Cellulosic Feedstocks Research Articles

New trends in mycosynthesis of cellulose nanocrystals promoted by gamma irradiation of sugarcane bagasse

AbstractThe biological synthesis of cellulose nanocrystals (CNCs) involves utilizing cellulose-degrading microorganisms or their hydrolytic enzymes as catalysts for the controlled degradation of cellulose, yielding CNCs. Chemical synthesis of CNCs involves acid hydrolysis conducted for 45 min at 45 °C using sulfuric acid (64%). Neurospora intermedia (Assiut University Mycological Center (AUMC) 14,359), Fusarium verticillioides (AUMC 14360), and Rhizopus oryzae (AUMC 14361) were employed in the preparation of CNCs. Before both chemical and biological treatments, sugarcane bagasse (SCB) was irradiated with doses of 100, 200, and 300 kGy, enhancing the yield of nanocellulose from the cellulosic feedstock. The resultant nanocellulose was initially assessed using UV–Vis spectroscopy, and the characterization was further refined through Dynamic Light Scattering analysis to delineate particle size distribution within the nanoscale and to evaluate stability. CNCs and chemically purified cellulose (CPC) displayed analogous Fourier Transform Infrared Spectroscopy but were markedly different from SCB. X-ray Diffraction patterns revealed a notably higher crystallinity of cellulose in nanocellulose, with larger crystallite dimensions compared to CPC and SCB. Transmission Electron Microscope investigations elucidated the morphology of the synthesized nanoparticles. In summary, the selection of F. verticillioides for nanocellulose production represents a promising and sustainable approach that combines effectiveness, environmental friendliness, and cost-efficiency in the synthesis of this valuable nanomaterial. Graphical abstract

A critical re‐analysis of biochar properties prediction from production parameters and elemental analysis

AbstractBiochar is the product of intentional pyrolysis of organic feedstocks. It is made under controlled conditions in order to achieve desired physico‐chemical characteristics. These characteristics ultimately affect biochar properties as a soil amendment. When biochar is used for carbon storage, an important property is its persistence in soil, often described by the proportion of biochar carbon remaining in soil after a 100 years (). We analyzed published data on 1230 biochars to re‐evaluate the effect of pyrolysis parameters on biochar characteristics and the possibility to predict from the maximum temperature reached during pyrolysis (HTT). We showed that biochar ash and nitrogen (N) contents were mostly affected by feedstock type. The oxygen to carbon (O:C) and hydrogen to carbon (H:C) ratios were mostly affected by the extent of pyrolysis (a combination of HTT and pyrolysis duration), except for non (ligno)cellulosic feedstocks (plastic waste, sewage sludge). The volatile matter (VM) content was affected by both feedstock type and the extent of pyrolysis. We demonstrated that HTT is the main driver of H:C ‐‐ an indicator of persistence ‐‐ but that it is not measured accurately enough to precisely predict H:C, let alone persistence. We examined the equations to estimate available in the literature and showed that calculated from HTT presented little agreement with calculated from H:C. The sign and magnitude of the bias depended on the equation used to calculate and the dispersion was usually large. This could lead to improper compensation of carbon emissions and wrong reporting of carbon sinks in national carbon accounting schemes. We recommend not to use HTT as a predictor for persistence and stress the importance to rapidly develop more accurate proxies of biochar C persistence in soil.

Surface functionalization of oil palm empty fruit bunch (OPEFB)-derived cellulose as a carboxyl platform for metal cations and dyes removal from aquatic media

