Decolorization and Biodegradation of basic violet dye by fungal- bacterial consortia

The present study was aimed to test the ability of Penicillium citrinum MTCC 8009, Aspergillus terreus MTCC 3006, Bacillus cohnii and their consortia to decolorize basic violet dye. Different parameters such as initial dye concentration, dye to inoculum ratio and period of incubation were studied for the decolourization of the dye. The developed fungalbacterial consortia exhibited maximum percent decolorization (92%) ability when compared to the treatment of dyes by individual microbes. Percent decolorization of basic violet dye (92%) was more efficient using fungal-bacterial (Penicillium citrinum and Bacillus cohnii) consortia than with individual cultures. Phyto-toxicity results indicated that bacterial-fungal consortia (Penicillium citrinum and Bacillus cohnii) treatment was believed to degrade the dyes to non-toxic intermediates. The FTIR analysis also revealed that decolorization of basic violet dyes was due to its degradation.


INTRODUCTION
Dyes are coloured substances, which are used along with a mordant to impart colour to fibres. There are about 10,000 different dyes that are used globally [18]. The textile industry releases about 10% of the above-mentioned value as their effluents in fresh water which was toxic to the environment [15]. The dyes from textile dyeing effluents pose a serious threat to environment even at a very low concentration (1mg/L). So, these effluents must be treated before they reach the environment [27]. Various treatment technologies have been investigated extensively such as the photochemical oxidation [20], membrane [28], chemical coagulation [1,24], adsorption [13,16,19] aerobic and anaerobic biological processes [11,14], nano filtration [8,26], electrocoagulation, ultra-sonic decomposition [3], pre-dispersed solvent extraction [10], ozonation [4,22], colloidal gas aphrons [21] and liquid-liquid extraction [6,12]. Although the aforesaid physico-chemical methods are economical, they generate sludge which are difficult to dispose-off and these sludges are secondary pollutants. There are viable technologies to treat the sludge. Biological treatments are being extensively investigated due to their low cost and efficient degradation process. Adsorption is one such biological method in which the microbial cells take-up the dyes on their external cell surface, and further they absorb the dyes into the cell. After this, biotransformation of the dye which involves enzymatic degradation takes place and as a result of this process the dye loses its toxicity. Several microorganisms belonging to different taxonomic groups like bacteria, fungi, yeast, algae, actinomycetes etc. have shown their capability to degrade dyes. Despite their great promise, both the bacteria and fungi have faced lot of challenges with respect to their ability to decolourise the dyes individually. On the other hand, some of them have resulted in the release of carcinogenic and mutagenic metabolites. The use of fungi in bioremediation of textile effluent was limited because of their slow growth rate and greater hydraulic retention time to decolorize the dyes completely.
An enhanced degradation and detoxification of the textile dyes could be possible with the synergetic actions of fungi and bacteria consortia which provide a better alternative technology for the removal of pollutants in the water [7,17,23]. In addition, rapid rates of decolorization proved that these synergetic consortia might be a powerful weapon to attack the dyes and completely mineralise them into non-toxic substances. Fungi despite of its slower growth rate is known to secrete various extra cellular enzymes like manganese peroxidase, lignin peroxidase and laccase which are non-specific to the dye molecules. These enzymes upon acting on the dye deforms the dye structure thereby reducing its toxicity. Thus, the fungalbacteria consortia coordination could be an additional advantage as they have inductive effects on various enzymes which could have improved action than in individual system. Fungal-bacterial consortia system could be further exploited in depth for the eco-friendly remediation of the textile effluents.

Chemicals
Veratryl alcohol, methyl red, ABTS, Nutrient Medium (NM), toluene, ethyl acetate, methanol and potato dextrose broth (PDB) were obtained from HiMedia Laboratories Pvt. Ltd., Mumbai. Basic violet dye was obtained from TIFAC-CORE department, Kumara guru College of Technology, Coimbatore. All chemicals used were of highest purity available and of an analytical grade. UV-Visible Spectrophotometer (Shimadzu, UV-1800) was used for measuring the absorbance of the solutions. F e b r u a r y 2 0 1 7 w w w . c i r w o r l d . c o m I S S N 2 3 2 1 -8 0 7 X V o l u m e 1 3 N u m b e r 3 J o u r n a l o f A d v a n c e s i n c h e m i s t r y

