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The main energy source for energy consumption since the 18th century was fossil fuels, mainly coal and petroleum. With world energy consumption estimated to increase 54% between 2001 and 2005, coupled with the phenomenon of global warming and global oil crisis, fossil fuels are depleting at very dangerous rates causing future generations not being able to sustain development. There is a need for an alternative energy source that could counter this predicament. (Carere et al, 2008)
Fossil fuels may be the main source for the energy consumption but it is very detrimental to the environment and overall public health. The extraction, processing and combustion of fossil fuels contribute to the pollution of soil, air and water. (Carere et al, 2008). Normal renewable energies such as solar energy are unreliable and may not be suitable for consumers' huge demands. Thus biofuels are considered as alternative energy sources due to the fact that they are environmentally friendly and renewable.
Bioethanol is the most widely used liquid biofuel in the world. Production of bioethanol has reached 41 billion liters in 2004. The largest producer of this fuel is Brazil, contributing to 37% of production. The demand of bioethanol is predicted to increase dramatically, and the efforts to reduce greenhouse gases emission can trigger the production of renewable sources. Therefore there is a need for the intensive research of this fuel. (Carere et al, 2008)
Bioethanol is liberated from agricultural feedstock. Upon combustion, the products formed are carbon dioxide and water which are clean and environment friendly. Therefore, it has the potential to improve sustainability and reduce greenhouse gas emissions. It is also deem more felicitous to fuel transportation as it has higher octane content than gasoline and does not modify current technologies. (Zaldivar et al, 2001) Agricultural feedstock is renewable because of its ability to photosynthesize from the sun's light energy.
Traditionally, bioethanol is produced from common crops such as maize, corn, sugar cane etc. These crops are part of our food supply and livestock feed, therefore these sources may not be enough to meet demands and in turn become very costly. There are also some other alternative cheaper substrates that can be utilized and are in abundance.
Lignocellulose is one of these cheaper and abundant resources we can consider but unfortunately it is too complex to be hydrolyze to simple sugars and can become a potential feedstock for the synthesis of bioethanol that could displace fossil fuel consumption. It is usually found in agricultural waste, industrial waste, forestry residues, municipal solid waste, etc. (Zaldivar et al, 2001). 50% of the biomass in the world is composed of lignocellulose, making it one of the most abundant sources in the world; an estimated 7.5 x 1010 tons are synthesized through photosynththetic process. (Carere et al, 2008) It is also compatible to be used as fuel synthesis as it is considered a waste. (Zaldivar et al, 2001).
The absence of a microorganism able to ferment all sugars after hydrolysis from lignocelluloses prevents the utilization of it. Thus metabolic engineering is considered to combine all enzymatic characteristics to one single microorganism, but first isolation of a microorganism that can degrade lignocelluloses is first implemented before slowly combining traits using genetic engineering to make it competent for large scale industrial production.
With large scale industrial production, fast and efficient production process and cheap resources are required to meet the demands of the consumers. Prior to the large scale of production of bioethanol, there are 5 basic steps. They are microbial fermentation of sugars which is usually performed by yeast fungal strains, distillation, dehydration and denaturation. Most of the time, microbial fermentation is coupled with saccharification and hydrolysis of the complex sugars to simple sugars before fermentation.
Enzymes produced from fungal strains such as Trichoderma sp are studied, so that further improvements can be imposed, resulting in production in high yield and higher affinity to cellulose, leading to more simple sugars products to undergo fermentation.
Hydrolysis of cellulose required high cost of enzymes. Thus the aim of this experiment is to improve lignocellulolytic microorganisms so that it will produce low cost lignocellulolytic enzymes.
