U. maydis combines important advantages of yeasts - a non-filamentous growth and resistance to hydromechani- cal stress - with the advantages of filamentous fungi - an inherent utilization of xylose and robustness against impurities from crude biomass feedstock. However, applying beech wood or other plant materials can inter- fere with the required nitrogen limitation. Consequently, composition of the applied material has to be evaluated when “pure” substrates such as glucose are replaced by complex sugar containing raw materials. Although U. maydis has proven itself to be insusceptible to possible impurities from pretreatment, it has been shown that pretreatment can significantly influence the subsequent fermentation process. In this study, online measurements with the RAMOS device have clearly demonstrated that chemical compounds from biomasspretreatment can inhibit microbial growth and the production of the desired product. Consequently, it is important to con- sider that a pretreatment method is only successful, if it does not inhibit the subsequent fermentation. Ultimately, mild pretreatment conditions and robust microbial pro- ducers such as Ustilago maydis will provide a good approach for a successful process development.
Substituting fossil fuels with biofuels can mitigate environmental pollution and climate change (Ho et al., 2019). Currently, more than 98% of the gasoline used in the United States is blended with bioethanol to generate a series of flex fuels such as E85, E15, and E10 for different vehicles (DOE, 2020). More than 95% of ethanol produced in the United States is from corn ethanol, whereas cellulosic ethanol accounts for less than 1% (RFA, 2017). This is because using lignocellulosic biomass for biofuel production still faces technical challenges, including the low coefficient of utilization of both cellulose and hemicellulose due to the chemical structural seal caused by lignin in the outer layer of the biomass cell wall (Ponnusamy et al. 2019). To address this issue, pretreatment is usually required as the first step of cellulosic ethanol production to dismantle the structural seal of lignin and expose more cellulose and hemicellulose to enzymes for saccharification (Kumar et al., 2009). In recent years, various pretreatment methods for lignocellulosic biomass have been developed, such as acid (Kuglarz et al., 2018; Sahoo et al., 2018), alkali (Kang et al., 2018; Tran et al., 2020), liquid hot water (LHW) (Yang et al., 2019), ammonia fiber explosion (Sousa et al., 2019), ionic liquid (Sundstrom et al., 2018), organic solvent (Yu et al., 2018), and physical assisted pretreatments (Bussemaker and Zhang, 2013; Ma et al., 2009), but most of these techniques are still at the laboratory stage. Only the dilute sulfuric acid pretreatment method has been applied in the industrial production of cellulosic ethanol.
An alternative form of this technique has also emerged namely hydrodynamic cavitation, generated by passing a liquid or slurry into a large transverse cavity directed to a very small cavity called a throttle valve which causes the constriction of the suspension. This process produces a pressure drop when it falls below the vapor pressure forming microbubbles that collapse when the pressure returns above the normal vapor pressure values. This collapse produces shock waves and increases the pressure and temperature which ultimately results in cell disruption (Lee and Han, 2015). The two mechanical pretreatment processes most used in the industry in batch or continuous mode for grinding of minerals, ceramics, powders, among other compounds, are the bead mill and the ball mill. In the field of biotechnology, these technologies have already been used for cellular disruption of some microorganisms and are affected by different parameters such as feed rate of the cell suspension, agitation speed, agitator design, diameter and size of the balls, as well as design of the grinding chamber. Besides biomass concentration, density of the suspension, and microorganism morphology, it is also necessary to take into account the interactions of the equipment with the biomass derived from different microorganisms (Montalescot et al., 2015; Postma et al., 2015). Overall, in spite of the effectiveness of these pretreatments for cellular disruption of microalgae biomass, their main drawback is that they do not directly affect the structure of the intracellular carbohydrates, and therefore, a further step would be needed to modify the starch structure. Moreover, these methods are also very energy-intensive. Hence, future studies are still needed and the results obtained through the combination of these methods with others could possibly promote or limit their application.