Degradation of dyes by microbial consortia
The fungal cultures from the stock were inoculated into 250 ml of Erlenmeyer flasks containing 100 ml of PDB and incubated for 8 days at 30°C under shaking condition. One loopful of 24h old grown Bacillus cohnii was inoculated into 100 ml of nutrient broth [NB] and incubated at 37°C for 24h under shaking condition. Penicillium citrinum and Bacillus cohnii consortia was prepared aseptically by transferring the 50 ml of 8 days grown Penicillium citrinum into 250 ml Erlenmeyer flasks containing 50 ml of log phase cells of Bacillus cohnii. Similar method was followed for consortia PA (Penicillium citrinum and Aspergillus terreus consortia) and consortia AB (Aspergillus terreus and Bacillus cohnii consortia). The pre-grown individual cultures and its developed consortia were then used as inoculums for further degradation studies. The dyes were subjected to degradation studies considering different parameters such as initial dye concentration, dye to inoculum ratio and the period of incubation.

Decolorization experiment
Decolorization of basic violet dye was carried out under shaking condition with 100 ml of PDB containing Aspergillus terreus and Penicillium citrinum in PDB, and Bacillus cohnii in NB. Initial dye concentrations used in the investigation were 20, 40, 60, and 100 mg/l of dye. A condition of 50% dye to inoculum ratio was prepared by aseptically transferring equal volumes of dye and inoculum. Similarly, for 75%, it was 37.5 ml of dye and 13.5 ml of inoculum or the developed consortia.
Dye to inoculum ratio (100%) was also prepared aseptically by transferring the loopful of culture or its developed consortia into the prepared dye solution. Aliquots of the cultured supernatant were withdrawn at regular intervals of time and % decolorization were calculated. % decolorization = Initial absorbance -Observed absorbance x100 Initial absorbance

Enzyme assay
The ligninolytic enzymes such as lignin peroxidase [25], laccase [2] and manganese peroxidase [9] were determined as reported in the literature.

Metabolite analysis
After decolorization of basic violet dye, the fungal mycelia were removed by filtration through Whatman filter paper no.1 while bacterial cells were removed by centrifugation at 10,000 rpm for 20 min. Similarly, the Penicillium citrinum-Bacillus cohnii consortium biomass were removed by filtration using mesh cloth followed by centrifugation of the filtrate at 10,000 rpm for 20 min. The supernatant thus obtainedwas subjected to TLC and FTIR analysis.

UV-Visible analysis of degraded products of basic violet dye
The maximum wavelength of the authentic and its degraded products were checked using UV-Visible spectrophotometer. UV-Visible spectra of authentic dye were compared with original dye to find the extent of degradation process.

FTIR analysis
FTIR analysis was performed in order to investigate the changes in surface functional groups of the degraded dye products before and after microbial treatment. FTIR analysis was done using Shimadzu spectrophotometer.

Toxicity studies
To determine the toxicity of the degraded samples, phyto-toxicity test was carried out on Vigna radiata. Ten healthy seeds of Vigna radiata were separately sowed into plastic pots containing 20 g of washed and oven dried sand. The toxicity study was carried out at room temperature 27°C by daily watering 5 ml of degraded dye solution. Simultaneously, control set was carried out at the same time by daily watering it with the dye solution that was not treated. Tests were done and the results are presented as an average.   The percentage decolourization of basic violet dye was evident from the Table 2 that on day 2, B. cohnii and A. terreus decolorized the dye to 52% and 48% at 75% and 100% dye to inoculum ratio whereas P. citrinum and B. cohnii degraded the basic violet dye to 48% at 50% dye to inoculum ratio, respectively. Then on day 6, it is clear that B. cohnii and A. terreus decolorized the dye to 69% and 59% at 50% and 75% dye to inoculum ratio whereas P. citrinum and B. cohnii removed the basic violet dye to 63% at 50% dye to inoculum ratio, respectively. It was apparent from Table 2 that on day 10, B. cohnii and A. Terreus decolourized the dye to 70% and 62% at 50% and 75% dye to inoculum ratio. Thus, it is clear from the Table 2 data that on day 10, the percentage decolourization was found to be maximum i.e., 79% when the dyes were treated with Penicillium citrinum and Bacillus cohnii consortia. A.terreus  42  30  20  52  38  28  68  48  38   B+A   35  42  39  42  42  40  48  46  40   P+A   27  29  26  34  32  30  40  38  32 From the Table 3 on day 2 basic violet dye was degraded to 42% dye by P. Citrinum and B. cohnii consortia at 50% dye to inoculums ratio while Bacillus cohnii and Aspergillus terreus decolorized the dyes to 42% and 39% at 75% and 100% dye to inoculum ratio, respectively. On day 6, table 3 data showed that Penicillium citrinum and Bacillus cohnii consortia at 50% dye to inoculum ratio decolourized the dye to 52% whereas the degradation was achieved only upto 42% and 40% by Bacillus cohnii and Aspergillus terreus consortia at 75% and 100% dye to inoculum ratio, respectively. It is also evident from the Table 3 that the percent decolourization decreases with an increase in dye concentration in all the days using Penicillium citrinum and Bacillus cohnii consortia. Penicillium citrinum and Bacillus cohnii consortia. showed maximum percent (68 %) decolorization on 10 th day with 50% dye to inoculum ratio.    biotransformation of basic violet dye into distinct non-toxic metabolites. These results also indicated that decolorization of the dye was by virtue of biodegradation.

