Microencapsulation of clostridium acetobutylicum cells and utilisation of samanea saman leaves for the production of biobutanol

MICROENCAPSULATION OF CLOSTRIDIUM ACETOBUTYLICUM CELLS AND UTILISATION OF SAMANEA SAMAN LEAF LITTER FOR THE PRODUCTION OF BIOBUTANOL SWETA RATHORE NATIONAL UNIVERSITY OF SINGAPORE 2013 MICROENCAPSULATION OF CLOSTRIDIUM ACETOBUTYLICUM CELLS AND UTILISATION OF SAMANEA SAMAN LEAF LITTER FOR THE PRODUCTION OF BIOBUTANOL SWETA RATHORE (B.Sc. (Pharm), Mumbai University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2013 Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. ACKNOWLEDGEMENTS I consider this as the most important page of my entire thesis as I list the names of all the people who have, in some way or the other, helped me reach the end of the scientific adventure that I ventured four years back. First and foremost, I would like to thank my supervisors, Associate Professor Chan Lai Wah and Associate Professor Paul Heng Wan Sia, for their attentive supervisor and invaluable guidance. This thesis would not have been possible without their encouragement and support. I am also grateful to National University of Singapore for providing me the opportunity and infrastructure to carry out my research work. Special thanks to the laboratory technologists, Mdm Teresa Ang, Ms Yong Sock Leng and Mdm Wong Mei Yin for providing technical and logistic assistance from time to time. I am thankful to my fellow GEANUS friends, past and present as well as the FYP students, Alvin, Jeanette and Eileen for helping with a part of this project. And last but not the least; I would like to express my heartfelt gratitude to the pillars of my life, my family. Their patience and support has motivated to face all the challenges in the four years with self-belief and positive attitude. Overall, this PhD journey has been an enriching experience inculcating in me to have a broader outlook towards science as well as life. Sweta Rathore 2013 CONTENTS SUMMARY .viii LIST OF TABLES . x LIST OF FIGURES .xii I. INTRODUCTION A. Biofuel A.1 Biobutanol B. Biobutanol production . B.1 Clostridium acetobutylicum . B.2 ABE fermentation B.3 Morphological changes in Cl. acetobutylicum during ABE fermentation B.4 Limitations of the conventional ABE batch fermentation process . C. Strategies to overcome limitations of ABE fermentation 10 C.1 Solvent recovery 10 C.2 Genetic/metabolic engineering 12 C.3 Advanced fermentation techniques 14 D. Cell immobilisation . 15 D.1 Immobilisation of solventogenic clostridia 16 i D.2 Limitations of conventional cell immobilisation methods used in ABE fermentation . 18 E. Microencapsulation as a cell immobilisation technique 19 E.1 Techniques used for microencapsulation of microbial cells 20 E.2 Polymers used for microencapsulation 23 F. Alternative fermentation substrates 28 F.1 Samanea saman tree (rain tree) 29 F.2 Structure of lignocellulosic substrate . 31 F.3 Pretreatment of lignocellulosic substrate . 34 F.4 Types of pretreatment . 35 F.5 Enzymatic hydrolysis of lignocellulosic substrate . 37 F.6 Strategies for detoxification of acid hydrolysate 38 II. HYPOTHESES AND OBJECTIVES . 42 III. EXPERIMENTAL 47 A. Materials 47 A.1 Model microorganism 47 A.2 Growth media 47 A.3 Fermentation medium 48 A.4 Encapsulating polymer and chemicals 48 A.5 Chemicals for assay of butanol by gas chromatography ii mass spectrometry 48 A.6 Lignocellulosic substrate 49 A.7 Cellulolytic enzyme 49 A.8 Chemicals used in assay of reducing sugars . 49 A.9 Chemicals used for dilute acid coupled with heat treatment of S. saman leaf litter. 49 A.10 Chemicals used for measuring the filter paper units (FPU) activity of Accelleraseđ 1500 . 50 A.11 Chemicals used for detoxification of acid hydrolysate of S. saman leaf litter . 50 B. METHODS 51 B.1 Preparation of growth media 51 B.2 Cultivation of Cl. acetobutylicum ATCC 824 . 51 B.2.1 Revival of Cl. acetobutylicum ATCC 824 . 