Multifaceted production strategies and applications of glucosamine: a comprehensive review.

Glucosamine (GlcN) and its derivatives are in high demand and used in various applications such as food, a precursor for the biochemical synthesis of fuels and chemicals, drug delivery, cosmetics, and supplements. The vast number of applications attributed to GlcN has raised its demand, and there is a growing emphasis on developing production methods that are sustainable and economical. Several: physical, chemical, enzymatic, microbial fermentation, recombinant processing methods, and their combinations have been reported to produce GlcN from chitin and chitosan available from different sources, such as animals, plants, and fungi. In addition, genetic manipulation of certain organisms has significantly improved the quality and yield of GlcN compared to conventional processing methods. This review will summarize the chitin and chitosan-degrading enzymes found in various organisms and the expression systems that are widely used to produce GlcN. Furthermore, new developments and methods, including genetic and metabolic engineering of Escherichia coli and Bacillus subtilis to produce high titers of GlcN and GlcNAc will be reviewed. Moreover, other sources of glucosamine production viz. starch and inorganic ammonia will also be discussed. Finally, the conversion of GlcN to fuels and chemicals using catalytic and biochemical conversion will be discussed.

Arabinose as an overlooked sugar for microbial bioproduction of chemical building blocks.

The circular economy is anticipated to bring a disruptive transformation in manufacturing technologies. Robust and industrial scalable microbial strains that can simultaneously assimilate and valorize multiple carbon substrates are highly desirable, as waste bioresources contain substantial amounts of renewable and fermentable carbon, which is diverse. Lignocellulosic biomass (LCB) is identified as an inexhaustible and alternative resource to reduce global dependence on oil. Glucose, xylose, and arabinose are the major monomeric sugars in LCB. However, primary research has focused on the use of glucose. On the other hand, the valorization of pentose sugars, xylose, and arabinose, has been mainly overlooked, despite possible assimilation by vast microbial communities. The present review highlights the research efforts that have explicitly proven the suitability of arabinose as the starting feedstock for producing various chemical building blocks via biological routes. It begins by analyzing the availability of various arabinose-rich biorenewable sources that can serve as potential feedstocks for biorefineries. The subsequent section outlines the current understanding of arabinose metabolism, biochemical routes prevalent in prokaryotic and eukaryotic systems, and possible products that can be derived from this sugar. Further, currently, exemplar products from arabinose, including arabitol, 2,3-butanediol, 1,2,3-butanetriol, ethanol, lactic acid, and xylitol are discussed, which have been produced by native and non-native microbial strains using metabolic engineering and genome editing tools. The final section deals with the challenges and obstacles associated with arabinose-based production, followed by concluding remarks and prospects.

Challenges and opportunities in producing high-quality edible mushrooms from lignocellulosic biomass in a small scale.

Mushrooms are high-value products that can be produced from lignocellulosic biomass. Mushrooms are the fruiting body of fungi and are domestically cultivated using lignocellulosic biomass obtained from agricultural byproducts and woody biomass. A handful of edible mushroom species are commercially cultivated at small, medium, and large scales for culinary and medicinal use. Details about different lignocellulosic biomass and their composition that are commonly used to produce mushrooms are outlined in this review. In addition, discussions on four major processing steps (i) producing solid and liquid spawn, (ii) conventional and mechanized processing lignocellulosic biomass substrates to produce mushroom beds, (iii) maintaining growth conditions in climate-controlled rooms, and (iv) energy requirements and managements to produce mushrooms are also provided. The new processing methods and technology outlined in this review may allow mushrooms to be economically and sustainably produced at a small scale to satisfy the growing food needs and create rural jobs. KEY POINTS: • Some of the challenges faced by small-scale mushroom growers are presented. This review is expected to stimulate more research to address the challenges.

Natural products as home-based prophylactic and symptom management agents in the setting of COVID-19.

