Deepti Tanjore

Engineering and two-stage evolution of a lignocellulosic hydrolysate-tolerant Saccharomyces cerevisiae strain for anaerobic fermentation of xylose from AFEX pretreated corn stover.

The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH.

Blending municipal solid waste with corn stover for sugar production using ionic liquid process

Municipal solid waste (MSW) represents an attractive cellulosic resource for sustainable fuel production. However, its heterogeneity is the major barrier to efficient conversion to biofuels. MSW paper mix was generated and blended with corn stover (CS). It has been shown that both of them can be efficiently pretreated in certain ionic liquids (ILs) with high yields of fermentable sugars. After pretreatment in 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]), over 80% glucose has been released with enzymatic saccharification. We have also applied an enzyme-free process by adding mineral acid and water directly into the IL/biomass slurry to induce hydrolysis. With the acidolysis process in 1-ethyl-3-methylimidazolium chloride ([C2C1Im]Cl), up to 80% glucose and 90% xylose are released. There is a correlation between the viscosity profile and hydrolysis efficiency; low viscosity of the hydrolysate generally corresponds to high sugar yields. Overall, the results indicate the feasibility of incorporating MSW as a robust blending agent for biorefineries.

Chapter 4:Dilute Acid and Hydrothermal Pretreatment of Cellulosic Biomass

Biomass pretreatment remains one of the most pressing challenges in terms of cost-effective production of biofuels. We present a short summary of pretreatments that re based on dilute acid and water. Water-only and dilute acid pretreatments can be effective in producing sugars from hemicellulose along with a solid residue enriched in cellulose that can be digested to glucose with high yields. Generally speaking, dilute acid is often favored because it realizes higher yields than water alone and produces mostly monomeric sugars, but water-only pretreatment can reduce the consequences of hydrolyzate conditioning to remove inhibitors, employ lower cost reaction vessels, and avoid the challenges of adding acid. The performance of water-only systems correlates well with the severity parameter, while the modified severity parameter is an effective tool in analyzing dilute acid performance. Kinetic models have also been applied to describe sugar release profiles from dilute acid pretreatments, with the parameters fit to match the data, but the models are not robust in terms of a’ priori predictions of performance. Feedstock features can have a significant effect on performance, with lignin amounts and makeup and mineral contents having potentially large effects. Economic studies clearly show that pretreatment is an expensive operation with pervasive impacts on the costs of other steps. Thus, although dilute acid and water-only pretreatments appear to be frontrunners currently, much more must be done to understand and advance pretreatment technologies to realize really low costs and high yields that are essential to production of commodity products.

Calorimetric evaluation indicates that lignin conversion to advanced biofuels is vital to improving energy yields

Energy density measurements using bomb calorimetry were applied along with mass yields to calculate energy yields from combinations of individual processes and lignocellulosic feedstocks. Sample preparation and the calorimetric method were fine-tuned for the biofuel process pathway prior to measuring the energy density of liquid fuels and catalysts and solid biomass types (untreated, pelletized, pretreated, and enzymatically hydrolyzed). To statistically establish the method, correlations between biomass composition and energy densities were tested. Strong correlations with lignin, hemicellulose, and ash concentrations were observed and statistically validated (Pearsons coefficient, r = 0.92 and −0.81, respectively). Finally, energy densities were applied along with mass yields on a process pathway including ionic liquid pretreatment (6 L) and saccharification (2 L) of three feedstocks. From switchgrass, eucalyptus, and mixed feedstocks, mass yields of 54.4, 62.0, and 61.7% led to energy yields that were observed to be 59.2, 55.9, and 61.0%, respectively. The disparity in change in mass and energy yields between switchgrass and eucalyptus was identified to have originated from the varied lignin removal during pretreatment. The overall energies recovered from 600 g of switchgrass, eucalyptus, and mixed feedstocks, were 9.8, 10.3, and 10.1 MJ, respectively. Calorimetry can promptly evaluate an integrated multi-process pathway to convert a discrete or mixed feedstock to sugars and other metabolites and eventually to advanced biofuels that can either be hydrocarbons or a mixture thereof. In this particular study, calorimetry and mass yields indicated that lignin removal led to lower energy yield to liquid fuels.