Methods Using literature data, an LCA of four different pre- treatment methods was carried out. Liquid hot water (LHW), steam explosion (SE), dilute acid (DA), and organosolv (OS) were chosen as the most common techniques with high scal- ability potential. Models were constructed using GaBi soft- ware. A cradle-to-gate analysis was selected with a common model of the corn stover growth and harvesting cycle being combined with the individual models for each pretreatment. Four impact categories were analyzed, and a selection has been discussed based on relevance to the biofuel production process.
This emphasises the fact that the unmodified Bligh and Dyer method was designed for wet samples (Bligh and Dyer, 1959). According to Chun- Zhao et al. (2013), high amounts of water in samples blocked non-polar hexane penetration and resulted in low algal oil recovery. It was observed in the extraction using a mixture of the chloroform: methanol solvent that the lyophilized sample allowed efficient lipid extraction from microalgal biomass. This pretreatment method increased the availability of the sample. This method is also considered as a method for cell disintegration. Kachel-Jakubowska and Szpryngiel (2008) reported in their investigation, that drying is a very important step in post-harvesting, as it has a key role in changing the chemical composition of rapeseed. It is signi- ficant to define the correct drying temperature and eliminate its negative impact on the compositional quality of biomass (Houghton et al., 2009). The drying temperature of 40°C be- fore lipid extraction with the hexane: methanol mixture in- creased the total lipid content relative to 60 and 90°C. These results show a rapid decrease in the content of lipids with increasing drying temperature. As could be seen, crude oil was degraded. When lipids were expozed to higher tempe- rature, oxidation occurred. According to Frankel (1993, 1998), oxidation is one of the most important reactions in lipid che- mistry and this process depends on storage temperature and proceeds slowly at room temperature. However, tempera- ture is one of the physical parameters that affect the rate of reaction. Therefore, the drying temperature had a significant impact on the biomass composition and lipid content (p<0.05). In the chloroform: methanol extraction, no effect of the drying temperature on the crude oil content was ob- served. The dependency of the lipid content on the organic solvent and pretreatment is shown in Fig. 3.
the industry proceed? Certainly, the development of cellulosic fuel refineries with an improved economic viability and environmental footprint will require an inte- grative chain approach. In our vision, the development of advanced lignocellulosic feedstocks for the industry will benefit from parallel developments in enzyme and fer- mentation technologies which maximize the yield and conversion of all fermentable biomass components. In this regard, numerous studies have demonstrated that at mild thermochemical pretreatments, the comple- mentation of cellulolytic cocktails with specialized xylan degrading enzymes greatly improves the release of mon- omeric xylose and enhances cellulose conversion [36, 38, 39]. Similarly, the derivation of pentoses into added-value ethanologens is seen by experts as a crucial step towards improving the productivity and product value of cellu- losic fuels [40–42]. Breeders can simultaneously comple- ment and potentiate these advances by creating cultivars with improved conversion efficiency, higher hemicellu- lose content, and competitive biomass yields. Small-scale cellulosic biomass refineries are constrained by the poor performance figures on economics and environmental efficiency of biomass-to-fuel conversion technologies, as well as the high costs associated with biomass collection and transportation. Conceivably, if improvements in the productivity and cost performance of biomass-to-fuel conversion systems derived from the use of highly digest- ible feedstocks can outweigh the high costs of biomass transportation inherent to small cellulosic ethanol biore- fineries, it should then be possible to realize projections advocating for small-scale biorefineries and the geo- graphic decentralization of cellulosic ethanol production.
Initially, studies focused on the use of PILs, a less studied inexpensive class of ILs. With a simple synthesis procedure and the ability to be distilled, these ILs would be ideal for biomass processing if the proper combination of cation and anion could be discovered. Through this research, it was found that a variety of protic cations with varying structural moieties, with acetate as the counterion, are incapable of dissolving cellulose. The reason for the insolubility is likely due to the increased strength of interaction between the cations and anions (relative to ILs known to be effective at dissolving cellulose), through increased hydrogen bonding, preventing the ions from effectively engaging the hydroxyl groups of cellulose causing dissolution. Surprisingly, a number of aprotic ILs studied with the acetate anion were also incapable of cellulose dissolution suggesting that the cation plays a larger role than previously believed in dissolution. For these ILs, the ability of the cation to hydrogen bond may be too weak to cause the disruption in the cellulose crystalline structure needed for dissolution.
