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.

Big data mining, rational modification, and ancestral sequence reconstruction inferred multiple xylose isomerases for biorefinery.

The isomerization of xylose to xylulose is considered the most promising approach to initiate xylose bioconversion. Here, phylogeny-guided big data mining, rational modification, and ancestral sequence reconstruction strategies were implemented to explore new active xylose isomerases (XIs) for Saccharomyces cerevisiae. Significantly, 13 new active XIs for S. cerevisiae were mined or artificially created. Moreover, the importance of the amino-terminal fragment for maintaining basic XI activity was demonstrated. With the mined XIs, four efficient xylose-utilizing S. cerevisiae were constructed and evolved, among which the strain S. cerevisiae CRD5HS contributed to ethanol titers as high as 85.95 and 94.76 g/liter from pretreated corn stover and corn cob, respectively, without detoxifying or washing pretreated biomass. Potential genetic targets obtained from adaptive laboratory evolution were further analyzed by sequencing the high-performance strains. The combined XI mining methods described here provide practical references for mining other scarce and valuable enzymes.

Understanding the structure and composition of recalcitrant oligosaccharides in hydrolysate using high-throughput biotin-based glycome profiling and mass spectrometry.

Novel Immunological and Mass Spectrometry Methods for Comprehensive Analysis of Recalcitrant Oligosaccharides in AFEX Pretreated Corn Stover. Lignocellulosic biomass is a sustainable alternative to fossil fuel and is extensively used for developing bio-based technologies to produce products such as food, feed, fuel, and chemicals. The key to these technologies is to develop cost competitive processes to convert complex carbohydrates present in plant cell wall to simple sugars such as glucose, xylose, and arabinose. Since lignocellulosic biomass is highly recalcitrant, it must undergo a combination of thermochemical treatment such as Ammonia Fiber Expansion (AFEX), dilute acid (DA), Ionic Liquid (IL) and biological treatment such as enzyme hydrolysis and microbial fermentation to produce desired products. However, when using commercial fungal enzymes during hydrolysis, only 75-85% of the soluble sugars generated are monomeric sugars, while the remaining 15-25% are soluble recalcitrant oligosaccharides that cannot be easily utilized by microorganisms. Previously, we successfully separated and purified the soluble recalcitrant oligosaccharides using a combination of charcoal and celite-based separation followed by size exclusion chromatography and studies their inhibitory properties on enzymes. We discovered that the oligosaccharides with higher degree of polymerization (DP) containing methylated uronic acid substitutions were more recalcitrant towards commercial enzyme mixtures than lower DP and neutral oligosaccharides. Here, we report the use of several complementary techniques that include glycome profiling using plant biomass glycan specific monoclonal antibodies (mAbs) to characterize sugar linkages in plant cell walls and enzymatic hydrolysate, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using structurally-informative diagnostic peaks offered by negative ion post-secondary decay spectra, gas chromatography followed by mass spectrometry (GC-MS) to characterize oligosaccharide sugar linkages with and without derivatization. Since oligosaccharides (DP 4-20) are small, it is challenging to mobilize these molecules for mAbs binding and characterization. To overcome this problem, we have applied a new biotin-coupling based oligosaccharide immobilization method that successfully tagged most of the low DP soluble oligosaccharides on to a micro-plate surface followed by specific linkage analysis using mAbs in a high-throughput system. This new approach will help develop more advanced versions of future high throughput glycome profiling methods that can be used to separate and characterize oligosaccharides present in biomarkers for diagnostic applications.

Transforming biorefinery designs with 'Plug-In Processes of Lignin' to enable economic waste valorization.

Biological lignin valorization has emerged as a major solution for sustainable and cost-effective biorefineries. However, current biorefineries yield lignin with inadequate fractionation for bioconversion, yet substantial changes of these biorefinery designs to focus on lignin could jeopardize carbohydrate efficiency and increase capital costs. We resolve the dilemma by designing 'plug-in processes of lignin' with the integration of leading pretreatment technologies. Substantial improvement of lignin bioconversion and synergistic enhancement of carbohydrate processing are achieved by solubilizing lignin via lowering molecular weight and increasing hydrophilic groups, addressing the dilemma of lignin- or carbohydrate-first scenarios. The plug-in processes of lignin could enable minimum polyhydroxyalkanoate selling price at as low as $6.18/kg. The results highlight the potential to achieve commercial production of polyhydroxyalkanoates as a co-product of cellulosic ethanol. Here, we show that the plug-in processes of lignin could transform biorefinery design toward sustainability by promoting carbon efficiency and optimizing the total capital cost.

