Cultivation and Characterization of a New Butanol-Acetone Producing Clostridium Species Project PI: Dr.

He Jianzhong Department of Civil and Environmental Engineering National University of Singapore, Singapore 117576 Email: jianzhong.he@nus.edu.sg ABSTRACT The global energy crisis has renewed interest in producing alternative liquid fuels from clean, renewable sources, e.g., generation of biofules via biological processes. In this study, a Clostridium sp. was obtained with soil inoculum from grassland in Singapore, which is capable of producing dominant butanol from monosugars (e.g., glucose and xylose). The 16S rRNA gene of the strain shows 99% identity with Clostridium sp. strain 8052, while both strains possess the capability to produce biosolvents such as acetone and butanol. In the presence of 30 g/L glucose, this strain could produce 8.60 g/L butanol in batch cultures with a butanol-to-acetone ratio of 2.7:1 and a butanol yield of 29%, the yield of which is the highest comparing with previous reported strain at similar conditions. No ethanol was detected during the fermentation, implying a simplified purification process of the products (butanol and acetone as end-products) in contrast to that of a typical Clostridium strain (e.g., acetone, butanol and ethanol as end-products). Butanol dehydrogenase (bdh) was found to be the functional gene responsible for butanol production. Interestingly, this strain could utilize xylan to produce hydrogen and fatty acids under mesophilic conditions, while most clostridium could only feed on simple sugars. When supplemented with 10 g/L xylan, this culture generated ~1.4 mmol hydrogen and ~10 mM butyric acid. Direct utilization of hemicellulosic feedstock by this newly discovered culture provides advantages over the traditional physicochemical pretreatment and enzymatic hydrolysis. Therefore, this new culture shows potential to convert the renewable lignocellulosic biomass into biofuels and serves as a candidate for industrial butanol production. Keywords: biofuel, butanol, lignocellulosic materials, Clostridium, butanol dehydrogenase genes Speakers Biography Dr. Jianzhong He is currently an Associate Professor in the Department of Civil and Environmental Engineering at the National University of Singapore. She received her Ph.D. degree from Georgia Institute of Technology in 2003. Prior to that, she obtained her M.S. and B.S. degrees from Tsinghua University and Harbin Institute of Technology in 1998 and 1995, respectively. She was a postdoctoral researcher at the University of California Berkeley for two years before joining the National University of Singapore as an assistant professor in 2005. Dr. He's research focuses on discovering novel microorganisms to transform and detoxify environmental contaminants, enhancing biodegradation by optimizing the growth of functional microbes, applying nucleic acid-based approach in laboratory cultures and in situ bioremediation, and converting biomass to biofuels (e.g., biobutanol).

Molecular engineering of membrane materials and fabrication for the separation of Acetone butanol ethanol (ABE) broths produced from non-food biomass Project PI: Prof. Neal CHUNG Tai-Shung Department of Chemical and Biomolecular Engineering National University of Singapore Email: chencts@nus.edu.sg Abstract: With rising energy demand in the modern lifestyle of people as well as increasing awareness of global warming and environmental protection, biofuel such as bioalcohols produced from non-food biomass has gained importance and worldwide attention. To produce fuel grade alcohols from fermentation broths, concentration and separation must be conducted to yield 99.5 % alcohol. It has been reported that separation and purification alone accounts for at least 40% (up to 80%) of the whole process cost in biofuel production utilizing conventional processes. Among several separation techniques, membrane-based pervaporation is emerging as one of the most direct, effective and feasible approaches for biofuel separation. Hybrid pervaporation membrane and distillation technologies to facilitate biofuel separation from biomass appear to be a very promising, economic and practical approach. In fact, not only can one take advantages of plenty distillation infrastructure already in place, but also leverage on membranes' unique features such as low energy consumption, minimum contamination and maintenance, superior performance to breakup azeotropic mixtures. Membrane is the key component for the success of pervaporation process. Therefore, in this project, we have conducted fundamental and applied research on molecular design and engineering of membrane materials with superior separation performance and chemical/mechanical stability for biofuel separation via novel concept of mixed matrix membranes, materials modifications, advanced single- and dual-layer hollow fiber configurations. The great potential and applications of various membrane materials specially designed to concentrate alcohols from the ABE fermentation solutions and to dehydrate water from highly concentrated alcohol/water mixtures are highlighted. Biography: PI: Prof Neal Chung received his PhD from SUNY at Buffalo (USA). He had worked for US industries for 15 years before joining NUS in 1995. He has received grants of more than US$40 million, produced over 31 PhD students, published 1 book, 17 book chapters, 30 patents and patent applications, 470 journal papers and 300 conference papers. He is a Subject Editor of Chemical Engineering Research and Design (CHERD) and editorial board member of 15 journals including J. Membrane Science, I & EC Research, Desalination, and many others. Prof Chungs research focuses on membranes for clean water, clean energy, biofuel, CO2 capture, osmotic energy and pharmaceutical separation. In 20052008, he worked as a Senior Consultant for Hyflux, led and built its membrane research team. He was a coinventor of Hyflux Kristal 600 ultrafiltration membranes which was commercialized worldwide. He has received numerous honors and awards for his research such as two IChemE Highly Recommended Awards in Energy and Water in 2009, the IES (Institution of Engineers Singapore) Prestigious Engineering in 2010 Achievement Award, the HyfluxSNIC (Singapore National Institute of Chemistry) Award in Environmental Chemistry, and the IChemE in Singapore Awards in Sustainability. He was appointed as Provosts Chair Professor and received the Engineering Research Leadership Award at the NUS Faculty of Engineering in 2011. He became a Fellow in the Singapore Academy of Engineering in 2012. In addition, he has served as a consultant for many companies and institutes around the world.

