Bioremediation of Tannery Wastewater by Chromium Resistant Fungal Isolate Fusarium Chlamydosporium Spfs2-g

The present study assessed the bioremediation potential of Fusarium chlamydosporium SPFS2-g isolated from tannery effluent enriched soil. The isolate exhibited minimum inhibitory concentration (MIC) for Cr(VI) as 500 ppm. The treatment of tannery wastewater with Fusarium chlamydosporium in shake flask experiment resulted in the reduction of chemical oxygen demand respectively after six days of treatment duration.


INTRODUCTION
Tanning industry is considered to be a major source of pollution and tannery wastewater in particular, is a potential environmental concern 1 .The wastewater emanating from tanneries is characterized by a strong color and is heavily polluted with high COD and biochemical oxygen demand (BOD) and inorganic impurities (sodium, calcium, nitrate, ammonia, sulfide, and chloride), dissolved and suspended solids, and other specific pollutants such as vegetable and/or synthetic tannins, sulfonated oils, chromium, Pentachlorophenol and surfactants 2,3 .These colored wastewaters hamper light penetration 4 , whereas high COD results in decreased dissolved oxygen in the aquatic ecosystem 5 .Similarly, chromium toxicity is also one of the major causes of environmental hazards caused by tannery effluents.Chromium exists in several oxidation states (I-VI), more stable as Cr(III) and Cr(VI).Cr(VI) is the toxic form of the element 6 which causes severe diarrhoea, ulcers, eye and skin irritation, kidney dysfunction and probably lung carcinoma 7 .Conventional methods for removing toxic Cr(VI) include chemical reduction followed by the precipitation under alkaline conditions, ion exchange, and adsorption on activated coal, alum, kaolinite, and ash 8 .Most of these methods have major disadvantages; for instance, a need for high energy use and large quantities of chemical reagents, incomplete metal removal and generation of large quantity of toxic waste sludge 9 .Furthermore, such processes may be ineffective or extremely expensive when the initial heavy metal concentrations are in the range of 10-100 mg L -1 as well as high organic load in the effluent 10 .
Microorganisms (bacteria/fungi) are the most important eco-friendly agents for the degradation and detoxification of industrial pollutants during the biological treatment of industrial wastewaters.Therefore, bioremediation is an alternative to conventional chemical and physical methods for wastewater treatment 11 .Fungi are known to tolerate heavy metals 12 .They are a versatile group, as they can adapt and grow under various extreme conditions of pH, temperature and nutrient availability, as well as high metal concentrations 13 .They offer the advantage of having cell wall material which shows excellent metal-binding properties 14 .Generally, microbial biomasses have evolved various measures to respond to heavy metals stress via processes such as transport across the cell membrane, biosorption to cell walls, entrapment in extracellular capsules, as well as precipitation and transformation of metals 15 .There are some studies which have been conducted employing bacteria for remediation of components of tannery wastewater 16,17 or fungal mycelia as bioabsorbent 18,19 .But, most of the studies are pollutant specific targeting only one or two specific pollutants mostly chromium.We could not find even a single study on remediation of COD, color and major ions of tannery wastewater.Therefore, the aim of the present study was to isolate and select fungal strain from tannery effluent enriched soil which can be able to remove the COD, color, Cr(VI) and other major ions from the leather industry wastewater.

Sample collection
The tannery wastewater used in the bioremediation studies was collected in polyethylene containers from the final discharge section of a tannery located in Central Leather Research Institute (CLRI) complex, Kapurthala Road, Jalandhar, India.On the other hand, for soil enrichment study, the tannery wastewater was collected from the chrome tanning stage.The collected samples were brought to the laboratory and stored in a refrigerator at 4 °C till their utilization.

Isolation and identification of potential fungal strain
For artificial soil enrichment experiments, an area of 1 square feet in a garden was selected where tannery effluent with gradually increasing concentrations was added for eighteen months.For the isolation of fungal strain the soil was serially diluted, centrifuged at 900 rpm for five minutes and the supernatant was used for further enrichment in modified Lee ' s minimal medium without glucose (0.25% KH 2 PO 4 , 0.20% MgSO 4 , 0.50% (NH 4 ) 2 SO 4 and 0.50% NaCl) containing tannery sludge as sole source of carbon in Erlenmeyer flasks for three days (150 rpm, 28 ºC).This process was repeated several times with fresh sludge amended minimal salt medium (MSM).The final set of flasks were used for subsequent plating and isolation of fungi on sludge containing MSM plates.The pH of the medium was maintained at 5.30 with 100 mM L -1 citrate-phosphate buffer 20 .The inoculated petriplates were incubated at 28 ºC for seven days.The fungal colonies appearing on MSM were picked and purified by repeated culturing on Potato Dextrose Agar (PDA) and were further identified by National Center of Fungal Taxonomy (NCFT), New Delhi.Out of the isolated fungi, in the present study Fusarium chlamydosporium SPFS2-g was used for the treatment of tannery wastewater (Fig. 1).

D e t e r m i n a t i o n o f m i n i m u m i n h i b i t o r y concentration (MIC) for fungal strain
The Cr-resistance of fungal isolate was evaluated on modified Lee ' s minimal medium (with 0.25% glucose) supplemented with 100, 200, 300, 400, 500, 600, and 700 ppm concentrations of hexavalent chromium.The petriplates were inoculated with 8 mm agar plugs from young fungal colonies, pre-grown on PDA and incubated at 28ºC for seven days 21 .The fungal growth was used as a measure of viability and it was determined by measuring the change in mycelia length with the help of measuring scale at 24 hours interval from the 3 rd day to the 7 th day post-inoculation 22 .The minimum inhibitory concentration for Cr(VI) [MIC Cr(VI)] was defined as the concentration of hexavalent chromium that inhibits visible growth of the fungal isolate.

