Environmental Biomolecular Sciences

1. EVALUATION OF NEW BIOPROCESSING TECHNIQUES TO ENHANCE UTILIZATION OF CELLULOSIC WASTESByClifford Louime, Ph.D.Environmental Biomolecular SciencesFlorida A&M UniversityTallahassee, Florida Environmental Biomolecular Sciences

2. Cellulosic Wastes or Biomass Feedstock: Agriculture, Forestry, Biomass Industry Trees, Grasses Agricultural Crops/Residues Municipal Solid Waste Animal wastes INTRODUCTION Environmental Biomolecular Sciences

3. INTRODUCTION • Biomass includes all plant and plant-derived material • Essentially all energy originally captured by photosynthesis: CO2 + H2O + Light + Chlorophyll  (CH2O) + O2 • Each year photosynthetic fixation of CO2 yields more than 1Mil tons of dry plant material worldwide carbohydrate Environmental Biomolecular Sciences

4. INTRODUCTION • Cellulose is the major component of plant cells • Cellulose is the most abundantly produced biopolymer in terrestrial environments Environmental Biomolecular Sciences

5. INTRODUCTION • Cellulose is a homopolymer consisting of glucose units joined by β-1,4 bonds • The degree of polymerization varies from 100 to 14,000 glucose moieties per molecule Glucose Cellobiose Environmental Biomolecular Sciences

6. BY-PRODUCTS Glucose Renewable Chemicals Plastics Chemical Intermediates Phenolics Adhesives Furfural Fatty Acids Carbon Black Dyes, Pigments Etc. Food Feed Fiber Renewable Fuels Ethanol Bio-Diesel Renewable Power Electricity Heat or CHP Environmental Biomolecular Sciences

7. Conversion Processes: • Enzymatic Fermentation • Gas/Liquid Fermentation • Acid hydrolysis • Gasification • Combustion • Co-firing Schematic of Conversion Process Environmental Biomolecular Sciences

8. Many of the products are now made from petroleum (petrochemicals) The basic molecules in petrochemicals are hydrocarbons. In plant resources, the basic molecules are carbohydrates, proteins, and plant oils Both plant and petroleum molecules can be processed to create building blocks for industry to manufacture a wide variety of consumer goods Plant resources, mostly for paper products and chemical feedstocks, now provide about 5 percent of manufacturing inputs Environmental Biomolecular Sciences

9. Statement of The Problem • One of the most important features of cellulose as a substrate for microorganisms is its insolubility • Cellulose fibrils are embedded in a matrix of other polymers, primarily including hemicelluloses, pectins, and proteins Environmental Biomolecular Sciences

10. Cellulose molecules are strongly associated through inter-and intramolecular hydrogen-bonds and van der Waals forces, that results in the formation of microfibrils, which in turn form fibers • These molecules form highly ordered crystalline domains interspersed by more disordered, amorphous regions • The degree of crystallinity in native cellulose is 60-90% • All this result in low turn-over rate Environmental Biomolecular Sciences

11. All microorganisms known to degrade cellulose release a battery of enzymes with different specificities: • Endoglucanases, which randomly hydrolyze ß-1,4 bonds within cellulose molecules, thereby producing reducing and non-reducing ends • Exoglucanases, which cleave cellobiose units from the nonreducing ends of cellulose polymers • ß-Glucosidases, which hydrolyze cellobiose and low-molecular-weight cellodextrins, thereby yielding glucose Environmental Biomolecular Sciences

12. MECHANISMS OF CELLULOSE HYDROLYSIS The enzymatic components act synergistically in the hydrolysis of crystalline cellulose. They catalyze the depolymerization via an acid-base mechanisms involving two residues: Inverting Mechanism Retaining Mechanism Environmental Biomolecular Sciences

13. CURRENT RESEARCH IN BIOLOGICAL PROCESSING • Goal: reduce the cost of using cellulase enzymes in the bio-products process by employing cutting-edge and efficient biochemical technologies: • Engineered Cellulase Mixtures • Protein Engineering • Advanced Cellulase Performance assays • Microbial Proteonomics Environmental Biomolecular Sciences

14. ENZYME ACTIVITY IMPROVEMENT • The current base is about 700 FPU per gram of enzyme: e.g. A 5X to 15X improvement will reduce the cost of enzyme from 3 to 10 per gallon of ethanol • There are many ways to improve the specific activity of the enzyme. These include the following: • Increase thermal stability of the enzyme • Decrease non-specific binding • Decrease feedback inhibition • Increase K catalyst • Enhance enzymatic Decrystallization Environmental Biomolecular Sciences

