Choo Yieng Hamilton

Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass

Switchgrass is a leading dedicated bioenergy feedstock in the United States because it is a native, high-yielding, perennial prairie grass with a broad cultivation range and low agronomic input requirements. Biomass conversion research has developed processes for production of ethanol and other biofuels, but they remain costly primarily because of the intrinsic recalcitrance of biomass. We show here that genetic modification of switchgrass can produce phenotypically normal plants that have reduced thermal-chemical (≤180 °C), enzymatic, and microbial recalcitrance. Down-regulation of the switchgrass caffeic acid O-methyltransferase gene decreases lignin content modestly, reduces the syringyl:guaiacyl lignin monomer ratio, improves forage quality, and, most importantly, increases the ethanol yield by up to 38% using conventional biomass fermentation processes. The down-regulated lines require less severe pretreatment and 300–400% lower cellulase dosages for equivalent product yields using simultaneous saccharification and fermentation with yeast. Furthermore, fermentation of diluted acid-pretreated transgenic switchgrass using Clostridium thermocellum with no added enzymes showed better product yields than obtained with unmodified switchgrass. Therefore, this apparent reduction in the recalcitrance of transgenic switchgrass has the potential to lower processing costs for biomass fermentation-derived fuels and chemicals significantly. Alternatively, such modified transgenic switchgrass lines should yield significantly more fermentation chemicals per hectare under identical process conditions.

Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations

BackgroundThe model bacterium Clostridium cellulolyticum efficiently degrades crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H2 and CO2, the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels production. Therefore genetic engineering will likely be required to improve the ethanol yield. Plasmid transformation, random mutagenesis and heterologous expression systems have previously been developed for C. cellulolyticum, but targeted mutagenesis has not been reported for this organism, hindering genetic engineering.ResultsThe first targeted gene inactivation system was developed for C. cellulolyticum, based on a mobile group II intron originating from the Lactococcus lactis L1.LtrB intron. This markerless mutagenesis system was used to disrupt both the paralogous L-lactate dehydrogenase (Ccel_2485; ldh) and L-malate dehydrogenase (Ccel_0137; mdh) genes, distinguishing the overlapping substrate specificities of these enzymes. Both mutations were then combined in a single strain, resulting in a substantial shift in fermentation toward ethanol production. This double mutant produced 8.5-times more ethanol than wild-type cells growing on crystalline cellulose. Ethanol constituted 93% of the major fermentation products, corresponding to a molar ratio of ethanol to organic acids of 15, versus 0.18 in wild-type cells. During growth on acid-pretreated switchgrass, the double mutant also produced four times as much ethanol as wild-type cells. Detailed metabolomic analyses identified increased flux through the oxidative branch of the mutants tricarboxylic acid pathway.ConclusionsThe efficient intron-based gene inactivation system produced the first non-random, targeted mutations in C. cellulolyticum. As a key component of the genetic toolbox for this bacterium, markerless targeted mutagenesis enables functional genomic research in C. cellulolyticum and rapid genetic engineering to significantly alter the mixture of fermentation products. The initial application of this system successfully engineered a strain with high ethanol productivity from cellobiose, cellulose and switchgrass.

Understanding long-term changes in microbial fuel cell performance using electrochemical impedance spectroscopy.

Changes in the anode, cathode, and solution/membrane impedances during enrichment of an anode microbial consortium were measured using electrochemical impedance spectroscopy. The consortium was enriched in a compact, flow-through porous electrode chamber coupled to an air-cathode. The anode impedance initially decreased from 296.1 to 36.3 Omega in the first 43 days indicating exoelectrogenic biofilm formation. The external load on the MFC was decreased in a stepwise manner to allow further enrichment. MFC operation at a final load of 50 Omega decreased the anode impedance to 1.4 Omega, with a corresponding cathode and membrane/solution impedance of 12.1 and 3.0 Omega, respectively. An analysis of the capacitive element suggested that most of the three-dimensional anode surface was participating in the bioelectrochemical reaction. The power density of the air-cathode MFC stabilized after 3 months of operation and stayed at 422 +/- 42 mW/m(2) (33 W/m(3)) for the next 3 months. The normalized anode impedance for the MFC was 0.017 kOmega cm(2), a 28-fold reduction over that reported previously. This study demonstrates a unique ability of biological systems to reduce the electron transfer resistance in MFCs, and their potential for stable energy production over extended periods of time.

