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Title of Thesis

Farooq Latif
Institute/University/Department Details
Institute of Chemistry/ University of the Punjab
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
lignocellulosic substrates, fungi, thermophilic cellulolytic fungi,mesophilic fungi, ultrafiltration, enzyme, sodium hydroxide

Potent thermophilic cellulolytic fungi were isolated frolocal habitats. These fungi, exhibited saccharifying ability showing clearance of cellulose medium in 5-6 days of incubation at 50°C.The isolates were namely: Aspergillus fumigatus (thermo tolerant), Chaetomium therophile,Humicola grisea, Sporotrichi thermophile, Torula thermophila, Malbranchea pulchella and Muco pusillus. Rhizospheres of grasses like Leptochloa fusca (kalla grass) and Cenchrus ciliaris, were rich sources for the occurrence of these fungi.

Seven thermophilic and three mesophilic fungi were screen for cellulase/xylanase production. A. fumigatus produced great levels of 0.4, 2.4, 3.7 and 0. 12 U/ml of EPase, CMCase, xylana and β-xylosidase, respectively when grown on 2% kallar grass. glucosidase was produced to a higher extent (0.47 (U/ml) by thermophile.M pulchella and M Pussilus showed poor enzy activities, whereas rest of the fungi showed moderate activit Among the mesophiles Chaetomium vergicephalum exhibited bet induction than T. reesei Rut C-30 when grown on 2% kallar grass The cellulase activity by the mesophiles was much lower than t thermophiles.

S. thermophile and C. vergicephalum because of their bett overall enzyme activities were selected for further studie Various lignocellulosic (LC) substrates were used for enzy production in solid state fermentation (SSF) and liquid ferment tion (LF). Quantitative cellulase production in general depend upon the particular inducer. In SSF, using S. thermophi.le_ great level of avicelase, CMCase and xylanase induction took- place when the substrate was rice straw, followed by Sesbania aculeat(dhancha) which induced β-glucosidase and β -xYlosidase to greate extent. However, in case of C vergicephalum, kallar grass induced more CMCase and xylanase, whereas dhancha was the substrate of choice for rest of the three enzymes. In LF wheanstraw along with rice straw and kallar grass were the substrat of choice for S. thermophile, whereas dhancha showed Bette prospects instead of wheat straw for C. vergicephallum. Compare to SSF, the cellulase production, was substantially higher case of LF. The production of β -glucosidase by S. thermophile was about 75 fold higher when the substrate was rice straw. It was found that S. thermophile produced higher cellulase titre in as compared to C. vergicephalum, whereas the latter produced marginally better enzyme titre than the former in SSF. S. thermopile was further selected for optimization studies in SSF a LF. In SSF, moisture (medium) level of 3: 1 (v/w) favoured high cellulase production. Moreover, the mineral medium concentratic when increased to 3-4 fold showed enhancement in cellular activity.

In LF, vegetative inoculum size of 5-10% (v/v) grown for hours was optimum for enzyme production in~. thermophile. medium pH set at 5.0-5.5 and a temperature of 45°C was benefic for higher yields. The fungus exhibited 4.6 and 8.3 times high FPase and β -glucosidase activities when grown on LC substrate (untreated kallar grass) than on pure cellulose (אּ-cellulose The order of induction by various substrates was; kallar grow (untreated) > kallar grass (treated with NaOH, 121° C, 1h) avicel >אּ-cellulose. Further studies enunciated that the fungus can tolerate high substrate -concentration (10%) It produce maximal activities at 8% substrate (kallar grass) concentration of 1.1 and 1.6 U/ml of FPase and f-glucosidase, whereas the extracellular protein and pH level were 3.1 mg/ml and 5.7, respectively after 9-11 days of incubation.