The abundant oil palm empty fruit bunch (OPEFB) as by-product of palm oil milling processes exhibits a potential as an alternative cellulose feedstock for bio-adsorbent. This study aimed to produce a highly carboxylated bio-adsorbent for direct industrial application from OPEFB-based cellulose via mercerization and followed by esterification with succinic anhydride (SA) to enhance its adsorptive capability towards hazardous heavy metal and dyes ions. The modification using SA provides the carbon backbone platform for carboxyl group attachment for the contaminants. The results showed that the carboxylated cellulose had a high carboxyl content (4.39 mmol/g). Carboxylated cellulose had a higher binding capacity for adsorbates, with removal rates of 94.7%, 97.85%, 40.9%, and 90.15% for dye, Pb2+, Cu2+, and Cd2+ cations, respectively, at pH 6, 4 hours reaction time, and at room temperature. In comparison, unmodified cellulose removed only 47%, 23.1%, 2.9%, and 7.5% for dye, Pb2+, Cu2+, and Cd2+ cations, respectively. The adsorption kinetics study revealed that the adsorption process followed the pseudo-second-order model. The adsorption isotherm of these two metal cations follows the model of Langmuir very well, while Cu2+ follows the Freundlich model. Our method produces bio-adsorbents with high carboxyl content and adsorption rate in a short reaction time using OPEFB as a green precursor material that is easily scalable for industrial use.

Investigating the Effects of the Physicochemical Properties of Cellulose-Derived Biocarbon on Direct Carbon Solid Oxide Fuel Cell Performance.

This paper presents a study of the characteristic effects of the physicochemical properties of microcrystalline cellulose and a series of biocarbon samples produced from this raw material through thermal conversion at temperatures ranging from 200 °C to 850 °C. Structural studies revealed that the biocarbon samples produced from cellulose had a relatively low degree of graphitization of the carbon and an isometric shape of the carbon particles. Based on thermal investigations using the differential thermal analysis/differential scanning calorimeter method, obtaining fully formed biocarbon samples from cellulose feedstock was possible at about 400 °C. The highest direct carbon solid oxide fuel cell (DC-SOFC) performance was found for biochar samples obtained via thermal treatment at 400-600 °C. The pyrolytic gases from cellulose decomposition had a considerable impact on the achieved current density and power density of the DC-SOFCs supplied by pure cellulose samples or biochars derived from cellulose feedstock at a lower temperature range of 200-400 °C. For the DC-SOFCs supplied by biochars synthesised at higher temperatures of 600-850 °C, the "shuttle delivery mechanism" had a substantial effect. The impact of the carbon oxide concentration in the anode or carbon bed was important for the performance of the DC-SOFCs. Carbon oxide oxidised at the anode to form carbon dioxide, which interacted with the carbon bed to form more carbon oxide. The application of biochar obtained from cellulose alone without an additional catalyst led to moderate electrochemical power output from the DC-SOFCs. The results show that catalysts for the reverse Boudouard reactions occurring in a biocarbon bed are critical to ensuring high performance and stable operation under electrical load, which is crucial for DC-SOFC development.

Preliminary hydrothermal treatment and steam explosion of cellulosic feedstock for the subsequent biotechnological transformation: A review

The use of renewable sources of cellulosic feedstock to produce high value-added products is a relevant issue. Cellulosic feedstock constitutes a natural matrix comprising cellulose (38–50 %), lignin (10–25 %), and hemicel-luloses (23–32 %). In order to break it down, pretreatment involving the removal of hemicelluloses and lignin is required. This process can change the chemical composition and structure of cellulose while increasing porosity. This review article provides an analysis of data on the hydrothermal treatment and steam explosion of cellulosic feedstock (sunflower straw, lawn grass, poplar sawdust, hay, reed, aspen, giant reed, silage, etc.) intended to convert it into substrates for the synthesis of biotechnological products (protein, biohydrogen, biogas, levulinic acid, methane, lactic acid, ethanol, and succinic acid). Hydrothermal treatment involves treating raw materials at 160–240 °С in water under high pressure. Pressure keeps water in a liquid state. During steam explosion, feedstock is treated with steam at a moderate temperature and pressure for a certain amount of time. Then, the pressure is rapidly released, and the fibers of cellulosic feedstock expand. The effectiveness of hydrothermal treatment and steam explosion depends both on the type of feedstock (chemical composition, solids concentration, and properties of solids) as well as on the conditions of hydrothermal treatment and steam explosion.

Spatially Varying Costs of GHG Abatement with Alternative Cellulosic Feedstocks for Sustainable Aviation Fuels.