Controlling accumulation of fermentation inhibitors in biorefinery recycle water using microbial fuel cells

BackgroundMicrobial fuel cells (MFC) and microbial electrolysis cells are electrical devices that treat water using microorganisms and convert soluble organic matter into electricity and hydrogen, respectively. Emerging cellulosic biorefineries are expected to use large amounts of water during production of ethanol. Pretreatment of cellulosic biomass results in production of fermentation inhibitors which accumulate in process water and make the water recycle process difficult. Use of MFCs to remove the inhibitory sugar and lignin degradation products from recycle water is investigated in this study.ResultsUse of an MFC to reduce the levels of furfural, 5-hydroxymethylfurfural, vanillic acid, 4-hydroxybenzaldehyde and 4-hydroxyacetophenone while simultaneously producing electricity is demonstrated here. An integrated MFC design approach was used which resulted in high power densities for the MFC, reaching up to 3700 mW/m2 (356 W/m3 net anode volume) and a coulombic efficiency of 69%. The exoelectrogenic microbial consortium enriched in the anode was characterized using a 16S rRNA clone library method. A unique exoelectrogenic microbial consortium dominated by δ-Proteobacteria (50%), along with β-Proteobacteria (28%), α-Proteobacteria (14%), γ-Proteobacteria (6%) and others was identified. The consortium demonstrated broad substrate specificity, ability to handle high inhibitor concentrations (5 to 20 mM) with near complete removal, while maintaining long-term stability with respect to power production.ConclusionUse of MFCs for removing fermentation inhibitors has implications for: 1) enabling higher ethanol yields at high biomass loading in cellulosic ethanol biorefineries, 2) improved water recycle and 3) electricity production up to 25% of total biorefinery power needs.

Down-regulation of the caffeic acid O-methyltransferase gene in switchgrass reveals a novel monolignol analog

BackgroundDown-regulation of the caffeic acid 3-O-methyltransferase EC 2.1.1.68 (COMT) gene in the lignin biosynthetic pathway of switchgrass (Panicum virgatum) resulted in cell walls of transgenic plants releasing more constituent sugars after pretreatment by dilute acid and treatment with glycosyl hydrolases from an added enzyme preparation and from Clostridium thermocellum. Fermentation of both wild-type and transgenic switchgrass after milder hot water pretreatment with no water washing showed that only the transgenic switchgrass inhibited C. thermocellum. Gas chromatography–mass spectrometry (GCMS)-based metabolomics were undertaken on cell wall aqueous extracts to determine the nature of the microbial inhibitors.ResultsGCMS confirmed the increased concentration of a number of phenolic acids and aldehydes that are known inhibitors of microbial fermentation. Metabolomic analyses of the transgenic biomass additionally revealed the presence of a novel monolignol-like metabolite, identified as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol (iso-sinapyl alcohol) in both non-pretreated, as well as hot water pretreated samples. iso-Sinapyl alcohol and its glucoside were subsequently generated by organic synthesis and the identity of natural and synthetic materials were confirmed by mass spectrometric and NMR analyses. The additional novel presence of iso-sinapic acid, iso-sinapyl aldehyde, and iso-syringin suggest the increased activity of a para-methyltransferase, concomitant with the reduced COMT activity, a strict meta-methyltransferase. Quantum chemical calculations were used to predict the most likely homodimeric lignans generated from dehydration reactions, but these products were not evident in plant samples.ConclusionsDown-regulation of COMT activity in switchgrass resulted in the accumulation of previously undetected metabolites resembling sinapyl alcohol and its related metabolites, but that are derived from para-methylation of 5-hydroxyconiferyl alcohol, and related precursors and products; the accumulation of which suggests altered metabolism of 5-hydroxyconiferyl alcohol in switchgrass. Given that there was no indication that iso-sinapyl alcohol was integrated in cell walls, it is considered a monolignol analog. Diversion of substrates from sinapyl alcohol to free iso-sinapyl alcohol, its glucoside, and associated upstream lignin pathway changes, including increased phenolic aldehydes and acids, are together associated with more facile cell wall deconstruction, and to the observed inhibitory effect on microbial growth. However, iso-sinapyl alcohol and iso-sinapic acid, added separately to media, were not inhibitory to C. thermocellum cultures.

Enhancement in current density and energy conversion efficiency of 3-dimensional MFC anodes using pre-enriched consortium and continuous supply of electron donors

Using a pre-enriched microbial consortium as the inoculum and continuous supply of carbon source, improvement in performance of a three-dimensional, flow-through MFC anode utilizing ferricyanide cathode was investigated. The power density increased from 170 W/m(3) (1800 mW/m(2)) to 580 W/m(3) (6130 mW/m(2)), when the carbon loading increased from 2.5 g/l-day to 50 g/l-day. The coulombic efficiency (CE) decreased from 90% to 23% with increasing carbon loading. The CEs are among the highest reported for glucose and lactate as the substrate with the maximum current density reaching 15.1A/m(2). This suggests establishment of a very high performance exoelectrogenic microbial consortium at the anode. A maximum energy conversion efficiency of 54% was observed at a loading of 2.5 g/l-day. Biological characterization of the consortium showed presence of Burkholderiales and Rhodocyclales as the dominant members. Imaging of the biofilms revealed thinner biofilms compared to the inoculum MFC, but a 1.9-fold higher power density.

