Chapter IV. Optimization of Cultural Conditions for Decolorization of Textile Dye Effluent

Transcription

1 Chapter IV Optimization of Cultural Conditions for Decolorization of Textile Dye Effluent The ability of the fungal system to degrade azo dyes depends on the structural characteristics of the dyes, temperature, ph, effects of various concentrations of carbon and nitrogen sources, inoculum size and presence of intermediates and is triggered by various parameters. The substituted rings of azo dyes are responsible for intense color, water solubility and resistance to degradation under conventional techniques (Forgacs et al., 2004). Fungi appear to be most appropriate in the treatment of colored effluents for removal, reduction and detoxification of industrial effluents. (Martinez et al., 2005). As fungal decolorization systems are greatly affected by nutrients as concentrations of carbon, nitrogen etc. and several environmental factors such as temperature, ph, effluent and oxygen concentrations, these conditions therefore need to be optimized for evolving a successful biodecolorization strategy. This chapter therefore, aims at optimization of several conditions as ph, temperature, inoculum size, effluent concentrations, concentration of medium constituents as glucose, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride and mode of cultivation for attaining maximum decolorization. Materials and Methods 10 6 spores / ml of R. oryzae were inoculated in a decolorization medium (Chapter III). Batch studies were conducted and the effect of various ph ( ), temperatures (25 45 C), effluent concentrations (1:1 1:80), inoculum size ( spores/ml) and medium components in g/l as glucose (0, 1 20), 69

2 ammonium chloride (0, ), calcium chloride (0, 0.5 3), potassium dihydrogen phosphate (0, 0.5 3), magnesium sulfate (0, 0.5 3) and mode of cultivation i.e. static and shaking on growth (DCM mg/50 ml) and decolorization (%) were examined in temporal sequence from 2 nd day onwards at every alternate day, up to 14 days considering the day of inoculation as 0 day. The experiments were conducted in triplicates. Statistical Analysis The data of the effect of optimized conditions on growth and decolorization by the isolates, as compared to before optimization was subjected to Paired T test using SPSS Results and Discussion Table 4.1 show the effect of various ph (3-8) on growth (mg/50 ml) and decolorization (%) by R. oryzae. R. oryzae attained maximum growth and decolorization at ph 4.5 (101 mg/50 ml, 85%), followed by ph 4 (79 mg/50 ml, 70%) and ph 5 (80 mg/50 ml, 69%) on 8 th day, with constant stationary phase. Least growth and decolorization were at ph 8 indicating alkaline ph not favorable for fungal decolorization. There was a considerable decrease in growth and decolorization, as ph was shifted towards alkaline i.e. from ph 5.5 to ph 8. Table 4.1 also indicates that R. oryzae can grow and decolorize the effluent at a wide ph range i.e. from 3 to 8 with optimum at ph 4.5. It is very evident from the examination that maximum growth and decolorization are correlated to each other. Maximum decolorization was achieved during the period of maximum growth. At times it was observed that after achieving maximum growth and decolorization, growth decreased but decolorization remained stable. 70

3 The results are similar to Jilani et al., 2010 showing acidic ph to be favourable for decolorization. ph is a critical factor for primary fungal growth and consequent dyestuff degradation by ligninases synthesized in secondary growth. As each microbial strain and its enzyme are highly specific to ph, decrease in decolorization occurs with changes in ph. Kadpan et al., 2000 suggested decolorization to be due to fungal ligolytic enzymes, proving it to be efficient at lower ph. The results of ph 4.5 to be optimum for decolorization are in agreement to the fact that in open aquifer, under sunlight and without any optimized conditions, the highest efficiency of decolorization of effluent was observed at acidic or low ph (Mahmoud et al., 2007). Similar results were also reported by Bhatt, 2008, proving acidic ph to optimal for degradation of lignin from paper and pulp mill effluents by fungi. Aksu et al., 2000 reported ph 2 to be optimum when dye uptake capacity of dried R. arrhizus was studied and a decrease dye uptake was observed with increase in ph. Similarly, Patricia et al., 2004 reported that Ascomycete yeast strain showed maximum decolorization of azo dyes in the acidic range. Higher decolorization at lower ph value may be due to the electrostatic attraction between negatively- charged dye anions and positively charged cell surface (Zille, 2005). On contrary Kumaran, 2011 have reported maximum decolorization at ph 7 and 8 by Pleurotus sajor-caju. Also P. chrysosporium showed maximum decolorization at ph 7 and 8. Perumal et al., 2000 have reported ph 6.5 to be optimum for growth and degradation by a Basidiomycete, Ganoderma lucidium. Fungi produce their own buffering system, a reason for better decolorization at low ph values. They may utilize accumulated acids later during their growth, leading to ph changes. Under acidic conditions, hydrogen ions react with different amino groups of dye structure and thus results in formation of ionic form of dyes which is less stable. The transformation of dye from neutral 71

