MICROBIAL TREATMENT OF OFFSET PRINTING INDUSTRY EFFLUENT

ABOUT AUTHORS:
Archana G. Kulkarni, R.S. Awasthi
Dept. of Microbiology, Shivaji Mahavidyalaya,
Renapur (M.S.)
*agnk.77@gmail.com

ABSTRACT
Out of 57 bacterial isolates only 3 bacterial isolates degraded azo dyes, observed in offset printing industry effluent. The strains were isolated from outlets of offset printing industry and then identified as Bacillus cereus, Bacillus subtilis and Micrococcus indicus. All three strains were further used for the treatment of effluent by optimizing the environmental and nutritional conditions. The physiochemical analysis of treated and untreated effluent was carried out by using standard methods as well as metals by Atomic Absorption Spectroscope and the analysis of degraded products by HPLC, LC MS. These bacteria appreciably mineralized the azo dyes viz. Scarlet red and Magenta, observed in the effluent. These bacteria thus could be used to device a simple but effective and viable microbial technology for treatment.

REFERENCE ID: PHARMATUTOR-ART-1746

INTRODUCTION
A significant proportion of the commercially available dyes are used each year in the textile, cosmetics, food, dyes (Y.Fu.et al., 2001) and printing industries.  The offset printing industry is one of such highly dye consuming industry. The offset printing end up in washing the equipments and this has lead to discharge of colored effluent which is very toxic for flora and fauna of receiving water body by increasing the COD and BOD levels. Though the effluent can be treated by various physicochemical methods but the interest has been focused on microbial degradation as an acceptable alternative (Arora ,S. et al., 2005 Yiguo.H. et al.,2007).Microbial degradation of azo dyes has been reported using different microorganisms (Raffi, et al., 1990; Latha,K. et al., 2004). This paper describes the isolation and identification of microorganisms, which degraded azo dyes and their application in the treatment of offset printing industry effluent.

MATERIALS AND METHODS
Collection of Effluent:
The offset printing industry effluents were collected from disposal site of five major printing industries within Latur city (MS) .All the samples were further used for decolorization studies.
All chemicals used in present study were standard analytical grade.

Isolation, Screening and Identification of Effective Microorganisms (EM):
A few studies have been conducted to isolate and identify bacterial species that are capable of reducing and degrading azo dyes (W. Sugiura et al., 1999).The serial dilution technique was adopted to isolate the microbial strains from printing industry effluents.1ml of the effluent was serially and different dilutions were plated on presterilised nutrient agar (Ram Chandra 2001).Plates were incubated at 28? ± 1?C in the incubator (Kumar’s bacteriological incubator-KI-05-03) for 24h.Morphologicaly distinct colonies were picked and purified by streaking.  Purified culture was then used for further decolorization studies i.e. screening of effective microorganisms.

A loop full of cell growth of all 57 different isolates was inoculated separately in 250 ml Erlenmeyer flask, containing 50 ml of presterilised nutrient broth with 2ml of effluent. All the flasks then incubated at 28? ± 1?C in the incubator (Kumar’s bacteriological incubator-KI-05-03) for 24h.  Screening was based on percent decolorization and time required for decolorization i.e. an optical density was measured on a UV-visible spectrophotometer Elico Ltd. SL159) at respective lmax 327nm and used for assessment of percent decolorization.

Percent decolorization was calculated by following formula-

% Decolorization = Initial Abs -Final Abs´100 

Initial Abs
On the basis of screening results only 3 bacterial strains were selected and identified by using microbial, biochemical characterization and 16s r-RNA technique. Identified strains then used for treatment of effluent by making their combinations.

Microbial Treatment of Effluent:
The decolorization potential of microorganisms either in pure form or consortium depend on the components of the medium and growth conditions. (S.Arora et al., 2007) therefore the nutritional and environmental parameters such as pH, temperature, carbon and nitrogen sources, effluent concentration etc. were optimized.

Assay of Decolorization:
The developed combinations were grown separately in 250 ml Erlenmeyer flask, containing 50 ml of presterilised nutrient broth with 2ml of effluent with all optimum conditions. All the flasks then incubated at 30?C (optimum temperature) for 24h.The colour and degradation was measured at 6h intervals up to 24h.Maximum percent decolorization shown by combination in 24h was selected as potent combination and proceed for degradation studies. An aliquot was centrifuged at 8000g for 15 minutes to separate the cell mass. Supernatant was used to determine the decolorization by measuring change in absorbance at respective lmax i.e. 327nm, quantitatively using UV-vis spectrophotometer (ELICO ltd. SL159).