Carbon-Negative Biofuel Production.

Achievement of the 1.5 °C limit for global temperature increase relies on the large-scale deployment of carbon dioxide removal (CDR) technologies. In this article, we explore two CDR technologies: soil carbon sequestration (SCS), and carbon capture and storage (CCS) integrated with cellulosic biofuel production. These CDR technologies are applied as part of decentralized biorefinery systems processing corn stover and unfertilized switchgrass grown in riparian zones in the Midwestern United States. Cover crops grown on corn-producing lands are chosen from the SCS approach, and biogenic CO2 in biorefineries is captured, transported by pipeline, and injected into saline aquifers. The decentralized biorefinery system using SCS, CCS, or both can produce carbon-negative cellulosic biofuels (≤-22.2 gCO2 MJ-1). Meanwhile, biofuel selling prices increase by 15-45% due to CDR costs. Economic incentives (e.g., cover crop incentives and/or a CO2 tax credit) can mitigate price increases caused by CDR technologies. A combination of different CDR technologies in decentralized biorefinery systems is the most efficient method for greenhouse gas (GHG) mitigation, and its total GHG mitigation potential in the Midwest is 0.16 GtCO2 year-1.

Ammonia Fiber Expansion (AFEX) Pretreatment of Lignocellulosic Biomass.

Lignocellulosic materials are plant-derived feedstocks, such as crop residues (e.g., corn stover, rice straw, and sugar cane bagasse) and purpose-grown energy crops (e.g., miscanthus, and switchgrass) that are available in large quantities to produce biofuels, biochemicals, and animal feed. Plant polysaccharides (i.e., cellulose, hemicellulose, and pectin) embedded within cell walls are highly recalcitrant towards conversion into useful products. Ammonia fiber expansion (AFEX) is a thermochemical pretreatment that increases accessibility of polysaccharides to enzymes for hydrolysis into fermentable sugars. These released sugars can be converted into fuels and chemicals in a biorefinery. Here, we describe a laboratory-scale batch AFEX process to produce pretreated biomass on the gram-scale without any ammonia recycling. The laboratory-scale process can be used to identify optimal pretreatment conditions (e.g., ammonia loading, water loading, biomass loading, temperature, pressure, residence time, etc.) and generates sufficient quantities of pretreated samples for detailed physicochemical characterization and enzymatic/microbial analysis. The yield of fermentable sugars from enzymatic hydrolysis of corn stover pretreated using the laboratory-scale AFEX process is comparable to pilot-scale AFEX process under similar pretreatment conditions. This paper is intended to provide a detailed standard operating procedure for the safe and consistent operation of laboratory-scale reactors for performing AFEX pretreatment of lignocellulosic biomass.

Sustainable feedstock for bioethanol production: Impact of spatial resolution on the design of a sustainable biomass supply-chain.

This study assesses the role of spatial-resolution and spatial-variations in environmental impacts estimation and decision-making for corn-stover harvesting to produce biofuels. Geospatial corn-stover yields and environmental impacts [global warming potential (GWP), eutrophication, and soil-loss] dataset for two study areas in Wisconsin and Michigan were generated through Environmental Policy Integrated Climate (EPIC) model and aggregated at different spatial-resolutions (i.e., 100; 1000; 10,000 ha). For each spatial-resolution, decision-making was accomplished using an optimization routine to minimize different environmental impacts associated with harvesting stover to meet varied biomass demands. The results of the study showed that selective harvesting at higher-resolution (or lower-aggregation level) can result in significantly lower environmental impacts, especially at low stover demand levels. Additionally, the increased spatial resolution had more impact in minimizing the environmental impacts of corn stover harvest under a more variable landscape such as terrains and its influences are more pronounced for soil-loss and eutrophication potential compared to GWP.

Mechanism-Guided Design of Highly Efficient Protein Secretion and Lipid Conversion for Biomanufacturing and Biorefining.

Bacterial protein secretion represents a significant challenge in biotechnology, which is essential for the cost-effective production of therapeutics, enzymes, and other functional proteins. Here, it is demonstrated that proteomics-guided engineering of transcription, translation, secretion, and folding of ligninolytic laccase balances the process, minimizes the toxicity, and enables efficient heterologous secretion with a total protein yield of 13.7 g L-1. The secretory laccase complements the biochemical limits on lignin depolymerization well in Rhodococcus opacus PD630. Further proteomics analysis reveals the mechanisms for the oleaginous phenotype of R. opacus PD630, where a distinct multiunit fatty acid synthase I drives the carbon partition to storage lipid. The discovery guides the design of efficient lipid conversion from lignin and carbohydrate. The proteomics-guided integration of laccase-secretion and lipid production modules enables a high titer in converting lignin-enriched biorefinery waste to lipid. The fundamental mechanisms, engineering components, and design principle can empower transformative platforms for biomanufacturing and biorefining.