4 compound to an ionic form facilitates its removal from effluent by several removal techniques (Prasad et al., 2007). Retardation of decolorization below ph 4 and above ph 5 may be due to fungi s inability to grow beyond its optimum ph for growth i.e. between Highest decolorization was attained at acidic ph in comparison to neutral or basic ph, as acidic ph is required for growth and decolorization (Bhatt, 2008). Moreover as decolorization increased, ph shifted towards acidic. This effect indicates that as consumption of glucose increases, the rate of organic acids production in medium also increases. (Chen et al., 2003). In case of ph change from lower to higher level, a decrease in decolorization efficacy was observed, perhaps because of polymerization of colored compounds. Table 4.2 shows the effect of various temperatures (25 45 C) on growth and decolorization by R. oryzae. R. oryzae showed maximum growth and decolorization at 37 C (80 mg/50 ml, 70%), followed by 30 C (60 mg/50 ml, 53%) and 45 C (49 mg/50 ml, 27%) on 8 th day. Least growth and decolorization were at 25 C. Least decolorization at this temperature showed that the maxima for enzyme activation had not been achieved and also due to low biomass production showing that C is favorable for fungal growth in comparison to 25 C. Decreasing temperature i.e 25 C stalked fungal growth and decreased enzymatic activity. Even at the increasing temperature 40 C, fungal growth rapidly decreased which consequently decreased decolorization extent. The results of the present study show that, dye removal is influenced by fluctuating temperature. Both growth and decolorization were inhibited at lower temperature i.e. 25 C. 37 C had the highest positive impact on the decolorization potency. Table 4.2 also indicates that R. oryzae can grow and decolorize the effluent at C, though to the varying extent. 72

5 Table 4.1 Effect of ph (3 8) on growth ( Gr - mg / 50ml) and decolorization (D - %) by R. oryzae ph R. oryzae Gr 4± ± ± ± ± ± ±0.57 D 8± ± ± ± ± ± ±1.20 Gr 8± ± ± ± ± ± ±1.52 D 17± ± ± ± ± ± ±0.57 Gr 22± ± ± ± ± ± ±1.00 D 35± ± ± ± ± ± ±1.00 Gr 39± ± ± ± ± ± ±2.00 D 41± ± ± ± ± ± ±1.00 Gr 30± ± ± ± ± ± ±2.00 D 10± ± ± ± ± ± ±1.00 Gr 35± ± ± ± ± ± ±2.64 D 17± ± ± ± ± ± ±1.57 Gr 25± ± ± ± ± ± ±2.51 D 24± ± ± ± ± ± ±1.00 Gr 22± ± ± ± ± ± ±1.15 D 11± ± ± ± ± ± ±1.00 Gr 20± ± ± ± ± ± ±2.08 D 13± ± ± ± ± ± ±1.00 Gr 26± ± ± ± ± ± ±1.00 D 2±1.73 5±1.68 7± ± ± ± ±1.00 Gr 11± ± ± ± ± ± ±1.00 D 3±2.00 7±1.73 9± ± ± ± ±1.00 ± - standard deviation (SD) 73

