Nasib Qureshi

Biomass to biofuels : strategies for global industries

Foreword. Preface. Contributors. PART I STRUCTURE OF THE BIOEVERGY BUSINESS. 1 Characteristics of Biofuels and Renewable Fuel Standards ( Alan C. Hansen, Dimitrios C. Kyritsis, and Chia fon F. Lee ). 1.1 Introduction. 1.2 Molecular Structure. 1.3 Physical Properties. 1.4 Chemical Properties. 1.5 Biofuel Standards. 1.6 Perspective. References. 2 The Global Demand for Biofuels: Technologies, Markets and Policies (Jurgen Scheffran). 2.1 Introduction. 2.2 Motivation and Potential of Renewable Fuels. 2.3 Renewable Fuels in the Transportation Sector. 2.4 Status and Potential of Major Biofuels. 2.5 Biofuel Policies and Markets in Selected Countries. 2.6 Perspective. References. 3 Biofuel Demand Realization ( Stephen R. Hughes and Nasib Qureshi ). 3.1 Introduction. 3.2 Availability of Renewable Resources to Realize Biofuel Demand. 3.3 Technology Improvements to Enhance Biofuel Production Economics. 3.4 US Regulatory Requirements for Organisms Engineered to Meet Biofuel Demand. 3.5 Perspective. Acknowledgments. References. 4 Advanced Biorefineries for the Production of Fuel Ethanol ( Stephen R. Hughes, William Gibbons, and Scott Kohl ). 4.1 Introduction. 4.2 Ethanol Production Plants Using Sugar Feedstocks. 4.3 Dedicated Dry-Grind and Dry-Mill Starch Ethanol Production Plants. 4.4 Dedicated Wet-Mill Starch Ethanol Production Plants. 4.5 Dedicated Cellulosic Ethanol Production Plants. 4.6 Advanced Combined Biorefineries. 4.7 Perspective. Acknowledgments. References. PART II DIESEL FROM BIOMASS. 5 Biomass Liquefaction and Gasification ( Nicolaus Dahmen, Edmund Henrich, Andrea Kruse, and Klaus Raffelt ). 5.1 Introduction. 5.2 Direct Liquefaction. 5.3 Biosynfuels from Biosyngas. 5.4 Perspective. References. 6 Diesel from Syngas ( Yong-Wang Li, Jian Xu, and Yong Yang ). 6.1 Introduction. 6.2 Overview of Fischer-Tropsch Synthesis. 6.3 Historical Development of the Fischer-Tropsch Synthesis Process. 6.4 Modern Fischer-Tropsch Synthesis Processes. 6.5 Economics. 6.6 Perspective. Acknowledgements. References. 7 Biodiesel from Vegetable Oils ( Jon Van Gerpen ). 7.1 Introduction. 7.2 Use of Vegetable Oils as Diesel Fuels. 7.3 Renewable Diesel. 7.4 Properties. 7.5 Biodiesel Production. 7.6 Transesteritication. 7.7 Biodiesel Purification. 7.8 Perspective. References. 8 Biofuels from Microalgae and Seaweeds ( Michael Huesemann, G. Roesjadi, John Benemann, and F. Blaine Metting ). 8.1 Introduction. 8.2 Biofuels from Microalgae: Products, Processes, and Limitations. 8.3 Biofuels from Seaweeds: Products, Processes, and Limitations. 8.4 Perspective. References. PART III ETHANOL AND BUTANOL. 9 Improvements in Corn to Ethanol Production Technology Using Saccharomyces cerevisiae ( Vijay Singh, David B. Johnston, Kent D. Rausch, and M.E. Tumbleson ). 9.1 Introduction. 9.2 Current Industrial Ethanol Production Technology. 9.3 Granular Starch Hydrolysis. 9.4 Corn Fractionation. 9.5 Simultaneous SSF and Distillation. 9.6 Dynamic Control of SSF Processes. 9.7 Cost of Ethanol. 9.8 Perspective. References. 10 Advanced Technologies for Biomass Hydrolysis and Saccharification Using Novel Enzymes ( Margret E. Berg Miller, Jennifer M. Brulc, Edward A. Bayer, Raphael Lamed, Harry J. Flint, and Bryan A. White ). 10.1 Introduction. 10.2 The Substrate. 10.3 Glycosyl Hydrolases. 10.4 The Cellulosome Concept. 10.5 New Approaches for the Identification of Novel Glycoside Hydrolases. 10.6 Perspective. References. 11 Mass Balances and Analytical Methods for Biomass Pretreatment Experiments ( Bruce S. Dien ). 11.1 Introduction. 11.2 Analysis of Feedstocks for Composition and Potential Ethanol Yield. 11.3 Pretreatment. 11.4 Enzymatic Extraction of Sugars. 11.5 Fermentation of Pretreated Hydrolysates to Ethanol. 11.6 Feedstock and Process Integration. 11.7 Perspective. Acknowledgments. References. 12 Biomass Conversion Inhibitors and In Situ Detoxification ( Z. Lewis Liu and Hans P. Blaschek ). 12.1 Introduction. 12.2 Inhibitory Compounds Derived from Biomass Pretreatment. 12.3 Inhibitory Effects. 12.4 Removal of Inhibitors. 12.5 Inhibitor-Tolerant Strain Development. 12.6 Inhibitor Conversion Pathways. 12.7 Molecular Mechanisms of In Situ Detoxification. 12.8 Perspective. Acknowledgments. References. 13 Fuel Ethanol Production From Lignocellulosic Raw Materials Using Recombinant Yeasts ( Grant Stanley and Barbel Hahn-Hagerdal ). 13.1 Introduction. 13.2 Consolidated Bioprocessing and Ethanol Production. 13.3 Pentose-Fermenting S. cerevisiae Strains. 13.4 Lignocellulose Fermentation and Ethanol Inhibition. 