Coronavirus disease (COVID-19) caused by the novel coronavirus (SARS-CoV-2) has rapidly spread across the globe affecting 213 countries or territories with greater than six million confirmed cases and about 0.37 million deaths, with World Health Organization categorizing it as a pandemic. Infected patients present with fever, cough, shortness of breath, and critical cases show acute respiratory infection and multiple organ failure. Likelihood of these severe indications is further enhanced by age as well as underlying comorbidities such as diabetes, cardiovascular, or thoracic problems, as well as due to an immunocompromised state. Currently, curative drugs or vaccines are lacking, and the standard of care is limited to symptom management. Natural products like ginger, turmeric, garlic, onion, cinnamon, lemon, neem, basil, and black pepper have been scientifically proven to have therapeutic benefits against acute respiratory tract infections including pulmonary fibrosis, diffuse alveolar damage, pneumonia, and acute respiratory distress syndrome, as well as associated septic shock, lung and kidney injury, all of which are symptoms associated with COVID-19 infection. This review highlights the potential of these natural products to serve as home-based, inexpensive, easily accessible, prophylactic agents against COVID-19.

Development of rapid bioconversion with integrated recycle technology for ethanol production from extractive ammonia pretreated corn stover.

High enzyme loading and low productivity are two major issues impeding low cost ethanol production from lignocellulosic biomass. This work applied rapid bioconversion with integrated recycle technology (RaBIT) and extractive ammonia (EA) pretreatment for conversion of corn stover (CS) to ethanol at high solids loading. Enzymes were recycled via recycling unhydrolyzed solids. Enzymatic hydrolysis with recycled enzymes and fermentation with recycled yeast cells were studied. Both enzymatic hydrolysis time and fermentation time were shortened to 24 h. Ethanol productivity was enhanced by two times and enzyme loading was reduced by 30%. Glucan and xylan conversions reached as high as 98% with an enzyme loading of as low as 8.4 mg protein per g glucan. The overall ethanol yield was 227 g ethanol/kg EA-CS (191 g ethanol/kg untreated CS). Biotechnol. Bioeng. 2017;114: 1713-1720. © 2017 Wiley Periodicals, Inc.

Toward high solids loading process for lignocellulosic biofuel production at a low cost.

High solids loadings (>18 wt%) in enzymatic hydrolysis and fermentation are desired for lignocellulosic biofuel production at a high titer and low cost. However, sugar conversion and ethanol yield decrease with increasing solids loading. The factor(s) limiting sugar conversion at high solids loading is not clearly understood. In the present study, we investigated the effect of solids loading on simultaneous saccharification and co-fermentation (SSCF) of AFEX™ (ammonia fiber expansion) pretreated corn stover for ethanol production using a xylose fermenting strain Saccharomyces cerevisiae 424A(LNH-ST). Decreased sugar conversion and ethanol yield with increasing solids loading were also observed. End-product (ethanol) was proven to be the major cause of this issue and increased degradation products with increasing solids loading was also a cause. For the first time, we show that with in situ removal of end-product by performing SSCF aerobically, sugar conversion stopped decreasing with increasing solids loading and monomeric sugar conversion reached as high as 93% at a high solids loading of 24.9 wt%. Techno-economic analysis was employed to explore the economic possibilities of cellulosic ethanol production at high solids loadings. The results suggest that low-cost in situ removal of ethanol during SSCF would significantly improve the economics of high solids loading processes. Biotechnol. Bioeng. 2017;114: 980-989. © 2016 Wiley Periodicals, Inc.

Fed-batch hydrolysate addition and cell separation by settling in high cell density lignocellulosic ethanol fermentations on AFEX™ corn stover in the Rapid Bioconversion with Integrated recycling Technology process.

The Rapid Bioconversion with Integrated recycling Technology (RaBIT) process uses enzyme and yeast recycling to improve cellulosic ethanol production economics. The previous versions of the RaBIT process exhibited decreased xylose consumption using cell recycle for a variety of different micro-organisms. Process changes were tested in an attempt to eliminate the xylose consumption decrease. Three different RaBIT process changes were evaluated in this work including (1) shortening the fermentation time, (2) fed-batch hydrolysate addition, and (3) selective cell recycling using a settling method. Shorting the RaBIT fermentation process to 11 h and introducing fed-batch hydrolysate addition eliminated any xylose consumption decrease over ten fermentation cycles; otherwise, decreased xylose consumption was apparent by the third cell recycle event. However, partial removal of yeast cells during recycle was not economical when compared to recycling all yeast cells.

Comparative lipid production by oleaginous yeasts in hydrolyzates of lignocellulosic biomass and process strategy for high titers.