6 Chen, 2002 and Chen et al., 2002 reported that optimum temperature for color removal of red azo dyes was 30 C and 35 C respectively in agreement to the result of 37 C to be optimum for R. oryzae. Mehna et al., 1995 reported that, a temperature from 25 C to 35 C was the optimum for color reduction. The results are quite contrary to the observations that elevated temperatures (50 C to 60 C) even supported the enzymatic activity and decolorization of polymeric dyes by different fungal strains as reported by Nyanhongo et al., Thongchai and Worrawit, 2000 explained that color reduction increased with increasing temperature due to higher respiration and substrate metabolism at the elevated temperature. Results are also contradictory to Erum and Ahmed, 2011 reporting maximal biomass production at 45 C. Table 4.2 Effect of temperature (25 45 ºC) on growth (Gr - mg / 50ml) and decolorization (D - %) by R. oryzae Temp (ºC) R. oryzae Gr - 9± ± ± ± ± ±1.00 D - 1±1.00 1±1.52 5±1.00 5±1.00 5±1.00 5±1.00 Gr 20± ± ± ± ± ± ±1.00 D 11± ± ± ± ± ± ±1.00 Gr 38± ± ± ± ± ± ±1.00 D 26± ± ± ± ± ± ±1.00 Gr 14± ± ± ± ± ± ±1.00 D 2± ± ± ± ± ± ±1.00 ± - standard deviation (SD) Table 4.3 shows the effect of various effluent concentrations on growth and decolorization by R.oryzae. 74

7 Maximum growth and decolorization were attained at 1:60 dilution (94 mg/50 ml, 86%) on 8 th day. Both growth and decolorization increased with increasing the effluent concentration from 1:5 reaching maximum at 1:60, therafter at 1:80 both growth and decolorization efficiency of the isolate was supressed. However, at 1:1 dilution, also both growth and decolorization could not be achieved showing the toxicity and hazardous effect of such effluent on fungi. Effluent is also inhibitory at lower concentrations. Zhang, 1977 observed that decolorization varies strongly with the proportion of the effluent in the medium, the effluent being inhibitory at higher concentrations and deprived of nutrients at lower concentrations. The present results are in corroborate to Pallerla and Chambers, 1997 and Bhatt, 2008 who reported lower decolorization at lower dilutions, which gradually increased at higher dilutions. Decrease in decolorization at at higher dilutions could be due to chromophoric compounds becoming less accessible as substrates. Also adsorption, the first step of color removal is significantly lower at reduced color levels, diminishing the interaction between chromophoric material and mycelia, eventually hampering the effective initiation of biodegradation (Driessel and Chrisotv, 2001). Ali et al., 2007 reported that elevated concentrations of dye from 50 to 200 ppm had inhibitory effect on fungal growth and decolorization. Buitron et al., 2004 reported that color removal of AR 151 dye was upto 99% using concentration of dye 50 mg/l. In contradiction to this, increase in concentration of dyes at times were found to facilitate higher decolorization, thereby indicating that most probably the higher concentration triggers the metabolizing properties of fungus (Arora and Chander, 2004) or dyes might have been used as an alternative carbon source other than glucose. Besides, decolorization of dyes at higher concentration may create an acidic situation which further facilitates their better removal by fungi (Aksu and Tezer 2000; Mansur et al., 2003; Baldrain, 2004). 75

8 Although decolorization was higher at 1:60 dilution, 1:10 was considered optimum for 2 reasons: with dilutions below and beyond 1:10, decolorization did not remain stable, making comparative studies for decolorization ambiguous. Also, when deciding between two dilutions, for industrial effluent treatment lower dilutions are considered to be the most effective (Knapp, 2001). Table 4.3 Effect of effluent concentration (1:1 1:80) on growth (Gr - mg / 50ml) and decolorization (D - %) by R. oryzae Effluent Concentration Gr D Gr 10± ± ± ± ± ± ±1.00 D 12± ± ± ± ± ± ±1.52 R. oryzae Gr 23± ± ± ± ± ± ±1.00 D 33± ± ± ± ± ± ±1.00 Gr 20± ± ± ± ± ± ±1.00 D 17± ± ± ± ± ± ±1.00 Gr 29± ± ± ± ± ± ±1.00 D 20± ± ± ± ± ± ±1.00 Gr 37± ± ± ± ± ± ±1.00 D 27± ± ± ± ± ± ±1.00 1:80 ± - standard deviation (SD) Gr 17± ± ± D