Speaker: Mr. Shi Guimin received his BTech in Chemical Engineering (1st Class Honors) from National University of Singapore (NUS) in 2010, where he started his research on biofuel separation with membrane processes. Before that, he had worked for Celanese chemical company in Singapore for several years. He is currently a research staff under the direction of Prof. Neal Chung while pursuing a Ph.D. degree at NUS. His main research areas are development of polybenzimidazole (PBI) hollow fiber membranes for biofuel separations with pervaporation and vapor permeation processes, vapor sorption studies and characterization of microstructure of membrane with positron annihilation lifetime spectroscopy.

Abstract : Production of lignocellulosic biomass is an important factor in cellulosic ethanol production. To meet the growing demands of renewable biofuel, it is important to generate sufficient amounts of feedstock biomass from non-food sources, and preferably using marginal lands. Switchgrass (Panicum virgatum L.) is a suitable plant species for this. However, research efforts for its genetic improvement have so far been inadequate. Enhancing its yield through biotechnology can be a useful and rapid approach to remedy this. It was shown earlier that suppression of the gene, Arabidopsis homology dependent gene silencing 1 (AtHOG1), can enhance biomass in Arabidopsis and rice. Therefore, we developed a simplified protocol for switchgrass genetic transformation and generated transgenic calluses harboring antisense constructs of AtHOG1 and its homologue from switchgrass (PvHOG1). We faced difficulties to regenerate transgenic plants exhibiting the intended gene suppression. This might be due to the fact that the gene may be essential for plant regeneration stage. To overcome this problem, we used a heatinducible gene promoter, namely, Heat Shock Promoter 81 (HSP81) from Arabidopsis to drive the gene suppression constructs. Recently, we succeeded in generating transgenic plants using such constructs to suppress PvHOG1. Various molecular and phenotypic characterizations of the transgenic plants are yet to be performed. Speaker's biography Professor Prakash Kumar obtained his early education in India, with BSc from Mysore University (St. Aloysius College, Mangalore, 1979) and MSc from Madras University (St. Josephs College Trichy, 1981). He went to the University of Calgary, Canada for his PhD in Developmental Plant Physiology (1988, where he was the recipient of the prestigious Izaac Walton Killam Memorial Pre-Doctoral scholarship). He joined the National University of Singapore (NUS) in September 1989 and is now a full professor at the Department of Biological Sciences, NUS. He has supervised research projects of 21 PhD, 7 MSc and about 30 BSc Honors students at the NUS between 1989 and 2011. Professor Kumar has established himself as a prominent scientist in the fields of plant developmental biology and biotechnology. His research contributions resulted in invitations to serve as Editor of three leading international journals in the fields of plant tissue culture and biotechnology. Professor Kumar has published about 70 scientific papers in international journals, with several of them in prestigious journals. He has coauthored a book and written 6 book chapters. Additionally, he has patented a method to enhance yield in plants. He is currently the team leader of a rice research program ($10 million over 5 years from the National Research Foundation, Singapore), in collaboration with the Temasek Life Sciences Laboratory, Singapore and the International Rice Research Institute (IRRI), Philippines. He has held several senior administrative roles at the NUS and served on several National and International committees.