Cellulosic biomass-based sustainable aviation fuels (SAFs) can be produced from various feedstocks. The breakeven price and carbon intensity of these feedstock-to-SAF pathways are likely to differ across feedstocks and across spatial locations due to differences in feedstock attributes, productivity, opportunity costs of land for feedstock production, soil carbon effects, and feedstock composition. We integrate feedstock to fuel supply chain economics and life-cycle carbon accounting using the same system boundary to quantify and compare the spatially varying greenhouse gas (GHG) intensities and costs of GHG abatement with SAFs derived from four feedstocks (switchgrass, miscanthus, energy sorghum, and corn stover) at 4 km resolution across the U.S. rainfed region. We show that the optimal feedstock for each location differs depending on whether the incentive is to lower breakeven price, carbon intensity, or cost of carbon abatement with biomass or to have high biomass production per unit land. The cost of abating GHG emissions with SAF ranges from $181 Mg-1 CO2e to more than $444 Mg-1 CO2e and is lowest with miscanthus in the Midwest, switchgrass in the south, and energy sorghum in a relatively small region in the Great Plains. While corn stover-based SAF has the lowest breakeven price per gallon, it has the highest cost of abatement due to its relatively high GHG intensity. Our findings imply that different types of policies, such as volumetric targets, tax credits, and low carbon fuel standards, will differ in the mix of feedstocks they incentivize and locations where they are produced in the U.S. rainfed region.

Dynamic covalent bonds of hemiacetals and acetals enables reprocessability and raw material recyclability of high performance cellulosic thermosetting resins

Despite covalent adaptable networks (CANs) imparting the reprocessing performance of crosslinked polymers, due to reversible bond exchange of the dynamic covalent bonds; it is still a huge challenge to design catalyst-free, fast reprocessing, controlled degradation and raw material recycling biomass base CANs. Here, for the first time, we prepared cellulose CANs base on hemiacetal and acetal bonds (ACCs) via aldehyde and hydroxyl reaction of the dialdehyde crosslinker with hydroxypropyl cellulose (HPC). By regulating the crosslink density and chemical structure of the crosslinked network, ACCs exhibit tunable thermal and mechanical properties. The crosslinked network structure of the ACCs benefits from the rapid bond exchange of the hemiacetal and acetal bonds at elevated temperatures, which allows for rapid stress relaxation and thus, superior reprocessing properties. In addition, it was found that the dynamic exchange of acetals in the ACCs is influenced by the chain length and flexibility of the crosslinker. Moreover, ACCs possess good degradation and cellulose feedstock recycling properties due to the acid sensitivity of the acetal bonds. This work provides an effective solution to the problems of resource waste caused by petroleum-based thermoset plastics.

Near-infrared spectroscopy and multivariate analysis as real-time monitoring strategy of TEMPO-mediated oxidation of cellulose fibers from different feedstocks

The regioselective oxidation of the primary hydroxyl groups of cellulose, usually mediated by the (2,2,6,6-tetramethylpiperidin-1-yl)oxyl radical (TEMPO), is highly popular in the scientific literature. However, the lack of efficient monitoring techniques imposes a severe limitation to its upscaling. This work involves a portable, user-friendly near-infrared spectroscopy device, optimized preprocessing techniques, and multivariate calibration to quickly estimate the carboxyl group content of modified cellulose (i.e., the extent of the oxidation). For that, bleached pulps from eucalyptus, pine, hemp, and sisal were submitted to TEMPO-mediated oxidation, varying the dosage of spent oxidizer (NaClO) and thus attaining samples of different values of carboxyl group content. These values were related to near-infrared spectra (908–1676 nm) by Partial Least Squares regression, yielding cross-validation coefficients (RCV2) above 0.97 for wood pulps, 0.95 for sisal pulp, and 0.91 for hemp pulp. Based on the residual prediction deviation, the model for each pulp was found to show good predictability. Nonetheless, the overall regression model, comprising the four different materials, was unreliable. In light of this, spectra were submitted to principal components analysis (PCA), hinting that pulps could be classified in terms of their hemicellulose to cellulose ratio. Considering all the statistical parameters, the overall proposal presented here begins with a PCA—Linear Discriminant Analysis model to classify the sample by its fiber type, subsequently selecting a specific regression model for that class. Overall, the presented models in this work allow the determination of the extent of oxidation of different cellulosic feedstocks, expressed as carboxyl content, in a fast and simple approach using a benchtop near-infrared equipment.

