Rebecca Garlock Ong

Take a Closer Look: Biofuels Can Support Environmental, Economic and Social Goals

The US Congress passed the Renewable Fuels Standard (RFS) seven years ago. Since then, biofuels have gone from darling to scapegoat for many environmentalists, policy makers, and the general public. The reasons for this shift are complex and include concerns about environmental degradation, uncertainties about impact on food security, new access to fossil fuels, and overly optimistic timetables. As a result, many people have written off biofuels. However, numerous studies indicate that biofuels, if managed sustainably, can help solve pressing environmental, social and economic problems (Figure 1). The scientific and policy communities should take a closer look by reviewing the key assumptions underlying opposition to biofuels and carefully consider the probable alternatives. Liquid fuels based on fossil raw materials are likely to come at increasing environmental cost. Sustainable futures require energy conservation, increased efficiency, and alternatives to fossil fuels, including biofuels.

Energy, wealth, and human development: Why and how biomass pretreatment research must improve

A high level of human development is dependent on energy consumption (roughly 4 kW per person), and most developed countries that have reached this level have done so through the extensive use of fossil energy. However, given that fossil resources are finite, in order for developed countries to maintain their level of development and simultaneously allow developing countries to reach their potential, it is essential to develop viable renewable energy alternatives. Of particular importance are liquid fuel replacements for petroleum, the fossil resource that primarily drives commerce and economic growth. The intent of this article is to remind our fellow biofuel researchers, particularly those involved in lignocellulosic pretreatment, of these global issues and the serious nature of our work. We hope that this will inspire us to generate and report higher quality and more thorough data than has been done in the past. Only in this way can accurate comparisons and technoeconomic evaluations be made for the many different pretreatment technologies that are currently being researched. The data that primarily influence biorefinery economics can be subdivided into three main categories: yield, concentration, and rate. For these three categories we detail the specific data that should be reported for pretreatment research. In addition, there is other information that is needed to allow for a thorough comparison of pretreatment technologies. An overview of these criteria and our comparison of the current state of a number of pretreatment technologies with respect to these criteria are covered in the last section.

Hydrogen peroxide presoaking of bamboo prior to AFEX pretreatment and impact on enzymatic conversion to fermentable sugars

Bamboo is a fast growing plant found worldwide that has high potential as an energy crop. This project evaluated the effectiveness of AFEX pretreatment for converting moso bamboo (Phyllostachys heterocycla var. pubescens) to fermentable sugars, both with and without pre-soaking in hydrogen peroxide. Pretreatment conditions including temperature, water loading, residence time, ammonia loading, and hydrogen peroxide loadings were varied to maximize hydrolysis yields. The optimal conditions for AFEX were 150°C, 0.8 or 2.0 (w/w) water loading, 10-30 min residence time, and 2.0-5.0 (w/w) ammonia loading. The optimal conditions for H-AFEX were same AFEX conditions with 0.7-1.9 (w/w) 30% (wt) hydrogen peroxide solutions loading. Using 15 FPU/g glucan cellulase and under optimal conditions, AFEX pretreatment achieved a theoretical sugars yield of 64.8-72.7% and addition of hydrogen peroxide presoaking increased the yield to 83.4-92.1%. It is about 5-fold and 7-fold increase in sugars yield for AFEX-treated and H-AFEX-treated bamboo respectively.

Linking plant biology and pretreatment: Understanding the structure and organization of the plant cell wall and interactions with cellulosic biofuel production

In order to more economically process cellulosic feedstocks using a biochemical pathway for fuel production, it is necessary to develop a detailed understanding of plant cell wall characteristics, pretreatment reaction chemistry, and their complex interactions. However given the large number of thermochemical pretreatment methods that are currently being researched and the extreme diversity of plant cell wall structure and composition, this prospect is extremely challenging. Here we present the current state of research at the interface between plant biology and pretreatment chemistry. The first two sections discuss the chemistry of the secondary plant cell wall and how different pretreatment methods alter the overall cell wall structure. The third section addresses how the characteristics of the cell wall and pretreatment efficacy are impacted by different factors such as plant maturity, classification, and plant fraction. The fourth section summarizes current directions in the development of novel plant materials for improved biochemical conversion. And the final section discusses the use of chemical pretreatments as a screening and analysis tool for rapid identification of amenable plant materials, and for expansion of the fundamental understanding of plant cell walls.

Controlling microbial contamination during hydrolysis of AFEX-pretreated corn stover and switchgrass: effects on hydrolysate composition, microbial response and fermentation.