Enhancing plant biomass for cellulosic ethanol production Project PI: Prof. Prakash Kumar Department of Biological Sciences National University of Singapore Email: dbskumar@nus.edu.sg

Developing Novel Biocatalysts for Cellulosic Ethanol Production

Senior Lecturer and Senior Manager, School of Life Sciences and Chemical Technology Ngee Ann Polytechnic Email: gan2@np.edu.sg Abstract Cellulosic ethanol is derived from lignocellulosic biomass such as agricultural waste, wood waste and grasses etc. Conversion of lignocellulosic biomass to ethanol involves biomass pretreatment to release cellulose, hemicellulose and lignin from the lignocellulose matrix, biomass hydrolysis to produce reducing sugars and sugar mixture fermentation to produce ethanol. In this project, novel biocatalysts for biomass hydrolysis and sugar mixture fermentation were developed. Two potential indigenous fungal strains were isolated and genetically improved for high-strength enzyme cocktail production. Oil palm empty fruit bunch and sago palm fiber were chemically pretreated, and they were then enzymatically hydrolyzed using the crude enzymes from these fungal strains. Sugar titer as high as 135.1 g/L was obtained with cellulose and hemicellulose conversion of above 84% with consistent high sugar yield of 770-844 mg/g biomass. The prepared crude enzyme perform equally well with the commercial enzymes without supplement of any commercial enzymes. The enzyme titer reach about 15 FPU/mL, making it possible for on-site enzyme production for a cost-effective and self-dependent cellulosic ethanol process. On the other hand, a novel genome shuffling method was developed and a hybrid yeast was developed, which could effectively ferment xylose and resulted in an ethanol titer of 62 g/L with ethanol yield of 0.47 g/g xylose and a productivity of 2 g/L/h. In addition, a xylose fermenting yeast was developed through rational metabolic engineering and the expression of a novel bacterial xylose isomerase. Both yeast strains are the potential biocatalysts for biomass hydrolysate fermentation and cellulosic ethanol production. Speakers biography Dr. Geng Anli is currently the President of the BioEnergy Society of Singapore (BESS). She is a Senior Manager and a Senior Lecturer at School of Life Sciences and Chemical Technology of Ngee Ann Polytechnic. Prior to joining Ngee Ann Polytechnic, she worked at the Institute of Environmental Science and Engineering (IESE) as a Research Scientist and group leader. Dr. Geng obtained her Ph. D in Chemical and Environmental Engineering from the National University of Singapore (NUS). She received her M. Eng and B. Eng degrees in Biochemical Engineering from Tianjin University, P. R. China. Dr. Geng has more than 20 years R&D experience, working extensively on environmental biotechnology, and industrial biotechnology. Her current research focus at Ngee Ann Polytechnic is developing novel microorganisms for the production of industrial enzymes, chemicals and fuels, and novel nutraceuticals. She obtained Ngee Ann Polytechnic Staff Excellence Award and Institution of Chemical Engineers (IChemE) Singapore Award on Sustainable Technology in 2012.

Project PI: Dr. Geng Anli

Next Generation Fuels: Upgrading of Biomass-derived Pyrolysis Oil Project PI: Dr. Chang Jie Institute of Chemical and Engineering Sciences (ICES), A*star Email: chang_jie@ices.a-star.edu.sg

Abstract:

Due to the depletion of fossil fuels and rising concern over environmental protection, the use of biomass as alternative resource to produce infrastructure-compatible fuels is appealing. Pyrolysis is one possible route by which the biomass wastes, not the food competitive biomass resources, can be converted effectively into low-cost liquid fuels, known as bio-oil. However, these raw bio-oils contain complex mixtures of reactive oxygenate compounds, for instance, carboxylic acids, aldehydes, ketones, furans, sugars, carbohydrates, and water. The presence of these compounds in bio-oils leads to undesired fuel properties, including instability, high acidity, high viscosity, and poor heating value. Therefore, the removal of oxygenate functional groups is an important step to obtain hydrocarbon biofuels, which can be potentially used without significant changes to current fuel distribution and utilization infrastructures. Various processes, such as cracking and hydrodeoxygenation(HDO) have been proposed. The common challenges for these researches are the catalyst life and performance, due to the notable lack of fundamental understandings on the mechanism of bio-oil catalytic upgrading, compared to the well-established knowledge to petroleum chemistry. In this project, we focused on the understanding of catalytic key issues in bio-oil HDO process and the development of novel catalysts for this specified application. According to the stability difference of various components in bio-oil, a two-step upgrading strategy, including prestabilization at low temperature (from low to room temperature) and deep deoxygenation at high temperature (ca. 400 oC), was adopted. Corresponding catalysts and catalytic mechanism will be discussed in the presentation. Biographies: Dr. Chang Jie is a scientist working in Heterogeneous Catalysis at Institute of Chemical and Engineering Sciences. His doctoral work at Institute of Coal Chemistry (ICC), Chinese Academy of Sciences focused on the kinetic studies on Fischer-Tropsch Synthesis (FTS). Before switching his interesting into biomass utilization, he was an Associate Professor in ICC and project manager in Synfuels China with the enthusiasm to coal conversion technologies. Currently, his research focuses on the development of new processes and corresponding catalysts for a future biorefinary, including bio-oil upgrading, bioethanol to olefins, and value-added chemicals from lignocelluloses. Dr. Wang holds a doctoral degree in Chemical Engineering from Yale University. While at Yale, he worked under Prof. Gary Hallers guidance on the synthesis, characterization and performance of novel mesoporous catalysts. The mesoporous supported transition metal catalysts, especially cobalt ones showed potential application in the growth of single walled carbon nanotubes (SWNT). After he obtained his Ph.D., he then joined the Institute of Chemical and Engineering Sciences, A-STAR Singapore as a research scientist. Dr. Chuan Wang's research interest lies in heterogeneous catalysis. His research primarily focuses on the development of mesoporous/microporous supported catalysts and their applications. He also works on the catalytic process of biomass to bio oil and special chemicals.

Thermal degradation of biomass to produce valuable products Project PI: Dr. Paul Sharratt Institute of Chemical and Engineering Sciences (ICES), A*star Email: paul_sharratt@ices.a-star.edu.sg Abstract Thermal degradation of Biomass to give solid, liquid and gaseous products has been known for thousands of years, but its high complexity means that research is still required to support the design of efficient industrial thermal degradation processes. This project has investigated the influence of conditions on the pyrolysis of various types of biomass initially whole and fractionated microalgal biomass and latterly other cellulosic materials. It has been found that thermal treatment of algal biomass holds little promise as a commercial route to fuels. However, some interesting results have been obtained through the treatment of cellulosic biomass so as to manipulate its acidity during thermal treatment and thereby to steer the reaction paths towards high yields of some higher value derivatives. Biographies PI: Dr. Paul Sharratt obtained his first degree in Cambridge, and was awarded a PhD for work in catalytic reaction engineering (1987) from the UMIST. He worked for Imperial Chemical Industries before returning to UMIST in 1991 where he was promoted to a full Chair in 2001. He was awarded a Royal Academy of Engineering / EPSRC Chair in Innovative Manufacturing for the period 2001-6 and until 2008 he was Professor of Sustainable Processing in the School of Chemical Engineering and Analytical Science (SCEAS). He holds an honorary chair in the Universidad Major de San Marcos in Lima, and is a fellow of the Institution of Chemical Engineers, and is a director of BRITEST Limited. He has been the Programme Manager for Process Science and Modelling at the Institute of Chemical and Engineering Sciences in Singapore since 2008. His programme of about 45 researchers works in innovative processing, analytics, chemometrics, reaction engineering, process systems, process design and operation of the kilo laboratory and a range of other pilot scale facilities. He has authored more than 50 journal papers and book chapters and 130 conference publications. Speaker: Ms Koh Cheng Yan is a Research Engineer at the Institute of Chemical and Engineering Sciences. She obtained her Bachelor degree in Chemical and Biomolecular Engineering from Nanyang Technological University in 2008 and is currently pursuing her Masters degree at the National University of Singapore. Her research interests include biomass characterization, biomass thermochemical conversion, process design and development.