51 B.2.2 Determination of suitable media for the growth of Cl. acetobutylicum ATCC 824 51 B.2.3 Determination of suitable anaerobic set-up for the growth of Cl. acetobutylicum ATCC 824 52 B.2.4 Determination of growth curve and morphology of Cl. acetobutylicum ATCC 824 53 iii B.2.5 Preparation of spore stock culture of Cl. acetobutylicum ATCC 824 54 B.2.6 Optimisation of heat shock treatment (HST) conditions for the revival of Cl. acetobutylicum ATCC 824 spores 55 B.2.7 Preparation of standardised inoculum of vegetative cells of Cl. acetobutylicum ATCC 824 56 B.3 Production of microspheres by emulsification method 57 B.3.1 Optimisation of production of gellan gum microspheres 58 B.3.2 Characterisation of the microspheres 62 B.4 Study of emulsification process on viability of Cl. acetobutylicum ATCC 824 vegetative cells/spores . 63 B.5 Method development for the assay of butanol by gas chromatography-mass spectrometry (GC-MS) . 63 B.6 Fermentation studies using Cl. acetobutylicum ATCC 824 cells . 66 B.7 Determination of viable count of cells liberated from microspheres into the fermentation medium 69 B.8 Comparison of reusability between free (non-encapsulated) cells and encapsulated cells of Cl. acetobutylicum ATCC 824 70 B.9 Pretreatment of S. saman leaf litter 70 B.10 Assay of fermentable sugars by DNS method 74 iv B.11 Determination of filter paper activity of Accelleraseđ 1500 . 76 B.12 Enzymatic hydrolysis of pretreated S. saman leaf litter . 77 B.13 Detoxification of acid hydrolysate of S. saman leaf litter 78 B.14 Fermentation of detoxified leaf hydrolysate by Cl. acetobutylicum ATCC 824 . 79 B.15 Statistical analysis . 80 IV. RESULTS AND DISCUSSION 82 PART ONE . 82 A. Cultivation of Cl. acetobutylicum ATCC 824 82 A.1 Suitable media for the growth of Cl. acetobutylicum ATCC 824 .83 A.2 Suitable set-up for the growth of Cl. acetobutylicum ATCC 824 86 A.3 Growth curve of Cl. acetobutylicum ATCC 824 in RCM 89 A.4 Morphological changes in Cl. acetobutylicum ATCC 824 cells during different phases of growth . 92 A.5 Optimisation of heat shock treatment for the revival of Cl. acetobutylicum ATCC 824 spores . 94 B. Optimisation of microsphere production using Design of Experiments (DoE) 96 v B.1 Influence of the variables on size 100 B.2 Influence of the variables on span . 101 B.3 Influence of the variables on aggregation index 102 B.4 Model equations and model adequacy . 102 B.5 Optimisation of formulation and process parameters in the production of microspheres with the desired properties 107 C. Effect of emulsification process on viability of Cl. acetobutylicum ATCC 824 vegetative cells and spores . 111 D. 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Bacterial acetone and butanol production by industrial fermentation in the Soviet Union: use of hydrolyzed agricultural waste for biorefinery. Applied Microbiology and Biotechnology 71, 587-597. \ 198 LIST OF PUBLICATIONS/PRESENTATIONS 199 PUBLICATIONS/PAPERS PRESENTED AT SCIENTIFIC MEETINGS Journal publications Rathore, S., Desai, P.M., Liew, C.V., Chan, L.W., Heng, P.W.S. (2012), Microencapsulation of microbial cells. Journal of Food Engineering, 116, 369-381. Manuscripts in preparation Rathore, S., Chan, L.W., Heng, P.W.S. (2013), Optimisation of pretreatment of Samanea saman leaf litter to obtain fermentable sugars for biofuel production Rathore, S., Chan, L.W., Heng, P.W.S. (2013), Feasibility study of microencapsulation of Clostridium acetobutylicum cells by emulsification method Rathore, S., Chan, L.W., Heng, P.W.S. (2013), Investigation of sporulation triggers in Clostridium acetobutylicum ATCC 824. Oral Presentations Rathore, S., Chan, L.W., Heng, P.W.S. (2010), Understanding growth characteristics of Clostridium acetobutylicum for the production of bioproducts. International Pharmatech Conference on Drug Delivery 2010, Kuala Lumpur, Malaysia. 200 Poster presentations Rathore, S., Chan, L.W., Heng, P.W.S. (2012), Evaluation of microencapsulation technique to immobilize Clostridium acetobutylicum cells for the bio production of butanol. 26th AAPS Annual meeting and exposition, Chicago, United States. Rathore, S., Chan, L.W., Heng, P.W.S. (2011), Optimisation of acid hydrolysis of an alternative lignocellulosic substrate using response surface methodology. 25th AAPS Annual meeting and exposition, Washington DC, United States. 201 [...]