15. SIGNIFICANCE OF THE STUDY • Economics is the major driver for development. But in biomass hydrolysis research, the following categories of impacts are also considered: • Criteria air pollutants • Greenhouse gas emissions • Landfill capacity use • Forest and watershed improvement • Rural employment and economic development • Energy diversity and security Environmental Biomolecular Sciences

16. OBJECTIVES OF THE STUDY • To evaluate the cellulose degrading potential of several microorganisms from the DOE Microbial genome Project • To clone, isolate and characterize the most efficient cellulase cDNA • To verify how Site-Directed Mutagenesis affects enzyme activity Environmental Biomolecular Sciences

17. EXPERIMENT I • Biodegradation and Utilization of Sugarcane Bagasse by Several Microorganisms Environmental Biomolecular Sciences

18. OBJECTIVES OF THE STUDY • To evaluate the cellulose degrading potential of several microorganisms from the DOE Microbial Genome Project, namely: Thermomonospora fusca, Cytophaga hutchinsonii and Cellulomonas fimi, grown on sugarcane bagasse as the carbon source Environmental Biomolecular Sciences

19. METHODOLOGY Cultures Grown on Bagasse T. Fusca Cytophaga Cellulomonas Samples Collected at different timepoints for a period of one year Indicators of Degradation Dry weight Sugars by DNS Method Organic Acids by HPLC Protein by Bradford Method Environmental Biomolecular Sciences

20. RESULTS AND DISCUSSION Sugarcane Bagasse Hydrolysis Mediated by several Microbial Strains as a Function of Time Environmental Biomolecular Sciences

21. *Total Activity ([µmol reducing sugars/min]) / ml of culture) ------------------------------------------------------------------------------------------------------ Sampling Time (Day) • Microorg • 0 1 2 4 7 15 30 60 90 120 365 • T. f. 0.00 0.06 0.21 0.50 1.30 0.60 1.00 0.80 0.70 0.60 X • C. f. 0.00 0.01 0.03 0.07 0.04 0.10 0.10 0.08 0.07 0.07 X • C. h. 0.00 0.02 0.04 0.10 0.03 0.30 0.20 0.08 0.08 0.07 X • **Specific Activity (mg protein/ml of culture) • ---------------------------------------------------------------------------------------------------- Sampling Time (Day) • T. f. 0.00 0.70 1.90 4.30 9.10 5.70 8.70 7.90 7.80 7.50 X • C. f. 0.00 0.01 0.06 0.15 0.10 0.08 0.08 0.06 0.06 0.06 X • C. h. 0.00 0.03 0.10 0.30 0.10 0.10 0.07 0.07 0.06 0.05 X • ***Dry weight (g) • ------------------------------------------------------------------------------------------------------ Sampling Time (Day) T. f. 1.00 0.994 0.979 0.950 0.870 0.940 0.900 0.920 0.930 0.940 X C. f. 1.00 0.999 0.997 0.993 0.996 0.990 0.990 0.992 0.993 0.993 X C. h. 1.00 0.998 0.996 0.990 0.970 0.970 0.980 0.991 0.992 0.993 X

22. CONCLUSIONS • Overall, C. hutchinsonii outperformed the other two microorganisms in two of the parameters tested • C. hutchinsonii released 5% more reducing sugars and 15% more protein (p<0.05) – Dry weight (p>0.05) • The higher total and specific activity of C. hutchinsonii could not be explained at that time due to the higher temperature optimum of T. fusca • Competitive inhibition might also explained some of behavior of the cultures Environmental Biomolecular Sciences

23. EXPERIMENT II • PCR Cloning, Isolation and Characterization of the Cellulase Gene from C. hutchinsonii Environmental Biomolecular Sciences

24. OBJECTIVES OF THE STUDY • To clone, isolate and characterize the β-1,4 Endoglucanase Gene from Cytophaga hutchinsonii Environmental Biomolecular Sciences

25. METHODOLOGY Grow Cultures of Cytophaga Bioinformatics Isolate DNA Clones Sequencing Design PCR Primers Clones Selection Run PCR Clone PCR Product into E.Coli Environmental Biomolecular Sciences

