccases, bacterial laccases are independent of pH and are efficient in wide pH range. Extracellular bacterial laccases were produced from wide range of genera including Bacillus sp, Escherichia sp, Pseudomonas sp and Stenotrophomonas sp [28-30]. The bacterial laccases were reported to decolorize and detoxify methanated distillery effluent and pulp paper mill waste [31]. Laccase mediated synthetic dyes biodegradation and decolourization is gaining considerable interest, as it is eco-friendly. Few reports have been proposed for the laccase mediated biodegradation of synthetic dyes [32-35]. However, laccase biodegradation of synthetic dyes require high capital cost which limits its application in commercial scale [36]. Therefore, it is important to optimize the bioprocess parameters for the maximum production of laccases at relatively cheaper cost. Response surface methodology (RSM) is used to optimize the parameters for enzyme production in order to improve the product yield, reducing the time and process cost. The optimization of medium for the production of laccase by response surface methodology has been reported in different fungal strains [37-39]. Laccase production optimization by RSM has been reported in actinomycetes Streptomyces psammoticus as well [40]. To the best of our knowledge this is first report on the optimization of laccase production from bacteria such as Pseudomonas stutzeri L1 and Acinetobacter baumannii L2. In the present study, laccase producing ability of newly isolated strains Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 was investigated. Optimization of extracellular laccase production was performed using one factor at a time optimization approach and full factorial response surface methodology approach. Decolorization and biodegradation (congo red and gentian violet) of the produced laccases were investigated as well. Phytotoxicity assay of the detoxified dyes was also investigated.
2. Materials and Methods 2.1. Samples collection The crude oil samples were collected from crude oil well head at oil reservoir at Karaikal, Tamilnadu, India (latitude: 10.7694 and longitude: 79.6155).The samples were collected in sterile screw capped vials and transported by using icebox from sites to laboratory. Samples were stored at 4°C until further analysis.
2.2.Isolation and screening of laccase producing bacteria The crude oil samples were used for the isolation of bacteria. The bacterial isolate were isolated by a pour plate technique using nutrient agar plates (Himedia, Mumbai). The plates were incubated at 37°C for 24 hours. Colonies were streaked and restreaked frequently until individual cultures were obtained. Isolated bacterial strains were screened for laccase production using 10 mM guaiacol as a substrate in nutrient agar plates [41]. The individual bacterial consortia were streaked on the plate and plates were incubated at 37°C for 72 hours. Colonies which produce the reddish brown or brown color on guaiacol containing nutrient agar plates were consider as a positive laccase producing bacterial strain [41]. An uninoculated plate was used as a control. The laccase producing bacterial strains were maintained in nutrient agar. 2.3.Identification of bacteria by 16S rDNA Sequence The laccase producing bacterial strains were identified by 16S rRNA gene sequencing. Genomic DNA of laccase producing bacterial strains was extracted using genomic DNA extraction Mini-kit (Himedia, Mumbai) according to manufacturer instructions. The eluted genomic DNA was PCR amplification by 16S rRNA gene sequencing by using universal
primers
specific
for
16S
AGAGTTTGATCCTGGCTCAG-3’
rRNA and
gene
forward
reverse
primer
primer
B27F
U1492R
5’5’-
GGTTACCTTGTTACGACTT-3’. The amplifications were performed using thermal cycler (Eppendorf, USA) and the amplification reaction consisted of an initial denaturation at 95ºC for 3 minutes, followed by 35 cycles of 95°C for 30 seconds, 50°C for 30 seconds and 72°C for 2 minutes, and a final extension step at 72°C for 10 minutes. Sequencing was performed by using Big Dye terminator cycle sequencing kit (Applied BioSystems, USA). Sequencing products were resolved on an Applied Biosystems model 3730XL automated DNA sequencing system (Applied BioSystems, USA). The sequences of bacterial isolates after sequencing were analysed using online NCBI BLAST tool program, http://www.ncbi.nib.gov/blast. Phylogenetic analysis was used for comparative genomics to show evolutionary relationships. The analysis was aligning of sequences using tools like CLUSTAL W version 2.0 and after alignment, phylogenetic tree was constructed using molecular evolutionary genetics analysis (MEGA v 5.05). The evolutionary history/phylogenetic analysis or relationship was inferred using the neighborjoining method [42]. 2.4.Optimization of culture conditions for laccase production 2.4.1. Process optimization – one factor at a time approach The production medium was optimized for various parameters such as agitation (static and shaking), days of incubation (1, 2, 3, 4 and 5 days), pH (5, 6, 7, 8, 9 and 10), carbon sources (lactose, glucose, starch, sucrose, fructose and maltose) and nitrogen sources (ammonium chloride, sodium nitrate, yeast extract and ammonium sulphate). For each experiment 1.6 ×104 CFU/mL cells of Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 were used as inoculum in 100 mL of medium. All the experiments were carried out in triplicate and average of three independent experiments values were taken.