Lignocellulose is composed of three components, mainly cellulose, hemicelluloses and lignin. Cellulose is composed of highly amorphous regions containing large voids, other irregularities and tightly packed crystalline regions. (Carere et al, 2008) It is a homopolysaccharide composed of Î²-D-glucopyranose units, linked by Î²-(1-4)-glycosidic bonds. Cellobiose is the smallest repetitive unit of cellulose and can be degraded into glucose residues. The enzymes that hydrolyze celluloses are divided into three major groups. They are endo-(1,4)-beta-D-glucan glucanhydrolases (endoglucanases), exo(1,4)-beta-D-glucancellobiohydrolases(exoglucanases) and beta-glucosidase. (Murray et al, 2004). Endoglucanase catalyze random cleavage of internal bonds of the cellulose chain, while exoglucanases degrade the chain ends that liberate cellobiose. Î’-glucosidases are only active on cello-oligosaccharides and cellobiose, finally liberating glucose monomers units. These monomers are then finally fermented to produce bioethanol. Hemicellulose is easier to hydrolyze compared to cellulose. (Jiang et al, 2010) Lignin is a polymer which cannot be utilized by any microorganism (Zaldivar et al, 2001).
Many microorganisms have the potential to produce cellulases to degrade lignocelluloses. One of these commercial strains is a filamentous fungus known as Trichoderma ressei, can produce some components of cellulases enzyme complex in large quantities, but the amount of Î²-glucosidase secreted by this fungal strain is inadequate which causes the accumulation of cellobiose. Cellobiose causes stress on the strain and end product inhibition of the enzymes. Cellulases produced from this strain have low specific activity, low thermal stability and high sensitivity to product inhibition. (Jiang et al, 2010)
There are also Northen and Southern blot analysis of bg1 and aven1 gene which is isolated for further analysis of hydrolysis of lignocelluloses. The bg1 gene encodes the putative intracellular beta-glucosidase which is similar to other fungal glucosidase is involved in cellulose degradation. There is another gene known as aven1 which encodes for a putative avenacinase, an enzyme which deglucosylates the anti-fungal saponin and avenacin, rendering it less toxic to the fungus. Both genes are found in the genome of Talaromyces emersonii (M.Collins et al, 2007). Through the study of both genes we can deduce the different components which induce or repress the genes that are involved in the degradation of cellulose.
Termite specie Coptotermes formosanus is also studied for its fascinating cellulose digestion. Unfortunately, the cellulase extracted from this organism has not been analyzed fully at molecular level because of the limits of culturing the organism itself (Tetsushi et al, 2004). There is also a cDNA library constructed from the mixed species of protists looking for functional genes for the production of cellulase.
Xylanase also plays a part in the degradation of hemicellulose. It degrades the plant cell wall which is composed of linear polysaccharides beta-1,4-xylan into xylose. Xylose will then be further fermented by yeast fungal strain to produce ethanol through XR-XDH pathway and XI pathway. (Zaldivar et al, 2001)
After all the enzymes have broken up lignocelluloses into different simple sugars, all sugars will be converted to ethanol through different metabolic pathways.
Large scale industrial production of cellulose needs high demand of cellulase from microorganisms. Therefore, the microorganism must have improved enzymatic activities, productivity and able to withstand product inhibition to reduce the cost (Cheng et al, 2009). Because of the low specific activity for cellulose, high productive strains are needed to produce exceptional enzyme-substrate ratio. Microorganism yield of enzymes can be improved using protein engineering or genetic engineering (Cheng et al, 2009). Protoplast fusion is used to improve the fungi causing gene recombination and genetic variation. Genome shuffling is used to allow the parent fungi strain to undergo multi parents crossing, allowing recombination of entire genome. Genome shuffling has more recombination compared to protoplast fusion.
The amount and the type of carbon and nitrogen sources can also affect the yield of enzymes, so fermentation media are prepared with different levels of sources which were used as substrates for enzymes production then cultivate the fungi in a shaking incubator, after that enzyme yield will be tested using these assays (Cheng et al, 2009). Environmental factors such as temperature, pH, oxygen levels and concentrations of nutrients and products in the medium can significantly affect microbial growth and cellulase production under solid state fermentation, and an accurate selection of levels of these factors can efficiently improve the yield of enzymes. A systematic study on the effect of media parameters such as media components to culture the strain considering their interaction effects under solid state fermentation is rare. (Mekala et al, 2008) Therefore, optimization of the media parameter was conducted to improve cellulase activity by D-64 wild type strain using a response surface Box-Behnken design.
