Improving Enzymes for Biomass Conversion: A Basic Research Perspective

The cost of enzymes for converting plant biomass materials to fermentable sugars is a major impediment to the development of a practical lignocellulosic ethanol industry. Research on enzyme optimization with the goal of reducing the cost of converting biomass materials such as corn stover into glucose, xylose, and other sugars is being actively pursued in private industry, academia, and government laboratories. Under the auspices of the Department of Energy Great Lakes Bioenergy Research Center, we are taking several approaches to address this problem, including “bioprospecting” for superior key enzymes, protein engineering, and high-level expression in plants. A particular focus is the development of synthetic enzyme mixtures, in order to learn which of the hundreds of known enzymes are important and in what ratios. A core set comprises cellobiohydrolase, endoglucanase, β-glucosidase, endoxylanase, and β-glucosidase. Accessory enzymes include esterases, proteases, nonhydrolytic proteins, and glycosyl hydrolases that cleave the less frequent chemical linkages found in plant cell walls.     

Reflections on the United States Military 1941-1987

This article, 'Reflections on the United States Military 1941-1987' written by my grandmother, Mary Mandels, illustrates her passion for life. Her outreach article was considered most appropriate for publication in this forum. Her career activities are outlined in the prior article 'Mary Elizabeth Hickox Mandels, 90, Bioenergy Leader' while her accomplishments were fully recognized, for instance, nationally through the American Chemical Society and through her induction into the Hall of Fame at the US Army Soldier Systems Center in Natick, Massachusetts. As illustrated, along with Dr Elwyn Reese at Natick's Pioneering Research Laboratory, she headed a bioengineering group that is particularly remembered for developing a process for the enzymatic conversion of waste cellulosic biomass into soluble sugars that could be fermented to ethanol for an alternate liquid fuel (gasohol). This technology remains a subject of interest with growing environmental concerns and an oil shortage crisis.     

Survey of genomics approaches to improve bioenergy traits in maize, sorghum and sugarcane

Bioenergy crops currently provide the only source of alternative energy with the potential to reduce the use of fossil transportation fuels in a way that is compatible with existing engine technology, including in developing countries. Even though bioenergy research is currently receiving considerable attention, many of the concepts are not new, but rather build on intense research efforts from 30 years ago. A major difference with that era is the availability of genomics tools that have the potential to accelerate crop improvement significantly. This review is focused on maize, sorghum and sugarcane as representatives of bioenergy grasses that produce sugar and/or lignocellulosic biomass. Examples of how genetic mapping, forward and reverse genetics, high-throughput expression profiling and comparative genomics can be used to unravel and improve bioenergy traits will be presented.     

A Survey of Bioenergy Research in Forest Service Research and Development

Forest biomass represents 25–30 % of the annual biomass available in the USA for conversion into bio-based fuels, bio-based chemicals, and bioproducts in general. The USDA Forest Service Research and Development (R&D) has been focused on producing products from forest biomass since its inception in 1905, with direct combustion, solid sawn lumber, pulp and paper, ethanol as fuel, and silvichemicals all among the mission areas of product research and development. The renewed national interest in biomass conversion to fuels and chemicals is supportive of the most critical need of USDA Forest Service R&D, uses for small-diameter trees and other forest biomass that needs to be removed in the fuel mitigation–fire suppression and forest restoration work of the USDA Forest Service. This paper will summarize the recent USDA Forest Service research on direct combustion, fuel pellets, and conversion of forest biomass to ethanol, both as stand-alone biorefinery processes and as an addition to the traditional wood pulping process.     

Status of peat and biomass gasification in Finland

Research and development into the gasification of solid fuels in Finland are focused on the utilization of indigenous fuels: peat and biomass. Development work is concentrated on the production of low-Btu gas. The Bioneer fixed-bed gasifier and the Pyroflow circulating fluidized-bed gasifier offer flexible and reliable operation coupled to boilers, dryers and lime kilns in the range 1–10 MW and 5–100 MW thermal effect, respectively.     