13.5 Perspective. Acknowledgments. References. 14 Conversion of Biomass to Ethanol by Other Organisms ( Siqing Liu ). 14.1 Introduction. 14.2 Desired Biocatalysts for Biomass to Bioethanol. 14.3 Gram-Negative Bacteria. 14.4 Gram-Positive Bacteria. 14.5 Perspective. Acknowledgments. References. 15 Advanced Fermentation Technologies ( Masayuki Inui, Alain A. Vertes and Hideaki Yukawa ). 15.1 Introduction. 15.2 Batch Processes. 15.3 Fed-Batch Processes. 15.4 Continuous Processes. 15.5 Immobilized Cell Systems. 15.6 Growth-Arrested Process. 15.7 Integrated Bioprocesses. 15.8 Consolidated Bioprocessing (CBP). 15.9 Perspective. References. 16 Advanced Product Recovery Technologies ( Thaddeus C Ezeji and Yebo Li ). 16.1 Introduction. 16.2 Membrane Separation. 16.3 Advanced Technologies for Biofuel Recovery: Industrially Relevant Processes. 16.4 Perspective. Acknowledgments. References. 17 Clostridia and Process Engineering for Energy Generation ( Nasib Qureshi and Hans P. Blaschek ). 17.1 Introduction. 17.2 Substrates, Cultures, and Traditional Technologies. 17.3 Agricultural Residues as Substrates for the Future. 17.4 Butanol-Producing Microbial Cultures. 17.5 Regulation of Butanol Production and Microbial Genetics. 17.6 Novel Fermentation Technologies. 17.7 Novel Product Recovery Technologies. 17.8 Fermentation of Lignocellulosic Substrates in Integrated Systems. 17.9 Integrated or Consolidated Processes. 17.10 Perspective. Acknowledgments. References. PART IV: HYDROGEN, METHANE, AND METHANOL. 18 Hydrogen Generation by Microbial Cultures ( Anja Hemschemeier, Katrin Mullner, Thilo Ruhle, and Thomas Happe ). 18.1. Introduction: Why Biological Hydrogen Production? 18.2. Biological Hydrogen Production. 18.3. Metabolic Basics for Hydrogen Production: Fermentation and Photosynthesis. 18.4. H 2 Production in Application: Cases in Point. 18.5. Perspective. References. 19 Engineering Photosynthesis for H 2 Production from H 2 O: Cyanobacteria as Design Organisms ( Nadine Waschewski, Gabor Bernat, and Matthias Rogner ). 19.1 The Basic Idea: Why Hydrogen from Water? 19.2 Realization: Three Mutually Supporting Strategies. 19.3 The Biological Strategy: How to Design a Hydrogen-Producing (Cyano-) Bacterial Cell. 19.4 Engineering the Environment of the Cells: Reactor Design. 19.5 How Much Can We Expect? The Limit of Natural Systems. 19.6 Perspective. Acknowledgments. References. 20 Production and Utilization of Methane Biogas as Renewable Fuel ( Zhongtang Yu, Mark Morrison, and Floyd L. Schanbacher ). 20.1 Introduction. 20.2 The Microbes and Metabolisms Underpinning Biomethanation. 20.3 Potential Feedstocks Used for Methane Biogas Production. 20.4 Biomethanation Technologies for Production of Methane Biogas. 20.5 Utilization of Methane Biogas as a Fuel. 20.6 Perspective. 20.7 Concluding Remarks. 20.8 Disclaimer. References. 21 Methanol Production and Utilization ( Gregory A. Dolan ). 21.1 Introduction. 21.2 Biomass Gasification: Mature and Immature. 21.3 Feedstocks: Diverse and Plentiful. 21.4 Biomethanol: ICEs, FFVs, and FCVs. 21.5 Case Study: Waste Wood Biorefinery. 21.6 Case Study: Two-Step Thermochemical Conversion Process. 21.7 Case Study: Mobile Methanol Machine. 21.8 Case Study: Scandinavia Leading the Way with Black Liquor Methanol Production. 21.9 Case Study: Methanol Fermentation through Anaerobic Digestion. References. PART V PERSPECTIVES. 22 Enhancing Primary Raw Materials for Biofuels ( Takahisa Hayashi, Rumi Kaida, Nobutaka Mitsuda, Masaru Ohme-Takagi, Nobuyuki Nishikuba, Shin-ichiro Kidou, and Kouki Yoshida ). 22.1 Introduction. 22.2 In-Fibril Modification. 22.3 In-Wall Modifications. 22.4 In-Planta Modifications. 22.5 In-CRES-T Modification. 22.6 A Catalogue of Gene Families for Glycan Synthases and Hydrolases. 22.7 Perspective. Acknowledgments. References. 23 Axes of Development in Chemical and Process Engineering for Converting Biomass to Energy ( Alain A. Vertes ). 23.1 Global Outlook. 23.2 Enhancement of Raw Material Biomass. 23.3 Conversion of Biomass to Fuels and Chemicals. 23.4 Chemical Engineering Development. 23.5 Perspective. References. 24 Financing Strategies for Industrial-Scale Biofuel Production and Technology Development Start-Ups ( Alain A. Vertes and Sarit Soccary Ben Yochanan ). 24.1 Background: The Financial Environment. 24.2 Biofuels Project: Steps in Value Creation and Required Funding at Each Stage. 24.3 Governmental Incentives to Support the Nascent Biofuel and Biomaterial Industry. 24.4 Perspective: What is the Best Funding Source for Each Step in a Companys Development? References. Index.