Medium used by Eggins and Pugh, E & P (1962), and Romanell et al (1975) induced higher cellulase and xylanase activity in S.thermophile. However, when each of the constituents in E & medium was increased from zero concentration to four fold, there was no noticeable increase in the cellulase activity compared the control. In fact, kallar grass (2%) resulted as a moss suitable substrate with its high ash content fulfilling the mineral requirements of the media to quite an extent. Enhancemer€€ of nitrogen contents to two fold in the medium resulted enhancement of cellulase activity. Various nitrogen source tested alone and in combination, depicted the medium (E & F components of yeast extract, אּ-asparagines and (NH ) 424 SO a most suitable for growth and Enzyme production.

It was evaluated that ultrafiltration of crude enzyme from S. thermophile grown on 6% kallar grass, increased the FPase glucosidase and extracellular protein activity by 4.3, 6.2 and 4.2 times, respectively, as compared to that of unconcentrate enzyme obtained from 2% kallar grass.

The characterization of cellulases revealed that pH level 5-5.5 was optimum for different enzymes. The optimum temperature for avicelase activity was 70° C, whereas CMCase, β-glucosidal and xylanase showed maximum activity at 65° C. The thermo stability of FPase at 60 and 70° C was highest for Humicola grisea i.e,80 oC and 60%, respectively, after 24 hours of incubation. Rest of the thermophilic fungi were also found to retain about 70 and 45% of FPase activity after incubation at above temperatures. Compare to this. mesophilic strain of T reesei Rut-30 showed 22 and 50 activity for FPase at 60 and 70° C, whereas C.vericephal showed no activity.

Analysis of six untreated LC substrates showed that the three salt tolerant plants had high ash (10-23%) and soluble sugar contents (9.6-12.9%). Out of the three pretreatments for each substrate, NaOH (2%) with autoclaving (121°C, 15 min resulted in maximum enzymatic accessibility. It was followed treatment with NaOH (2--3%) at room temperature. and steaming high temperatures (200°C). Among the different substrates when straw and Panicum maximum resulted in maximum enzymatic access bility of 89 and 86%, respectively (based on pretreated fibre However, based on the raw materials. NaOH treatment at row temperaure was the method of choice. Mass balance studies show that although harsher pretreatment conditions resulted in high enzymatic accessibility because of greater deliginification a increase in polysaccharidc3 along with structural modification but at the expense of greater fibre losses. Steaming in genera resulted in greater fibre recovery than NaOH with autoclaving poor delignification showed overall low hydrolysis yields. Bas on the raw materials, wheat straw was enzymatically solubility up to 56, 53 and 31%, when the substrate was treated with 2% NaoH at room temperature. 2% NaoH along with autoclaving and steam at 190°C, respectively. Other substrates to follow the accessibility were in the order of Pancium maximum > kallar grass bagasse > Atriplex gmnicol > poplar.