9 Table 4.4 shows the effect of various inoculum sizes on growth and decolorization by R. oryzae. R. oryzae showed maximum growth and decolorization at an inoculum size of 10 8 spores/ ml (81 mg/ 50 ml, 76%), followed by 10 9 spores/ml (67 mg/50 ml, 56%) and spores/ml (51 mg/50 ml and 56%) 8 th day. Both growth and decolorization increased with increase in inoculum size from 10 4 spores/ml reaching maxima at 10 8 spores/ml and decreasing thereafter. The present result suggested positive correlation between inoculum size and decolorization by Ganoderma lucidum (Perumal et al., 2000). Erum and Ahmed, 2010 have reported that maximum decolorization was obtained with 2% inoculum for Aspergillus sp. Also inoculum size of 1, 5 and 10% had adverse effect on dye removal efficiency. There was no significant increase in decolorization with increasing inoculum size from 5 to 10%. Best decolorization obtained at 10 8 spores/ml is due to availability of nutrients and effect of population density on the hydrodynamic characteristics of the medium, which consequently contribute to the final decolorization phenomena (Shahvali et al., 2000). Table 4.5 shows effect of various glucose concentrations (0-20 g/l) on growth and decolorization by R. oryzae. Maximum growth and decolorization were at 5 g/l of glucose concentration (119 mg/50 ml, 82%), followed by 2g/L of glucose (73 mg/50 ml, 76%) and 10g/L of glucose (68 mg/50 ml, 72%) on 8 th day. In absence of glucose the isolates exhibited neither growth nor decolorization. It was observed that the weight of mycelium increases with the increasing concentration of glucose upto 5g/L, whereas 10, 15 and 20g/L concentration showed retarding effect on 77

10 mycelial growth. This may be due to the effect of osmotic pressure on the cell contents. (Usmani et al., 2006). Table 4.4 Effect of inoculum size ( spores / ml) on growth (Gr - mg / 50ml) and decolorization (D - %) by R. oryzae Inoculum Size 10 Gr 14± ± ± ± ±1.00 9±1.00 9±1.00 D 10± ± ± ± ± ± ± Gr 19± ± ± ± ± ± ±1.00 D 15± ± ± ± ± ± ± Gr 34± ± ± ± ± ± ±1.00 D 23± ± ± ± ± ± ±1.52 R. oryzae 10 Gr 25± ± ± ± ± ± ±1.00 D 14± ± ± ± ± ± ±1.52 Gr 28± ± ± ± ± ± ± D 12± ± ± ± ± ± ±1.00 Gr 21± ± ± ± ± ± ± D 11± ± ± ± ± ± ± Gr 16± ± ± ± ± ± ±1.00 D 34± ± ± ± ± ± ±1.00 ± - standard deviation (SD) The reason why low decolorization appeared when glucose concentration was low proves that low glucose concentration could not meet the growth requirements of fungi. Decrease in decolorization potential beyond 5g/L may be due to accumulation of simple carbon compound acting as a co-metabolic substrate in media. When glucose concentration was much higher i.e. 20 g/l, fungal mycelia degraded only glucose rather than degrading dye molecules which are complex in nature (Ramsay and Goode, 2004). 78

11 Table 4.5 Effect of glucose concentration (0 20 g/l) on growth ( Gr - mg/ 50ml) and decolorization ( D - %) by R. oryzae Glucose (g/l) Gr D Gr 14± ± ± ± ± ± ±1.00 D 18± ± ± ± ± ± ±1.57 Gr 34± ± ± ± ± ± ±1.00 D 36± ± ± ± ± ± ±2.56 R. oryzae Gr 64± ± ± ± ± ± ±1.00 D 52± ± ± ± ± ± ±1.00 ± - standard deviation (SD) Gr 31± ± ± ± ± ± ±1.00 D 9± ± ± ± ± ± ±1.52 Gr 19± ± ± ± ± ± ±0.57 D 7± ± ± ± ± ± ±1.00 Gr 9± ± ± ± ± ± ±1.00 D 5± ± ± ± ± ± ±1.00 Two opinions have been argued: one deems that dyes are not a carbon source since organisms obtain energy from glucose instead of dyes and glucose can enhance the decolorization performance of biological systems, while another deems that glucose can inhibit the decolorizing ability or activity (Wang et al., 2009). As with dyes, the chromophores and the auxochromes cannot be degraded without addition of a carbonaceous co-substrate. Levels of such co - substrates are low in effluent and so they need to be added, but the amount added varies with the organism and the effluent. 79