Oleaginous yeasts can convert sugars to lipids with fatty acid profiles similar to those of vegetable oils, making them attractive for production of biodiesel. Lignocellulosic biomass is an attractive source of sugars for yeast lipid production because it is abundant, potentially low cost, and renewable. However, lignocellulosic hydrolyzates are laden with byproducts which inhibit microbial growth and metabolism. With the goal of identifying oleaginous yeast strains able to convert plant biomass to lipids, we screened 32 strains from the ARS Culture Collection, Peoria, IL to identify four robust strains able to produce high lipid concentrations from both acid and base-pretreated biomass. The screening was arranged in two tiers using undetoxified enzyme hydrolyzates of ammonia fiber expansion (AFEX)-pretreated cornstover as the primary screening medium and acid-pretreated switch grass as the secondary screening medium applied to strains passing the primary screen. Hydrolyzates were prepared at ∼18-20% solids loading to provide ∼110 g/L sugars at ∼56:39:5 mass ratio glucose:xylose:arabinose. A two stage process boosting the molar C:N ratio from 60 to well above 400 in undetoxified switchgrass hydrolyzate was optimized with respect to nitrogen source, C:N, and carbon loading. Using this process three strains were able to consume acetic acid and nearly all available sugars to accumulate 50-65% of cell biomass as lipid (w/w), to produce 25-30 g/L lipid at 0.12-0.22 g/L/h and 0.13-0.15 g/g or 39-45% of the theoretical yield at pH 6 and 7, a performance unprecedented in lignocellulosic hydrolyzates. Three of the top strains have not previously been reported for the bioconversion of lignocellulose to lipids. The successful identification and development of top-performing lipid-producing yeast in lignocellulose hydrolyzates is expected to advance the economic feasibility of high quality biodiesel and jet fuels from renewable biomass, expanding the market potential for lignocellulose-derived fuels beyond ethanol for automobiles to the entire U.S. transportation market. Biotechnol. Bioeng. 2016;113: 1676-1690. © 2016 Wiley Periodicals, Inc.

Conversion of apple pomace waste to ethanol at industrial relevant conditions.

Apple pomace samples were evaluated for conversion to ethanol at industrial relevant conditions. Biomass degradation efficiency by commercial enzymes was evaluated at 20 % solid loading for dilute sulfuric acid, calcium oxide, and autoclave without any chemical (control) apple pomace samples. The control and calcium oxide-pretreated pomace provided similar sugar yields, while dilute sulfuric acid pretreatment resulted in reduced sugar yields. The control and calcium oxide-pretreated pomace hydrolysate were fermented to ethanol using a native Saccharomyces cerevisiae yeast strain, producing 38.8 and 36.9 g/L of ethanol, respectively. When control apple pomace sample loading was increased from 20 to 30 %, 57.5 and 50.1 g/L of glucose and fructose was produced, respectively. Lastly, we found that unhydrolyzed solids (UHS) present during fermentation had little effect on ethanol yield, as 53.6 and 53.8 g/L of ethanol were produced with and without UHS, respectively. Overall, ethanol yields were 134 g per kg of dry apple pomace. A complete process mass balance for enzyme hydrolysis and ethanol fermentation is provided in this manuscript. These results show that apple pomace is an excellent feedstock for producing ethanol that could be either used as biofuel or as beverage.

Assessment of bacterial and fungal (hemi)cellulose-degrading enzymes in saccharification of ammonia fibre expansion-pretreated Arundo donax.

This study reports enzymatic hydrolysis of the biomass of the giant reed (Arundo donax L.) after ammonia fibre expansion (AFEX) pretreatment. In particular, the capacity of the arabinofuranosidase from the fungus Pleurotus ostreatus recombinantly expressed in Pichia pastoris rPoAbf, its evolved mutant rPoAbf F435Y/Y446F and the endo-cellulase from Streptomyces sp. G12 CelStrep recombinantly expressed in Escherichia coli to enhance the hydrolysis of AFEX-treated A. donax was investigated, using the corn stover as reference feedstock. The investigated enzymes were assayed using a mixture of purified cellulases (CBHI, CBHII, EGI and ßG), endoxylanases (LX3, LX4) and accessory hemicellulases (LarbF and LßX) as reference enzyme mixture and substituting EGI with rCelStrep and LarbF with rPoAbf or rPoAbf F435Y/Y446F. The use of rPoAbf F435Y/Y446F in the substitution of LarbF led to improvements in sugar conversion, giving a glucan, xylan and arabinan conversion after 72 h of around 62, 63 and 80 %, respectively, similar or higher than those (44, 66 and 55 %) achieved by 72 h hydrolysis with commercial enzymes Novozymes Cellic®, Ctec3 and Htec3. The enzymes rPoAbf, rPoAbf F435Y/Y446F and rCelStrep were also investigated for their effect on hydrolysis of AFEX-pretreated A. donax by addition to commercial enzyme mixture Novozymes Cellic®, Ctec3 and Htec3, and it was shown that the addition of rPoAbf and its evolved mutant rPoAbf F435Y/Y446F enhanced both xylan and arabinan conversions, which achieved 80 % after 6 days of saccharification with rPoAbf F435Y/Y446F.

Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis.

Substrate binding is typically one of the rate-limiting steps preceding enzyme catalytic action during homogeneous reactions. However, interfacial-based enzyme catalysis on insoluble crystalline substrates, like cellulose, has additional bottlenecks of individual biopolymer chain decrystallization from the substrate interface followed by its processive depolymerization to soluble sugars. This additional decrystallization step has ramifications on the role of enzyme-substrate binding and its relationship to overall catalytic efficiency. We found that altering the crystalline structure of cellulose from its native allomorph I(ß) to III(I) results in 40-50% lower binding partition coefficient for fungal cellulases, but surprisingly, it enhanced hydrolytic activity on the latter allomorph. We developed a comprehensive kinetic model for processive cellulases acting on insoluble substrates to explain this anomalous finding. Our model predicts that a reduction in the effective binding affinity to the substrate coupled with an increase in the decrystallization procession rate of individual cellulose chains from the substrate surface into the enzyme active site can reproduce our anomalous experimental findings.

Comparative metabolic profiling revealed limitations in xylose-fermenting yeast during co-fermentation of glucose and xylose in the presence of inhibitors.

During lignocellulosic ethanol fermentation, yeasts are exposed to various lignocellulose-derived inhibitors, which disrupt the efficiency of hexose and pentose co-fermentation. To understand the metabolic response of fermentation microbes to these inhibitors, a comparative metabolomic investigation was performed on a xylose-fermenting Saccharomyces cerevisiae 424A (LNH-ST) and its parental strain 4124 with and without three typical inhibitors (furfural, acetic acid, and phenol). Three traits were uncovered according to fermentation results. First, the growth of strain 424A (LNH-ST) was more sensitive to inhibitors than strain 4124. Through metabolomic analysis, the variance of trehalose, cadaverine, glutamate and g-aminobutyric acid (GABA) suggested that strain 424A (LNH-ST) had a lower capability to buffer redox changes caused by inhibitors. Second, lower ethanol yield in glucose and xylose co-fermentation than glucose fermentation was observed in strain 424A (LNH-ST), which was considered to be correlated with the generation of xylitol, as well as the reduced levels of lysine, glutamate, glycine and isoleucine in strain 424A (LNH-ST). Accumulation of glycerol, galactinol and mannitol was also observed in strain 424A (LNH-ST) during xylose fermentation. Third, xylose utilization of strain 424A (LNH-ST) was more significantly disturbed by inhibitors than glucose utilization. Through the analysis of fermentation and metabolomic results, it was suggested that xylose catabolism and energy supply, rather than xylose uptake, were the limiting steps in xylose utilization in the presence of inhibitors.

Identification of oleaginous yeast strains able to accumulate high intracellular lipids when cultivated in alkaline pretreated corn stover.

Microbial oil is a potential alternative to food/plant-derived biodiesel fuel. Our previous screening studies identified a wide range of oleaginous yeast species, using a defined laboratory medium known to stimulate lipid accumulation. In this study, the ability of these yeasts to grow and accumulate lipids was further investigated in synthetic hydrolysate (SynH) and authentic ammonia fiber expansion (AFEX™)-pretreated corn stover hydrolysate (ACSH). Most yeast strains tested were able to accumulate lipids in SynH, but only a few were able to grow and accumulate lipids in ACSH medium. Cryptococcus humicola UCDFST 10-1004 was able to accumulate as high as 15.5 g/L lipids, out of a total of 36 g/L cellular biomass when grown in ACSH, with a cellular lipid content of 40 % of cell dry weight. This lipid production is among the highest reported values for oleaginous yeasts grown in authentic hydrolysate. Preculturing in SynH media with xylose as sole carbon source enabled yeasts to assimilate both glucose and xylose more efficiently in the subsequent hydrolysate medium. This study demonstrates that ACSH is a suitable medium for certain oleaginous yeasts to convert lignocellullosic sugars to triacylglycerols for production of biodiesel and other valuable oleochemicals.