Fungal inoculum preparation
For tannery wastewater bioremediation studies, the fungal inoculum was prepared in the form of mycelial pellets.Erlenmeyer flasks (250 ml capacity) containing 100 ml potato dextrose broth (PDB) and streptopenicillin (100 ppm) were inoculated with mycelial discs 19 .These flasks were incubated at 30 ºC for 5 days in orbital shaker at 150 rpm.The mycelium thus obtained was filtered by cheesecloth and air-dried on sterilized petriplates.Fungal pellets were prepared by cutting in approximately 1.5-2.0mm size.The fungal pellets (2% w/v) were inoculated in combined tannery effluent amended with 0.1% glucose and 0.1% ammonium nitrate.The pH was maintained at 5.30 and the flasks were incubated at 30 ºC in a shaker for six days at 150 rpm.The wastewater samples were collected at different time intervals (2d, 4d and 6d) and reduction in COD, color, Cr(VI) and other pollution parameters were measured.

Physico-chemical analysis of the tannery wastewater
The tannery wastewater samples were analyzed for physico-chemical parameters as per standard methods for wastewater analysis.Chemical oxygen demand (COD) and total suspended solids (TSS) were determined according to American Public Health Association (APHA) methods 23 .Color was measured spectrophotometrically (465 nm) according to the method of Bajpai et al. 24 .The hexavalent chromium [Cr(VI)] was determined colorimetrically using the diphenylcarbazide (DPC) method 23 .
Other parameters of the wastewater e.g.pH, electrical conductivity (EC), and total dissolved solids (TDS) were measured using Multi Parameter Water Analyzer Kit (WTW, Germany).Sodium, chloride and nitrate ions were measured by Thermo Scientific Orion DUAL STAR ion meter while turbidity was measured by Digital Turbidity Meter (Environmental and Scientific Instruments Co., India).
The data obtained in the study were analyzed by Duncan's Multiple Range Test using SPSS Inc. (v 17.0) software.The differences between means were considered significant at values of pd"0.05.

Isolation and Screening of fungi
The fungal strain Fusarium chlamydosporium SPFS2-g was isolated from tannery effluent enriched soil by serial dilution technique.The isolate exhibited MIC for Cr(VI) as 500 ppm.The results indicated that some native fungi have a marked adaptation to heavy metals under constant metal stress for a long time, and the toxic metals were even used as micronutrients by these growth stimulated fungi 25 .Similar to our findings, Aspergillus niger, Aspergillus lentulus, Penicillium sp., and Fusarium solani isolated from contaminated sites have been reported to tolerate 1000 ppm Cr(VI) 17,26,27 .The tolerance to Cr(VI) occur by various mechanisms such as transport across the cell membrane, biosorption to cell walls and entrapment in extracellular capsules, precipitation, complexation and oxidation-reduction reactions 15 .
Many microorganisms could develop potential to biodegrade the recalcitrant pollutants when they are exposed to polluted environment 31 extended time duration.On this line, the treatment of tannery wastewater with Fusarium chlamydosporium SPFS2-g isolated from tannery wastewater affected soil resulted in significant reduction of pollution parameters after six days, concomitant with the increase in fungal dry weight (Table 2; Fig. 2).The   32 .The tannery wastewater also registered reduction in pH from 5.30 to 4.60, due to release of organic acids by the fungal isolate.The acidic environment facilitated the bisorption of Cr(VI) ions.Many researchers have also reported highly acidic pH for Cr(VI) biosorption 33,34 .In aqueous solution, chromium ions generally exist in two stable oxidation states, trivalent and hexavalent.The former exists as Cr 3+ , Cr(OH) 2+ and Cr(OH) 2 + in the pH range 1.0-6.0 and starts precipitating as Cr(OH) 3 at a pH value > 6.0, while Cr 6+ forms H 2 CrO 4 and HCrO 4 " species at pH 2.0-3.0 35 .Thus, at low pH values oxyanionic species of Cr 6+ is likely to be attracted by the positively charged functional groups present on the fungal cell surface.
Similarly, the reduction in TSS and turbidity was ascribed to entrapment of suspended solid particles by the filamentous fungi 36 .Whereas, reduction in NO 3 -, Na + and Cl -ions might be attributed to utilization of these ions for growth by the fungal isolate 37 .

CONCLUSION
Fusarium chlamydosporium SPFS2-g isolated from tannery effluent enriched soil exhibited detoxification of tannery wastewater.The treatment of tannery wastewater with Fusarium chlamydosporium resulted in the reduction of COD, color, Cr(VI), total suspended solids (TSS), turbidity, Na + , Cl -, and NO 3 -in the order of 71.80, 64.69, 100, 36.47,22.77, 11.69, 27.87 and 62.33%, respectively after six days of duration.As the bioremediation activity is highly regulated by cell metabolism, which in turn is controlled by media components such as carbon and nitrogen sources and their ratio in addition to other process parameters like pH, incubation temperature and aeration.Thus, in a future there is a need to conduct process parameter optimization studies to improve the bioremediation efficiency of the isolate.