The laccase production medium was used as suggested by Sivakumar et al. [43]. The composition of the production medium is (in g.L-1) starch (20), yeast extract (2), KH2PO4 (1.0), Na2HPO4 (0.05), MgSO4 (0.5), CaCl2 (0.01), FeSO4 (0.01), MnSO4 (0.001), ZnSO4 (0.001), CuSO4 (0.002). The bacterial culture was harvested at every 1 day interval. The culture supernatant was obtained by centrifugation (REMI, India) of overnight cultures such as laccase production medium at 10,000 g at 4ºC for 10 minutes and this supernatant was used to crude extracellular laccase enzyme activity. 2.4.2. Process optimization - Response surface methodology CCD approach Based on the one factor at a time optimization studies, the pH, starch and yeast extract were chosen as the key parameters to be optimized. Minitab v16.0 (United States of America) was used to design the RSM experiments. A full factorial CCD (face centered, unblocked) was used. The process variables such as pH (6 - 10), starch (1 - 3 %) and yeast extract (0.1 - 0.3%) were selected as the factors to be optimized, while the laccase activity (U) was selected as the response variable. The design matrix in non-coded and coded units is presented in Table 1. Temperature (37 °C) and agitation speed (150 rpm) were kept constant. An initial inoculum size containing 1.6 ×104 CFU/mL cells was used in all the experiments. RSM experiments were performed in duplicates and the center point experiments were performed in 6 batches for statistical reproducibility. All the samples were analyzed after 4 days of batch incubations. The statistical analysis, in the form of analysis of variance (ANOVA), of the laccase production efficiencies was performed by the Minitab v16.0 software. A quadratic equation (Equation 1) was derived to determine the optimum conditions based on the responses (laccase activity) after 30 days: Y=β0+β1A+β2B+β3C+β11A2+β22B2+β33C2+β12AB+β13AC+β23BC
2.5.Laccase activity measurement Extracellular laccase activity was measured spectrophotometrically with guaiacol (2 mM) as substrate. The reaction mixture contains 1 mL of crude laccase, 1 mL of 2 mM guaiacol and 3 mL of 0.1 M sodium acetate buffer (pH 5.0). Enzyme blank contains 1 mL of distilled water instead of crude laccase [44]. The reaction mixture was incubated at 30º C for 15 minutes and absorbance was measured at 450nm by using UV spectrophotometer (Elico, Double Beam UV-VIS Spectrophotometer SL 210, India). Enzyme activity was expressed as International Units (IU), where 1 IU is defined as amount of enzyme required to oxidize 1 µM of guaiacol per min. The laccase activity in U/mL is calculated using the extinction coefficient of guaiacol (12,100 M-1 cm-1) at 450 nm by the formula: E.A = (A* V) / (t* e * v), where E.A = enzyme activity (U/mL), A = absorbance at 450nm, V = total volume of reaction mixture (mL), t = incubation time (min) and e = extinction coefficient (M-1cm-1), v = enzyme volume (mL).
2.6.Decolorization of synthetic dyes The identified laccase producing bacterium Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 were tested for their ability to decolorize congo red and gentian violet (100ppm- 500ppm). For decolorization experiment hundred milliliters of the optimized laccase production medium was taken in 250 mL conical flasks inoculated with individual strains L1 and L2 with the different concentration 100 ppm to 500 ppm of each dye. The inoculated flasks were incubated at 37ºC for 5 days. The culture was harvested at every 1
day interval. Culture supernatant of decolorization experiment flask was obtained by centrifugation (10,000 g at 4ºC for 10 min) from the overnight cultures [33]. This supernatant was used to measure the decolorization of synthetic dyes by UV spectrophotometer.
