A.niger is a commonly found fungus.

It is a network of brownish black mycelia and can easily grow on milk and sugar containing spent tea. It can easily produce bioethanol by its versatile enzymes. A. niger which can be isolated from different sources is responsible for bioethanol production (Ali and El-Dein, 2008). Presence of milk and sucrose in large quantities are responsible for its growth on spent tea. Cellulase is an induced enzyme and its production increases with increase in fungal biomass over the incubation period and as simple sugar in the substrate diminished. There is an increase in sacchrification from 0 to 24 h. Cellulase enzymes of A. niger are responsible for the hydrolysis of cellulose which results into ethanol after fermentation. There is a progressive increase in enzyme activity from 24 to 144 h after incubation (Nieves et al., 1998). Cellular hemi-cellulase enzymes can be used in fermentation. The sucrose is enzymatically converted into ethanol (Gray et al., 2006). Cellulase, hemicellulase and pectinase enzymes are responsible for conversion of sugars of plant origin into ethanol (Bhat, 2000). The ethanol producing enzymes are cellulose and hemicellulosedegrading. These may be isolated from A. niger (Thygesen et al., 2003). Cellulose changes into glucose by using the enzymes of fungi. It is then converted into ethanol. Hemicellulse hydrolyzed into xylose and mannose etc. These are converted into ethanol by enzymes. More than 90 - 95% of ethanol cannot be obtained by fermentation. Some other by-products like acetic acid and glycerol are also possible. The xylose is converted into ethanol by enzymes like xylose isomerases (Nag, 2008). Cellular hemi-cellulases can be used in fermentation. Sucrose is enzymatically converted into ethanol (Gray et al., 2006) Isolation and identification of A. niger from spent tea Samples of spent tea containing milk and sugar were placed in a beaker undisturbed for 3 - 5 days. After 3 - 5 days, a net work of brownish black A. niger was observed. A sample of A. niger containing tea inoculums was mixed with AR Grade chloroform in a ratio of 1:4. This sample was mixed thoroughly on magnetic stirrer and filtered. The aqueous layer was separated from non-aqueous layer. Bioethanol Production For bioethanol production A. niger was used. The non aqueous filtered sample was analyzed on GC-MS for ethanol. The results of GC-MS showed ethanol (m/z 45, abundance 1513472 and ethanol was 57.49%). There were some strong evidences for the use of fermentation for bioethanol production (Ali and El-Dein, 2008; Zakpaa et al., 2009). Table 4 shows results of identification, confirmation of growth temperature, shape, colony morphology, mobility (Kausar, 2004), spore formation, H2S test (Tabassum, 2003; Kausar, 2004) and gram staining, (Pandey, 1994) of A. niger. It showed that A. niger is excellent for bioethanol production from spent tea. Spent tea contains sugar in sufficient quantity. Skory et al. (1997) examined 19 Aspergillus and 10 Rhizopus strains for their ability to ferment sugars. An appreciable level

of ethanol has been produced by them. A. niger can easily grow on milk and sugar already present in spent tea. Hence, it can easily produce ethanol. Cellular hemicellulases are enzymes that can be used in fermentation. The sucrose was enzymatically converted into ethanol (Gray et al., 2006). The ethanol producing enzymes are cellulose and hemicellulose-degrading. These were isolated from A. niger (Thygesen et al., 2003; Villena and Gutierrez-Correa, 2006). A. niger can be isolated from different sources for bioethanol production (Li et al., 2009). The sample containing ethanol was subjected to distillation. The pure sample was analyzed on GC (Gas Chromatograph Model Varian CP-3800). Result showed that when minimum time was 2.358 min, peak area was 199986.96 Vs, height was 35341.77 V, area percentage was 100% and BL area /height (s)