Nth-plant scenario for blended pellets of Miscanthus, Switchgrass, and Corn Stover using multi-modal transportation: Biorefineries and depots in the contiguous U.S.

The sustainability of the biofuel industry depends on the development of a mature conversion technology on a national level that can take advantage of the economies of scale: the nth-plant. This study addresses the logistic challenge of mobilizing national cellulosic feedstock supplies for a sustainable bioenergy industry. A Mixed Integer Linear Programming (MILP) model was developed and updated to deliver on-spec biomass that considers both a desired quantity and quality at the biorefinery. Our supply chain analysis includes multi-modal transport (truck and rail), varying depot and biorefinery sizes, and feedstock blends of corn stover (harvested by either a two- or three-pass method), switchgrass, and miscanthus. The following US states: Illinois, Kansas, Missouri, North Carolina, Oklahoma, Georgia, and Texas were identified as key locations for producing accessible miscanthus. Based on our most optimistic scenario, using trucks as the only transportation mode in 2040 with a cost target of $79/dt, corn stover, switchgrass, and miscanthus could help meet 48% of the EPA target, 173 million dry tons that translate into 7.8 billion GGE. The addition of rail transportation for biomass delivery to biorefineries could help meet 79% of the EPA target, 283 million dry tons that translate into 12.7 billion GGE.

Influence of Impurities in the Chemical Processing Chain of Biomass on the Catalytic Valorisation of Cellulose towards γ-Valerolactone

The conversion of lignocellulosic biomass to valuable chemicals such as levulinic acid and γ-valerolactone is a promising approach for achieving a sustainable circular economy. However, the presence of impurities during the stepwise chemical processing chain of the biomass feedstock can significantly impact both the hydrolysis and hydrogenation steps implemented to convert the cellulosic feedstock to levulinic acid and further to γ-valerolactone, respectively. This review article explores the effects of those impurities by classifying them into two groups, namely endogenous and exogenous types, based on whether they originate directly from the raw lignocellulosic biomass or arise during its multi-step chemical processing. Endogenous impurities include heavy metals, alkali metals, alkaline earth metals, proteins, and side products from the downstream treatment of cellulose, while exogenous impurities are introduced during physical pre-treatments such as ball milling or during the hydrolysis step, or they might originate from the reactor setup. The specific catalyst deactivation by carbonaceous species such as humins and coke is considered. The mechanisms of impurity-induced catalyst deactivation and by-product formation are thoroughly discussed. Additionally, strategies for minimizing the detrimental effects of impurities on biomass conversion and enhancing catalytic efficiency and stability are also proposed.

Hydrolysis of ionic liquid–treated substrate with an Iocasia fonsfrigidae strain SP3-1 endoglucanase

Recently, we reported the discovery of a novel endoglucanase of the glycoside hydrolase family 12 (GH12), designated IfCelS12A, from the haloalkaliphilic anaerobic bacterium Iocasia fonsfrigidae strain SP3-1, which was isolated from a hypersaline pond in the Samut Sakhon province of Thailand (ca. 2017). IfCelS12A exhibits high substrate specificity on carboxymethyl cellulose and amorphous cellulose but low substrate specificity on b-1,3;1,4-glucan. Unlike some endoglucanases of the GH12 family, IfCelS12A does not exhibit hydrolytic activity on crystalline cellulose (i.e., Avicel™). High-Pressure Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC) analyses of products resulting from IfCelS12-mediated hydrolysis indicate mode of action for this enzyme. Notably, IfCelS12A preferentially hydrolyzes cellotetraoses, cellopentaoses, and cellohexaoses with negligible activity on cellobiose or cellotriose. Kinetic analysis with cellopentaose and barely b-d-glucan as cellulosic substrates were conducted. On cellopentaose, IfCelS12A demonstrates a 16-fold increase in activity (KM = 0.27 mM; kcat = 0.36 s−1; kcat/KM = 1.34 mM−1 s−1) compared to the enzymatic hydrolysis of barley b-d-glucan (KM: 0.04 mM, kcat: 0.51 s−1, kcat/KM = 0.08 mM−1 s−1). Moreover, IfCelS12A enzymatic efficacy is stable in hypersaline sodium chlorids (NaCl) solutions (up to 10% NaCl). Specifically, IfCel12A retains notable activity after 24 h at 2M NaCl (10% saline solution). IfCelS12A used as a cocktail component with other cellulolytic enzymes and in conjunction with mobile sequestration platform technology offers additional options for deconstruction of ionic liquid–pretreated cellulosic feedstock.Key points• IfCelS12A from an anaerobic alkaliphile Iocasia fronsfrigidae shows salt tolerance• IfCelS12A in cocktails with other enzymes efficiently degrades cellulosic biomass• IfCelS12A used with mobile enzyme sequestration platforms enhances hydrolysis