... acid formation As the cells enter the stationary phase, they begin to swell and accumulate reserve material in the form of granulose These cells in this phase are known as clostridial cells, which subsequently form the forespore cells (mother cells containing the endospores) The cells in the stationary phase are involved in conversion of acids to solvents Finally, the endospore is released from the forespore... butanol yield The fermentation efficiency of the encapsulated spores was however much higher than that of the free cells in subsequent cycles Significant cell leakage from the microspheres was observed at the end of the fermentation process The microspheres served as nurseries for the generation of new cells Both encapsulated and liberated cells contributed to butanol production The potential of S saman leaf... gellan gum microspheres and fermentation profile of encapsulated and liberated cells 129 Figure 23 Viability and fermentation profiles of free Cl acetobutylicum ATCC 824 cells (equivalent to the number of liberated cells from the microspheres during the course of fermentation) 133 Figure 24 Photographs of microspheres with Cl acetobutylicum ATCC 824 cells at the periphery of gellan gum microspheres... The purpose of the present study was to provide insights on applicability of microencapsulation using gellan gum, as a cell immobilisation method for Clostridium acetobutylicum ATCC 824 cells for biobutanol production Secondly, an investigation on the use of leaf litter from Samanea saman tree, as a lignocellulosic substrate for biobutanol production, was attempted The combination of these methods were... in the optimisation study for the microencapsulation process 98 Table 14 Coefficient estimate, sum of squares and their respective p-values for the three responses 106 Table 15 Effect of emulsification process on the viability of vegetative cells and spores of Cl acetobutylicum ATCC 824 112 Table 16 Results from the fermentation optimisation studies of free (non-encapsulated) cells of. .. by liberated cells to butanol production 128 I Reusability of free (non-encapsulated) vegetative cells/ spores and encapsulated spores of Cl acetobutylicum ATCC 824 135 PART TWO 143 A Potential of Samanea saman leaf litter as a source of fermentable sugars for biobutanol production 143 A.1 Recovery of total fermentable sugars from S saman leaves 144 A.2 Determination of filter paper... address the issues of low butanol yield and high production cost of biobutanol production The factors affecting the production of gellan gum microspheres by emulsification technique were investigated using full factorial design, followed by derivation of optimised process conditions The viability of Cl acetobutylicum ATCC 824 cells was adversely affected by the emulsification process The spore form was... defined region of space while preserving their activity for repeated or continuous use (Karel et al., 1985) In a cell recycle system, the cells and the fermentation products are first separated using a filter and then the cells are returned to the fermentor (Tashiro et al., 2005) The separation of the cells from the toxic metabolic products in the fermentation medium allows attainment of high viable... toxicity, the high cost of the fermentation substrates for ABE fermentation is another area of concern Traditionally, food crops or food by-products like corn, potatoes, maize and molasses were used as fermentation substrates (Jones and Woods, 1986) Increase in the demand and price of these food crops has hindered the large scale economical production of biobutanol 9 C Strategies to overcome limitations of. .. technique, either nitrogen or the fermentation gases (carbon dioxide and hydrogen) are sparged into the fermentation medium The formation and bursting of the gas bubbles cause the surrounding fermentation liquid to vibrate and the gases capture the volatile butanol The gases are then separated from the fermentation medium and butanol isolated by condensation (Ezeji et al., 2007a; Zheng et al., 2009b) The gases . NATIONAL UNIVERSITY OF SINGAPORE 2013 MICROENCAPSULATION OF CLOSTRIDIUM ACETOBUTYLICUM CELLS AND UTILISATION OF SAMANEA SAMAN LEAF LITTER FOR THE PRODUCTION OF BIOBUTANOL . MICROENCAPSULATION OF CLOSTRIDIUM ACETOBUTYLICUM CELLS AND UTILISATION OF SAMANEA SAMAN LEAF LITTER FOR THE PRODUCTION OF BIOBUTANOL SWETA RATHORE. from Samanea saman tree, as a lignocellulosic substrate for biobutanol production, was attempted. The combination of these methods were aimed to address the issues of low butanol yield and