生物能源专刊序言

生物能源作为可再生能源,有望减少能源供给中对石油的依赖程度。近年来,我国生物能源的发展非常迅速,已经成为继巴西和美国后的第三大燃料乙醇生产国和消费国。为促进生物能源相关技术研究的发展,本期“生物能源”专刊收录了我国生物能源专家学者在燃料乙醇、生物柴油、微生物油脂、生物燃料系统分析等领域的最新研究进展。     

Regulation of D-xylose utilization by hexoses in pentose-fermenting yeasts

The aldopentose D-xylose is one of the most abundant sugars in plant biomass and its efficient microbial utilization is of fundamental importance in the overall bioconversion of lignocellulosic materials into liquid fuels and chemicals. The discovery of pentose-fermenting yeasts in the early 1980's led to world wide interest because of the perceived potential for improved D-xylose fermentation to enhance the prospect of biomass conversions. However, the utilization of D-xylose by pentose-fermenting yeasts can be adversely affected by the hexoses, mainly D-glucose and D-mannose, which are usually present in high amounts in lignocellulosic hydrolysates. Research in the past several years has uncovered some of the regulatory effects of D-glucose on D-xylose utilization. However, much remains unknown about the mechanisms responsible for these effects. This review summarizes the current state of knowledge on the induction, repression and inactivation of D-xylose utilization in pentose-fermenting yeasts.     

2013生物能源专刊序言

生物能源作为可再生能源,可以替代部分石化能源,有望缓解能源供给中对石油的依赖程度.本期专刊结合第6届国际生物能源会议,包括综述和研究报告两部分,报道了我国生物能源专家学者在燃料乙醇、生物柴油、微生物油脂、生物燃料标准、航空生物燃料等领域的最新研究进展.     

Metabolic engineering for the utilization of carbohydrate portions of lignocellulosic biomass

The petrochemical industry has grown to meet the need for massive production of energy and commodities along with an explosive population growth; however, serious side effects such as greenhouse gas emissions and global warming have negatively impacted the environment. Lignocellulosic biomass with myriad quantities on Earth is an attractive resource for the production of carbon-neutral fuels and chemicals through environmentally friendly processes of microbial fermentation. This review discusses metabolic engineering efforts to achieve economically feasible industrial production of fuels and chemicals from microbial cell factories using the carbohydrate portion of lignocellulosic biomass as substrates. The combined knowledge of systems biology and metabolic engineering has been applied to construct robust platform microorganisms with maximum conversion of monomeric sugars, such as glucose and xylose, derived from lignocellulosic biomass. By comprehensively revisiting carbon conversion pathways, we provide a rationale for engineering strategies, as well as their features, feasibility, and recent representative studies. In addition, we briefly discuss how tools in systems biology can be applied in the field of metabolic engineering to accelerate the development of microbial cell factories that convert lignocellulosic biomass into carbon-neutral fuels and chemicals with economic feasibility.     

Plants to power: bioenergy to fuel the future

Bioenergy should play an essential part in reaching targets to replace petroleum-based transportation fuels with a viable alternative, and in reducing long-term carbon dioxide emissions, if environmental and economic sustainability are considered carefully. Here, we review different platforms, crops, and biotechnology-based improvements for sustainable bioenergy. Among the different platforms, there are two obvious advantages to using lignocellulosic biomass for ethanol production: higher net energy gain and lower production costs. However, the use of lignocellulosic ethanol as a viable alternative to petroleum-based transportation fuels largely depends on plant biotechnology breakthroughs. We examine how biotechnology, such as lignin modification, abiotic stress resistance, nutrition usage, in planta expression of cell wall digestion enzymes, biomass production, feedstock establishment, biocontainment of transgenes, metabolic engineering, and basic research, can be used to address the challenges faced by bioenergy crop production.     