Replacing All Fossil Fuels With Nuclear-Enabled Hydrogen, Cellulosic Hydrocarbon Biofuels, and Dispatchable Electricity

Abstract We describe a roadmap using three sets of technologies to enable base-load nuclear reactors to replace all fossil fuels in a low-carbon world. The technologies integrate nuclear, wind, solar, hydroelectricity and biomass energy sources. Base-load nuclear reactors with large-scale heat storage enable dispatchable electricity to the grid. The low-cost heat storage enables buying excess wind and solar electricity to charge heat storage for later electricity production while providing assured generating capacity. Nuclear hydrogen production facilities at the scale of global oil refineries produce hydrogen to replace natural gas (gaseous fuel) as a chemical feedstock and heat source. Single sites may have tens of modular reactors produced in a local factory to lower costs by converting to a manufacturing model for reactor construction. Nuclear heat and hydrogen convert cellulosic biomass into drop-in liquid hydrocarbon biofuels to replace fossil-fuel gasoline, diesel, jet fuel, and hydrocarbon feed stocks for the chemical industry. External heat and hydrogen inputs increase the quantities of biofuels that can be produced per unit of cellulosic feedstock, thus assuring sufficient biomass feed stocks to replace all crude oil without major impacts on food and fiber prices. The biofuel production system enables the removal of large quantities of carbon dioxide from the atmosphere that is sequestered as carbon char in the soil while recycling plant nutrients (potassium, phosphorous, etc.) to assure agricultural and forest sustainability.

Longitudinal genome-wide association study reveals early QTL that predict biomass accumulation under cold stress in sorghum.

Sorghum bicolor is a promising cellulosic feedstock crop for bioenergy due to its high biomass yields. However, early growth phases of sorghum are sensitive to cold stress, limiting its planting in temperate environments. Cold adaptability is crucial for cultivating bioenergy and grain sorghum at higher latitudes and elevations, or for extending the growing season. Identifying genes and alleles that enhance biomass accumulation under early cold stress can lead to improved sorghum varieties through breeding or genetic engineering. We conducted image-based phenotyping on 369 accessions from the sorghum Bioenergy Association Panel (BAP) in a controlled environment with early cold treatment. The BAP includes diverse accessions with dense genotyping and varied racial, geographical, and phenotypic backgrounds. Daily, non-destructive imaging allowed temporal analysis of growth-related traits and water use efficiency (WUE). A genome-wide association study (GWAS) was performed to identify genomic intervals and genes associated with cold stress response. The GWAS identified transient quantitative trait loci (QTL) strongly associated with growth-related traits, enabling an exploration of the genetic basis of cold stress response at different developmental stages. This analysis of daily growth traits, rather than endpoint traits, revealed early transient QTL predictive of final phenotypes. The study identified both known and novel candidate genes associated with growth-related traits and temporal responses to cold stress. The identified QTL and candidate genes contribute to understanding the genetic mechanisms underlying sorghum's response to cold stress. These findings can inform breeding and genetic engineering strategies to develop sorghum varieties with improved biomass yields and resilience to cold, facilitating earlier planting, extended growing seasons, and cultivation at higher latitudes and elevations.