An economic assessment of the biomasspretreatment methods available in literature must be made in order to determine their feasibility when designing an industrial biomass-based process. A quantitative economic analysis of some pretreatment methods is given by Eggeman and Elander (2005). Within this work, five different biomasspretreatment technologies were evaluated in order to compare them in terms of capital costs: dilute acid, hot water, ammonia fibre explosion (AFEX), ammonia recycle percolation (ARP), and lime. As biomass corn stover was used. Two additional pretreatment cases were considered as reference: ‘No Pretreatment’ involved the dilution of the biomass to 20 wt% solids prior to enzymatic hydrolysis. For the ‘Ideal Pretreatment’, the biomass was diluted to 20 wt% prior to hydrolysis, and the yield of glucose and xylose sugars after enzymatic hydrolysis were assumed to be 100% of the theoretical value.
However, the precise mechanism underpinning the facil- itation of biomass deconstruction as catalyzed by iron ions, either in Fe 2+ ion form [1,6] or in ferric ion form (hereinafter referred to as Fe 3+ ) [4-6], remains unknown. Adding to the technical difficulty of studying the role of Fe 2+ ions in biomasspretreatment is its transient nature. Fe 2+ ions can easily be oxidized to Fe 3+ ions by exposure to air. Thus continuous argon or nitrogen gas needs to be used to purge the Fe 2+ ion-containing solutions until it is transferred to a sealed container, such as reactors used in pretreatment. Consequently, there is a scarcity of informa- tion about which components of biomass, a complex matrix of celluloses, hemicelluloses and lignins, are affected by iron cocatalysts and which types of chemical bonds are actively engaged during catalytic deconstruction. The aim of our research was to identify the factors that may contribute to metal-enhanced efficiency during dilute acid/Fe 2+ ion pretreatment and initiate the exploration of their mechanisms by using model cellulose substrates (fil- ter paper (FP) and cotton linter (CL)) as well as model bio- mass feedstock (corn stover). To achieve this goal, we employed high-performance liquid chromatography (HPLC), dinitrosalicylic acid (DNS) assay, Fourier
The choice of mill for mechanical pretreatment can be determined based on biomass moisture content. Knife mills and hammer mills are suitable for dry samples but do little to disrupt cell walls and are generally not used for pretreatment purposes, but are important for reduc- ing particle sizes to increase biomass flowability (Fig. 4). Ball mills, extruders, and disk (disk) mills are the major scalable methods used for pretreatment. These unit oper- ations are scalable and adapted for dry and wet samples (Fig. 4). Ball mills grind using shear and compressive forces. Ball milling reduces cellulose crystallinity as well as particle size [37, 38]. Since the ball milling can be done with high slurry concentration, it reduces reactor volume and capital costs. However, long milling times and high processing costs, including power usage, can make ball milling impractical on an industrial scale . Extrud- ers provide shear force, heating, and mixing, which can achieve thermomechanical and chemical pretreatments at the same time. Single-screw and twin-screw extrud- ers have been widely studied for biomasspretreatment [40, 41]. However, screw extrusion requires a high energy
As discussed before, the hybrid pretreatment method developed here resulted in a process with delignifica- tion yields as high as 86% (as calculated with the Eq. 1). Besides the yield, the percentage of impurities (e.g., cellu- losic and hemicellulosic sugars and ash) in the recovered lignin is also important when considering the utilization of lignin in a biorefinery to produce fuels, chemicals, or materials. The carbohydrate and inorganic ash contents of the different lignin fractions are presented in Fig. 1. Lignin purity remained high throughout the whole range of pretreatment conditions evaluated. Ash content was minimal and did not exceed 0.17% w/w; and the cellulose content remained low, between 0.11% and 0.31% w/w. Hemicellulose sugars were slightly higher, but they never exceeded 3.25%; the lowest carbohydrate content was obtained with 1% sulfuric acid. Overall, purity of > 96% was achieved in all samples, thus offering a very efficient fractionation of high-quality lignin. High purity and low ash content (especially low sulfur content) are unique qualities of organosolv lignin compared to Kraft pulp- ing . Due to the efficient delignification by the novel hybrid pretreatment method, lignin depositions on the biomass were not observed (see “ Assessing the role of explosive discharge” section).