was 5.6587. The results of GC-MS and GC showed that spent tea can produce bioethanol on large scale. The biomass polysaccharide part has been considered as safe source of energy for the production of fuel ethanol. The enzyme based application is advantageous over chemical treatments due to its higher conversion efficiency, the absence of substrate loss due to chemical modifications and the use of more moderate and non-corrosive physical-chemical operating conditions, such as lower reaction temperatures, neutral pH and the use of biodegradable and non-toxic reagents. Biomass degradation is a complex multi-enzymatic process. Cellulose digestibility by cellulases alters the organization and the chemical interactions amongst the cellulose, hemicellulose and lignin. Pending on process conditions, there is formation of significant sugars and lignin degradation products that are inhibitory to ethanol fermentation. Bon and Ferrara (2007) studied bioethanol production via enzymatic hydrolysis of cellulosic biomass (Bon and Ferrara, 2007). They reported that the rate of saccharification is directly proportional to substrate concentration up to optimal substrate concentration. This is because random collisions between the substrate and enzyme active sites happen more frequently. Beyond the optimum, the active sites are saturated so that higher substrate concentration has no effect on rate of saccharification. Saccharification increase with substrate concentration and also increase in reducing sugars with increase in substrate. Temperature has a complex effect on enzyme activity. It affects the speed of molecules, the activation energy of the catalytic reaction and thermal stability of the enzyme. Generally, saccharification increase with temperature up to the optimum after which it declines. The increase in temperature is due to corresponding increase in kinetic energy and the decline after the optimum due to enzyme denaturation. The pH of a solution has several effects on the structure and activity of enzymes and hence saccharification and fermentation. Enzymes are amphoteric molecules containing a large number of acidic and basic groups, mainly situated on their surface. The charges on these groups are different. The variation is according to their acid dissociation constants, with the pH of the solution. Thus pH affects the reactivity of the catalytically active groups. The relative low pH provides acidic condition which prevents bacterial contamination during fermentation (Zakpaa et al., 2009). The fermentation waste can be used as fertilizer. Bioethanol technology is very useful because it can be used to produce 4 billion gallons of alcohol fuel enough to replace 460 million barrels of oil.

Cellulase production by Aspergillus niger Cellulase was produced in corncobs based broth medium (CBB). CBB is a modified Mandel broth medium as described by Jeffries (1987) and consisted of the following per liter: (NH4)2SO4 1.4 g, KH2PO4 2.04 g, CaCl2 0.3 g, MgSO4.7H2O 0.3 g, Citric acid 0.25 g, Tween 80 2 ml, Avicel cellulose 10 g, yeast 1 g and Trace metal stock solution 1 ml. The trace metal stock solution consisted of thefollowing chemicals per 500 ml: FeSO4 2.55 g, MnSO4.H2O 0.93 g, ZnSO4.H20 1.78 g, Co(NO3)2.6H2O 1.25 g, and Conc. HCl 5 ml. Two hundred and fifty milliliters of CBB was inoculated with 3.02107 spores of A. niger and incubated on shaker (G24 Environmental incubator shaker, New Brumswick Co. USA) at 120 rpm at 25oC for 6 days period. The broth was centrifuged at 10000 g for 10 min and the supernatant was used as crude enzyme in saccharification tests. To determine enzyme activity over the period, 5 ml aliquots were collected at 24 h interval. Optimization tests The method of Baig et al. (2004) was adopted. One percent corncobs suspension was prepared by suspending 10 g of the substrate in 1 liter 50 mM citrate buffer pH 5.5 and autoclaved at 121oC for 20 min. Exactly 15 ml of the substrate suspension were put in each stopped 100 ml Erlenmeyer flasks and 5 ml of culture filtrate obtained from A. niger culture were added to each. Saccharification was performed in a water bath shaker at 272C for 24 h. The resultant supernatants following centrifugation (2500 g, 15 min) were assayed for total reducing sugars by DNS method (www.glue.umd.edu/~nsw/ench485/lab4a.htm). The sugars released were expressed as equivalent of glucose. The optimum temperature of saccharification was determined by incubating the reaction mixture at different temperature ranging from 25 to 60C. The optimum pH was determined by adjusting the pH of the reaction mixture from 3.5 to 6.5. The optimum substrate concentration was determined by preparing substrate suspension 1 to 6%. Yeast inoculum preparation The yeast inoculum was prepared as described by Scholar and Benedikte (1999) and Suh et al. (2007). One gram of dry bakers yeast purchased from Kumasi central market was grown on Yeast Peptone Dextrose (YPD) agar plate at 30oC for 48 h to activate the yeast and check for contaminates. A loopful of the yeast colony was transferred from the agar plate into 100 ml of 5% YPD broth and incubated at room temperature on a shaker at 130 rpm for 48 h. Seven milliliters of the broth (from calculation) was centrifuged at 4500 rpm for 5 min. The supernatant was decanted, and the pellet was resuspended in 10 ml of sterile distilled water twice, centrifuged and the supernatant decanted. The pellet was resuspended in 1/10th of 50 mM citrate buffer of working for each flask and was used as its inoculum. Simultaneous saccharification and fermentation (SSF) The SSF was carried out as described by Dowe and McMillian (2008). Six grams of corncobs powder, 1.0 g yeast extract and 2.0 g