12 The results are in agreement to Kumaran and Dharani, 2011 and Senthilkumar et al., 2011 proving increased decolorization with increased glucose concentration, the carbon source most readily used for fungi. Glucose acts as the primary nutrient in decolorization of dyes. Jilani et al., 2011 reported the decolorization ability of WRF decreases rapidly in absence of glucose, suggesting that fungi need extreme energy sources to promote the growth and biodegradation process. Nagarathnamma and Bajpai, 1999 and Perumal et al., 2000 have shown considerable decolorization in absence of glucose, an observation in contrast to the present findings. Hence, for developing efficient biotreatment process for dye effluents, the amount of usable carbohydrates present in the effluent is an important factor. Table 4.6 shows effect of various NH4Cl concentrations (0 1.2 g/l) as N source on growth and decolorization by R. oryzae. Maximum growth and decolorization were achieved at 0.30 g/l of NH4Cl concentration (78mg/50 ml, 64%), followed by 0.15 g/l (60 mg/50 ml, 56%) and 0.60 g/l (52 mg/50 ml, 43%) on 8 th day respectively. Above 0.30 g/l, both growth and decolorization decreased with marginal growth and decolorization at 1.2 g/l concentration. In absence of NH4Cl the isolate showed minimal growth and decolorization. The effect of nitrogen source is inferred in the form of ammonium ions in the dye effluent. Nitrogen in the form of ammonium ions acts as nutrient for the growth of fungal mycelium. The rate of decolorization of dye molecules depends on the rate of breaking of azo ( - N=N - ) bonds in the dye molecule. The results are in accordance to Cripps et al., 1990 that illustrates more efficient decolorization and biodegradation of azo and reactive dyes by P. chrysosporium in nitrogen-sufficient conditions than in nitrogen-limited conditions. An inhibitory effect was recorded at higher levels of nitrogen concentrations. These 80

13 results are in harmony with those of Sanghi et al., 2006 who also reported inhibition of dye decolorization at higher concentrations. It appears that nitrogen of the dye molecules secure the nitrogen demand. Various authors have observed high lignolytic and decolorization activities in N sufficient conditions (Vasdev et al., 1995; Mester et al., 1996; Ben Hamman et al., 1997). Vahabzadeh et al., 2004 reported that the rate of decolorization increased with increasing nitrogen concentrations upto a certain level and further increasing concentration decreased the rate of decolorization since the breakage of azo bonds decreased due to the presence of easily accesbile excess nitrogen in the form of ammonium ions. Table 4.6 Effect of NH4Cl concentration (0 1.2 g/l) on growth (Gr - mg / 50ml) and decolorization (D - %) by R. oryzae. NH 4Cl (g/l) 0 Gr ±0.00 6±0.00 9±0.00 9±0.00 D - 1±0.00 1±0.00 2±0.00 3±0.00 3±0.00 3± Gr 15± ± ± ± ± ± ±1.00 D 19± ± ± ± ± ± ±1.00 R. oryzae 0.30 Gr 25± ± ± ± ± ± ±1.00 D 13± ± ± ± ± ± ± Gr 12± ± ± ± ± ± ±1.00 D 2±2.08 5± ± ± ± ± ± Gr 8± ± ± ± ± ± ±1.00 D 2± ± ± ± ± ± ±1.00 ± - standard deviation( SD) Table 4.7 show the effect of various CaCl2 concentrations ( g/l) on growth and decolorization by R. oryzae. Maximum growth and decolorization were at concentration 1.5 g/l (71 mg/50 ml, 55%), followed by 2 g/l (60 mg/50 ml, 54%) and 2.5 g/l (43 mg/50 ml, 42%) 81