solid with some glucose and xylose contents which were released during the hydrothermal pretreatment. Amounts of glucose and xylose released during the pretreatment were dependent on the process conditions. During the pretreatment part of monomers (hexoses pyranosidic structures and pentoses furanosidic structures) were con- verted into hydroxymethylfurfural (HMF) and furfural. These compounds are considered inhibitory for the fer- mentation. Parameters such as temperature or residence time influence the products through the kinetics of the reaction; therefore knowing the kinetics is a key factor to predict the product yields. Kinetic modeling plays an important role in the design, development, and operation of many chemical processes. Kinetic data are also im- portant in the design and evaluation of the processes to hydrolyze cellulosic materials to glucose for fermenta- tion to ethanol or a variety of other chemical intermedi- ates. Several by-products were formed during the pre- treatment. These included: acetic acid (formed during deacetylation of hemicellulose), furfural (can be de- graded to formic acid) and HMF (can be degraded to formic and levulinic acids). Reaction rate Kc of acetic acid, furfural, and HMF were modeled using data from this study. The kinetic parameters including the activa- tion energy (E) and the constant (A H ) were estimated and
starting material) decreases as a function of increasing biomass loading. Higher quantities of ally guaiacol are produced from low sulfonate alkali lignin (2 g/kg) than from kraft lignin (1 g/kg). Other products like methyl guaiacol, ethyl guaiacol, vinyl guaiacol, vanillin, guaiacyl acetone are also present at smaller concentrations. Pro- duction of these minor products is observed to increase with increases in biomass loading from 3 wt% to 10 wt %, but is observed to decrease on further increasing the biomass loading to 20 wt%. Similar quantities of guaiacyl acetone are produced on dissolution of both kraft lignin and low sulfonate alkali lignin. Higher quantities of ethyl guaiacol, vinyl guaiacol and vanillin were produced from kraft lignin, whereas a higher quantity of methyl guaicol was obtained from low sulfonate alkali lignin. As these technical lignins were derived from softwood [29,30] and contain very small quantities of S-lignin in the original feedstocks, syringyl compounds were not significant. Similar compounds were observed by Stark et al. from the oxidative depolymerization of beech lignin  and by Reichert on electrolysis oxidative cleavage of alkali lignin .
For pretreatment experiments, the concept of reactor severity [24,26,29,30] or severity factor is commonly used to characterize the combination of time and temperature in a given reactor. Severity is a nonlinear combination of reactor temperature and time. We did not use severity in this work. For all experiments the heating and static times in the ASE 350 reactor were held constant (7 minutes and 6 minutes, respectively), while the cell temperature varied. Although the severity factor could be calculated a number of ways (the static time alone, sum of the heating and static times, the sum of the fraction of the heating time above a certain temperature and the static time), the results would be the same for all screening data presented in this work. Because the calculation of severity explicitly excludes any consideration of the ratio of acid to biomass, it is unlikely that other reactor geometries operating at the same severities used in this work (for example, a flow- through reactor with near-instantaneous heating or a microwave system), or even the same reactor system op- erated in a different flow mode would provide identical conversion data. We believe the concept of severity is most useful for interpreting data in a single-reactor geometry and operating mode.
