Dr. Taous Khan Department of Pharmacy, COMSATS, Abbottabad, Pakistan

1. Dr. Taous Khan Department of Pharmacy, COMSATS, Abbottabad, Pakistan Wastes from Various Sectors of Pakistan: Potential Raw Materials for Biofuels & Biomaterials

2. INTRODUCTION • Generation of wastes is increasing due to: • – growing urbanisation & • – changes in life style • ~1.6 billion metric tons of solid waste is produced per year • A lot of money is used managing this waste • – Asian countries spent ~US$25 billion per year in early 1990s • – expected to rise to ~US$50 billion by 2025 Mahar et al., Proceedings of the International Conference on Sustainable Solid Waste Management, 5 -7 September 2007, Chennai, India. pp.34-41 Department of Pharmacy, COMSATS University, Abbottabad

3. INTRODUCTION • Threat to our already degraded environment • Advances in biotechnology, genetics, chemistry & engineering • – new manufacturing concepts • – converting waste materials to valuable fuels & biomaterials 3 Kyungpook National University, Biochemical Engineering Lab

4. Wastes from various sectors of Pakistan • Large amount of waste materials produced from various sectors of Pakistan • – industrial sectors • – agricultural sectors • These materials are a source of: • – environmental pollution • – water pollution • – different diseases COMSATS University, Abbottabad 4

5. Wastes from various sectors of Pakistan • These materials are: • – rich in various elements including carbon & nitrogen • – can be used as substrate for microbial growth • – production of useful metabolic products • Thus, these waste materials can be a good source for production of bioenergy & biomaterials • – their use can address several societal needs • – will lead to a new manufacturing paradigm Department of Pharmacy, COMSATS, Abbottabad 5

6. Wastes from various sectors of Pakistan • In America • – 2500 MW electricity • – generated by waste-to-energy plants Department of Pharmacy, COMSATS, Abbottabad 6

7. Physical Composition of Wastes • Three general categories of solid waste in Pakistan: • – biodegradable e.g. food & animal wastes, leaves, grass & wood • – non-biodegradable e.g., plastic, rubber, textile waste, metals, stones • – recyclable material e.g., paper, card board, and bones Department of Pharmacy, COMSATS, Abbottabad 7

8. Physical Composition of Wastes Figure. Physical composition of solid wastes in Pakistan (% Weight). Mahar et al., Proceedings of the International Conference on Sustainable Solid Waste Management, 5 -7 September 2007, Chennai, India. pp.34-41 Department of Pharmacy, COMSATS, Abbottabad 8

9. Wastes from various sectors of Pakistan Figure. Various sources of waste materials from Pakistan. Department of Pharmacy, COMSATS, Abbottabad 9

10. Potential Products from Wastes from various Sectors of Pakistan Figure. Various products that can be produced from waste materials from Pakistan. Department of Pharmacy, COMSATS, Abbottabad 10

11. A case study–Production of WSOS & BC from Wastes of Korean Breweries • WSOS & BC successfully produced from synthetic media • Expensive process, hindering their commercial applications • Alternate, cheaper culture media for production of these products • Waste from beer fermentation broth • – large-scale availability from Korean breweries • – contains carbon, nitrogen, sulfur & ethanol Department of Pharmacy, COMSATS, Abbottabad 11

12. Composition of WBFB Table. Detailed composition of the waste from beer fermentation broth. Department of Pharmacy, COMSATS, Abbottabad 12

13. Importance of Oligosaccharides • Medical • Infectious & inflammatory diseases • Metabolic & cardiovascular disorders • Transplantation & neutralization of toxins • Cancer immunotherapy • Food • Pre-biotics • Bulking agents in diet foods • Agriculture • Activation of plant cell machinery • Fertilizers Department of Pharmacy, COMSATS, Abbottabad 13

14. Bacterial Cellulose Figure. Organic constituents of wood. Department of Pharmacy, COMSATS, Abbottabad 14

15. Bacterial Cellulose • Pure, free from lignin, pectin & hemicellulose • High degree of crystallinity & water retention value • High tensile strength and moldability • Biodegradable (eco-friendly materials) Department of Pharmacy, COMSATS, Abbottabad 15

16. Applications of Bacterial Cellulose Figure. Important applications of bacterial cellulose. http://www.rish.kyoto-u.ac.jp/houga/researches/2006m05a.jpg Department of Pharmacy, COMSATS, Abbottabad 16

17. Gluconacetobacter hansenii PJK • Gluconacetobacterhansenii PJK – Gram-negative bacteria – originally isolated from rotten apples • Capable of producing – water soluble oligosaccharides (WSOS) – bacterial cellulose (BC) 17 Department of Pharmacy, COMSATS, Abbottabad

18. Production of BC & WSOS by G. hansenii PJK Figure. Proposed biosynthetic pathway for the simultaneous production of BC & WSOS from glucose by G. hansenii PJK 18 Department of Pharmacy, COMSATS, Abbottabad

19. Pre-treatment of WBFB Figure. Schematic representation for the processing of WBFB. 19 Department of Pharmacy, COMSATS, Abbottabad

20. Bioreactors Configuration (a) (b) Figure. Schematic diagrams of jar fermenters used for the production of WSOS and BC by G. hansenii PJK; 5 L jar fermenter (a) and 5 L jar fermenter equipped with a spin filter (b). 20 Department of Pharmacy, COMSATS, Abbottabad

21. Bioreactors Configuration (a) (b) Figure 19. Selected pictures of the 2 L jar fermenter equipped with a spin filter; empty fermenter (a) and with 1.6 L of culture broth. 21 Department of Pharmacy, COMSATS, Abbottabad