Isolated strain, D-64 is a cellulose degrading fungi, the wild strain secretes cellulases and it has a potential to become better than commercialized fungal strain T.ressei. To improve the strain to reach industrial scale, random mutagenesis was done to mutate the genes of the strain, creating mutants which have better cellulases activities and better repression resistance. Mutants were then assayed under shake flask liquid state fermentation. Optimization of the media was done to create a high cellulases liberation environment for the fungal strain. By considering all these factors, further improvement of the fungal strain cellulases activities were done to achieve industrial level.
Method and Materials
2.1 Cultivated Fungal Strains and Sporulation Media
The isolated D64 fungus strain which was chosen from a selection of a variety of wild type strains for cellulolytic enzyme production. All control and mutant strains were grown and maintained on Potato Dextrose Agar Plates (39 g/L PDA) and Potato Dextrose Cellulose Agar Plates (19.5 g/L PDA, 0.5 %(w/v) Cellulose, 2 %(w/v) Bacto Agar) for 7 days at room temperature till mycelium were fully developed and spores were mature. Commercial strain RUT-C30 was obtained from the American Type Culture Collection (ATCC) and cultured using Potato Dextrose Agar Plates (39g/L PDA) for comparison of cellulase activity with D64 fungus strain. All strains are sub-cultured every month.
2.2 Basal media
The Mandel's and Weber's medium was prepared with the following composition (g/L): urea, 0.3; peptone, 0.75; yeast extract 0.25; (NH4)2SO4, 1.4; KH2PO4, 2.0; CaCl2, 0.3; MgSO4.H2O, 0.3 and trace element 1ml. 0.1% (v/v) Tween 80 was added to the liquid Mandel's medium.
Trace Element solution (g/L): FeSO4.7H2O, 5; MnSO4.4H2O, 1.6; ZnSO4.7H2O, 1.4 and CoCl2.6H2O, 20.0 (Mandels et al, 1976). The medium and trace elements were autoclaved separately.
2.3 Screening Agar media
Selection medium contained Basal Medium (BM) described by Mandels and Weber and 0.1 % (v/v) Triton X-100, 2 % (w/v) Bacto Agar and 1.0 % (w/v) phosphoric acid swollen cellulose (according to the Walseth) supplemented with 0.5 %(w/v) 2-deoxy-D-glucose (SM1) or 1 %(w/v) D-glucose (SM2) or 2 %(w/v) D-glucose (SM3) acting as an catabolite repression. Restriction of radial colony growth after spore plating/inoculation was obtained by adding 0.1 % (v/v) Triton X-100 in the media. All media are autoclaved at 115 ÌŠC for 15 minutes. The clear zones produced from the fungal strain colonies after approximately 7 days were measured and the ratio of the clear zones diameter: the colonies diameters were measured.
2.4 Shake Flask Fermentation Cultivation
Both seed media and fermentation media were pre-prepared before shake flask cultivation. Seed media was the combination of Mandel's and Weber's medium with 1 % (w/v) lactose and sometimes with the addition of glucose as a carbon source.
Fermentation media was the combination of Mandel's and Weber's medium with combination of cellulose or solid substrates such as wheat bran as the carbon sources and the inducer of cellulase activity of the strain. All media were autoclaved a 121 ÌŠC for 20 minutes.
Shake flask experiments were carried out in 250 ml Erlenmeyer flask, 2 ml spore suspension (108 spore/ml) was added to 100ml seed medium and incubated in an orbital shaking incubator at 30 ÌŠC, 200 rpm for 48 hours. Successful seed culture was indicated by the viscous medium at that time. 10 % seed was then inoculated into 30 ml fermentation in 125ml Erlenmeyer flask and the flasks were incubated at 30 ÌŠC and 150 rpm for 5 days.
There were 2 parallel experiments for each strain cultured. The amount of inoculation seed was also based on the cell density of the seed culture. The cell density of seed for all strains cultivated in fermentation medium should be relatively similar.
2.5 Enzyme Assays
After 7 days of culture in fermentation media, the samples were withdrawn and centrifuged at 8060 rpm, 10 minutes at 23 ÌŠC and the supernatants were assayed for enzyme acitivties and the soluble protein content.