and wheat straw. Altogether, our results confirm that the steam explosion pretreatment represents a versatile pretreatment promoting the glucose release from cellu- lase-catalysed hydrolysis of woody and non-woody spe- cies. The chemical analyses have demonstrated that the pretreatment significantly removes the hemicellulosic fraction, facilitated by the considerable reduction in fer- ulic acids cross-links, greatly contributing towards the improved cellulose accessibility. Hence, the pretreated residues contain mostly cellulose and lignin, regardless the biomass type. The cellulose structure itself is also altered by the pretreatment, especially through changes in crystallinity. The pretreated poplar indeed showed a less ordered cellulose structure while the opposite pat- tern was observed after pretreatment of miscanthus and wheat straw samples in spite of similar enzymatic saccharification performances. Thus, it is clear that the cellulose crystallinity cannot be correlated with the higher susceptibility to cellulase-catalysed hydrolysis observed for the pretreated LCBs. Moreover, signifi- cant modifications of lignin structure and its redistri- bution occur, also favouring the cellulose accessibility due to the most exposed cell wall structure as shown by the microscopy images. In light of our results, a pos- sible redistribution mechanism of lignin can originate from: (i) the homolytic cleavage of β-O-4 ′ interlinkages during pretreatment, leading to their depolymerization while (ii) β-β ′ and β-5 ′ linkages are mostly preserved in the residues, regardless the LCB, suggesting that some reorganization/recondensation reactions also took place after pretreatment. Finally, the pretreatment selectively degraded the S-type lignin fragments in miscanthus and wheat straw samples while the pretreated poplar
The concentration of glucan in the solid fraction and the DM in enzyme hydrolysis are important factors in cellulosic ethanol production as the high viscosity of the fibrous DM fraction determines an upper limit of poten- tial sugars and hence ethanol concentration in the fer- mentation broth. At bioethanol demonstration plants enzymatic hydrolysis is typically operated at a maximum 30% DM . The higher the glucan concentration in the fiber fraction, the higher the potential ethanol concen- tration. The relationship between glucan concentration in the solid fraction and potential ethanol concentration mainly applies to the fermentation of C6 sugars only (when the liquid fraction is separated) and the C5 sugars are used for other purposes such as molasses or biogas production . In principle, high solubilization of the biomass is of course an advantage to the enzymatic li- quefaction process. The presence of less water insoluble fiber reduces the viscosity of the process stream from the HTT pretreatment and reduces complications of en- zymatic hydrolysis at high DM concentrations . However, more severe HTT pretreatments leading to in- creased biomass solubilization also cause a rise in the concentration of inhibitory compounds.
Nowadays, biogas plants are substrates main- ly composed of lignocellulose that is included in such materials as agricultural and forestry wastes, municipal solid waste, and herbaceous energy crops [Bhatia et al. 2017]. Lignocelluloses are composed of cellulose, hemicellulose, lignin, as well as of organic and inorganic compounds. The structure of lignocellulose is such that cellulose forms a skeleton surrounded by hemicellulose and lignin, functioning as a matrix and encrust- ing materials. Cellulose and hemicellulose should be converted to monosaccharides in order to en- hance the fermentation and biogas production. The structure of lignocellulosic biomass hinders its decomposition. The inherent properties of bio- mass are crystallinity of cellulose, its accessible surface area, protection by lignin and hemicellu-
The use of energy crops (maize straw, wheat straw, barley straw etc.) as substrate for renewable energy production (e.g. biogas) is more efficient when it is degraded by different hydrolysis methods. However, fibers contained inside energy crops (e.g. cellulose and hemicellulose) are only hardly and slowly degraded by anaerobic bacteria. The slow degradation of these substances can decrease the methane yields of agricultural biogas plants.In the present study, we investigated the efficiency of combined pretreatment (different concentrations H 2 SO 4 + 30 minutes at 121 0 C)
We previously reported that AHP pretreatment cata- lyzed by copper(II) 2,2΄-bipyridine complexes (Cu(bpy)) can result in significant improvements in the enzymatic digestibility of a number of biomass feedstocks including switchgrass, silver birch, and most notably hybrid poplar . The improved cellulose digestibility correlated with increased lignin removal as well as modifications to cellu- lose that included oxidative depolymerization, the intro- duction of carboxylate groups, and the solubilization and/ or oxidative degradation of only up to 5% of the glucan in the original biomass. In the present manuscript we de- scribe our investigation into the key parameters that impact the effectiveness of Cu(bpy)-catalyzed AHP pre- treatment on hybrid poplar heartwood as quantified by glucose and xylose release during the subsequent enzym- atic digestion of the pretreated biomass. Importantly, we report that the presence of catalytic amounts of Cu(bpy) during AHP pretreatment greatly improves process per- formance and decreases the required H 2 O 2 loading, pre-