peptone were placed in each 250 ml Erlenmeyer flask, 50 mM citrate buffer, pH 5.0 added to form 80 ml suspensions and fitted with two-hole silicone bungs. The flasks and their accessories were autoclaved at 121oC for 20 min. After cooling to room temperature,10 ml of the A. niger culture filtrate and 10 ml yeast inoculums were also introduced into each flask. For each flask sterile syringe was inserted into a tube fixed in one hole and fermentation lock device fixed to the tube in other hole of the bung. The flasks were incubated at room temperature on shaker at 110 rpm for 24 h. Five ml aliquots were drawn from the flasks at 4, 6, 8, 12 and 24 h with the inserted sterile syringes. The syringes were disposed off after every sampling. The aliquots were centrifuged at 10000 g for 10 min and the supernatant kept frozen for gas chromatographic analysis for ethanol. RESULTS AND DISCUSSION A niger, the best cellulolytic fungi isolated from soil sampled from Ejura farms was cultured on CBB for 6 days on shaker at 120 rpm. Aliquots of 5 ml were sampled at 24 h interval and assayed for enzyme activities. There was progressive increase in enzyme activity from 24 to 144th h after incubation. Cellulase is an induced enzyme and its production increased with increase in fungal biomass over the incubation period and as simple sugar in the substrate diminished (Lynd et al., 2002). The slow down in rate for hydrolysis must be due to the action of the enzymes been slowed down by obstacles that interfere with their path or a loss in activity and/or processivity making them less effective (Yang et al., 2006). The effects of substrate concentration, temperature and pH on release of reducing sugars were also carried out. The rate of saccchariffication is directly proportional to substrate concentration up to the optimal substrate concentration. This is because random collisions between the substrate and enzyme active sites happen more frequently. Beyond the optimum, the active sites are saturated so higher substrate concentration has no effect on rate of saccharification (www.canacad.ac.jp:3445/BiologyIBHL/541). There was increase in reducing sugars with increase in substrateconcentration. The highest mean glucose concentration of 3.1105 mg/ml was recorded for substrate concentration of 6% and was significantly different (p<0.05). Substrate concentration of 1% released the least reducing sugars concentration. The glucose concentration for 6% substrate concentration was higher and significantly different from 5% substrate concentration, which suggests that 6% is or below optimum substrate concentration. Temperature has complex effect on enzyme activity and hence saccharification. It affects the speed of molecules; the activation energy of the catalytic reaction and thermal stability of the enzyme. Generally saccharification increased with temperature up to the optimum after which it declines. The increase with temperature is due to corresponding increase in kinetic energy and the decline after the optimum due enzyme denaturation (Shuler and Kargi, 1997; Chaplin and Bucke, 1990a; www.blurtit.com/science/chemistry). Effect of temperature

on saccharification is shown on Figure 4. Saccharification increased from 25oC to maximum at 40oC after which it decreased up to 60oC. Temperature (40oC) recorded the highest release of reducing sugars and it was significantly different (p<0.05). Thus the optimum temperature is 40oC, implying that the isolate is mesophile (Shuler and et al. (2007) reported of A. niger with optimum tempe- rature of 40oC on coir waste and sawdust. Fewer studies of enzymes from mesophilic fungi are available (Baig et al., 2003). Saccharification was least significant (p<0.05) at 60oC. The decrease from 50 to 60oC was sharp due to the fact that enzyme denaturation is much faster (Shuler and Kargi, 1997; Chaplin and Bucke, 1990a; www.blurtit.com/science/chemistry). The pH of a solution has several effects on the structure and activity of enzymes and hence saccharification. Enzymes are amphoteric molecules containing a large number of acid and basic groups, mainly situated on their surface. The charges on these groups vary, according to their acid dissociation constants, with the pH of the solution. Thus pH affects the reactivity of the catalytically active groups (Chaplin and Bucke, 1990b). Figure 5 illustrates the effect of pH on release of reducing sugars from the substrate. Saccharification increased from pH 3.5 to a maximum of 4.0 after which it decreased up to 6.5. The highest saccharification which was significantly different (p<0.05) was recorded at pH 4.0. This makes the isolate acidophile (Nester et al.,2001). Saccharification was least significant (p<0.05) at pH 6.5, thus the optimum pH was 4.0. The relative low pH provides acidic condition which prevents bacterial contamination during fermentation (Olofsson et al., 2008). Figure 6 shows the trend of ethanol production over 24 h period at 25oC and 110 rpm. SSF was carried out with 10 ml of A. niger filtrate. Ethanol concentrations of the fermentations were determined at 4, 6, 8, 12 and 24 h after inoculation. The mean ethanol concentration was 0.4326 g/l. There was increase in ethanol production over the period. There might be increase in saccharification over the period making glucose available to S. cerevisae for fermentation. Olofsson et al. (2008) reported that enzymatic hydrolysis of the solid fraction has a large control over the total rate of ethanol production in SSF. Also the ethanol concentration and other byproducts in the broth might not have reached the inhibitory level of the yeast over the period. The highest concentration of 0.642 g/l was recorded at 24th h. Comparison to similar works in literature is difficult because ethanol concentration was not cited and they differ in either in type of pretreatment if any and detoxification, substrate concentration, fermentation strain, temperature or mode of operation which affects the final ethanol concentration as reported in work by Olofsson et al. (2008).