Enzymatic assays were measured according to Mandels et al.
2.5.1 Filter Paper Assay
Filter paper activity was assayed by incubating the suitable diluted (5 times or 10 times) enzyme (0.5 ml) with 1.0 ml citrate buffer (50 mM, pH 4.8) containing filter paper Whatman No. 1 (50 mg, 1 x 6 cm). The reaction mixture was incubated at 50 ÌŠC for 60 minutes.
2.5.2 Endoglucanase Assay
Endoglucanase activity was carried out in a reaction mixture of 1.5ml containing 0.5ml of suitably diluted (10 times and 40 times) enzyme and 1.0 ml of 2 % (w/v) carboxymethyl cellulose solution in citrate buffer (50 mM, pH 4.8). This mixture was incubated at 50 ÌŠ C for 30 minutes.
2.5.3 Xylanase Assay
Xylanase activity was determined under the similar conditions as described for endoglucase activity, except that 1 % (w/v) xylan in 50 mM phosphate buffer, pH 6.5 was used as substrate and the mixture was incubated at 40 ÌŠ C for 10 minutes.
2.5.4 Î²-glucosidase Assay
Î² - glucosidase activity was estimated using pNPG as substrate. The total of assay mixture (1 ml) consisting of 0.9 ml of 1 mg/ml pNPG in citrate buffer (50 mM, pH 4.8) and 0.1 ml of suitably diluted (10 times and 40 times) enzyme was incubated at 50 ÌŠ C for 10 minutes. The p-nitrophenol liberated was measured at 400 nm after a light green colour was produced with 2 ml of 1 M sodium carbonate solution.
2.5.5 Determination of Reducing Sugars Concentration
All reducing sugars were determined by dinitrosalicylic acid (DNS) method. One unit (IU) of enzyme activity was defined as the amount of enzyme required to liberate 1 Âµmol of glucose, xylose or other reducing sugars produced from the appropriate substrates per min under assay conditions.
2.5.6 Laccase Assay
Laccase enzyme assay was done with diluted crude enzymes added with ABTS substrate added and absorbance readings were recorded at every minute from 0 minute - 3 minutes at 420 nm.
Protein was estimated according to the method of Bradford (1976).
Spores of D64 fungal strain from one-week-old PDA plates were washed using 0.05 % (v/v) Tween80 sterilized water with the spore density of 108/ml were treated with either ethyl methane sulfonate (EMS) and Ultra-Violet radiation at 254 nm as described below at different dose rate. Killing effect was determined by serial dilution plating in comparison with untreated spore suspension. Only the mutagenized spores exhibiting 5 % to 20 % survival percentage were used for mutant screening. The disposition of getting the mutants was described in Figure 1.
2.6.1 Ethyl Methane Sulfonate Method
EMS mutagenesis was conducted by adding 0.05 ml EMS to 1 ml spore suspension After 40 minutes of incubation under room temperature, 2 ml of peptone-glycerol solution and 7 ml NaCl soltuion was added to 1 ml of the treated spore suspension.
2.6.2 Ultra-Violet Irradiation Method
UV irradiation was made by exposing the washed spores under 254 nm at 10 cm for a variety of timings (10 seconds - 30 minutes) in the UV emitting money detector, after the process, 2 ml of peptone-glycerol solution and 7 ml of NaCl was added 1 ml of the treated spore suspension.
Treated spore suspension mixture was further diluted to 103 spore density and were cultured on screening medium (103 and 104 spore density). The rest of the mixture was stored in 1.5 ml sterile eppendorf tubes and stored in -80 ÌŠ C.
Figure 1. A brief flowchart of strain improvement using mutagenesis method applied to D-64 strain.
2.7 Optimization of D-64 fungal strain
The biomass production of cellulase activity is influenced by various process variables including media components as a parameter. The amount of wheat bran and the amount of cellulose were identified as significant parameters. The levels of these variables were optimized for enhancing the cellulase yield using a response surface Box-Behnken experiment design. The design matrix with 13 experimental runs in two blocks with 2 replicates of the midpoint is shown in Table 1. The variables selected for optimization, i.e., amount of wheat bran and amount of cellulose, were coded as X1 and X2 respectively.