Crude laccase (culture filtrate) was also used to study the decolorization of congo red and gentian violet. Crude laccase (41.76 U/mL) was incubated with 100 ppm of each dyes and incubated at 37ºC for 24 h. The percentage of decolorization was calculated with reference to the control samples that were not treated with the enzyme or culture. The supernatant was also treated with the inhibitor 1 mM EDTA and tested for decolorization to check whether decolorization is due to enzyme or other metabolites. The percentage of dye decolorization was calculated by the following formula: (
)×100
2.7.Phytotoxicity assay The Phytotoxicity tests were conducted to evaluate the toxicity of the untreated and treated dye on Vigna radiata. The Phytotoxicity tests were conducted using the protocol suggested by Ayed et al. [45]. Ten seeds of Vigna radiata were sowed into a plastic pot. These pots were sprayed with a solution of 5mL containing congo red (100ppm) and gentian violet (100ppm). L1, L2 and mixed consortia treated congo red and gentian violet containing solution were added to the pot. 5 mL of sterile distilled water was used as the control. Germination, length of the root and shoot were monitored during the entire course of the experiments. The experiments were carried out at room temperature (27ºC).
3. Results 3.1.Isolation, screening and identification of laccase producing bacteria The laccase producing bacteria were isolated from crude oil sample and the results were shown in Fig. 1. Total bacterial count was 41× 104 CFU/mL and five different bacterial colonies Pseudomonas stutzeri L1, Acinetobacter baumannii L2, Pseudomonas aeroginosa L3, Chelatococcus caeni L4 and Achromobacter xylosoxidans L5 were observed. The L1, L2, L3, L4 and L5 individual bacterial colonies were screened for laccase and the results were shown in Fig 1. In the present study L1 and L2 strains were further investigated for their ability to oxidize the laccase substrate and identified as potential laccase producing bacteria. The isolated strains L1 and L2 were characterized for optimization studies to enhance the production of laccase. The parameters including incubation days, agitation, pH, carbon sources and nitrogen sources were carried out to examine the production of laccase by individual isolates L1 and L2. The bacterial isolates were amplified and sequenced by 16S rDNA gene sequencing. The amplified DNA sequences were analyzed using NCBI database and L1, L2, L3, L4 and L5 strains were found as Pseudomonas stutzeri, Acinetobacter baumannii, Pseudomonas aeruginosa, Chelatococcus caeni and Achromobacter xylosoxidans respectively. The sequence similarity and phylogenetic tree were constructed and shown in Fig. 2. The nucleotides sequences data have been deposited in GenBank under the sequence accession numbers KU708859, KU708860, KU708862, KU708863 and KU708865. 3.2.Bioprocess parameters optimization for the production of laccase. The optimum conditions for agitation, pH, carbon source and nitrogen source for the production of laccase were studied. The maximum laccase production by the L1 1.2 U/mL
and L2 1.9 U/mL was higher in shaking condition when compared to static condition L1 0.44 U/mL and L2 0.69 U/mL were shown in Fig 3. The optimization of pH in the production medium was investigated by L1 and L2 the results were shown in Fig 4. The pH 5,6,7,8,9 and10 shows in L1 strain laccase activity 0.57 U/mL, 0.74 U/mL, 0.68 U/mL, 3.69 U/mL, 0.66 U/mL and1.2 U/mL respectively. The L2 strain shows laccase activity 1.07 U/mL, 0.97 U/mL, 1.57 U/mL, 3.39 U/mL, 1.31 U/mL and1.65 U/mL for pH 5, 6, 7, 8, 9 and10 respectively. The optimization of carbon sources for bacterial growth and maximum laccase production were studied. Six different carbon sources such as lactose, glucose, starch, sucrose, fructose and maltose were tested at a range of 2% (w/v) for laccase production by L1 and L2. L1 strain shows laccase activity 7.33 U/mL, 7.6 U/mL, 9.41 U/mL, 1.28 U/mL, 1.58 U/mL and 7.31 U/mL respectively. L2 Strain shows laccase activity 6.79 U/mL, 9.11 U/mL, 11.67 U/mL, 1.59 U/mL, 10.75 U/mL and 7.57 U/mL respectively. Among these carbon sources tested, starch provided a maximum laccase activity which was about 9.41 U/mL in L1 and 11.67 U/mL were observed in L2 shown in Fig 4. The optimization of nitrogen sources for bacterial growth and laccase production were studied. Four different nitrogen sources such as ammonium chloride, sodium nitrate, yeast extract and ammonium sulphate were tested at 0.2% (w/v) for laccase production by L1 and L2. L1 strain shows laccase activity 12.03 U/mL, 16.7 U/mL, 29.41 U/mL and 11.28 U/mL. L2 strain shows 16.79 U/mL, 19.11 U/mL, 41.76 U/mL and 18.95 U/mL respectively. Among these nitrogen sources investigated, yeast extract provided a maximum laccase activity or production of 29.41 U/mL in L1 and 41.76 U/mL were observed in L2. Surface plots obtained from the response surface methodology – full factorial design were shown in Fig 5. It can be observed from Fig 5, that the interaction between starch and the yeast extract was significant.