26. RESULTS AND DISCUSSION • DNA Sequence analysis of the cloned DNA fragment coding for the cellulase gene identified an open reading frame (ORF) of 2934 bases • This sequence has 31% homology with endB gene coding for the endoglucanase EGB from Pseudomonas sp. • PROSITE patterns present in this chain GLYCOSYL_HYDROL_F9_1. : GGWYDAGD • Cel9A consists of a catalytic domain of 480aa and C-terminal adjoining Ig-like domain

27. CONCLUSIONS • All known cellulases have the following structure • C. hutchinsonii has no CBD • CBD has been considered as the limiting factor in cellulose hydrolysis Sig CBD Catalytic Domain CBD linker linker Sig Catalytic Domain Environmental Biomolecular Sciences

28. EXPERIMENT III • Location, Formation and Biosynthetic Regulation of Cellulase in Cytophaga hutchinsonii Environmental Biomolecular Sciences

29. OBJECTIVES OF THE STUDY • To find out where the cellulase gene is located and how it is being regulated, getting thereby additional insight into the mechanism of cellulose hydrolysis by Cytophaga hutchinsonii Environmental Biomolecular Sciences

30. PERIPLASM SPHEROBLAST French Press CYTOPLASM MEMBRANE PROCEDURE FOR SEPARATION OF VARIOUS CELL FRACTIONS METHODOLOGY CELLS Centrifugation SUPER PELLET SUPER Shock Environmental Biomolecular Sciences

31. RESULTS AND DISCUSSION

32. Cellulose Fiber Bacteria Cells Fig. 4: Microscopic observation of C. hutchinsonii grown on cellulose revealed no level of cell adhesion by this microorganism to the cellulose fibers. Picture taken after 96h incubation when most of the activity was measured

33. CONCLUSIONS • The cellulase enzyme of C. hutchinsonii is located in the soluble portion of the cell, the periplasm • Glucose and cellobiose act as cellulase suppressors and cellulose as inducers • Cell adhesion was negligible Environmental Biomolecular Sciences

34. EXPERIMENT IV • Expression and Characterization of Cel9A, a β-1,4 Endoglucanase from Cytophaga hutchinsonii Environmental Biomolecular Sciences

35. OBJECTIVES OF THE STUDY • To investigate the properties of Cel9A, a β-1,4 Endoglucanase from Cytophaga hutchinsonii • and possible role of this protein in the degradation of plant biomass Environmental Biomolecular Sciences

36. METHODOLOGY Expression Induction with IPTG Run on Q-Sepharose Culture Growth in Fermenter Fractions Collection Inoculum with Antibiotics Filtration and Column Loading Concentrate Best Fractions Competent Cells Biochemicals Assays Environmental Biomolecular Sciences

37. RESULTS AND DISCUSSION Environmental Biomolecular Sciences

38. RESULTS AND DISCUSSION NCBI BLAST RESULTS FOR MULTIPLE SEQUENCE ALIGNMENT unk|VIRT7738|Blast_submission EYDLSGGWHDCGDHVKFGQTEFYSAYMLLKGYAEFPAGYGDYYAYDYQGYK tr|Q97KK3 L DLTGGFHDAGDHVKFGLPQAYAASTLGWAYYEFKDSFVKKGQD------sp|P26221|GUN4_THEFU L DLTGGWYDAGDHVKFGFPMAFTATMLAWGAIESPEGYIRSGQM------sp|P28622|GUN4_BACS5 H DLTGGWYDAGDHVKFGLPMAYSAAVLAWTVYEYREAYEEAELL------tr|Q870A8 V DLTGG-YDAGDNVKFNFPQASALTILAYGAYKWKDGYKQAGQW------ **:** :*.**:***. . : * : .: unk|VIRT7738|Blast_submission TSGSWSFEGTGHAPNGIPDILDEVKHATDFFIKCAKDATTFYYQVGQGDP tr|Q97KK3 -----------------KYMLNILKHFTDYFLKCYPNKTTFYYQCGDGTT sp|P26221|GUN4_THEFU -----------------PYLKDNLRWVNDYFIKAHPSPNVLYVQVGDGDA sp|P28622|GUN4_BACS5 -----------------DDMLDQIKWATDYFLKAHTGPNEFWAQVGDGNA tr|Q870A8 -----------------EYMKDLLKWGMDYFLKCHTDKYVLYGQVGNGSL : : :: *:*:*. . :: * *:* Environmental Biomolecular Sciences

39. CONCLUSIONS • Cel9A showed a pH optimum at 6-6.5 and retained >50% of its activity from pH 5 to pH 9 • Cel9A hydrolyzes CMC, but thermostability decreases markedly above 50ºC • Analysis of CMC and filter paper hydrolysis suggests that Cel9A is a non-processive enzyme with endo-cellulase activities Environmental Biomolecular Sciences