22. Measurement of WSOS, BC & Cells Figure. Schematic representation for the measurement of WSOS, BC and Cells from the culture broth.. 22 Department of Pharmacy, COMSATS, Abbottabad

23. Culture Conditions Table. Detailed fermentation conditions used for the production of WSOS and BC by G. hansenii PJK. 23 Department of Pharmacy, COMSATS, Abbottabad

24. WSOS & BC Production using WFBF Effect of dilution ratio Figure. Production of WSOS & BC by G. hansenii PJKin baffled flasks shaken at 100 rpm at 30 oC for 7 days, using the WBFB diluted with distilled water, in various ratios. 24 Department of Pharmacy, COMSATS, Abbottabad

25. WSOS & BC Production using WFBF Effect of impeller speed Figure. Production of WSOS & BC by G. hansenii PJKin a 5 L jar fermenter (3 L working volume), an aeration rate of 1 vvm, a pH of 4.5-5.5 and impeller speeds of 300 rpm. 25 Department of Pharmacy, COMSATS, Abbottabad

26. WSOS & BC Production using WFBF Effect of impeller speed Figure. Production of WSOS & BC by G. hansenii PJKin a 5 L jar fermenter (3 L working volume), an aeration rate of 1 vvm, a pH of 4.5-5.5 and impeller speeds of 500 rpm. Department of Pharmacy, COMSATS, Abbottabad 26

27. WSOS & BC Production using WFBF Effect of impeller speed Figure. Production of WSOS & BC by G. hansenii PJKin a 5 L jar fermenter (3 L working volume), an aeration rate of 1 vvm, a pH of 4.5-5.5 and impeller speeds of 600 rpm. Department of Pharmacy, COMSATS, Abbottabad 27

28. Overproduction of WSOS obtained in present study Table. Comparison of production of WSOS obtained in present study with products of related chemical nature reported in literature. [1]. Jung et al., Enzyme Microb. Technol., 37 (2005) 354. [2]. Courtois et al., J. Carbohydr. Chem., 12 (1993) 448. [3]. Michaud et al., Int. J. Biol. Macromol., 17 (1995) 369. [4]. Michaud et al., Int. J. Biol. Macromol., 16 (1994) 301. [5]. Heyraud et al., Carbohydr. Res., 240 (1993) 71. Department of Pharmacy, COMSATS, Abbottabad 28

29. Structure of WSOS Figure. Schematic representation of various techniques applied for structure determination of WSOS. 29 Department of Pharmacy, COMSATS, Abbottabad

30. Structure of WSOS Structure of WSOS Figure. Chemical structures and fragmentation scheme of the major WSOS produced from WBFB. Department of Pharmacy, COMSATS, Abbottabad 30

31. Structure of WSOS • Chemical structure of WSOS : • Mixture of oligomers of glucuronic acid • Molecular weights less than 1000 • α- Linked rather than β Department of Pharmacy, COMSATS, Abbottabad 31

32. Potential applications of WSOS • WSOS produced from WBFB: • Glucuronan oligosaccharide • Glucuronanpolysaccharides: • Carriers for various drugs • Solvents, stabilizers, binders and swelling agents • Medical properties of glucuronan polysaccharides • Bleeding stoppage during surgery • Prevention of post-surgical adhesions • Antibacterial activity • Bone regeneration Department of Pharmacy, COMSATS, Abbottabad 32

33. Physical properties of WSOS Figure. Schematic representation for the study of physical properties of WSOS. Department of Pharmacy, COMSATS, Abbottabad 33

34. Physical properties of WSOS • Physical characteristics of WSOS from the defined medium: • Flake-type with porous surfaces • Free from adhered microbial cells contamination • No problems in its practical applications. • Thermal stability better or comparable to the other microbial polysaccharides • Can proceed for development into a valuable product • Good emulsifying activity with short term stability • Useful as an emulsifier in combination with a stabilizer • Alone in situations where only short-term emulsion stability is desired. Department of Pharmacy, COMSATS, Abbottabad 34

35. Conclusion • Production of WSOS and BCby G. hansenii PJK from the WBFB : • WBFB – worthy substitute for the conventional defined medium • Better production yield of WSOS compared to synthetic medium • Physico-chemical properties of WSOS : • Few structural differences from the WSOS from a defined medium • The presence of O-acetyl and O-methyl groups • Lack of unsaturation in the terminal unit • Similar or even better thermal and emulsifying characteristics • Potential for development for commercial applications Department of Pharmacy, COMSATS, Abbottabad 35

36. Our Current Research in Pakistan • Currently we are working to evaluate various waste materials from different industrial & agricultural sectors of Pakistan for the production of BC Department of Pharmacy, COMSATS, Abbottabad 36

37. Publications • From this work • Taous Khan, S.H. Hyun, J.K. Park, Production of glucuronan oligosaccharidesusing the waste of beer fermentation broth as a basal medium, Enzyme and Microbial Technology, 42, 89-92 (2007). • Taous Khan, J.K. Park, The structure and physical properties of glucuronic acid oligomers produced by a Gluconacetobacterhansenii strain using the waste from beer fermentation broth, Carbohydrate Polymers, 73(3), 438–445 (2008). • J.H. Ha, O. Shehzad, S. Khan, S.Y. Lee, Taous Khan, J.K. Park, Production of bacterial cellulose by a static cultivation using the waste from beer culture broth, Korean Journal of Chemical Engineering, 25(4), 812–815 (2008). Department of Pharmacy, COMSATS, Abbottabad 37

38. THANKS