Microbial conversion of sugars from plant biomass to lactic acid or ethanol

Concerns for our environment and unease with our dependence on foreign oil have renewed interest in converting plant biomass into fuels and 'green' chemicals. The volume of plant matter available makes lignocellulose conversion desirable, although no single isolated organism has been shown to depolymerize lignocellulose and efficiently metabolize the resulting sugars into a specific product. This work reviews selected chemicals and fuels that can be produced from microbial fermentation of plant-derived cell-wall sugars and directed engineering for improvement of microbial biocatalysts. Lactic acid and ethanol production are highlighted, with a focus on engineered Escherichia coli .     

High-throughput microplate technique for enzymatic hydrolysis of lignocellulosic biomass

Several factors will influence the viability of a biochemical platform for manufacturing lignocellulosic based fuels and chemicals, for example, genetically engineering energy crops, reducing pre-treatment severity, and minimizing enzyme loading. Past research on biomass conversion has focused largely on acid based pre-treatment technologies that fractionate lignin and hemicellulose from cellulose. However, for alkaline based (e.g., AFEX) and other lower severity pre-treatments it becomes critical to co-hydrolyze cellulose and hemicellulose using an optimized enzyme cocktail. Lignocellulosics are appropriate substrates to assess hydrolytic activity of enzyme mixtures compared to conventional unrealistic substrates (e.g., filter paper, chromogenic, and fluorigenic compounds) for studying synergistic hydrolysis. However, there are few, if any, high-throughput lignocellulosic digestibility analytical platforms for optimizing biomass conversion. The 96-well Biomass Conversion Research Lab (BCRL) microplate method is a high-throughput assay to study digestibility of lignocellulosic biomass as a function of biomass composition, pre-treatment severity, and enzyme composition. The most suitable method for delivering milled biomass to the microplate was through multi-pipetting slurry suspensions. A rapid bio-enzymatic, spectrophotometric assay was used to determine fermentable sugars. The entire procedure was automated using a robotic pipetting workstation. Several parameters that affect hydrolysis in the microplate were studied and optimized (i.e., particle size reduction, slurry solids concentration, glucan loading, mass transfer issues, and time period for hydrolysis). The microplate method was optimized for crystalline cellulose (Avicel) and ammonia fiber expansion (AFEX) pre-treated corn stover.     

Improved method of analysis of biomass sugars using high-performance liquid chromatography

The precise quantitative analysis of biomass derived sugars is a very important step in the conversion of biomass feedstocks to fuels and chemicals. However, the most accurate method of biomass sugar analysis is based on the gas chromatography analysis of derivatized sugars either as alditol acetates or trimethylsilanes. The derivatization method is time-consuming but the alternative HPLC method cannot resolve most sugars found in biomass hydrolysates. We have demonstrated for the first time that by careful manipulation of the HPLC mobile phase, biomass monomeric sugars (arabinose, xylose, fructose, glucose, mannose, and galactose) can be analyzed quantitatively and there is excellent baseline resolution of all the sugars. This was demonstrated for both standard sugars and corn stover hydrolysates. Our method can also be used to analyze dimmeric sugars (cellobiose and sucrose).     

Biomass deconstruction to sugars

The production of biofuels from lignocellulosic biomass relies on the depolymerization of its polysaccharide content into fermentable sugars. Accomplishing this requires pretreatment of the biomass to reduce its size, and chemical or physical alteration of the biomass polymers to enhance the susceptibility of their glycosidic linkages to enzymatic or acid catalyzed cleavage. Well-studied approaches include dilute and concentrated acid pretreatment and catalysis, and the dissolution of biomass in organic solvents. These and recently developed approaches, such as solubilization in ionic liquids, are reviewed in terms of the chemical and physical changes occurring in biomass pretreatment. As pretreatment represents one of the major costs in converting biomass to fuels, the factors that contribute to pretreatments costs, and their impact on overall process economics, are described.     