A bacterial pioneer produces cellulase complexes that persist through community succession

Cultivation of microbial consortia provides low-complexity communities that can serve as tractable models to understand community dynamics. Time-resolved metagenomics demonstrated that an aerobic cellulolytic consortium cultivated from compost exhibited community dynamics consistent with the definition of an endogenous heterotrophic succession. The genome of the proposed pioneer population, ‘Candidatus Reconcilibacillus cellulovorans’, possessed a gene cluster containing multidomain glycoside hydrolases (GHs). Purification of the soluble cellulase activity from a 300litre cultivation of this consortium revealed that ~70% of the activity arose from the ‘Ca. Reconcilibacillus cellulovorans’ multidomain GHs assembled into cellulase complexes through glycosylation. These remarkably stable complexes have supramolecular structures for enzymatic cellulose hydrolysis that are distinct from cellulosomes. The persistence of these complexes during cultivation indicates that they may be active through multiple cultivations of this consortium and act as public goods that sustain the community. The provision of extracellular GHs as public goods may influence microbial community dynamics in native biomass-deconstructing communities relevant to agriculture, human health and biotechnology.Cultivation of a cellulolytic consortium reveals successional community dynamics and the presence of multidomain glycoside hydrolases assembled into stable complexes distinct from cellulosomes, which are produced by a potential pioneer population.

Demonstrating a separation-free process coupling ionic liquid pretreatment, saccharification, and fermentation with Rhodosporidium toruloides to produce advanced biofuels

Achieving low cost and high efficiency lignocellulose deconstruction is a critical step towards widespread adoption of lignocellulosic biofuels. Certain ionic liquid (IL)-based pretreatment processes effectively reduce recalcitrance of lignocellulose to enzymatic degradation but require either costly separations following pretreatment or novel IL compatible processes to mitigate downstream toxicity. Here we demonstrate at benchtop and pilot bioreactor scales a separation-free, intensified process for IL pretreatment, saccharification, and fermentation of sorghum biomass to produce the sesquiterpene bisabolene, a precursor to the renewable diesel and jet fuel bisabolane. The deconstruction process employs the IL cholinium lysinate ([Ch][Lys]), followed by enzymatic saccharification with the commercial enzyme cocktails Cellic CTec2 and HTec2. Glucose yields above 80% and xylose yields above 60% are observed at all scales tested. Unfiltered hydrolysate is fermented directly by Rhodosporidium toruloides – with glucose, xylose, acetate and lactate fully consumed during fermentation at all scales tested. Bisabolene titers improved with scale from 1.3 g L−1 in 30 mL shake flasks to 2.2 g L−1 in 20 L fermentation. The combined process enables conversion of saccharified IL-pretreated biomass directly to advanced biofuels with no separations or washing, minimal additions to facilitate fermentation, no loss of performance due to IL toxicity, and simplified fuel recovery via phase separation. This study is the first to demonstrate a separation-free IL based process for conversion of biomass to an advanced biofuel and is the first to demonstrate full consumption of glucose, xylose, acetate, and lactic acid in the presence of [Ch][Lys].

A Systems View of Lignocellulose Hydrolysis

With a growing need to produce low-carbon fuels and chemicals for sustainable economic development, there is growing interest in utilizing lignocellulosic biomass as a feedstock resource for the emerging bioeconomy. Lignocellulosic biomass is currently available in large quantities as crop and forest residues or organic wastes, and could be produced at high yields by planting dedicated energy crops, but is not as easily processed by biochemical or thermochemical conversion technologies as other bio-based feedstocks such as sugars, starches, or oils. Many approaches to utilizing lignocellulosic resources and converting them into value added products first require deconstruction the plant cell wall polymers into their constitutive sugars for fermentation or chemical conversion, and often recover lignin as a co-product. This process of plant cell wall deconstruction, or hydrolysis, includes both pretreatment technologies that degrade the lignin, and saccharification technologies that produce sugar monomers and oligomers from sugar polymers such as cellulose and hemicellulose. This chapter reviews leading technologies for deconstructing lignocellulosic biomass, including mechanical, chemical, and biological approaches, and evaluates the advantages and tradeoffs among them.