Table 1: Box-Behnken experiment design matrix with amount %(w/v) of cellulose (X1) and Wheat Bran (X2) for different trials.
XI Cellulose (%)
X2 Wheat Bran (%)
3.1 Mutagenesis and screening of mutants
Wild type fungal strain D-64 was subjected to mutagenic factors and were all treated successfully with either UV-irradiation (first round mutagenesis) or ethyl methyl sulfonate (second round mutagenesis). The mutant colonies were predicted to be able to produce wider clear zones on screening medium in the earlier growing days and have higher cellulase activities compared to D-64 wild type strain.
The mutants were observed to exhibit morphological changes during sub-culturing on sporulation plates. From Photograph 1, it was observed there was a change in the hyphal structure with comparison of the wild type strain and the mutant strain U20-1. After each round of mutagenesis, semi-quantitative plates clearing assays on phosphoric-acid-swollen cellulose plates (Photograph 2 and Photograph 3) were done followed by a detailed assessment using shake flask cultures. After all the mutants have gone through detailed assessment, the most promising mutants will then undergo further rounds of mutagenesis.
First Round Mutant U20-1
Wild Type D-64 StrainC:\Users\aLiS0n\Desktop\FYP INTERNSHIP (2010)\alee's plates\On PDA\Originals\CIMG1061.JPG
Photograph 1: Comparison of the morphology of wild type D-64 strain with its first generation mutant U20-1.
3.1.1 First Round Mutagenesis
During first round mutagenesis, 25 strains were selected out of 250 mutated strains after semi-quantitative plates clearing assays on SM1. All 25 strains underwent second round of semi-quantitative plates clearing assay after sub-culturing on SM2 to further compare the size of the clear zones surrounding the colonies. (Photograph 2) The 25 strains also underwent shake flask fermentation assay for cellulase production. After assessment of semi quantitative plate clearing assays with strong catabolism repressor and shake flask fermentation enzyme assay, 2 potential strains were isolated.
Mutant U20-1C:\Users\aLiS0n\Desktop\FYP INTERNSHIP (2010)\alee's plates\Clear Zones!\20-1.jpg
Photograph 2: : First round mutants of D-64 fungal strain displaying clear zones on 1.0 % phosphoric acid swollen cellulose supplemented with 1 % (w/v) D-glucose.
3.1.2 Second Round Mutagenesis
Second mutagenesis was conducted and a total of 20 strains were selected out of 200 mutated strains after semi-quantitative plates clearing assay on SM1 and SM2. Potential mutants which were able to produce exceptional clear zones on both screening media were re-inoculated in to SM3 with 2 % (w/v) glucose as a stronger catabolism repressor. Photograph 3 showed some second round mutants displaying clear zones in SM3. After assessment of semi quantitative plate clearing assays with strong catabolism repressor and shake flask fermentation enzyme assay, 5 potential strains were isolated. C:\Users\aLiS0n\Desktop\FYP INTERNSHIP (2010)\alee's plates\Clear Zones!\CTG 2 E20-1 2.8 - 117, 192, 201 back.JPG
Mutant E201Photograph 3: Second round mutants of D-64 fungal strain displaying clear zones on 1.0 % phosphoric acid swollen cellulose supplemented with 2 % (w/v) D-glucose.
Enzyme production in shake flask fermentation assessment
3.2.1 First Round Mutagenesis
Table 2 shows the enzyme activities of first round mutagenesis results after shake flask fermentation assessment. 2 potential strains were selected, U20-1 and UH. U20-1 is observed to have 1.3 times FPase activity, 1.2 times CMCase activity, 1.5 times Î²-glucosidase activity and 1.8 times xylanase activity of control strain D-64 wild type strain. On the other hand, UH is observed to have exceptional Î²-glucosidase activity, which is 3.8 times of the control strain.
Table 2: Enzyme activities of first round mutants and wild type D-64 strain produced in shake flask 1% cellulose fermentation medium.