3.3.Decolorization of synthetic dyes Two different synthetic dyes such as congo red and gentian violet were used for dye decolorization study and the results were shown in Fig. 6 and Fig. 7. The synthetic dye congo red were decolorized by L1 up to 84% and L2 showed 89% decolorization as well as treated with both L1 and L2 mixed consortia for congo red decolorization showed 97% on 5th day (Fig. 6). The gentian violet were decolorized by L1 up to 83% and L2 showed 90% decolorization as well as treated with both L1 and L2 mixed consortia for gentian violet decolorization showed 95% on 5th day (Fig. 7). Also the culture supernatant of L2 showed 70% of congo red and 84% of gentian violet decolorization within 24 h (Fig 8). The culture supernatant was treated with laccase enzyme inhibitor such as EDTA (10 mM), there was no change in color which indicating that the decolorization of congo red and gentian violet was due to laccase enzyme not by any other metabolites. 3.4.Phytotoxicity assay The phytotoxicity for untreated and treated dye effluent on Vigna radiata was evaluated and the results were shown in Fig 9. The untreated congo red dye effluent showed 30% germination, L1 treated effluent showed 70% germination, L2 treated effluent showed 90% germination and L1 and L2 mixed consortia degraded dye effluent showed 100% germination. The untreated gentian violet dye effluent showed 40% germination, L1 treated effluent showed 80% germination, L2 treated effluent showed 90% germination and L1 and L2 mixed consortia degraded dye effluent showed 100% germination. The shoot and root are grown better in the degraded dye effluent than the untreated dye effluent. 4. Discussion Laccase enzyme has wide range of applications such as pulp delignification, [46] bioremediation, [47] ethanol production, [48] biosensors, [49, 50] wine clarification, [51]
detergent manufacturing, transformation of antibiotics and steroids [52] and herbicide degradation [53]. In the present study indigenous laccase producing bacteria were isolated in the crude oil sample collected from oil reservoir, Karaikal, Tamilnadu, India. Crude oil is naturally unrefined petroleum product which contains mixture of hydrocarbons. Bacteria are adapted to this condition are able to degrade the hydrocarbons and toxic pollutants. Laccase received much attention from researchers in the last decade due to its ability to oxidize both phenolic and non-phenolic lignin related compounds and also environmental pollutants. Most of the laccases are derived from fungi (especially white rot fungi). However, the major disadvantage of fungal laccases is that they are acidic in nature and could not be used for the treatment of textile effluents (due to the alkaline nature of the textile effluents). Bacterial laccases with wide pH range can work better than the fungal laccases. Bacterial laccases are highly active and more stable at high temperature, pH and chloride concentrations [54-57]. Laccases from S. psammoticus and S. ipomoea showed high activity at the alkaline pH 7.0 to 8.0 found in wastewater, and tolerance to high concentrations of sodium chloride [58, 59].
