Lee R. Lynd

Microbial Cellulose Utilization: Fundamentals and Biotechnology

SUMMARY Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for “consolidated bioprocessing” (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.

Beneficial Biofuels—The Food, Energy, and Environment Trilemma

Exploiting multiple feedstocks, under new policies and accounting rules, to balance biofuel production, food security, and greenhouse-gas reduction. Recent analyses of the energy and greenhouse-gas performance of alternative biofuels have ignited a controversy that may be best resolved by applying two simple principles. In a world seeking solutions to its energy, environmental, and food challenges, society cannot afford to miss out on the global greenhouse-gas emission reductions and the local environmental and societal benefits when biofuels are done right. However, society also cannot accept the undesirable impacts of biofuels done wrong.

How biotech can transform biofuels

For cellulosic ethanol to become a reality, biotechnological solutions should focus on optimizing the conversion of biomass to sugars.

Fuel ethanol from cellulosic biomass.

Ethanol produced from cellulosic biomass is examined as a large-scale transportation fuel. Desirable features include ethanols fuel properties as well as benefits with respect to urban air quality, global climate change, balance of trade, and energy security. Energy balance, feedstock supply, and environmental impact considerations are not seen as significant barriers to the widespread use of fuel ethanol derived from cellulosic biomass. Conversion economics is the key obstacle to be overcome. In light of past progress and future prospects for research-driven improvements, a cost-competitive process appears possible in a decade.

A comparison of liquid hot water and steam pretreatments of sugar cane bagasse for bioconversion to ethanol

Sugar cane bagasse was pretreated with either liquid hot water (LHW) or steam using the same 25 l reactor. Solids concentration ranged from 1% to 8% for LHW pretreatment and was > or = 50% for steam pretreatment. Reaction temperature and time ranged from 170 to 230 degrees C and 1 to 46 min, respectively. Key performance metrics included fiber reactivity, xylan recovery, and the extent to which pretreatment hydrolyzate inhibited glucose fermentation. In four cases, LHW pretreatment achieved > or = 80% conversion by simultaneous saccharification and fermentation (SSF). > or = 80% xylan recovery, and no hydrolyzate inhibition of glucose fermentation yield. Combined effectiveness was not as good for steam pretreatment due to low xylan recovery. SSF conversion increased and xylan recovery decreased as xylan dissolution increased for both modes. SSF conversion, xylan dissolution. hydrolyzate furfural concentration, and hydrolyzate inhibition increased, while xylan recovery and hydrolyzate pH decreased, as a function of increasing LHW pretreatment solids concentration (1-8%). These results are consistent with the notion that autohydrolysis plays an important. if not exclusive, role in batch hydrothermal pretreatment. Achieving concurrently high (greater than 90%) SSF conversion and xylan recovery will likely require a modified reactor configuration (e.g. continuous percolation or base addition) that better preserves dissolved xylan.

Recent progress in consolidated bioprocessing.

Consolidated bioprocessing, or CBP, the conversion of lignocellulose into desired products in one step without added enzymes, has been a subject of increased research effort in recent years. In this review, the economic motivation for CBP is addressed, advances and remaining obstacles for CBP organism development are reviewed, and we comment briefly on fundamental aspects. For CBP organism development beginning with microbes that have native ability to utilize insoluble components of cellulosic biomass, key recent advances include the development of genetic systems for several cellulolytic bacteria, engineering a thermophilic bacterium to produce ethanol at commercially attractive yields and titers, and engineering a cellulolytic microbe to produce butanol. For CBP organism development, beginning with microbes that do not have this ability and thus requiring heterologous expression of a saccharolytic enzyme system, high-yield conversion of model cellulosic substrates and heterologous expression of CBH1 and CBH2 in yeast at levels believed to be sufficient for an industrial process have recently been demonstrated. For both strategies, increased emphasis on realizing high performance under industrial conditions is needed. Continued exploration of the underlying fundamentals of microbial cellulose utilization is likely to be useful in order to guide the choice and development of CBP systems.

Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield

We report engineering Thermoanaerobacterium saccharolyticum, a thermophilic anaerobic bacterium that ferments xylan and biomass-derived sugars, to produce ethanol at high yield. Knockout of genes involved in organic acid formation (acetate kinase, phosphate acetyltransferase, and L-lactate dehydrogenase) resulted in a strain able to produce ethanol as the only detectable organic product and substantial changes in electron flow relative to the wild type. Ethanol formation in the engineered strain (ALK2) utilizes pyruvate:ferredoxin oxidoreductase with electrons transferred from ferredoxin to NAD(P), a pathway different from that in previously described microbes with a homoethanol fermentation. The homoethanologenic phenotype was stable for >150 generations in continuous culture. The growth rate of strain ALK2 was similar to the wild-type strain, with a reduction in cell yield proportional to the decreased ATP availability resulting from acetate kinase inactivation. Glucose and xylose are co-utilized and utilization of mannose and arabinose commences before glucose and xylose are exhausted. Using strain ALK2 in simultaneous hydrolysis and fermentation experiments at 50°C allows a 2.5-fold reduction in cellulase loading compared with using Saccharomyces cerevisiae at 37°C. The maximum ethanol titer produced by strain ALK2, 37 g/liter, is the highest reported thus far for a thermophilic anaerobe, although further improvements are desired and likely possible. Our results extend the frontier of metabolic engineering in thermophilic hosts, have the potential to significantly lower the cost of cellulosic ethanol production, and support the feasibility of further cost reductions through engineering a diversity of host organisms.

Likely Features and Costs of Mature Biomass Ethanol Technology

Analysis is undertaken motivated by the question: “What are the likely features and cost of a facility producing ethanol from cellulosic biomass at a level of maturity comparable to a refinery?” This question is considered with respect to cost reductions arising from increased scale, lower-cost feedstock, and process improvements in pretreatment and biological conversion, but not other process steps. An “advanced technology” scenario is developed that represents our estimate of the most likely features of mature biomass ethanol technology. A “bestparameter” scenario, intended to be indicative of the potential for R&D-driven cost reductions, is also developed based on the best values for individual process parameters reported in the literature. Both scenarios involve large plants (2.7 million dry t feedstock/yr). Feedstock costs are taken to be

Consolidated bioprocessing for bioethanol production using Saccharomyces cerevisiae.

38.60/delivered dry t for the advanced scenario and

High Ethanol Titers from Cellulose by Using Metabolically Engineered Thermophilic, Anaerobic Microbes

34.00/delivered dry t for the best-parameter scenario. Projected selling prices, including operating costs and capital recovery corresponding to a 14.2% return on investment, are 50¢/gal (pure ethanol basis) for the advanced technology case and 34¢/gal for the best-parameter case. These are markedly lower than the 118¢/gal selling price projected for base-case technology, with the largest share of cost reductions due to improved conversion technology. Key conversion technology improvements include, in order of importance, consolidated bioprocessing, advanced pretreatment, elimination of seed reactors, and faster rates. First-law thermodynamic efficiencies based on the biomass high heating value and production of ethanol and electricity are 61.2% for the advanced case and 69.3% for the best-parameter case, as compared to 50.3% currently. Combining advanced ethanol production technology of the type presented here with advanced gas turbine-based power generation is a promising direction for future analysis and may offer still further cost reductions and efficiency increases.