1 % (w/v) Cellulose fermentation medium
1.4 Â± 0.35
6.5 Â± 8.03
0.7 Â± 0.12
0.7 Â± 0.02
5.2 Â± 1.77
2.7 Â± 0.02
1.8 Â± 0.3
7.6 Â± 4.67
1.1 Â± 0.02
3.2.2 Second Round Mutagenesis
Table 3 shows the enzyme activities of second round mutagenesis results after shake flask fermentation assessment. Enzyme activities of the mutants are observed to have higher enzyme activities of FPase, CMCase, Î²-glucosidase or xylanase compared to wild D-64 strain.
Second generation mutant E49 which was considered as an all-rounder in producing the higher activities of FPase (1.9 IU/ml), CMCase (13.9 IU/ml), Î²-glucosidase (1.0 IU/ml) and xylanase (5.1 IU/ml) compared to our D-64 wild type strain. D-64 wild type strain and its mutants were observed to have higher Î²-glucosidase activity compared to T.ressei RUT-C30 commercial strain, but the FPase activity was lower. EH30 second generation mutant strain was also observed to have the highest Î²-glucosidase activity (3.0 IU/ml), 3-fold increase from the D-64 wild type fungal strain. E110 second generation mutant was observed to have the highest xylanase activity (16.4 IU/ml), 5-fold increase from the D-64 wild type strain and 3-fold increase from the parent strain U20-1 first round mutant.
Both wild type and mutant strains were observed to have no significant difference in cell growth by measuring wet biomass weight. This shows that the enzyme activities are improved due to the improvement of enzyme secretion/production instead of increase of cell growth.
Table 3: Enzyme Activities and protein contents of wild strain D-64, its mutants and T.ressei RUT-C30 produced in shake flask fermentation assessment in 1%(w/v) cellulose fermentation media.
1 % (w/v) Cellulose Fermentation Media
Enzyme Activities (IU/ml)
RUTC30 Commercial Strain
2.5 Â± 0.23
18.9 Â± 0.82
0.1 Â± 0.08
5.5 Â± 0.59
D64 Wild Type Strain
1.4 Â± 0.35
6.5 Â± 8.03
0.7 Â± 0.12
2.9 Â± 0.91
First Generation Mutants
1.8 Â± 0.30
7.6 Â± 4.67
1.1 Â± 0.02
5.2 Â± 4.12
0.7 Â± 0.02
5.2 Â± 1.77
2.7 Â± 0.02
3.4 Â± 4.12
Second Generation Mutants
1.9 Â± 0.12
13.9 Â± 2.09
1.0 Â± 0.07
5.1 Â± 0.42
1.6 Â± 0.14
12.7 Â± 1.05
1.4 Â± 0.21
16.4 Â± 0.50
1.5 Â± 0.04
10.0 Â± 0.04
0.9 Â± 0.09
13.2 Â± 0.06
0.6 Â± 0.05
8.3 Â± 0.06
2.6 Â± 0.05
2.0 Â± 0.00
0.8 Â± 0.31
9.4 Â± 0.05
3.0 Â± 0.03
3.8 Â± 0.08
3.3 Optimization of D64 Strain
Based on results of preliminary experiments, the levels of cellulose and wheat bran were factors that affect the maximal yield of cellulase to design the medium composition used in present studies. For each run, the experimental responses that was conducted with enzymatic assays were shown in Table 3. The maximum response obtained was from run 8, with the parameters of 2.5 % (w/v) cellulose and 3.4 % (w/v) wheat bran. The maximal yields of cellulase obtained from run 8 are the FPase activity of 3.23 IU/ml, CMCase activity of 31.21 IU/ml, Î²-glucosidase activity of 4.28 IU/ml, xylanase activity of 13.63 IU/ml with a total protein content of 4.25 mg/ml respectively. From Table 5, mutant strains of D-64 fungal strain were cultured in the optimized media scaled down. It is observed that the activities of the strains have increased drastically compared to Table 3.
Table 4. Box-Behnken experiment design matrix with observed and predicted responses for different trials with observed enzyme activities for each trial.