Laccase which was at first derived from the rhizosphere of rice (Azospirillum lipoferum) [60]. Previously laccase producing bacteria are isolated from seawater, [61] river sludge/top-soil containing organic litter, [62] soil contaminated with dyes and lignocellulosic wastes [63, 64] and soil samples containing sawdust and dairy effluents [33]. The pH of the culture medium strongly affects many enzymatic reactions and influences the secretion of enzyme in the medium [65]. The optimum pH for production of laccase by white rot fungi was found to be 4.0 to 6.0 [66]. But in the present study, pH 8 was found to be the optimum pH for the growth and extracellular laccase production by
L1 and L2 Fig. 4. The maximum laccase production was observed in pH 8 for L1 3.39 U/mL and L2 3.69 U/mL. Hence, the laccase produced by the isolates L1 and L2 are suitable to treat alkaline industrial effluents/wastewater as well. Nitrogen sources are essential for the appropriate growth and metabolism of microorganisms and enzyme production. The use of costeffective nitrogen sources is important for the production of laccase as these can significantly reduce the cost [67,68]. Among the tested nitrogen source, both the isolates L1 29.41 U/mL and L2 41.76 U/mL gave highest yield, with respect to yeast extract Fig. 4. Yeast extract is the commonly used as a nitrogen source for cell growth and to enhance hydrogen photo-production by Rhodobacter sphaeroides. [69]. Synthetic dyes commonly present in industrial (such as textiles, printing, paper, plastics and leather) effluents [70]. Colored industrial effluent contains different of synthetic dyes including azo dyes which are mostly toxic, mutagenic and carcinogenic in nature. Congo red is a benzidine based azo dye which is used in various industries such as textile, paper and pulp, and leather industries [71]. Synthetic dyes are very complicated to remove because of their complex aromatic structure. In the present study the L1 and L2 has the ability to decolorize the both dyes up to 84% to 90%. It indicates that the strain L2 has the higher decolorization capacity and also mixed consortia. The dye decolorization efficiency of the isolates (pure and mixed consortia) was decreased while increasing the dye concentration which could be attributed by toxicity of the dye. The higher concentration of the dye was found to be lethal to bacterial cells in many instances [72-73]. Increase in dye concentration, decreased the decolorization rate and biodegradation of congo red by Bacillus sp [72]. Enterobacter sp growth was inhibited by higher concentrations (50 and 100 mg L-1) of reactive red 195 [73].
On one hand, 29.41 U/mL and 41.76 U/mL laccase activity was observed in the pure cultures of isolates L1 and L2, respectively. 23 ×104 CFU/mL of individual bacterial cultures L1 and L2 were used for the decolorization study. On the other hand, the mixed consortia had higher laccase activity (58.22 U/mL). Mixed consortia contain 23 ×104 CFU/mL of individual bacterial cultures of L1 and L2. Higher dye decolourizing efficiency of the mixed consortia (among the tested pure and mixed cultures) could be influenced by higher laccase activity of the mixed consortia. Most of the organic dyes are harmful and affect aquatic life [74]. The removal of dyes from industrial effluents prior being discharged into the water bodies is most important in view of health and environmental protection [75]. Gentian violet dye is a carcinogen at several different organ sites [76]. Phytotoxicity assay reveals that treated dyes with the strain L1, L2 and mixed consortia have up to 100% germination efficiency (Fig. 8). A majority of industrial processes to treat dye contaminated wastewater are ineffective and too expensive [77]. Therefore, the development of laccase enzyme mediated decolorization or degradation is an attractive solution due to their potential capability in degrading dyes of different chemical structure as well its eco-friendly biodegradation of toxic pollutants. 5. Conclusion In this study two potent laccase producing bacteria Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 were isolated from crude oil sample. These strains show the laccase activity in guaiacol containing nutrient agar plates which was confirmed by oxidation of the laccase substrate. Biomolecular identification by 16S rRNA gene sequencing confirms that the laccase producing bacteria were Pseudomonas stutzeri and Acinetobacter baumannii. The optimization of various parameters shows the maximum laccase activity were observed by L1 on 4th day (1.2 U/mL), pH 8 (3.39 U/mL), starch 2%
(w/v) (9.41 U/mL), yeast extract 0.2% (w/v) (29.41 U/mL). While at the same conditions, L2 showed maximum laccase activity on 4th day (1.9 U/mL), pH 8 (3.69 U/mL), starch 2% (w/v) (11.67 U/mL), yeast extract 0.2% (w/v) (41.76 U/mL). The synthetic dye congo red were decolorized by L1 showed 84% and L2 showed 89% decolorization as well as treated with both L1 and L2 mixed consortia showed 97% on 5th day. Gentian violet were decolorized by L1showed 83% and L2 showed 90% decolorization as well as treated with both L1 and L2 mixed consortia for gentian violet decolorization showed 95% on 5th day. The culture supernatant of L2 (41.76 U/mL) showed 70% of congo red and 84% of gentian violet decolorization were observed within 24 hours. It can be concluded that these laccase producing Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 strains can be used to decolorize and detoxify the textile effluents and assist in wastewater treatment. It can be used for bioremediation of textile effluent as well.