14 on 8 th day respectively. Neither increasing concentrations nor decreasing concentrations were found to be completely suppressive for decolorization. Table 4.7 Effect of CaCl2 concentration (0.5 3 g/l) on growth (Gr - mg / 50ml) and decolorization (D - %) by R. oryzae CaCl 2 (g/l) 0.5 D 24± ± ± ± ± ± ±1.00 Gr 13± ± ± ± ± ± ± D 17± ± ± ± ± ± ±1.00 Gr 20± ± ± ± ± ± ±1.00 R.oryzae 1.5 D 26± ± ± ± ± ± ±1.00 Gr 42± ± ± ± ± ± ± Gr 31± ± ± ± ± ± ±1.00 D 17± ± ± ± ± ± ± D 10± ± ± ± ± ± ±1.52 Gr 11± ± ± ± ± ± ±1.00 ± - standard deviation (SD) 3 D 7± ± ± ± ± ± ±1.00 Gr 12± ± ± ± ± ±1.00 8±1.52 Nagarathnamma and Bajpai, 1999 have also reported 1.5 g/ L of CaCl2 concentration optimum for decolorization by R.oryzae. Increasing concentrations of CaCl2 were not found to be inhibitory, an observation which is in agreement to Jefferies et al.,1981 and Bhatt, 2008 who reported that increasing concentrations of Ca 2+, Mg 2+ and Mn 2+ are not inhibitory but the balance between these metals are critical. Table 4.8 show the effect of various KH2PO4 concentrations (0.5 3 g/l) on growth and decolorization by R. oryzae. 82

15 Maximum growth and decolorization were at 2% KH2PO4 concentration (89 mg/50 ml, 66%), followed by 1.5% concentration (54 mg/50 ml, 49%) and 1% concentration (60 mg/50 ml, 44%) on 8 th day respectively. Table 4.8 Effect of KH2PO4 concentration (0.5 3 g/l) on growth (Gr - mg / 50ml) and decolorization (D - %) by R. oryzae KH 2PO 4 (g/l) 0.5 Gr 11± ± ± ± ± ± ±1.00 D 3± ± ± ± ± ± ± Gr 29± ± ± ± ± ± ±1.00 D 16± ± ± ± ± ± ±2.51 R.oryzae 1.5 Gr 1± ±2.00 4±2.64 4±1.00 1±1.00 7± ±1.00 D 18± ± ± ± ± ± ± Gr 40± ± ± ± ± ± ±1.00 D 27± ± ± ± ± ± ±1.00 Gr 16± ± ± ± ± ± ± D 9± ± ± ± ± ± ±0.00 ± - standard deviation (SD) 3 Gr 14± ± ± ± ± ± ±1.00 D 8± ± ± ± ± ± ±0.00 At limiting concentrations there was not remarkable change in decolorization, an observation in contrast to Bhatt, 2008, who reported severe reduction in growth and decolorization at limiting concentrations. Jefferies et al., 1981 reported reduction of decolorization by almost 50% with limiting phosphorous concentration. Limiting or higher concentration of KH2PO4 had no major effect on decolorization. The effect was a minor decrease in decolorization. Inorganic phosphorous concentrations that permit primary growth, are inhibitory to various secondary metabolic functions in bacteria and fungi, hence the decrease in decolorization (Weinberg, 1977). 83