How microbes tolerate ethanol and butanol.

New robust biocatalysts are needed to depolymerize or hydrolyze recalcitrant heterogeneous lignocellulosic biomass polymers into monomers and to convert the mixed substrates into biofuels. The ideal biocatalysts should be able to tolerate inhibitory compounds released from biomass hydrolysis and increased concentrations of the final products: ethanol or butanol. The solvent tolerance trait plays an important role in cost-effective recovery processes. Here we provide an overview of the literature of fermenting microbes in response to increased ethanol or butanol concentrations, aimed to provide insight on how microbes deal with and adapt to the ethanol and butanol stress.

Butanol Production from Corn Fiber Xylan Using Clostridium acetobutylicum

Acetone, butanol, and ethanol (ABE) were produced from corn fiber arabinoxylan (CFAX) and CFAX sugars (glucose, xylose, galactose, and arabinose) using Clostridium acetobutylicum P260. In mixed sugar (glucose, xylose, galactose, and arabinose) fermentation, the culture preferred glucose and arabinose over galactose and xylose. Under the experimental conditions, CFAX (60 g/L) was not fermented until either 5 g/L xylose or glucose plus xylanase enzyme were added to support initial growth and fermentation. In this system, C. acetobutylicum produced 9.60 g/L ABE from CFAX and xylose. This experiment resulted in a yield and productivity of 0.41 and 0.20 g/L·h, respectively. In the integrated hydrolysis, fermentation, and recovery process, 60 g/L CFAX and 5 g/L xylose produced 24.67 g/L ABE and resulted in a higher yield (0.44) and a higher productivity (0.47 g/L·h). CFAX was hydrolyzed by xylan‐hydrolyzing enzymes, and ABE were recovered by gas stripping. This investigation demonstrated that integration of hydrolysis of CFAX, fermentation to ABE, and recovery of ABE in a single system is an economically attractive process. It is suggested that the culture be further developed to hydrolyze CFAX and utilize all xylan sugars simultaneously. This would further increase productivity of the reactor.