Acknowledgement This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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Figures Fig. 1. Screening of laccase production on nutrient agar containing 10 mM guaiacol Fig. 2. Phylogenic tree of 16S rRNA gene sequence of Pseudomonas stutzeri L1, Acinetobacter baumannii L2, Pseudomonas aeroginosa L3, Chelatococcus caeni L4 and Achromobacter xylosoxidans L5 and taxonomy Fig. 3. Time course study to determine maximum laccase activity by Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 in shaking and static condition (A) L1 Shaking (B) L2 Shaking (C) L1 Static (D) L2 Static Fig. 4. Effect of pH, carbon and nitrogen sources to determine the maximum laccase activity by Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 (A) L1 (B) L2, SU-sucrose, FR-fructose, ST-starch, LA-lactose, MA-maltose, AC- ammonium chloride, SN-sodium nitrate, YE-yeast extract, AS-ammonium sulphate Fig. 5. 3-D surface plots for the optimization of laccase production by Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 (a) starch versus yeast extract, (b) pH versus yeast extract and (c) pH versus starch. Fig. 6. Decolorization of congo red by laccase producing bacterial strain (a) Pseudomonas stutzeri L1 (b) Acinetobacter baumannii L2 (c) Mixed consortia L1 and L2 Fig. 7. . Decolorization of gentian violet by laccase producing bacterial strain (a) Pseudomonas stutzeri L1 (b) Acinetobacter baumannii L2 (c) Mixed consortia L1 and L2 Fig. 8. Decolorization of (A) congo red and (B) gentian violet by crude laccase Fig. 9. Phytotoxicity study on vigna radiata CR-UT- congo red untreated, CR-L1- congo red L1 treated, CR-L2- congo red L2 treated, CR-M- congo red mixed consortium treated, GVUT-gentian violet untreated, GV-L1- gentian violet L1 treated, GV-L2-gentian violet L2 treated, GV-M-gentian violet mixed consortium treated
L1
L2
Fig. 1. Screening of laccase production on nutrient agar containing 10 mM guaiacol
Fig. 2. Phylogenic tree of 16S rRNA gene sequence of Pseudomonas stutzeri L1, Acinetobacter baumannii L2, Pseudomonas aeroginosa L3, Chelatococcus caeni L4 and Achromobacter xylosoxidans L5 and taxonomy
Fig. 3. Time course study to determine maximum laccase activity by Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 in shaking and static condition (A) L1 Shaking (B) L2 Shaking (C) L1 Static (D) L2 Static
Fig. 4. Effect of pH, carbon and nitrogen sources to determine the maximum laccase activity by Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 (A) L1 (B) L2, SU-sucrose, FR-fructose, ST-starch, LA-lactose, MA-maltose, AC- ammonium chloride, SN-sodium nitrate, YE-yeast extract, AS-ammonium sulphate
Fig. 5. 3-D surface plots for the optimization of laccase production by Pseudomonas stutzeri L1 and Acinetobacter baumannii L2 (a) starch versus yeast extract, (b) pH versus yeast extract and (c) pH versus starch.
Fig. 6. Decolorization of congo red by laccase producing bacterial strain (a) Pseudomonas stutzeri L1 (b) Acinetobacter baumannii L2 (c) Mixed consortia L1 and L2
Fig. 7. Decolorization of gentian violet by laccase producing bacterial strain (a) Pseudomonas stutzeri L1 (b) Acinetobacter baumannii L2 (c) Mixed consortia L1 and L2
Fig. 8. Decolorization of (A) congo red and (B) gentian violet by crude laccase
Fig. 9. Phytotoxicity study on vigna radiata CR-UT- congo red untreated, CR-L1- congo red L1 treated, CR-L2- congo red L2 treated, CR-M- congo red mixed consortium treated, GVUT-gentian violet untreated, GV-L1- gentian violet L1 treated, GV-L2-gentian violet L2 treated, GV-M-gentian violet mixed consortium treated
List of Tables Table 1: Laccase production parameters optimization and experimental runs based on 23 – central composite design.
Table 1: Laccase production parameters optimization and experimental runs based on 23 – central composite design.
Std Order
pH
Starch (%, wt/vol)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
6 10 6 10 6 10 6 10 6 10 8 8 8 8 8 8 8 8 8 8
1 1 3 3 1 1 3 3 2 2 1 3 2 2 2 2 2 2 2 2
Yeast extract (%, wt/vol) 0.1 0.1 0.1 0.1 0.3 0.3 0.3 0.3 0.2 0.2 0.2 0.2 0.1 0.3 0.2 0.2 0.2 0.2 0.2 0.2