The Renewable Fuel Standard May Limit Overall Greenhouse Gas Savings by Corn Stover-Based Cellulosic Biofuels in the U.S. Midwest: Effects of the Regulatory Approach on Projected Emissions.

The Renewable Fuel Standard (RFS) program specifies a greenhouse gas (GHG) reduction threshold for cellulosic biofuels, while the Low Carbon Fuel Standard (LCFS) program in California does not. Here, we investigate the effects of the GHG threshold under the RFS on projected GHG savings from two corn stover-based biofuel supply chain systems in the United States Midwest. The analysis is based on a techno-economic framework that minimizes ethanol selling price. The GHG threshold lowers the lifecycle GHG of ethanol: 34.39 ± 4.92 gCO2 MJ-1 in the RFS-compliant system and 46.30 ± 10.05 gCO2 MJ-1 in the non RFS-compliant system. However, hypothetical biorefinery systems complying with the RFS will not process the more GHG-intensive corn stover, and thus much less biofuel will be produced compared to the non RFS-compliant system. Thus, taken as a whole, the non RFS-compliant system would achieve more GHG savings than an RFS-compliant system: 10.7 TgCO2 year-1 in the non RFS-compliant system compared with 4.4 TgCO2 year-1 in the RFS-compliant system. These results suggest that the current RFS GHG reduction threshold may not be the most efficient way to carry out the purposes of the Energy Security and Independence Act in the corn stover-based biofuel system: relaxing the threshold could actually increase the overall GHG savings from corn stover-based biofuels. Therefore, the LCFS-type regulatory approach is recommended for the corn stover-based cellulosic biofuel system under the RFS program. In addition, our calculation of the GHG balance for stover-based biofuel accounts for SOC losses, while the current RFS estimates do not include effects on SOC.

Incorporating anaerobic co-digestion of steam exploded or ammonia fiber expansion pretreated sugarcane residues with manure into a sugarcane-based bioenergy-livestock nexus.

The co-digestion of pretreated sugarcane lignocelluloses with dairy cow manure (DCM) as a bioenergy production and waste management strategy, for intensive livestock farms located in sugarcane regions, was investigated. Ammonia fiber expansion (AFEX) increased the nitrogen content and accelerated the biodegradability of sugarcane bagasse (SCB) and cane leaf matter (CLM) through the cleavage of lignin carbohydrate crosslinks, resulting in the highest specific methane yields (292-299 L CH4/kg VSadded), biogas methane content (57-59% v/v) and biodegradation rates, with or without co-digestion with DCM. To obtain comparable methane yields, untreated and steam exploded (StEx) SCB and CLM had to be co-digested with DCM, at mass ratios providing initial C/N ratios in the range of 18 to 35. Co-digestion with DCM improved the nutrient content of the solid digestates, providing digestates that could be used as biofertilizer to replace CLM that is removed from sugarcane fields during green harvesting.

Correction to: 'The effect of alkali-soluble lignin on purified core cellulase and hemicellulase activities during hydrolysis of extractive ammonia-pretreated lignocellulosic biomass'.

[This corrects the article DOI: 10.1098/rsos.171529.].

The effect of alkali-soluble lignin on purified core cellulase and hemicellulase activities during hydrolysis of extractive ammonia-pretreated lignocellulosic biomass.