Process optimization for saccharification and fermentation of the Organic Fraction of Municipal Solid Waste (OFMSW) to maximize ethanol production performance

The Organic Fraction of Municipal Solid Waste (OFMSW) contains paper and cardboard, an attractive cellulosic feedstock for bioethanol production. Waste paper/cardboard (WPC) was subjected to mild acid treatment in solids loadings from 20 to 27.5 %. Different doses of commercial enzyme cocktail and various adjuvants facilitating enzymatic saccharification were tested. Enzyme dosing of 3.75 FPU/g fiber with very slow fed-batch saccharification of 27.5 % (w/v) solids from model lignin-free paper pulp resulted in 12.6 % (w/v) total sugar after 74 h. Use of cheap non-catalytic waste protein adjuvants including Soymeal, Protisyr, Feather meal strongly improved enzymatic saccharification and reduced the overall process cost by reducing commercial enzyme dosing. After fermentation with the industrial yeast strain BMD44 (Cellusec® 1.0) an ethanol titer of 5.77 % (v/v) was obtained using a reduced enzyme loading of 2.5 FPU/g of actual industrial WPC fiber. These results show the potential of economically profitable second-generation bioethanol production with WPC fiber.

Simultaneous suppression of lignin, tricin and wall-bound phenolic biosynthesis via the expression of monolignol 4-O-methyltransferases in rice.

Grass lignocelluloses feature complex compositions and structures. In addition to the presence of conventional lignin units from monolignols, acylated monolignols and flavonoid tricin also incorporate into lignin polymer; moreover, hydroxycinnamates, particularly ferulate, cross-link arabinoxylan chains with each other and/or with lignin polymers. These structural complexities make grass lignocellulosics difficult to optimize for effective agro-industrial applications. In the present study, we assess the applications of two engineered monolignol 4-O-methyltransferases (MOMTs) in modifying rice lignocellulosic properties. Two MOMTs confer regiospecific para-methylation of monolignols but with different catalytic preferences. The expression of MOMTs in rice resulted in differential but drastic suppression of lignin deposition, showing more than 50% decrease in guaiacyl lignin and up to an 90% reduction in syringyl lignin in transgenic lines. Moreover, the levels of arabinoxylan-bound ferulate were reduced by up to 50%, and the levels of tricin in lignin fraction were also substantially reduced. Concomitantly, up to 11 μmol/g of the methanol-extractable 4-O-methylated ferulic acid and 5-7 μmol/g 4-O-methylated sinapic acid were accumulated in MOMT transgenic lines. Both MOMTs in vitro displayed discernible substrate promiscuity towards a range of phenolics in addition to the dominant substrate monolignols, which partially explains their broad effects on grass phenolic biosynthesis. The cell wall structural and compositional changes resulted in up to 30% increase in saccharification yield of the de-starched rice straw biomass after diluted acid-pretreatment. These results demonstrate an effective strategy to tailor complex grass cell walls to generate improved cellulosic feedstocks for the fermentable sugar-based production of biofuel and bio-chemicals.

Biosynthesis of Bacterial Nanocellulose from Low-Cost Cellulosic Feedstocks: Effect of Microbial Producer.

Biodegradable bacterial nanocellulose (BNC) is a highly in-demand but expensive polymer, and the reduction of its production cost is an important task. The present study aimed to biosynthesize BNC on biologically high-quality hydrolyzate media prepared from miscanthus and oat hulls, and to explore the properties of the resultant BNC depending on the microbial producer used. In this study, three microbial producers were utilized for the biosynthesis of BNC: individual strains Komagataeibacter xylinus B-12429 and Komagataeibacter xylinus B-12431, and symbiotic Medusomyces gisevii Sa-12. The use of symbiotic Medusomyces gisevii Sa-12 was found to have technological benefits: nutrient media require no mineral salts or growth factors, and pasteurization is sufficient for the nutrient medium instead of sterilization. The yield of BNCs produced by the symbiotic culture turned out to be 44-65% higher than that for the individual strains. The physicochemical properties of BNC, such as nanofibril width, degree of polymerization, elastic modulus, Iα allomorph content and crystallinity index, are most notably dependent on the microbial producer type rather than the nutrient medium composition. This is the first study in which we investigated the biosynthesis of BNC on hydrolyzate media prepared from miscanthus and oat hulls under the same conditions but using different microbial producers, and showed that it is advisable to use the symbiotic culture. The choice of a microbial producer is grounded on the yield, production process simplification and properties. The BNC production from technical raw materials would cover considerable demands of BNC for technical purposes without competing with food resources.