Culture condition for ethanol production A synthetic medium containing yeast nitrogen base glucose broth and a complex medium containing the pretreated corn cobs were used for ethanol production. The synthetic medium was prepared by dissolving 6.7g of yeast nitrogen and 10g of glucose in a litre of distilled water. The complex medium was prepared with corn cobs at a concentration of 10g/litre supplemented with 0.1% FeNH4 (SO4)2, 0.25% (NH4)2 HPO4, 0.3% urea and 0.5% peptone. The pH of the medium was adjusted to 5.0 using 1 normal HCl and NaOH, sterilized in an autoclave and filtered. Another set of media containing 100g/L of glucose and corn cobs were similarly prepared. The media were dispensed into 500ml Erlenmeyer flasks each containing 200ml of the medium. The flasks containing synthetic medium were inoculated with Saccharomyces cerevisiae while those containing complex medium were inoculated with both Saccharomyces cerevisiae and Aspergillus niger. The flasks were incubated at ambient temperatures on an orbital shaker set at 250rpm for 5 days (Abouzied and Reddy, 1986). Quantitative determination of ethanol production At 24 hour intervals, samples were taken aseptically from the fermentation media to determine growth, residual sugar and ethanol concentrations. The growth was determined by measuring the cell density (optical density) at 650nm using spectrophotometer, the residual sugar was determined using dinitrosalicylic acid (DNS) method described by Miller (1959). Finally, ethanol was determined after standard distillation using the method described by AOAC (1974). RESULTS AND DISCUSSION The results of the ethanol yield from the synthetic medium containing glucose and complex medium containing corn cobs are shown in Table 1. At 1% and 10% glucose concentration, the synthetic medium gave a maximum ethanol yield of 3.45% and 6.23% respectively all at 72 hours fermentation period. While at the same concentrations of corn cobs, the complex medium yielded 4.17% and 6.17% ethanol. It was observed that at all concentrations of substrates, the ethanol yield increased steadily reaching the peak after 72 hours of fermentation and then declined. The corn cobs at 1% concentration gave a better ethanol yield compared to glucose, while at 10%, the ethanol yield was comparable to that of glucose. This shows that the corn cobs can serve as one of the cheaper substrates for ethanol production (Schugerl,1994; Joshi et al., 2001; Akin-Osanaiye et al., 2008).

Enzyme Production Enzyme such as amylase and cellulase were produced by Aspergillus niger and Trichoderma viride in potato dextrose broth incubated at room temperature for seven days Enzyme Assay After incubation, fermented media was filtered and centrifuged, supernatant was collected for enzyme assay. Amylase was assayed by starch plate method and cellulase was assayed by DNS method (Dinitrosalicylic acid). Two Step Enzymatic Hydrolysis 10% of biomass was boiled in distilled water. Incase of cassava, fruit pulp, rice extract, sweet potato, potato, sugar cane beet and rice straw samples are chopped, boiled and filtered. Extract was then sterilized, after sterilization 5% of enzymes was added for hydrolysis and incubated for 3 hours at 37 oC. In case of saw dust, coconut pith, groundnut mill waste, rice husk, leaf litter, wood bark, maize husk and waste paper (25 gm) in 250ml distilled water are boiled as a whole and kept for sterilization. After sterilization 5% of enzymes was added for hydrolysis at 37oC for 3 hours of incubation and filtered aseptically. Fermentation Hydrolyzed and filtered extracts were fermented using Sacharomyces sp for seven days of incubation at room temperature in rotary shaker Distillation Primary distillation was carried in rotary vacuum flask at 80oC (boiling point of ethanol) and fraction is collected. Specific Gravity Method Ethanol is determined by specific gravity method.