Enzyme Activity IU/ml
XI Cellulose (%)
X2 Wheat Bran (%)
2.2 Â± 0.02
26.2 Â± 0.01
3.1 Â± 0.03
8.6 Â± 0.01
1.7 Â± 0.01
27.3 Â± 0.03
4.9 Â± 0.05
59.8 Â± 0.10
0.6 Â± 0.01
14.9 Â± 0.00
1.2 Â± 0.01
3.1 Â± 0.01
2.8 Â± 0.01
23.9 Â± 0.01
2.2 Â± 0.09
14.1 Â± 0.01
0.4 Â± 0.01
6.3 Â± 0.03
0.1 Â± 0.16
4.9 Â± 0.02
1.3 Â± 0.07
17.9 Â± 0.03
2.3 Â± 0.01
1.8 Â± 0.01
1.0 Â± 0.03
15.9 Â± 0.00
0.7 Â± 0.16
3.3 Â± 0.02
3.2 Â± 0.09
31.2 Â± 0.01
4.3 Â± 0.01
13.6 Â± 0.01
2.2 Â± 0.01
24.6 Â± 0.02
1.1 Â± 0.01
4.2 Â± 0.01
2.3 Â± 0.03
27.3 Â± 0.00
1.1 Â± 0.04
5.7 Â± 0.01
2.3 Â± 0.01
24.3 Â± 0.00
1.3 Â± 0.03
8.6 Â± 0.05
2.2 Â± 0.04
23.9 Â± 0.00
1.5 Â± 0.01
13.3 Â± 0.01
2.3 Â± 0.01
22.0 Â± 0.04
1.0 Â± 0.03
13.4 Â± 0.01
Table 5: Enzyme activities of the D-64 mutant strains (first generation and second generation) which were cultured in 1 % (w/v) cellulose, 1.4 % (w/v) wheat bran shake flask fermentation media.
1 % (w/v) Cellulose 1.4% (w/v) Wheat Bran
Enzyme Activity (IU/ml)
( Control ) U20-1
2.3 Â± 0.08
24.5 Â± 0.045
4.3 Â± 0.05
20.2 Â± 0.05
2.9 Â± 0.05
25.9 Â± 0.01
6.3 Â± 0.05
22.8 Â± 0.31
2.1 Â± 0.01
21.9 Â± 0.04
5.1 Â± 0.03
23.1 Â± 0.02
2.4 Â± 0.05
26.6 Â± 0.01
4.0 Â± 0.01
21.6 Â± 0.07
( Control ) UH
2.3 Â± 0.01
24.7 Â± 0.01
4.7 Â± 0.05
14.9 Â± 0.32
3.5 Â± 0.14
34.0 Â± 0.07
7.2 Â± 0.17
15.2 Â± 0.05
3.1 Â± 0.05
31.0 Â± 0.00
7.1 Â± 0.04
13.4 Â± 0.02
4.1 Mutagenesis and screening of mutants
The objective of the research was to improve a fungal strain which has the potential to reach industrial scale. Technical and economical constraints were considered during the process. The selection process was based on 3 criteria.
Firstly the enzyme activities of the mutant strain should be increased under fixed conditions such as the amount of nutrients/inducers present in the fermentation media.
Secondly, the new mutant should be as stable as the control strain. The variability of the strain should be as low as possible. All standard errors calculated next the the activities in each table mentioned above were less than < 3.0. Therefore, almost all of the strains remain stable after many repeats.
Lastly, the strain should be able to be further improved. General experimental regulations were applied. 99% killing effect is imposed on the selection of mutants on semi-quatitative plate clearing assay in the beginning to avoid accumulation of silent mutations (Durand and Clanet, 1987). Mutants that have reduced sporulation were eliminated. All screening media and shake flask fermentation media were under defined conditions, thus also eliminating auxotrophic strains (Durand and Clanet, 1987).