Engineering natural isolates of Saccharomyces cerevisiae for consolidated bioprocessing of cellulosic feedstocks

Saccharomyces cerevisiae has gained much attention as a potential host for cellulosic bioethanol production using consolidated bioprocessing (CBP) methodologies, due to its high-ethanol-producing titres, heterologous protein production capabilities, and tolerance to various industry-relevant stresses. Since the secretion levels of heterologous proteins are generally low in domesticated strains of S. cerevisiae, natural isolates may offer a more diverse genetic background for improved heterologous protein secretion, while also displaying greater robustness to process stresses. In this study, the potential of natural and industrial S. cerevisiae strains to secrete a core set of cellulases (CBH1, CBH2, EG2, and BGL1), encoded by genes integrated using CRISPR/Cas9 tools, was evaluated. High levels of heterologous protein production were associated with a reduced maximal growth rate and with slight changes in overall strain robustness, compared to the parental strains. The natural isolate derivatives YI13_BECC and YI59_BECC displayed superior secretion capacity for the heterologous cellulases at high incubation temperature and in the presence of acetic acid, respectively, compared to the reference industrial strain MH1000_BECC. These strains also exhibited multi-tolerance to several fermentation-associated and secretion stresses. Cultivation of the strains on crystalline cellulose in oxygen-limited conditions yielded ethanol concentrations in the range of 4–4.5 g/L, representing 35–40% of the theoretical maximum ethanol yield after 120 h, without the addition of exogenous enzymes. This study therefore highlights the potential of these natural isolates to be used as chassis organisms in CBP bioethanol production.Key points• Process-related fermentation stresses influence heterologous protein production.• Transformants produced up to 4.5 g/L ethanol, ~ 40% of the theoretical yield in CBP.• CRISPR/Cas9 was feasible for integrating genes in natural S. cerevisiae isolates.

Profiling of the β-glucosidases identified in the genome of Penicillium funiculosum: insights from genomics, transcriptomics, proteomics, and homology-modeling studies.

The enzymatic conversion of lignocellulosic biomass to bioethanol depends on efficient enzyme systems with β-glucosidase as one of the key components. In this study, we performed in-depth profiling of the various β-glucosidases present in the genome of the hypercellulolytic fungus Penicillium funiculosum using genomics, transcriptomics, proteomics, and molecular dynamics simulation approaches. Of the eight β-glucosidase genes identified in the P. funiculosum genome, three were predicted to be extracellular based on signal peptide prediction and abundance in the secretome. Among the three secreted β-glucosidases, two belonged to the GH3 family and one belonged to the GH1 family. Homology models of these proteins predicted a deep and narrow active site for the GH3 β-glucosidases (PfBgl3A and PfBgl3B) and a shallow open active site for the GH1 β-glucosidase (PfBgl1A). The enzymatic assays indicated that P. funiculosum-secreted proteins showed high β-glucosidase activities with prominent bands on the 4-methylumbelliferyl β-D-glucopyranoside zymogram. To understand the contributory effects of each of the three secreted β-glucosidases (PfBgls), the corresponding gene was deleted separately, and the effect of the deletion on the β-glucosidase activity of the secretome was examined. Although not the most abundant, PfBgl3A was found to be one of the most important β-glucosidases, as evidenced by a 42% reduction in β-glucosidase activity in the ΔPfBgl3A strain. Our results advance the understanding of the genetic and biochemical nature of all β-glucosidases produced by P. funiculosum and pave the way to design a superior biocatalyst for the hydrolysis of lignocellulosic biomass. IMPORTANCE Commercially available cellulases are primarily produced from Trichoderma reesei. However, external supplementation of the cellulase cocktail from this host with exogenous β-glucosidase is often required to achieve the desired optimal saccharification of cellulosic feedstocks. This challenge has led to the exploration of other cellulase-producing strains. The nonmodel hypercellulolytic fungus Penicillium funiculosum has been studied in recent times and identified as a promising source of industrial cellulases mainly due to its ability to produce a balanced concoction of cellulolytic enzymes, including β-glucosidases. Various genetic interventions targeted at strain improvement for cellulase production have been performed; however, the β-glucosidases of this strain have remained largely understudied. This study, therefore, reports profiling of all eight β-glucosidases of P. funiculosum via molecular and computational approaches. The results of this study provide useful insights that will establish the background for future engineering strategies to transform this fungus into an industrial workhorse.