Simultaneous application of predictive model and least cost formulation can substantially benefit biorefineries outside Corn Belt in United States: A case study in Florida

Previously, a predictive model was developed to identify optimal blends of expensive high-quality and cheaper low-quality feedstocks for a given geographical location that can deliver high sugar yields. In this study, the optimal process conditions were tested for application at commercially-relevant higher biomass loadings. We observed lower sugar yields but 100% conversion to ethanol from a blend that contained only 20% high-quality feedstock. The impact of applying this predictive model simultaneously with least cost formulation model for a biorefinery location outside of the US Corn Belt in Lee County, Florida was investigated. A blend ratio of 0.30 EC, 0.45 SG, and 0.25 CS in Lee County was necessary to produce sugars at high yields and ethanol at a capacity of 50 MMGY. This work demonstrates utility in applying predictive model and LCF to reduce feedstock costs and supply chain risks while optimizing for product yields.

Short-chain ketone production by engineered polyketide synthases in Streptomyces albus

Microbial production of fuels and commodity chemicals has been performed primarily using natural or slightly modified enzymes, which inherently limits the types of molecules that can be produced. Type I modular polyketide synthases (PKSs) are multi-domain enzymes that can produce unique and diverse molecular structures by combining particular types of catalytic domains in a specific order. This catalytic mechanism offers a wealth of engineering opportunities. Here we report engineered microbes that produce various short-chain (C5–C7) ketones using hybrid PKSs. Introduction of the genes into the chromosome of Streptomyces albus enables it to produce >1 g · l−1 of C6 and C7 ethyl ketones and several hundred mg · l−1 of C5 and C6 methyl ketones from plant biomass hydrolysates. Engine tests indicate these short-chain ketones can be added to gasoline as oxygenates to increase the octane of gasoline. Together, it demonstrates the efficient and renewable microbial production of biogasolines by hybrid enzymes.Mutating natural enzymes is effective in broadening the substrate or product range, but generally leads to reduced titers. Here the authors engineer hybrid polyketide synthases for efficient production of short-chain ketones from plant biomass hydrolysates in Streptomyces, which can increase the octane of gasoline.

In situ Rheological Method to evaluate Feedstock Physical Properties throughout Enzymatic Deconstruction

Feedstock physical properties determine not only downstream flow behavior, but also downstream process yields. Enzymatic treatment of pretreated feedstocks greatly dependent on upstream feedstock physical properties and choice of pre-processing technologies. Currently available enzyme assays have been developed to study biomass slurries at low concentrations of ≤ 1% w/w. At commercially relevant biomass concentrations of ≥ 15% w/w, pretreated feedstocks have sludge-like properties, where low free water restricts movement of unattached enzymes. This work is an account of the various steps taken to develop a method that helps identify the time needed for solid-like biomass slurries transition into liquid-like states during enzymatic hydrolysis. A pre-processing technology that enables feedstocks in achieving this transition sooner will greatly benefit enzyme kinetics and thereby overall process economics. Through this in situ rheological properties determining method, we compared a model feedstock, Avicel®PH101 cellulose, and acid pretreated corn stover. We determined that 25% (w/w) Avicel when treated with Novozymes Cellic®CTec2 (80 mg protein/g glucan) can reduce from solid-like to liquid-like state in 5.5 h, as the phase angles rise beyond 45° at this time. The same slurry needed 5.3 h to achieve liquid-like state with Megazyme endoglucanase (40 mg protein/g glucan). After 10.8 h, CTec2 slurry reached a phase angle of 89° or complete liquid-like state but Megazyme slurry peaked only to 64.7°, possibly due to inhibition by cello-oligomers. Acid pretreated corn stover at 30% (w/w) with a CTec2 protein loading of 80 mg/g glucan exhibited a solid-like to liquid-like transition at 37.8 h, which reflects the combined inhibition of low water activity and presence of lignin. The acid pretreated slurry also never achieved complete liquid-like state due to the presence of biomass residue. This method is applicable in several scenarios comparing varying combinations of pre-processing technologies, feedstock types, pretreatment chemistries, and enzymes. Using this method, we can generate a process chain with optimal flow behavior at commercially-relevant conditions.