BackgroundMicrobial conversion of lignocellulosic feedstocks into biofuels remains an attractive means to produce sustainable energy. It is essential to produce lignocellulosic hydrolysates in a consistent manner in order to study microbial performance in different feedstock hydrolysates. Because of the potential to introduce microbial contamination from the untreated biomass or at various points during the process, it can be difficult to control sterility during hydrolysate production. In this study, we compared hydrolysates produced from AFEX-pretreated corn stover and switchgrass using two different methods to control contamination: either by autoclaving the pretreated feedstocks prior to enzymatic hydrolysis, or by introducing antibiotics during the hydrolysis of non-autoclaved feedstocks. We then performed extensive chemical analysis, chemical genomics, and comparative fermentations to evaluate any differences between these two different methods used for producing corn stover and switchgrass hydrolysates.ResultsAutoclaving the pretreated feedstocks could eliminate the contamination for a variety of feedstocks, whereas the antibiotic gentamicin was unable to control contamination consistently during hydrolysis. Compared to the addition of gentamicin, autoclaving of biomass before hydrolysis had a minimal effect on mineral concentrations, and showed no significant effect on the two major sugars (glucose and xylose) found in these hydrolysates. However, autoclaving elevated the concentration of some furanic and phenolic compounds. Chemical genomics analyses using Saccharomyces cerevisiae strains indicated a high correlation between the AFEX-pretreated hydrolysates produced using these two methods within the same feedstock, indicating minimal differences between the autoclaving and antibiotic methods. Comparative fermentations with S. cerevisiae and Zymomonas mobilis also showed that autoclaving the AFEX-pretreated feedstocks had no significant effects on microbial performance in these hydrolysates.ConclusionsOur results showed that autoclaving the pretreated feedstocks offered advantages over the addition of antibiotics for hydrolysate production. The autoclaving method produced a more consistent quality of hydrolysate, and also showed negligible effects on microbial performance. Although the levels of some of the lignocellulose degradation inhibitors were elevated by autoclaving the feedstocks prior to enzymatic hydrolysis, no significant effects on cell growth, sugar utilization, or ethanol production were seen during bacterial or yeast fermentations in hydrolysates produced using the two different methods.

Strategies for the production of cell wall-deconstructing enzymes in lignocellulosic biomass and their utilization for biofuel production

Summary Microbial cell wall‐deconstructing enzymes are widely used in the food, wine, pulp and paper, textile, and detergent industries and will be heavily utilized by cellulosic biorefineries in the production of fuels and chemicals. Due to their ability to use freely available solar energy, genetically engineered bioenergy crops provide an attractive alternative to microbial bioreactors for the production of cell wall‐deconstructing enzymes. This review article summarizes the efforts made within the last decade on the production of cell wall‐deconstructing enzymes in planta for use in the deconstruction of lignocellulosic biomass. A number of strategies have been employed to increase enzyme yields and limit negative impacts on plant growth and development including targeting heterologous enzymes into specific subcellular compartments using signal peptides, using tissue‐specific or inducible promoters to limit the expression of enzymes to certain portions of the plant or certain times, and fusion of amplification sequences upstream of the coding region to enhance expression. We also summarize methods that have been used to access and maintain activity of plant‐generated enzymes when used in conjunction with thermochemical pretreatments for the production of lignocellulosic biofuels.

Water sorption in pretreated grasses as a predictor of enzymatic hydrolysis yields

This work investigated the impact of two alkaline pretreatments, ammonia fiber expansion (AFEX) and alkaline hydrogen peroxide (AHP) delignification performed over a range of conditions on the properties of corn stover and switchgrass. Changes in feedstock properties resulting from pretreatment were subsequently compared to enzymatic hydrolysis yields to examine the relationship between enzymatic hydrolysis and cell wall properties. The pretreatments function to increase enzymatic hydrolysis yields through different mechanisms; AFEX pretreatment through lignin relocalization and some xylan solubilization and AHP primarily through lignin solubilization. An important outcome of this work demonstrated that while changes in lignin content in AHP-delignified biomass could be clearly correlated to improved response to hydrolysis, compositional changes alone in AFEX-pretreated biomass could not explain differences in hydrolysis yields. We determined the water retention value, which characterizes the association of water with the cell wall of the pretreated biomass, can be used to predict hydrolysis yields for all pretreated biomass within this study.