16 Table 4.9 shows effect of various concentrations of MgSO4 on growth and decolorization by R. oryzae. Maximum growth and decolorization was attained at 2 g/l concentration (80 mg/50 ml, 79%), followed by 2.5 g/l (60 mg/50 ml, 62%) and 1.5 g/l (60 mg/50 ml, 58%) on 8 th day respectively. Table 4.9 Effect of MgSO4 concentration (0.5 3 g/l) on growth (Gr - mg / 50ml) and decolorization (D - %) by R. oryzae MgSO 4 (g/l) Gr 7± ± ± ± ± ± ± D 9± ± ± ± ± ± ±1.00 Gr 15± ± ± ± ± ± ± D 25± ± ± ± ± ± ±1.00 R. oryzae 1.5 Gr 14± ± ± ± ± ± ±1.00 D 10± ± ± ± ± ± ± Gr 24± ± ± ± ± ± ±1.00 D 20± ± ± ± ± ± ± Gr 13± ± ± ± ± ± ±1.00 D 15± ± ± ± ± ± ±1.00 ± - standard deviation (SD) 3 Gr 7± ± ± ± ± ± ±1.00 D 11± ± ± ± ± ± ±1.00 The observations are in contrast to Miranda et al., 1996 who reported 0.5 g/l of MgSO4 to be optimum for decolorization by A. niger. Contradictory to this, Ramsay and Goode, 2004 and Vahabzadeh et al., 2004, reported that increase in concentration above 0.1% resulted in decrease in decolorization. Table 4.10 shows the effect of mode of cultivation (static and shaking) on growth and decolorization by R. oryzae. 84

17 Maximum decolorization was exhibited under shaking conditions (73%) whereas maximum growth was found at static condition (298 mg/50 ml), clearly indicating the importance of aeration in decolorization. Table 4.10 Effect of mode of cultivation on growth (Gr - mg/50ml) and decolorization (D - %) by R. oryzae Mode of Cultivation R. oryzae Shaking Static ± - standard deviation (SD) Gr 44± ± ± ± ± ± ±1.00 D 21± ± ± ± ± ± ±1.00 Gr 87± ± ± ± ± ± ±1.00 D - 4±0.00 5± ± ± ± ±1.00 Under static conditions, suspended particles of dye settle down at the bottom of culture flask, whereas in shaking conditions there was prominent removal of solids if suspended. The observation is in contrast to Svobodova et al., 2006 who suggested a correlation between the ability of I. lacteus static cultures to decolorize a textile industry effluent more effectively than agitated cultures. Results show that the time required for decolorization of effluent was on the 8 th day in accordance to the results of Knapp et al., 1997 who reported that agitation resulted in better decolorization as dye decolorization requires O2 either for generation of H2O2 for peroxidases or for the direct action of laccases. The problem with static mode of cultivation could be poor oxygen transfer, lack of homogeneity and poor nutrient distribution. On the contrary, static cultures of P. chrysosporium and Plerotus ostreus have given better decolorization than shaking cultures (Kim et al., 1996; Bakshi et al., 1999). Table 4.11 indicates the optimized conditions that have been obtained for both growth and decolorization by R. oryzae. 85

18 Table 4.11 Optimized conditions obtained by R. oryzae for maximum growth and decolorization Sr. Cultural Conditions Optimized Value No. 1 ph Temperature 37 C 3 Effluent Concentration 1:10 4 Inoculum size 10 8 spores / ml 5 Glucose (g/l) 5 6 Ammonium Chloride (g/l) Calcium Chloride Pottasium dihydrogen Phosphate (g/l) 2 9 Magnesium Sulfate (g/l) 2 10 Mode of Cultivation Shaking (decolorization), Static (growth) Table 4.12 indicates growth and decolorization by R. oryzae both under control - unoptimized (C) and test - optimized (T) conditions. With optimized conditions R. oryzae exhibited maximum growth (122mg/50 ml) on 2 nd day and decolorization of 81% on 4 th day. Maximum growth and decolorization achieved under unoptimized conditions were 51 mg/50 ml and 56% respectively on 8 th day. Hence, optimized conditions led to 1.44 fold increase in growth and 2.39 fold increase in decolorization with reduction in time of decolorization from 8 th day to 4 th day. 86

19 Table 4.12 Growth (Gr - mg / 50ml) and decolorization (D - %) under control and test conditions by R. oryzae R. oryzae C T Gr 16± ± ± ± ± ± ±1.00 D 19± ± ± ± ± ± ±1.00 Gr 122± ± ± ± ± ± ±1.00 D*** 21± ± ± ± ± ± ±1.00 C-Control, T-Test, ***- p ± - standard deviation (SD) Fig. 4.1 shows the untreated effluent and the decolorized effluent treated with R. oryzae on 4 th day. C T Fig. 4.1 Decolorization of effluent by R. oryzae under optimized conditions C Control, T Treated (4 th day) 87