The degradation was monitored by High Performance Liquid Chromatography (HPLC). After complete decolorization by combination (BM), culture broth was centrifuged at 12000 rpm for 30 minutes and equal volume of ethyl acetate was used for extraction. The extracts were dried over anhydrous Na2SO4 and evaporated to dryness in rotary evaporator. The crystals obtained were dissolved in small volume of HPLC grade methanol and used for analysis. HPLC (Simadzu) was carried out on column (YMC ODS, 150 mm x 4.6 mm, 5- μ, ID: E-AC-1/07/COL/22) with 0.05 % TFA (Tri fluro acetic acid) in acetonitrile as mobile phase at flow rate of 1.4 mL/min at wavelength 220 nm and performance of MBR i.e. treated effluent by consortium results in visually decolorization and the degraded products were confirmed by using Liquid Chromatography and Mass Spectrum (LCMS) (Simadzu) on column (YMC ODC,50x4.6mm,3μ ID: E-AC-1/07/COL/26) with 0.05 % TFA (Tri fluro acetic acid) in acetonitrile as mobile phase at flow rate of 1.2mL/min at wavelength 220nm.

The screening of the effluent sample by HPLC and comparison study on LC MS was done under polar mobile phase. Though M+ for monomeric unit of dye is not observed in LC MS analysis however M+=550 for the fragmented dimeric unit a Scarlet red (ret. time- 3.95) was observed in LCMS and molecular fragments of Magenta (M+=284) (ret. Time 3.4) was observed in LC MS analysis indicates the presence of said dyes in the offset printing industry effluent. To conclude the effluent consists of unconsumed mixture of Scarlet red, Magenta dye, some degraded organic aromatic fragments along with Kerosene and suspended particles in water (Graph: 1, 2, 3).

All the assays were performed in triplicate and compared with uninoculated controls.

Physicochemical Analysis of Effluent:
The untreated and treated effluent was analyzed after 0, 6, 12, 18 and 24h of incubation. The effluent was analyzed for pH, COD, BOD, DO, TS, TDS, TSS, and different metal ions like Cu, Fe, Mn, Zn and Cd as per standard methods. (APHA, 1995).

RESULTS AND DISCUSSION
Isolation Studies:
After screening for decolorization, out of 57 bacterial isolates only three of them had shown the maximum decolorization activity within 24h .Based on microbial, biochemical characterization three bacterial strains were tentatively identified and confirmed by 16s rRNA identification (courtesy, NCCS Pune). The strains are Bacillus cereus,Bacillus subtilis, and Micrococcus indicus.  (Table:1,)    

Though the variety of microorganisms are capable for dye degradation or dye removal the effectiveness of microbial decolorization depends on the adoptability and the activity of selected microorganisms (R.G.Sartale et al., 2009) and usually a consortium of microorganisms posses more ability to biodegradation and mineralization than that of an individual bacterial strain due to their synergistic metabolic activity (M.S. Khehra et al., 2005).

In the present study different combinations of microorganisms were used to understand their capability of decolorization and one potent combination of Bacillus cereus and Micrococcus indicus (BM) was selected on the basis of percent decolorization and time required for decolorization. 78.29 % activity was showed by BM within 24h. Therefore this combination was further used for microbial treatment of effluent (Table: 2).

Decolorization activity was monitored for 24h with an interval of 6h and it was observed that the decolorization increased till 24^th hour. This was achieved by optimizing the nutritional and environmental conditions. After repetitive study of microbial treatment the treated effluent was further used for the final chemical analysis i.e. HPLC and LC MS to trace out the residual chemical contents. Consequently, LC MS and HPLC analysis showed no presence of dyes Scarlet red and Magenta (LC MS retention time 3.95, 3.4 respectively). Only some untraceable organic compounds (not ionizing on LCMS) were found in very low and negligible concentrations. 

The degraded effluent was further characterized for physicochemical characterization as well, and it was found that, the values of different parameters decreased significantly. The heavy metals present in effluent i.e. Cu, Fe, Mn, Zn and Cd decreased by 51%, 65%, 60% 58% and 91% respectively and this was checked on Atomic Absorption spectrophotometer (Chemito AA203). (Table: 3)

CONCLUSION
Experiment aimed for removal of biologically hazardous dye contents from effluent of offset printing industry, showed remarkable results i.e. the developed combination BM has the ability to mineralize the dyes present in the effluent and can be used to device a simple but effective and viable microbial technology for treatment.

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