Identification of developmental stage and anatomical fraction contributions to cell wall recalcitrance in switchgrass

BackgroundHeterogeneity within herbaceous biomass can present important challenges for processing feedstocks to cellulosic biofuels. Alterations to cell wall composition and organization during plant growth represent major contributions to heterogeneity within a single species or cultivar. To address this challenge, the focus of this study was to characterize the relationship between composition and properties of the plant cell wall and cell wall response to deconstruction by NaOH pretreatment and enzymatic hydrolysis for anatomical fractions (stem internodes, leaf sheaths, and leaf blades) within switchgrass at various tissue maturities as assessed by differing internode.ResultsSubstantial differences in both cell wall composition and response to deconstruction were observed as a function of anatomical fraction and tissue maturity. Notably, lignin content increased with tissue maturity concurrently with decreasing ferulate content across all three anatomical fractions. Stem internodes exhibited the highest lignin content as well as the lowest hydrolysis yields, which were inversely correlated to lignin content. Confocal microscopy was used to demonstrate that removal of cell wall aromatics (i.e., lignins and hydroxycinnamates) by NaOH pretreatment was non-uniform across diverse cell types. Non-cellulosic polysaccharides were linked to differences in cell wall response to deconstruction in lower lignin fractions. Specifically, leaf sheath and leaf blade were found to have higher contents of substituted glucuronoarabinoxylans and pectic polysaccharides. Glycome profiling demonstrated that xylan and pectic polysaccharide extractability varied with stem internode maturity, with more mature internodes requiring harsher chemical extractions to remove comparable glycan abundances relative to less mature internodes. While enzymatic hydrolysis was performed on extractives-free biomass, extractible sugars (i.e., starch and sucrose) comprised a significant portion of total dry weight particularly in stem internodes, and may provide an opportunity for recovery during processing.ConclusionsCell wall structural differences within a single plant can play a significant role in feedstock properties and have the potential to be exploited for improving biomass processability during a biorefining process. The results from this work demonstrate that cell wall lignin content, while generally exhibiting a negative correlation with enzymatic hydrolysis yields, is not the sole contributor to cell wall recalcitrance across diverse anatomical fractions within switchgrass.

Cellulose-hemicellulose interactions at elevated temperatures increase cellulose recalcitrance to biological conversion

It has been previously shown that cellulose-lignin droplets’ strong interactions, resulting from lignin coalescence and redisposition on cellulose surface during thermochemical pretreatments, increase cellulose recalcitrance to biological conversion, especially at commercially viable low enzyme loadings. However, information on the impact of cellulose–hemicellulose interactions on cellulose recalcitrance following relevant pretreatment conditions are scarce. Here, to investigate the effects of plausible hemicellulose precipitation and re-association with cellulose on cellulose conversion, different pretreatments were applied to pure Avicel® PH101 cellulose alone and Avicel mixed with model hemicellulose compounds followed by enzymatic hydrolysis of resulting solids at both low and high enzyme loadings. Solids produced by pretreatment of Avicel mixed with hemicelluloses (AMH) were found to contain about 2 to 14.6% of exogenous, precipitated hemicelluloses and showed a remarkably much lower digestibility (up to 60%) than their respective controls. However, the exogenous hemicellulosic residues that associated with Avicel following high temperature pretreatments resulted in greater losses in cellulose conversion than those formed at low temperatures, suggesting that temperature plays a strong role in the strength of cellulose–hemicellulose association. Molecular dynamics simulations of hemicellulosic xylan and cellulose were found to further support this temperature effect as the xylan–cellulose interactions were found to substantially increase at elevated temperatures. Furthermore, exogenous, precipitated hemicelluloses in pretreated AMH solids resulted in a larger drop in cellulose conversion than the delignified lignocellulosic biomass containing comparably much higher natural hemicellulose amounts. Increased cellulase loadings or supplementation of cellulase with xylanases enhanced cellulose conversion for most pretreated AMH solids; however, this approach was less effective for solids containing mannan polysaccharides, suggesting stronger association of cellulose with (hetero) mannans or lack of enzymes in the mixture required to hydrolyze such polysaccharides.

Balancing energy, conservation, and soil health requirements for plant biomass

The global importance of plant biomass for mitigating water and wind erosion, sustaining soil organic carbon (SOC), and providing animal feed and bedding is well recognized, but those needs are no longer the only factors influencing crop residue management decisions. As fossil energy sources diminish, the need for cellulosic derived liquid fuels is going to increase. Supplying bio-based fuels while simultaneously meeting food, feed, and fiber demands of more than nine billion people will require tremendous grain and biomass yield increases, as well as innovative crop residue management strategies for efficient agricultural operations. Our goal is to examine the challenges farmers, conservationists, and land managers face as they strive to manage crop residues without degrading soil health. Harvesting a portion of our nations crop residues, such as corn (Zea mays L.) stover, to simultaneously provide liquid fuels and enhance agricultural operations will occur, provided it is done in a manner that sustains critical ecosystem and soil health services within the landscape. Harvest rates must be site-specific at subfield scales (Bonner et al. 2014a, 2014b) to ensure a sufficient amount of plant biomass remains at every harvest location to protect the soil surface from wind and water erosion and to sustain…