Comparative genomics of xylose-fermenting fungi for enhanced biofuel production.

Cellulosic biomass is an abundant and underused substrate for biofuel production. The inability of many microbes to metabolize the pentose sugars abundant within hemicellulose creates specific challenges for microbial biofuel production from cellulosic material. Although engineered strains of Saccharomyces cerevisiae can use the pentose xylose, the fermentative capacity pales in comparison with glucose, limiting the economic feasibility of industrial fermentations. To better understand xylose utilization for subsequent microbial engineering, we sequenced the genomes of two xylose-fermenting, beetle-associated fungi, Spathaspora passalidarum and Candida tenuis. To identify genes involved in xylose metabolism, we applied a comparative genomic approach across 14 Ascomycete genomes, mapping phenotypes and genotypes onto the fungal phylogeny, and measured genomic expression across five Hemiascomycete species with different xylose-consumption phenotypes. This approach implicated many genes and processes involved in xylose assimilation. Several of these genes significantly improved xylose utilization when engineered into S. cerevisiae, demonstrating the power of comparative methods in rapidly identifying genes for biomass conversion while reflecting on fungal ecology.

Multifaced application of AFEX-pretreated biomass in producing second-generation biofuels, ruminant animal feed, and value-added bioproducts.

Lignocellulosic biomass holds a crucial position in the prospective bio-based economy, serving as a sustainable and renewable source for a variety of bio-based products. These products play a vital role in displacing fossil fuels and contributing to environmental well-being. However, the inherent recalcitrance of biomass poses a significant obstacle to the efficient access of sugar polymers. Consequently, the bioconversion of lignocellulosic biomass into fermentable sugars remains a prominent challenge in biorefinery processes to produce biofuels and biochemicals. In addressing these challenges, extensive efforts have been dedicated to mitigating biomass recalcitrance through diverse pretreatment methods. One noteworthy process is Ammonia Fiber Expansion (AFEX) pretreatment, characterized by its dry-to-dry nature and minimal water usage. The volatile ammonia, acting as a catalyst in the process, is recyclable. AFEX contributes to cleaning biomass ester linkages and facilitating the opening of cell wall structures, enhancing enzyme accessibility and leading to a fivefold increase in sugar conversion compared to untreated biomass. Over the last decade, AFEX has demonstrated substantial success in augmenting the efficiency of biomass conversion processes. This success has unlocked the potential for sustainable and economically viable biorefineries. This paper offers a comprehensive review of studies focusing on the utilization of AFEX-pretreated biomass in the production of second-generation biofuels, ruminant feed, and additional value-added bioproducts like enzymes, lipids, proteins, and mushrooms. It delves into the details of the AFEX pretreatment process at both laboratory and pilot scales, elucidates the mechanism of action, and underscores the role of AFEX in the biorefinery for developing biofuels and bioproducts, and nutritious ruminant animal feed production. While highlighting the strides made, the paper also addresses current challenges in the commercialization of AFEX pretreatment within biorefineries. Furthermore, it outlines critical considerations that must be addressed to overcome these challenges, ensuring the continued progress and widespread adoption of AFEX in advancing sustainable and economically viable bio-based industries.

Pressure Indicator Composite Films via Compressive Deformation of a Translucent Matrix Containing a Contrasting Filler.

A neglected mechanism for pressure-responsive color change is demonstrated using cellulose acetate composites prepared by direct (solvent) immersion annealing (DIA), with different loadings of activated charcoal filler. Namely, compressive plastic deformation of the translucent cellulose acetate leads to a decrease in the optical path length and a concomitant increase in the visibility of the opaque contrasting filler. Composites bearing 1-7 wt% activated charcoal exhibited a linear relationship between applied pressure and resulting pressure mark brightness in the range of 12-56 MPa. Comparison of pressure mark patterns with cross-sectional scanning electron microscopy (SEM) supports the importance of the porous morphology arising from DIA for the tuning of the pressure indicator sensitivity. A simple ball drop test is used to illustrate the robustness and utility of these indicators in optical impact assessment.

Continuous SSCF of AFEX™ pretreated corn stover for enhanced ethanol productivity using commercial enzymes and Saccharomyces cerevisiae 424A (LNH-ST).