Removing alkali-soluble lignin using extractive ammonia (EA) pretreatment of corn stover (CS) is known to improve biomass conversion efficiency during enzymatic hydrolysis. In this study, we investigated the effect of alkali-soluble lignin on six purified core glycosyl hydrolases and their enzyme synergies, adopting 31 enzyme combinations derived by a five-component simplex centroid model, during EA-CS hydrolysis. Hydrolysis experiment was carried out using EA-CS(-) (approx. 40% lignin removed during EA pretreatment) and EA-CS(+) (where no lignin was extracted). Enzymatic hydrolysis experiments were done at three different enzyme mass loadings (7.5, 15 and 30 mg protein g-1 glucan), using a previously developed high-throughput microplate-based protocol, and the sugar yields of glucose and xylose were detected. The optimal enzyme combinations (based on % protein mass loading) of six core glycosyl hydrolases for EA-CS(-) and EA-CS(+) were determined that gave high sugar conversion. The inhibition of lignin on optimal enzyme ratios was studied, by adding fixed amount of alkali-soluble lignin fractions to EA-CS(-), and pure Avicel, beechwood xylan and evaluating their sugar conversion. The optimal enzyme ratios that gave higher sugar conversion for EA-CS(-) were CBH I: 27.2-28.2%, CBH II: 18.2-22.2%, EG I: 29.2-34.3%, EX: 9.0-14.1%, ßX: 7.2-10.2%, ßG: 1.0-5.0% (at 7.5-30 mg g-1 protein mass loading). Endoglucanase was inhibited to a greater extent than other core cellulases and xylanases by lignin during enzyme hydrolysis. We also found that alkali-soluble lignin inhibits cellulase more strongly than hemicellulase during the course of enzyme hydrolysis.

Ethanol production potential from AFEX™ and steam-exploded sugarcane residues for sugarcane biorefineries.

BACKGROUND: Expanding biofuel markets are challenged by the need to meet future biofuel demands and mitigate greenhouse gas emissions, while using domestically available feedstock sustainably. In the context of the sugar industry, exploiting under-utilized cane leaf matter (CLM) in addition to surplus sugarcane bagasse as supplementary feedstock for second-generation ethanol production has the potential to improve bioenergy yields per unit land. In this study, the ethanol yields and processing bottlenecks of ammonia fibre expansion (AFEX™) and steam explosion (StEx) as adopted technologies for pretreating sugarcane bagasse and CLM were experimentally measured and compared for the first time. RESULTS: Ethanol yields between 249 and 256 kg Mg-1 raw dry biomass (RDM) were obtained with AFEX™-pretreated sugarcane bagasse and CLM after high solids loading enzymatic hydrolysis and fermentation. In contrast, StEx-pretreated sugarcane bagasse and CLM resulted in substantially lower ethanol yields that ranged between 162 and 203 kg Mg-1 RDM. The ethanol yields from StEx-treated sugarcane residues were limited by the aggregated effect of sugar degradation during pretreatment, enzyme inhibition during enzymatic hydrolysis and microbial inhibition of S. cerevisiae 424A (LNH-ST) during fermentation. However, relatively high enzyme dosages (> 20 mg g-1 glucan) were required irrespective of pretreatment method to reach 75% carbohydrate conversion, even when optimal combinations of Cellic® CTec3, Cellic® HTec3 and Pectinex Ultra-SP were used. Ethanol yields per hectare sugarcane cultivation area were estimated at 4496 and 3416 L ha-1 for biorefineries using AFEX™- or StEx-treated sugarcane residues, respectively. CONCLUSIONS: AFEX™ proved to be a more effective pretreatment method for sugarcane residues relative to StEx due to the higher fermentable sugar recovery and enzymatic hydrolysate fermentability after high solids loading enzymatic hydrolysis and fermentation by S. cerevisiae 424A (LNH-ST). The identification of auxiliary enzyme activities, adequate process integration and the use of robust xylose-fermenting ethanologens were identified as opportunities to further improve ethanol yields from AFEX™- and StEx-treated sugarcane residues.

Water-soluble phenolic compounds produced from extractive ammonia pretreatment exerted binary inhibitory effects on yeast fermentation using synthetic hydrolysate.

Biochemical conversion of lignocellulosic biomass to liquid fuels requires pretreatment and enzymatic hydrolysis of the biomass to produce fermentable sugars. Degradation products produced during thermochemical pretreatment, however, inhibit the microbes with regard to both ethanol yield and cell growth. In this work, we used synthetic hydrolysates (SynH) to study the inhibition of yeast fermentation by water-soluble components (WSC) isolated from lignin streams obtained after extractive ammonia pretreatment (EA). We found that SynH with 20g/L WSC mimics real hydrolysate in cell growth, sugar consumption and ethanol production. However, a long lag phase was observed in the first 48 h of fermentation of SynH, which is not observed during fermentation with the crude extraction mixture. Ethyl acetate extraction was conducted to separate phenolic compounds from other water-soluble components. These phenolic compounds play a key inhibitory role during ethanol fermentation. The most abundant compounds were identified by Liquid Chromatography followed by Mass Spectrometry (LC-MS) and Gas Chromatography followed by Mass Spectrometry (GC-MS), including coumaroyl amide, feruloyl amide and coumaroyl glycerol. Chemical genomics profiling was employed to fingerprint the gene deletion response of yeast to different groups of inhibitors in WSC and AFEX-Pretreated Corn Stover Hydrolysate (ACSH). The sensitive/resistant genes cluster patterns for different fermentation media revealed their similarities and differences with regard to degradation compounds.