Selection of the mutants was also followed in a defined procedure. From the selection of mutants on screening medium using semi-quatitative plate clearing assay followed by enzyme production in shake flask assessment. All mutants have met all 3 criteria during the selection process. The result was obtaining second generation mutant strains E49 (FPase activity of 1.9 IU/ml) as the next potential strain for further improvement and test for hydrolysis of lignocellulosic biomass until it is capable to reaching the industrial scale. The rest of second generation mutant strains can be further improved by using genome shuffling among themselves and create a new recombinant mutant strain that could have higher activities of the 5 main enzymes assayed.
4.2 Optimization of D-64 Strain
The cost of cellulase enzyme is a major factor that is involved in the processing of biomass to bioethanol. The production cost of cellulases can be brought down if cheaper substrates are used, as the cost of substrate is a major fraction of production cost. The parameter showed the highest influence on cellulase production by T.ressei in the optimization studies was the concentration of inducer in the medium (Mekala, 2008)
The present research requires me to improve a new strain that has the potential to break down lignocellulosic biomass. As "solid state fermentation" is utilized to produce cellulases from biomass, we conducted an assessment by understanding what carbon or nitrogen sources played a part in maximal production of enzymes. Therefore, different amounts of components of the media could play a part in affecting the efficiency of the production of enzymes and microbial growth.
D-64 fungal strain was able to produce a high FPase activity of 3.23 IU/ml in 2.5 % (w/v) cellulose and 3.4 % (w/v) wheat bran mixed with basal media, which is favourable as cellulase fermentation needed longer duration for production and obtaining maximal activities.
After converting the optimized parameter to lower proportions to save costs, the mutants were assessed with the optimized shake flask fermentation media. (Table 5) As compared to Table 3 with only 1 % (w/v) cellulose fermentation, the FPase activity has increased 2 times, CMCase activity has increased 2.5 times, Î²-glucosidase activity has increased almost 3.5 times and xylanase activity have increased 1.5 times. This proves the reliability of the optimized parameters.
In conclusion, the improvement of lignocellulolytic fungal strains is challenging. The fact that fungus is a species that is not stable genetically and time is an important factor as to culturing them and grasping their characteristics and morphologies. Once the fungal strain changes its characteristics, more time is wasted on the grasping the characteristics back again and getting improvement of enzyme activity cannot be continued. So every step in all methods, from the making of every media to incubation factors has to be precise and accurate or the pitfall would result in the fungal strains' optimum characteristics not being isolated.
After finishing up this final year project, the objective of improving the fungal strain was reached, but the potential have not reached the industrial scale level. Two generations of mutants were generated, each of which led to the improvement of the control strain regardless of which enzyme. The strain that was show great potential to be further improved is E49 second generation mutant strain due to the higher activities of all 5 main enzymes. The 4 mutants strains (E110, E183, EH29, EH30) could be potentially improved, and if they could undergo genome shuffling to create fusants that could also have higher activities of all 5 main enzymes.
Not only have the inducer concentrations in media as a parameter for optimization of the D-64 wild type strained, other environmental parameters such as the temperature, pH, oxygen level etc could be considered. These parameters can become another milestone for optimizing the conditions for maximal cellulase production. The most optimized parameters of 2.5 %(w/v) cellulose and 3.4 %(w/v) wheat bran coupled with the most optimized environmental parameters can make significant difference to the cellulase activity of the fungal strain.
Future works involved would be the obtaining more generations of mutants from the isolated mutants and conduct genome shuffling on the mutant strains itself, these experiments can create even more potential strains and ultimately reaching industrial scale. Other optimization parameters such as environmental factors can also be studied to create the optimum environment and parameter for the production of cellulase activity for the degradation of complex lignocelluloses. Identification of the strain must be carried on and genetic works could be done to the strain to construct cDNA genomic library of the functional gene analysis of critical genes for cellulolytic degradation. With all these done, it will definitely help to develop an economical industry for bioethanol production.
In the end, the final goal is the final efficiency of the process of converting lignocelluloses to bioethanol. With this goal fulfilled, the bioethanol industry can reap great advantages and meet with huge consumers' demands and at the same time, reduce the cost of production and protect the environment. By applying sophisticated and skilful science practical skills to improve a lignocellulolytic microorganism, the degradation of lignocelluloses could be become more refined, and ultimately reaching industrial scale.