Metabolic engineering of microbial pathways for advanced biofuels production

Production of biofuels from renewable resources such as cellulosic biomass provides a source of liquid transportation fuel to replace petroleum-based fuels. This endeavor requires the conversion of cellulosic biomass into simple sugars, and the conversion of simple sugars into biofuels. Recently, microorganisms have been engineered to convert simple sugars into several types of biofuels, such as alcohols, fatty acid alkyl esters, alkanes, and terpenes, with high titers and yields. Here, we review recently engineered biosynthetic pathways from the well-characterized microorganisms Escherichia coli and Saccharomyces cerevisiae for the production of several advanced biofuels.     

Effect of adding surfactant for transforming lignocellulose into fermentable sugars during biocatalysing

Fuel ethanol is one of the most important alternative fuels used as a substitute for fossil fuel. Lignocellulose is the most abundant biomass resource for the production of fuel ethanol. However, the hydrolysis of lignocellulose requires high enzyme loading. In order to strengthen the process of enzyme hydrolysis of lignocellulose, surfactant-polyethylene glycol (PEG) was applied to the catalysis of lignocellulose into fermentable sugars. The effect of PEG on both the enzymatic hydrolysis and adsorption of cellulose were investigated. The addition of surfactant obviously facilitated enzymatic hydrolysis. In particular, upon addition of PEG4000, the enzyme catalytic efficiency increased by 51.06%. Meanwhile, the adsorption quantity of cellulase decreased by 11.25%. In addition, the mechanism of the effect of PEG on enzymatic hydrolysis and cellulase adsorption is discussed.     

Engineering biocatalysts for production of commodity chemicals

Lignocellulosic biomass is an attractive alternate to petroleum for production of both fuels and commodity chemicals. This conversion of biomass would require a new generation of microbial biocatalysts that can convert all the sugars present in the biomass to the desired compounds. In this review, the critical factors that need to be considered in engineering such microbial biocatalysts for cost-effective fermentation of sugars are discussed with specific emphasis on commodity chemicals such as lactic acid, succinic acid and acetic acid.     

Hybrid thermochemical processing: fermentation of pyrolysis-derived bio-oil

Thermochemical processing of biomass by fast pyrolysis provides a nonenzymatic route for depolymerization of biomass into sugars that can be used for the biological production of fuels and chemicals. Fermentative utilization of this bio-oil faces two formidable challenges. First is the fact that most bio-oil-associated sugars are present in the anhydrous form. Metabolic engineering has enabled utilization of the main anhydrosugar, levoglucosan, in workhorse biocatalysts. The second challenge is the fact that bio-oil is rich in microbial inhibitors. Collection of bio-oil in distinct fractions, detoxification of bio-oil prior to fermentation, and increased robustness of the biocatalyst have all proven effective methods for addressing this inhibition.     

Bioenergy futures in Sweden – system effects of CO2 reduction and fossil fuel phase‐out policies

Bioenergy could contribute both to the reduction of greenhouse gases and to increased energy security, but the extent of this contribution strongly depends on the cost and potential of biomass resources. For Sweden, this study investigates how the implementation of policies for CO₂ reduction and for phase out of fossil fuels in road transport affect the future utilization of biomass, in the stationary energy system and in the transport sector, and its price. The analysis is based on the bottom‐up, optimization MARKAL_Sweden model, which includes a comprehensive representation of the national energy system. For the analysis, the biomass supply representation of MARKAL_Sweden is updated and improved by the use of, e.g., forestry forecasting modeling and through construction of detailed biomass supply curves. A time horizon up to 2050 is applied. The results indicate a potential for significantly higher use of bioenergy. In the main analysis scenario, in which CO₂ reduction of 80% by 2050 is imposed on the Swedish energy system, the total bioenergy utilization increases by 63% by 2050 compared to 2010. The largest increase occurs in the transport sector, which by 2050 accounts for 43% of the total primary bioenergy use. The high demand and strong competition significantly increase biomass prices and lead to the utilization of higher cost biomass sources such as stumps and cultivated energy forest, as well as use of pulpwood resources for energy purposes.