Expression of a heterologous xylose transporter in a Saccharomyces cerevisiae strain engineered to utilize xylose improves aerobic xylose consumption

The goal of this investigation was to determine the effect of a xylose transport system on glucose and xylose co-consumption as well as total xylose consumption in Saccharomyces cerevisiae. We expressed two heterologous transporters from Arabidopsis thaliana in recombinant xylose-utilizing S. cerevisiae cells. Strains expressing the heterologous transporters were grown on glucose and xylose mixtures. Sugar consumption rates and ethanol concentrations were determined and compared to an isogenic control strain lacking the A. thaliana transporters. Expression of the transporters increased xylose uptake and xylose consumption up to 46% and 40%, respectively. Xylose co-consumption rates (prior to glucose depletion) were also increased by up to 2.5-fold compared to the control strain. Increased xylose consumption correlated with increased ethanol concentration and productivity. During the xylose/glucose co-consumption phase, strains expressing the transporters had up to a 70% increase in ethanol production rate. It was concluded that in these strains, xylose transport was a limiting factor for xylose utilization and that increasing xylose/glucose co-consumption is a viable strategy for improving xylose fermentation.

Bioproduction of butanol in bioreactors: New insights from simultaneous in situ butanol recovery to eliminate product toxicity

Simultaneous acetone butanol ethanol (ABE) fermentation by Clostridium beijerinckii P260 and in situ product recovery was investigated using a vacuum process operated in two modes: continuous and intermittent. Integrated batch fermentations and ABE recovery were conducted at 37°C using a 14‐L bioreactor (7.0 L fermentation volume) containing initial substrate (glucose) concentration of 60 g/L. The bioreactor was connected in series with a condensation system and vacuum pump. Vacuum was applied continuously or intermittently with 1.5 h vacuum sessions separated by 4, 6, and 8 h intervals. A control ABE fermentation experiment was characterized by incomplete glucose utilization due to butanol toxicity to C. beijerinckii P260, while fermentation coupled with in situ recovery by both continuous and intermittent vacuum modes resulted in complete utilization of glucose, greater productivity, improved cell growth, and concentrated recovered ABE stream. These results demonstrate that vacuum technology can be applied to integrated ABE fermentation and recovery even though the boiling point of butanol is greater than that of water. Biotechnol. Bioeng. 2011; 108:1757–1765.

Process integration for simultaneous saccharification, fermentation, and recovery (SSFR): Production of butanol from corn stover using Clostridium beijerinckii P260

A simultaneous saccharification, fermentation, and recovery (SSFR) process was developed for the production of acetone-butanol-ethanol (AB or ABE), of which butanol is the main product, from corn stover employing Clostridium beijerinckii P260. Of the 86 g L(-1) corn stover provided, over 97% of the sugars were released during hydrolysis and these were fermented completely with an ABE productivity of 0.34 g L(-1)h(-1) and yield of 0.39. This productivity is higher than 0.31 g L(-1)h(-1) when using glucose as a substrate demonstrating that AB could be produced efficiently from lignocellulosic biomass. Acetic acid that was released from the biomass during pretreatment and hydrolysis was also used by the culture to produce AB. An average rate of generation of sugars during corn stover hydrolysis was 0.98 g L(-1)h(-1). In this system AB was recovered using vacuum, and as a result of this (simultaneous product recovery), 100% sugars were used by the culture.

Prolonged conversion of n‐butyrate to n‐butanol with Clostridium saccharoperbutylacetonicum in a two‐stage continuous culture with in‐situ product removal

n‐Butanol was produced continuously in a two‐stage fermentor system with integrated product removal from a co‐feed of n‐butyric acid and glucose. Glucose was always required as a source of ATP and electrons for the conversion of n‐butyrate to n‐butanol and for biomass growth; for the latter it also served as a carbon source. The first stage generated metabolically active planktonic cells of Clostridium saccharoperbutylacetonicum strain N1‐4 that were continuously fed into the second (production) stage; the volumetric ratio of the two fermentors was 1:10. n‐Butanol was removed continuously from the second stage via gas stripping. Implementing a two‐stage process was observed to dramatically dampen metabolic oscillations (i.e., periodical changes of solventogenic activity). Culture degeneration (i.e., an irreversible loss of solventogenic activity) was avoided by periodical heat shocking and re‐inoculating stage 1 and by maintaining the concentration of undissociated n‐butyric acid in stage 2 at 3.4 mM with a pH‐auxostat. The system was successfully operated for 42 days during which 93% of the fed n‐butyrate was converted to n‐butanol at a production rate of 0.39 g/(L × h). The molar yields Yn‐butanol/n‐butyrate and Yn‐butanol/glucose were 2.0, and 0.718, respectively. For the same run, the molar ratio of n‐butyrate to glucose consumed was 0.358. The molar yield of carbon in n‐butanol produced from carbon in n‐butyrate and glucose consumed (Yn‐butanol/carbon) was 0.386. These data illustrate that conversion of n‐butyrate into n‐butanol by solventogenic Clostridium species is feasible and that this can be performed in a continuous system operating for longer than a month. However, our data also demonstrate that a relatively large amount of glucose is required to supply electrons and ATP for this conversion and for cell growth in a continuous culture. Biotechnol. Bioeng. 2012; 109:913–921.