Fig.6-Germination of Vigna radiata seedling on Penicillium citrinum-Bacillus cohnii treated dye solution
Basic violet dye (100 mg/l) strongly inhibited the germination of Vigna radiata. On the contrary, degraded products of the dye did not inhibit the germination of the seedlings (Fig.7). Complete germination (100%) as well as significant growth in the plumule and radicals were observed for the plants grown in (Penicillium citrinum-Bacillus cohnii) a treated dye consortium metabolites as compared to authentic dye. Results of the present investigation indicates that the dye was toxic to these plants, while the metabolites formed after consortium degradation was less toxic, which signifies the detoxification of dye by Penicillium citrinum-Bacillus cohnii consortium. These results also underline the importance of fungal-bacterium synergism for bioremediation of textile effluent in terms of both decolorization and detoxification. Plant bioassays have been used to establish the toxicity levels of dye, and its degraded products on common agricultural crops. The assessment of toxicity of dyes, and its degraded products is often great concern as most of them exert toxic effect on plants and animals when released in stream water. Rubine GFL dye was decolorized to 78% using fungal-bacteria consortia of Pseudomonas sp. and Aspergillus ochraceus [5].They also reported that dye degrading enzymes such as azoreductase, veratyl oxidase, tyrosinase, laccase, and NADH-DCIP reductase enzymes in the medium were responsible for the decolorization of Rubine GFL dye. In the present study, microbial consortia consisting of Penicillium citrinum and Bacillus cohnii showed maximum percent (92%) decolorization for basic violet dye. Ligniniolytic enzymes such as lignin peroxidase, laccase and manganese peroxidase were detected in the medium and these enzymes were believed to be responsible for the degradation of basic violet dye. Further, FTIR analysis for the dye and its degraded product showed the disappearance of major peaks at their functional group and a similar results reported in the literature [5]. Results of phytotoxicity of the present study are in good agreement with their work using fungal-bacterial consortia.

CONCLUSION
Fungal-bacterial synergetic consortium was applied for the degradation of basic violet dye in submerged conditions. Results revealed that the synergetic metabolic activities of Bacillus cohnii and Penicillium citrinum the consortium led to complete decolorization of basic violet dye. An enhanced efficiency of Penicillium citrinum-Bacillus cohnii consortia could be due to constitutive levels of ligninolytic enzymes (laccase, lignin peroxidase and manganese peroxidase). Results of the present investigation paves the way for the decolorization of dye with co-culture approach that could alleviate the pollution problems due to synthetic dyes. Thus in this present work the degradation was found to be more efficient i.e., 92 % of degradation in fungal bacterial (Penicillium citrinum and B. cohnii) consortia at the day 10 for 50% of dye to inoculum ratio when compared with the individual cultures. An increase in the concentration of dye increases the contact time for efficient degradation. FTIR analysis proved that control effluent showed specific peaks at 3464 cm -1 for OH stretching while in degraded product the peaks were obtained at 3387 cm -1 for N-H stretching. The disappearance of major peaks at the alkane groups further supports the degradation process. Phototoxicity studies conducted for authentic basic violet dye showed an inhibition of germination of Vigna radiata seedlings while complete germination was observed for the plants grown in the presence of degraded products which are less toxic or non-toxic to Vigna radiata. F e b r u a r y 2 0 1 7 w w w . c i r w o r l d . c o m I S S N 2 3 2 1 -8 0 7 X V o l u m e 1 3 N u m b e r 3 J o u r n a l o f A d v a n c e s i n c h e m i s t r y Dr.Ramalingam, P. Professor, Department of Biotechnology, Kumaraguru College of Technology, Coimbatore, Tamil Nadu, India, received his doctorate from Anna University and Gulbarga University. His area of research focus on wastewater treatment and bioremediation. He is active reviewer in many National and International Journals. He worked as a Principal Investigator in many R&D projects funded by many well known funding agencies.