Commercial cellulase preparation of Trichoderma reesei VT D-79125 with its FPase, supplemented with β-glucosidase Aspergillus niger (Miles Kalli) in a ratio of 1:1.4 and at 16.27 U/g substrate showed increase in saccharification of 2, 5 a 10% kallar grass to 10.4, 6.3 and 6.0%, respecively. With 2 and 1.7 fold increase in these enzyme concentrations the overa yields attained were 67 and 60% from 5 and 10% kallar grass aft 48 hours of hydrolysis Compared to this enzyme filtra obtained, from 4% kallar grass by Q. thermophile, yielded 60 a 33% reducing sugars. However, the estim.3.ted enzyme titre for FPase and β-glucosidase of 7.2: 14 and 3.6:7.0 U/g of substrat respectively was substantially lower to that of supplemented reesei cellulase, for these substrate concentrations. This can attributed to better synergistic enzyme system as well as the stability of the cellulases of S. thermophile. The concentrate enzyme filtrate of S.thermophile after ultrafiltration, in the ratio of FPase β-glucosidase of 24:80 U/g substrate, hydroys 5% kallar grass to 70% reducing sugars after 70 hours. However maximum yields of 75% were attained by T. reesei, but only When the cellulase preparation was supplemented with β-glucosidal from A. niger in the ratio 33:54 U/gm of substrate. The control from S.thermophile (unconcentrated) and T. reesei with supplementation yielded 55 and 60% reducing sugars, respective The composition of sugars determined by HPLC showed high relative yield of glucose and oligosaccharides in the hydroly zates from S.thermophile. The saccharification of filter paper was poor using enzyme filtrate of S.. thermophile,. However because of potent p-glucosidase, the sugar composition Showed almost 99% of glucose in the hydrolyzates. In order to elucidate the enzyme system from S. thermophile and I. reesei, freeze dri€.. enzymes used at equivalent proportions of FPU/g of substrat resulted in only a slightly higher saccharification of 5% kalla grass by the latter preparation. However, the saccharification was much higher when the substrate was filter paper, in the case. HPLC of sugars from the hydr0lysis of these substrate depicted higher relative glucose and oligosaccharides from kalla grass by S. thermopile, whereas higher xylose content result, from T. reesei The hydrolyzates from Filter paper depicted apa€€.. from glucose and a small amount of oligosaccharide component, substantial amount of cellobiose in the hydrolyzate from the latter enzyme preparation. No cellobiose was formed by the S. thermophile preparation. The significance of these findings in the fact that in a fixed proportions of FPU's used T. reese enzyme system contains a strong endo-glucanase along with xylanase, whereas a greater level of β-glucosidase is present S. thermophile. The optimum FPU/g of substrate yielding maxim'saccharification results were 30 and 35 for S. thermophile and' reesei enzyme sources.

Saccharifying ability of thermophilic fungi was evaluate by using culture filtrate obtained from 2% kallar grass. Amoy the thermophiles highest reducing sugar yield was obtained from Chaetomium thermophile (69.2%) from 5% kallar grass. All the fungi showed considerable saccharification rate up to 40 hours with the dilute enzymes. Freeze dried enzymes from T. reese (mesophile) showed the maximum yield at 75% _but at much higher enzyme concentrations. The HPLC elucidated the enzyme system of these fungi Glucose sugar was obtained at higher concentration from C. thermophile > reesei S. Thermophile > H. grisea > T. thermophila > A. fumigaytus > Mpulchella. The potential of these fungi to carry hydrolysis at 60°C in relation to their thermostable enzyme system was studied Enzymes from C. thermophile exhibited 55% of saccharification up to 40 hours, whereas rest 0f the fungal enzymes did not show any noteworthy increase after 2 hours of incubation. c. thermophile showed 1.4 times faste saccharificaton rate but could not increase the overa11 yield it Contrast T. reesei enzymes resulted in 1nactivation after hours and depicted lowering in sugar yield of 46.5% reducing sugars, compared to that at 50° C, whereas, C. thermophile showe an over all decline in yield of 11.2%, only.