Life cycle inventory of Miscanthus production on a commercial farm in the US.

There has been considerable interest in use of Miscanthus (Miscanthus x giganteus) as a feedstock for bioenergy production due to its potential to reduce greenhouse gas emissions associated with cellulosic feedstock production and more recently for alternative uses as a biomass crop. To date, data on Miscanthus production in the US has been based on small scale research plots due to the lack of commercial scale production fields. Research plot yields are often much higher than commercial fields for a variety of reasons including reduced spatial variability and location on better quality farmland. The objectives of this study were to quantify the inputs for production of Miscanthus at the commercial farm scale, evaluating methods to characterize fuel use for establishment and management of Miscanthus production and using satellite data to characterize spatial yield variation of production fields. We logged energy use on agricultural machinery from Miscanthus production planted on more than 1000 ha of land and modeled N2O emissions and changes in soil carbon using DayCent. Although fuel use was higher for land preparation in fields with perennial vegetation, fuel to harvest Miscanthus dominated greenhouse gas (GHG) emissions (>90%) from agriculture machinery for crop management. The N2O emissions and changes in soil carbon were the largest source and sink of GHG emissions associated with Miscanthus production, respectively. Although ~ 50% of the established lands had Miscanthus yields < 5 Mg/ha, yields needed to be > 5 Mg/ha for ΔSOC to be positive. Given the large impact of yield on ΔSOC, net GHG for Miscanthus production with yields of 5 to 25 Mg/ha ranged ~130 to -260 kg CO2e/Mg biomass. Use of both energy use for Miscanthus harvest and satellite imagery were good methods to characterize spatial variability of commercial production fields. This demonstrates the potential to use this within field yield data to better understand factors driving subfield yield variability and use of satellite data to quantify early yield predictions.

Hydrogen production by a novel Klebsiella pneumoniae strain from sheep rumen uses corn straw as substrate

Lignocellulosic biomass (LB)-based hydrogen-biofuel represents a promising, sustainable, clean, and renewable energy resource for meeting the world's ever-increasing energy demand while reducing environmental problems caused by combustion of fossil energy and accumulation of crop straw. However, due to the lack of strains that can directly produce hydrogen from cheap cellulose feedstocks, the high cost of hydrogen production from biomass fermentation has become a key problem limiting its large-scale production. Therefore, finding fermentation strains that can ferment cellulose feedstocks directly has become the key to overcoming the bottleneck that limits the biomass-based hydrogen production. In this study, we reported for the first time that the Klebsiella pneumoniae Y7-3 cellulose-degrading strain isolated from the rumen of sheep in Inner Mongolia was able to directly produce high-yield hydrogen with cheap straw as raw materials. Anaerobic fermentation with 50 g L−1 of corn straw as the carbon source could produce 1253.72 ± 55.67 mL/L of hydrogen in 24 h. To further improve the hydrogen production from corn straw, some regulatory factors were used. 30 mg/L FeSO4, NiCl2 (70 mg/L) or formic acid (5 g L−1) increased the hydrogen production by 25.72%, 17.89% or 331.07%, respectively. This work provides a new insight for microbial hydrogen production.