Pilot scale conversion of wheat straw to ethanol via simultaneous saccharification and fermentation

The production of ethanol from wheat straw (WS) by dilute acid pretreatment, bioabatement of fermentation inhibitors by a fungal strain, and simultaneous saccharification and fermentation (SSF) of the bio-abated WS to ethanol using an ethanologenic recombinant bacterium was studied at a pilot scale without sterilization. WS (124.2g/L) was pretreated with dilute H2SO4 in two parallel tube reactors at 160°C. The inhibitors were bio-abated by growing the fungus aerobically. The maximum ethanol produced by SSF of the bio-abated WS by the recombinant Escherichia coli FBR5 at pH 6.0 and 35°C was 36.0g/L in 83h with a productivity of 0.43gL(-1)h(-1). This value corresponds to an ethanol yield of 0.29g/g of WS which is 86% of the theoretical ethanol yield from WS. This is the first report on the production of ethanol by the recombinant bacterium from a lignocellulosic biomass at a pilot scale.

Butyric acid from anaerobic fermentation of lignocellulosic biomass hydrolysates by Clostridium tyrobutyricum strain RPT-4213.

A novel Clostridium tyrobutyricum strain RPT-4213 was found producing butyrate under strict anaerobic conditions. This strain produced 9.47 g L(-1) butyric acid from MRS media (0.48 g/g glucose). RPT-4213 was also used to ferment dilute acid pretreated hydrolysates including wheat straw (WSH), corn fiber (CFH), corn stover (CSH), rice hull (RHH), and switchgrass (SGH). Results indicated that 50% WSH with a Clostridia medium (Ct) produced the most butyric acid (8.06 g L(-1), 0.46 g/g glucose), followed by 50% SGH with Ct (6.01 g L(-1), 0.44 g/g glucose), however, 50% CSH Ct showed growth inhibition. RPT-4213 was then used in pH-controlled bioreactor fermentations using 60% WSH and SGH, with a dilute (0.5×) Ct medium, resulting 9.87 g L(-1) butyric acid in WSH (yield 0.44 g/g) and 7.05 g L(-1) butyric acid in SGH (yield 0.42 g/g). The titer and productivity could be improved through process engineering.

High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell

BackgroundThe field of plasmid-based functional proteomics requires the rapid assay of proteins expressed from plasmid libraries. Automation is essential since large sets of mutant open reading frames are being cloned for evaluation. To date no integrated automated platform is available to carry out the entire process including production of plasmid libraries, expression of cloned genes, and functional testing of expressed proteins.ResultsWe used a functional proteomic assay in a multiplexed setting on an integrated plasmid-based robotic workcell for high-throughput screening of mutants of cellulase F, an endoglucanase from the anaerobic fungus Orpinomyces PC-2. This allowed us to identify plasmids containing optimized clones expressing mutants with improved activity at lower pH. A plasmid library of mutagenized clones of the celF gene with targeted variations in the last four codons was constructed by site-directed PCR mutagenesis and transformed into Escherichia coli. A robotic picker integrated into the workcell was used to inoculate medium in a 96-well deep well plate, combining the transformants into a multiplexed set in each well, and the plate was incubated on the workcell. Plasmids were prepared from the multiplexed culture on the liquid handler component of the workcell and used for in vitro transcription/translation. The multiplexed expressed recombinant proteins were screened for improved activity and stability in an azo-carboxymethylcellulose plate assay. The multiplexed wells containing mutants with improved activity were identified and linked back to the corresponding multiplexed cultures stored in glycerol. Spread plates were prepared from the glycerol stocks and the workcell was used to pick single colonies from the spread plates, prepare plasmid, produce recombinant protein, and assay for activity. The screening assay and subsequent deconvolution of the multiplexed wells resulted in identification of improved CelF mutants and corresponding optimized clones in expression-ready plasmids.ConclusionThe multiplex method using an integrated automated platform for high-throughput screening in a functional proteomic assay allows rapid identification of plasmids containing optimized clones ready for use in subsequent applications including transformations to produce improved strains or cell lines.