The results have shown great merits for the microbial conversion of waste land plants into sugars, which can be fermented to various products. Use of thermophilic fungi for cellulas production and biomass conversion has generated interests further elucidation of their behaviour and genetic structure.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
336.24 KB
2 1 Introduction 8
342.31 KB
3 2 Review of Literature 22
1061.48 KB
4 3 Materials and Methods 60
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  3.1 Fungal Species 60
  3.2 Medium and Carbon Sources for Isolation and Production of Cellulases 60
  3.3 Isolation and Taxonomy of Thermophilic Fungi 61
  3.4 Fermentation Methods 62
  3.5 Inoculum Preparation 63
  3.6 Enzyme Assays 64
  3.7 Optimization of Culture Condutions in solid state and liquid fermentations 66
  3.8 Ultrafiltration of crude enzyme 68
  3.9 Enzyme Parameters 68
  3.10 Pretreatment of Lignocellulosic substrates 68
  3.11 Steam Extraction 69
  3.12 Sodium Hydroxide Pretreatment 69
  3.13 Analysis of total polysaccharides by 70
  3.14 Analysis of soluble sugars 70
  3.15 ASH Contents 70
  3.16 Enzymatic Accessibility 70
  3.17 Cellulase sources for hydrolysis 71
  3.18 Substrate and Pretreatment 72
  3.19 Enzymatic Hydrolysis 72
  3.20 Sugar Analysis by Chemical Methods 73
  3.21 Analysis of Sugars by HPLC 73
5 4 Results 75
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  4.1 Isolation of thermophilic fungi 75
  4.2 Cellulase and xylanase production by 77
  4.3 Cellulase and xylanase production by lignocellulosic (LC) substrates 82
  4.4 Solid state fermentation by sporotrichum thermophile 83
  4.5 SSF by chaetomium vergicephalum 85
  4.6 Liquid Fermentation (LF) by S. thermophile 87
  4.7 LF by C. vergicephalum 89
  4.8 Comparison of SSF with LF in S. thermophile 91
  4.9 Comparisons of SSF with LF in C. vergicephalum 93
  4.10 Optimization of culture conditions for S. thermophile in SSF 94
  4.11 Moisture Level 94
  4.12 Nutrient Concentration 94
  4.13 Optimization of Culture Conditions for S. thermophile In LF 96
  4.14 size and Age of Inoculum 96
  4.15 Temperature 97
  4.16 pH 98
  4.17 Cellulosic and LC substrates 99
  4.18 Substrate Concentration 100
  4.19 Synthetic Media 102
  4.20 Mineral Salts and Orgainc Niogen 103
  4.21 Miscellaneous Additives 107
  4.22 Concentration of Cellulases by Ultrafiltration 107
  4.23 Cellulase production by using a fermentor 110
  4.24 Enzyme Parameters 112
  4.25 pH 112
  4.26 Temperature 112
  4.27 Thermostability 113
  4.28 Pretreatment of LC substrates 114
  4.29 Kallar Grass ( Leptochloa Fusca ) 114
  4.30 Green panic ( panicum maximum ) 116
  4.31 River salt bush ( Atriplex amnicola ) 117
  4.32 Bagasse ( Saccarum officinarum ) 119
  4.33 Wheat straw ( Panicum sativa ) 120
  4.34 Poplar ( populus alba ) 121
  4.35 Comparison of Untreated Substrates 122
  4.36 Comparison of pretreated Substrates 123
  4.37 Steaming at High Temperature 123
  4.38 Alkali Treatment at Room Temperature 126
  4.39 Alkali Treatment with Autoclaving 129
  4.40 Saccharification of Kallar Grass by 132
  4.41 T. reesei Cellulase supplemented with B-glucosidase 132
  4.42 Increased Enzyme Concentration 133
  4.43 Saccharification of Kallar Grass by Crude Enzyme 134
  4.44 Increased Enzyme Titre by S. thermophile 134
  4.45 Saccharification of Kallar Grass and Filter Paper 134
  4.46 Use of Concentrated Enzyme 140
  4.47 Comparision of Cellulases From S. Thermophile and T. Reesei 144
  4.48 Use of Equal Filter Paper Units ( FPU€™s ) 144
  4.49 Optimum Enzyme Concentration ( FPU€™s per gram substrate ) 150
  4.50 Saccharification by thermophilic fungi 152
  4.51 Hydrolysis at 50°C 152
  4.52 Use of Enzyme Filtrate at High Temperature 156
6 5 Discussion1 60
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  5.1 Isolation and Screening of Thermophilic Cellulolytic Fungi on Lignocellulose (LC ) 160
  5.2 Optimisation of Culture Conditions and Enzyme Characterisation 164
  5.3 Pretreatment of LC substrates 172
  5.4 Saccharification of LC by Fungal Enzyme Preparations 177
7 6 References 186
661.36 KB