High productivity processes are critical for commercial production of cellulosic ethanol. One high productivity process-continuous hydrolysis and fermentation-has been applied in corn ethanol industry. However, little research related to this process has been conducted on cellulosic ethanol production. Here, we report and compare the kinetics of both batch SHF (separate hydrolysis and co-fermentation) and SSCF (simultaneous saccharification and co-fermentation) of AFEX™ (Ammonia Fiber Expansion) pretreated corn stover (AFEX™-CS). Subsequently, we designed a SSCF process to evaluate continuous hydrolysis and fermentation performance on AFEX™-CS in a series of continuous stirred tank reactors (CSTRs). Based on similar sugar to ethanol conversions (around 80% glucose-to-ethanol conversion and 47% xylose-to-ethanol conversion), the overall process ethanol productivity for continuous SSCF was 2.3- and 1.8-fold higher than batch SHF and SSCF, respectively. Slow xylose fermentation and high concentrations of xylose oligomers were the major factors limiting further enhancement of productivity.

Profiling of diferulates (plant cell wall cross-linkers) using ultrahigh-performance liquid chromatography-tandem mass spectrometry.

Recalcitrance of grasses to enzymatic digestion arises to a significant degree from a complex array of phenolic crosslinks between cell wall polysaccharide chains that inhibit their conversion to biofuels and lower their nutritive value for animal feed applications. Polysaccharide esters of ferulic acid are abundant in plant cell walls. Crosslinks between polysaccharides are formed through oxidative dehydrodimerization of ferulates, producing dehydrodiferulates (henceforth termed diferulates). Such ferulates and diferulates further crosslink plant cell walls by radical coupling cross-reactions during lignification. Although cell wall digestibility can be improved by cell wall metabolic engineering, or post-harvest by various pretreatment processes, a more comprehensive understanding of the role and impact of ferulate crosslinking on polysaccharide hydrolysis would be accelerated by availability of analytical methods that can distinguish the various diferulates released during biomass pretreatments, many of which are isomers. In this report, we present an ultrahigh-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) strategy for comprehensive separation and identification of diferulate isomers. Collision-induced dissociation (CID) mass spectra of [M + H](+) ions distinguished various isomers without requiring derivatization. Characteristic product ions for 8-O-4-, 8-8-non-cyclic, 8-8-cyclic, 8-5-cyclic, 8-5-non-cyclic, and 5-5-linked isomers were identified. All diferulates were identified either as di-acids in extracts of NaOH-hydrolyzed corn stover, or as a diverse group of diferulate mono- and di-amides in extracts of Ammonia Fiber Expansion (AFEX™)-treated corn stover. This approach allows for direct analysis of released diferulates with minimal sample preparation, and can serve as the foundation for high-throughput profiling and correlating pretreatment conditions with biomass digestibility in biorefineries producing biofuels and biochemicals.

Corrigendum: Microbiome variation across populations of desert halophyte Zygophyllum qatarensis.

[This corrects the article DOI: 10.3389/fpls.2022.841217.].

Complex physiology and compound stress responses during fermentation of alkali-pretreated corn stover hydrolysate by an Escherichia coli ethanologen.

The physiology of ethanologenic Escherichia coli grown anaerobically in alkali-pretreated plant hydrolysates is complex and not well studied. To gain insight into how E. coli responds to such hydrolysates, we studied an E. coli K-12 ethanologen fermenting a hydrolysate prepared from corn stover pretreated by ammonia fiber expansion. Despite the high sugar content (∼6% glucose, 3% xylose) and relatively low toxicity of this hydrolysate, E. coli ceased growth long before glucose was depleted. Nevertheless, the cells remained metabolically active and continued conversion of glucose to ethanol until all glucose was consumed. Gene expression profiling revealed complex and changing patterns of metabolic physiology and cellular stress responses during an exponential growth phase, a transition phase, and the glycolytically active stationary phase. During the exponential and transition phases, high cell maintenance and stress response costs were mitigated, in part, by free amino acids available in the hydrolysate. However, after the majority of amino acids were depleted, the cells entered stationary phase, and ATP derived from glucose fermentation was consumed entirely by the demands of cell maintenance in the hydrolysate. Comparative gene expression profiling and metabolic modeling of the ethanologen suggested that the high energetic cost of mitigating osmotic, lignotoxin, and ethanol stress collectively limits growth, sugar utilization rates, and ethanol yields in alkali-pretreated lignocellulosic hydrolysates.