Conversion of lignocellulosic agave residues into liquid biofuels using an AFEX™-based biorefinery.

BACKGROUND: Agave-based alcoholic beverage companies generate thousands of tons of solid residues per year in Mexico. These agave residues might be used for biofuel production due to their abundance and favorable sustainability characteristics. In this work, agave leaf and bagasse residues from species Agave tequilana and Agave salmiana were subjected to pretreatment using the ammonia fiber expansion (AFEX) process. The pretreatment conditions were optimized using a response surface design methodology. We also identified commercial enzyme mixtures that maximize sugar yields for AFEX-pretreated agave bagasse and leaf matter, at ~ 6% glucan (w/w) loading enzymatic hydrolysis. Finally, the pretreated agave hydrolysates (at a total solids loading of ~ 20%) were used for ethanol fermentation using the glucose- and xylose-consuming strain Saccharomyces cerevisiae 424A (LNH-ST), to determine ethanol yields at industrially relevant conditions. RESULTS: Low-severity AFEX pretreatment conditions are required (100-120 °C) to enable efficient enzymatic deconstruction of the agave cell wall. These studies showed that AFEX-pretreated A. tequilana bagasse, A. tequilana leaf fiber, and A. salmiana bagasse gave ~ 85% sugar conversion during enzyme hydrolysis and over 90% metabolic yields of ethanol during fermentation without any washing step or nutrient supplementation. On the other hand, although lignocellulosic A. salmiana leaf gave high sugar conversions, the hydrolysate could not be fermented at high solids loadings, apparently due to the presence of natural inhibitory compounds. CONCLUSIONS: These results show that AFEX-pretreated agave residues can be effectively hydrolyzed at high solids loading using an optimized commercial enzyme cocktail (at 25 mg protein/g glucan) producing > 85% sugar conversions and over 40 g/L bioethanol titers. These results show that AFEX technology has considerable potential to convert lignocellulosic agave residues to bio-based fuels and chemicals in a biorefinery.

Anaerobic co-digestion of multiple agricultural residues to enhance biogas production in southern Italy.

To valorize agricultural wastes and byproducts in southern Italy, anaerobic co-digestion of six feedstocks (citrus pulp, olive pomace, cattle manure, poultry litter, whey, and corn silage) was studied to produce biogas for renewable energy generation. Both batch and semi-continuous co-digestion approaches were adopted to carry out the investigation. The feedstocks were mixed at different percentages according to their availabilities in southern Italy. The batch anaerobic co-digestion demonstrated that six studied feedstock mixtures generated an average of 239 mL CH4/g VS loading without significant difference between each other, which concluded that the feedstock mixtures can be used for biogas production. Considering the feedstock availability of citrus pulp and olive pomace in Sicily, three feedstock mixtures with the highest volatile solids concentration of citrus pulp (42% citrus pulp, 17% corn silage, 4% cattle manure, 8% poultry litter, and 18% whey; 34% citrus pulp, 8% olive pomace, 17% corn silage, 4% cattle manure, 8% poultry litter, and 18% whey; and 25% citrus pulp, 16% olive pomace, 17% corn silage, 4% cattle manure, 8% poultry litter, and 18% whey, respectively) were selected to run the semi-continuous anaerobic digestion. Under the stabilized culture condition, the feed mixture with 42% citrus pulp, 17% corn silage, 4% cattle manure, 8% poultry litter, and 18% whey presented the best biogas production (231 L methane/kg VS loading/day). The corresponding mass and energy balance concluded that all three tested feedstock mixtures have positive net energy outputs (1.5, 0.9, and 1.2 kWh-e/kg dry feedstock mixture, respectively).

Cellulosic biofuel contributions to a sustainable energy future: Choices and outcomes.

Cellulosic crops are projected to provide a large fraction of transportation energy needs by mid-century. However, the anticipated land requirements are substantial, which creates a potential for environmental harm if trade-offs are not sufficiently well understood to create appropriately prescriptive policy. Recent empirical findings show that cellulosic bioenergy concerns related to climate mitigation, biodiversity, reactive nitrogen loss, and crop water use can be addressed with appropriate crop, placement, and management choices. In particular, growing native perennial species on marginal lands not currently farmed provides substantial potential for climate mitigation and other benefits.