40. EXPERIMENT V • Site-Directed Mutagenesis and Computer Modeling of Cel9A, a β-1,4 Endoglucanase from Cytophaga hutchinsonii Environmental Biomolecular Sciences

41. OBJECTIVES OF THE STUDY • To locate conserved residues in relation to the catalytic cleft based on known Family9 computer models. • To mutate conserved residues within the catalytic domain; and • To determine the enzyme-substrate complex contributing to the catalytic mechanism. Environmental Biomolecular Sciences

42. METHODOLOGY FAM9 catalytic domain is characterized by the following conserved sequence: GGWYDAGD • Quick Change Mutagenesis • This method uses the two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions • FORWARD PRIMER • Seq 5’ TCCGGCGGATGGCATGATTGCGGTGATCACGTAAAATTCGGGC • PRIMER CCGGCGGATGGCATGCTTGCGGTGCTCACGTAAAATTCGGGC • COMP3’ GGCCGCCTACCGTACTAACGCCACGAGTGCATTTTAAGCCCA • FRAME1 Ser Gly Gly Trp His Asp Cys Gly AspHis Val Lys Phe Gly Gln • PRIMER Ser Gly Gly Trp His Ala Cys Gly Ala His Val Lys Phe Gly Gln • REVERSE PRIMER • COMP3’ GCCCGAATTTTACGTGATCACCGCAATCATGCCATCCGCCGGA • PRIMER GCCCGAATTTTACGTGAGCACCGCAAGCATGCCATCCGCCGG • Seq3’ CGGGCTTAAAATGCACTAGTGGCGTTAGTACGGTAGGCGGCCT • FRAME4 Cys Pro Asn Phe Thr . Ser Pro Gln Ser Cys His Pro Pro Glu • PRIMER Cys Pro Asn Phe Thr . AlaPro Gln Ala Cys His Pro Pro Glu Environmental Biomolecular Sciences

43. RESULTS AND DISCUSSION Environmental Biomolecular Sciences

44. Environmental Biomolecular Sciences

45. CONCLUSIONS • The overall model produced a good fit. Cel9A residues, Y204, Y406 and D504 which line the tunnel have counterparts as do the proposed catalytic residues • Asp appears to be involved in the enzyme-substrate complex contributing to the catalytic mechanism • Mutation of Asp with Ala resulted in at least 70% loss of enzyme activity Environmental Biomolecular Sciences

46. GENERAL CONCLUSIONS AND RECOMMENDATIONS • Cytophaga does not grow well in liquid media. Cells might require close contact to the substrate? • Cytophaga might be polar: Zeta Potential? • Cytophaga differs from known cellulose degraders: Is CBD imperative for cellulose hydrolysis? • What mechanism does Cytophaga use to colonize its insoluble substrates? • Possible mode of action based on my data Environmental Biomolecular Sciences

47. ACKNOWLEDGEMENTS • This research was supported by a grant from the United States Department of Agriculture and by TITLE III Programs • I would like to acknowledge: • AND Dr. Michael Abazinge Dr. Larry Robinson, Provost Dr. Jennifer Cherrier Dr. Henry N. Williams, Director of ESI Dr. Elijah Johnson Dr. Richard Gragg, Associate Director Dr. Lekan Latinwo ESI Faculty, staff and students Dr. Christopher Ikediobi Dr. John West, Chemistry Dr. James Bouyer, Chemistry Dr. Nazarius Lamango, Pharmacy Dr. Oghenekome Onokpise, CESTA Dr. David Wilson, Cornell University Dr. Lonnie Ingram, University of Florida Dr. Ginger Clark, University of Florida Dr. David Moraga Amador, University of Florida Dr. Verian Thomas, CESTA Dr. Jiang Lu, CESTA Environmental Biomolecular Sciences

48. QUESTIONS?

49. Why Sugarcane?

50. Methods of Generating Energy from Biomass • In order of complexity of the processes involved: • Direct combustion: Co-firing • Thermochemical Processing: Pyrolysis, gasification and liquefaction • Biological Processing: Natural processes such as anaerobic Digestion and fermentation The immediate product of some of the processes is heat – for chemical processing or district heating, or to generate steam for power production. For other processes the product is solid, liquid or gaseous fuel: Charcoal, liquid fuel as a petrol substitute or additive, gas for sale or for power generation using either steam or gas turbines