Optimization on the conversion of cassava waste-starch by Aspergillus niger TISTR 3352. The investigation of cassava waste starch conversion by A. niger TISTR 3352 was carried out in submerge cultivation. The cultivation medium containing of cassava waste supplemented with nitrogen source and adjust initial pH to 5.5. The fungal spore suspension was inoculated into the medium and cultivate by shaking flask at 200 rpm, 30 oC for 72 or 96 hours. After cultivation, the reducing sugar was measured to indicate of the conversion efficiency. For optimization of the conversion, the following parameters were investigated. To find an optimum cassava waste content in the cultivation medium. The amount of cassava waste was varying, 15, 30, 45, 60 and 70% (w/v) (wet weight) in the cultivation medium. To find an optimum fungal inoculum size. 10% (v/v) of the various concentration of the fungal spore suspension, 106, 107, and 108 spore/ml were inoculated into the cultivation medium containing of 45% (w/v) (wet weight) cassava waste. To find an optimum amount of ammonium sulfate, the nitrogen source supplemented in the cultivation medium. The various concentration of ammonium sulfate, 0.2, 0.4, and 0.8% (w/v) were supplemented in the cultivation medium containing of 45% (w/v) (wet weight) cassava waste. Analysis of reducing sugar The reducing sugar obtained from the conversion process was analyzed according to Nelson-Somogyi method (Somogyi, 1952). The study on conversion of cassava waste-lignocellulose by cellulase from Trichoderma reesei TISTR 3080. Optimization for the appropriate condition of cellulase production by Trichoderma reesei TISTR 3080 by using cassava waste lignocellulose as substrate had done. Cassava waste-lignocellulose was obtained by squeezing out the soluble starch from the fibrous residue of the hydrolysis of cassava waste by using A. niger (Figure 1). After obtaining the appropriate condition for cellulase production, the study on the properties of the fungal enzyme was conducted as the following. To find an optimum pH for conversion of cellulose by the fungal enzyme. The various pH, 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0 in the hydrolysis reaction were investigated in enzyme activity of the fungal enzyme. The hydrolysis was performed by using 15% (w/v) (wet weight) cassava waste-lignocellulose in liquid medium and shaking at 150 rpm at 30 oC. To find an optimum temperature for conversion of cellulose by using the fungal cellulase enzyme. The various temperature, 30, 35, 40, 45, 50, 55, 60, 65 and 70 oC were used to incubate for the hydrolysis reactions. To find an optimum incubation time for conversion of cassava waste-lignocellulose by the fungal enzyme. The reaction of hydrolysis of cellulose by the fungal enzyme was incubated in the various period of time 0, 3, 6, 9,12, and 15 hours. The reducing sugar was analyzed at intervals of time in all experiments to indicate the efficiency of conversion and the

activity of the fungal enzyme. Result and Discussion Optimization on the conversion of cassava waste-starch by Aspergillus niger TISTR 3352. The optimization on cassava waste content in the cultivation medium for conversion by A. niger. After cultivation by shaking flask at 200 rpm at 30 oC for 72 hours, the maximum reducing sugar were obtained in closed values among the experiments cassava waste content of 45, 60 and 70% (w/v) which were 44.91, 45.50 and 42.87 g/L, respectively (Figure 2). According to the result, the cassava content of 45%(w/v) (wet weight was selected as an appropriate cassava content in the cultivation medium. The optimization of fungal inoculum size to inoculate in the cultivation medium for conversion of cassava waste. After cultivation by shaking flask at 200 rpm at 30 oC for 96 hours, the maximum reducing sugar obtained from the cultivation medium using of inoculation of 10% (v/v) fungal spore at concentration 107 spore/mL which was reached to 52.60 g/L at 72 hours. Therefore, inoculum size of 10% (v/v) fungal spore concentration 107 spore/ml was selected as an appropriate fungal spore inoculum size (Figure 3). The optimization of ammonium sulfate, the nitrogen source supplemented in the cultivation medium. After cultivation by shaking flask at 200 rpm at 30 oC for 96 hours, the maximum reducing sugar was observed from the cultivation medium supplemented with ammonium sulfate 0.2% (w/v) which was 52.60 g/l at 72 hours (Figure 4).