Conversion of residual organics in corn stover-derived biorefinery stream to bioenergy via a microbial fuel cell.

A biorefinery process typically uses about 4-10 times more water than the amount of biofuel generated. The wastewater produced in a biorefinery process contains residual sugars, 5-furfural, phenolics, and other pretreatment and fermentation byproducts. Treatment of the wastewater can reduce the need for fresh water and potentially add to the environmental benefits of the process. Use of microbial fuel cells (MFCs) for conversion of the complete range of phenolic compounds and furan aldehyde derivatives present in a postfermentation biorefinery stream is reported here. The consortium was capable of removing the molecules simultaneously with sugars, which were present at 2 orders of magnitude higher concentrations. Organic loading in a fed-batch MFC affected Coulombic efficiency, which decreased from 40% at 0.66 g/L loading to 1.8% at 66.4 g/L loading. Power density increased with loading reaching 1180 mW/m(2) at 5.3 g/L (8% dilution), but decreased thereafter. Excessive loading leads to poor electrogenic performance; therefore, operation of an MFC at an intermediate loading using dilution and recirculation of the process stream can enable effective treatment with bioenergy recovery.

Investigating microbial fuel cell bioanode performance under different cathode conditions.

A compact, three‐in‐one, flow‐through, porous, electrode design with minimal electrode spacing and minimal dead volume was implemented to develop a microbial fuel cell (MFC) with improved anode performance. A biofilm‐dominated anode consortium enriched under a multimode, continuous‐flow regime was used. The increase in the power density of the MFC was investigated by changing the cathode (type, as well as catholyte strength) to determine whether anode was limiting. The power density obtained with an air‐breathing cathode was 56 W/m3 of net anode volume (590 mW/m2) and 203 W/m3 (2160 mW/m2) with a 50‐mM ferricyanide‐based cathode. Increasing the ferricyanide concentration and ionic strength further increased the power density, reaching 304 W/m3 (3220 mW/m2, with 200 mM ferricyanide and 200 mM buffer concentration). The increasing trend in the power density indicated that the anode was not limiting and that higher power densities could be obtained using cathodes capable of higher rates of oxidation. The internal solution resistance for the MFC was 5–6 Ω, which supported the improved performance of the anode design. A new parameter defined as the ratio of projected surface area to total anode volume is suggested as a design parameter to relate volumetric and area‐based power densities and to enable comparison of various MFC configurations. Published 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009

Energy Production from Food Industry Wastewaters Using Bioelectrochemical Cells

Conversion of waste and renewable resources to energy using microbial fuel cells (MFCs) is an upcoming technology for enabling a cleaner and sustainable environment. This chapter assesses the energy production potential from the US food industry wastewater resource. It also reports on an experimental study investigating conversion of wastewater from a local milk dairy plant to electricity. An MFC anode biocatalyst enriched on model sugar and organic acid substrates was used as the inoculum for the dairy wastewater MFC. The tests were conducted using a two-chamber MFC with a porous three dimensional anode and a Pt/C air-cathode. Power densities up to 690 mW/m2 (54 W/m3) were obtained.

Ethanol and High-Value Terpene Co-Production from Lignocellulosic Biomass of Cymbopogon flexuosus and Cymbopogon martinii

Cymbopogon flexuosus, lemongrass, and C. martinii, palmarosa, are perennial grasses grown to produce essential oils for the fragrance industry. The objectives of this study were (1) to evaluate biomass and oil yields as a function of nitrogen and sulfur fertilization, and (2) to characterize their utility for lignocellulosic ethanol compared to Panicum virgatum (switchgrass). Mean biomass yields were 12.83 Mg lemongrass ha-1 and 15.11 Mg palmarosa ha-1 during the second harvest year resulting in theoretical biofuel yields of 2541 and 2569 L ethanol ha-1 respectively compared to reported 1749–3691 L ethanol ha-1 for switchgrass. Pretreated lemongrass yielded 198 mL ethanol (g biomass)-1 and pretreated palmarosa yielded 170 mL ethanol (g biomass)-1. Additionally, lemongrass yielded 85.7 kg essential oil ha-1 and palmarosa yielded 67.0 kg ha-1 with an estimated value of USD