An update on enzymatic cocktails for lignocellulose breakdown

Alternative energy sources have received increasing attention in recent years. The possibility of adding value to agricultural wastes, by producing biofuels and other products with economic value from lignocellulosic biomass by enzymatic hydrolysis, has been widely explored. Lignocellulosic biomass, as well as being an abundant residue, is a complex recalcitrant structure that requires a consortium of enzymes for its complete degradation. Pools of enzymes with different specificities acting together usually produce an increase in hydrolysis yield. Enzymatic cocktails have been widely studied due to their potential industrial application for the bioconversion of lignocellulosic biomass. This review presents an overview of enzymes required to degrade the plant cell wall, paying particular attention to the latest advances in enzymatic cocktail production and the main results obtained with cocktails used to degrade a variety of types of biomass, as well as some future perspectives within this field.     

Use of lignocellulose biomass for endoxylanase production by Streptomyces termitum

Actinobacteria isolates from Brazilian Cerrado soil were evaluated for their ability to produce enzymes of the cellulolytic and xylanolytic complex using lignocellulose residual biomass. Preliminary semiquantitative tests, made in Petri plates containing carboxymethylcellulose and beechwood xylan, indicated 11 potential species producing enzymes, all belonging to the genus Streptomyces. The species were subsequently grown in pure substrates in submerged fermentation and analyzed for the production of enzymes endoglucanase, β-glucosidase, endoxylanase, and β-xylosidase. The best results were obtained for endoxylanase enzyme production with Streptomyces termitum(UFLA CES 93). The strain was grown on lignocellulose biomass (bagasse, straw sugarcane, and cocoa pod husk) that was used in natura or acid pretreated. The medium containing sugarcane bagasse in natura favored the production of the endoxylanase that was subsequently optimized through an experimental model. The highest enzyme production 0.387?U?mL^?1, (25.8 times higher), compared to the lowest value obtained in one of the trials, was observed when combining 2.75% sugar cane bagasse and 1.0?g?L^?1 of yeast extract to the alkaline medium (pH 9.7). This is the first study using S. termitum as a producer of endoxylanase.     

Hydrodynamic cavitation as a strategy to enhance the efficiency of lignocellulosic biomass pretreatment

Hydrodynamic cavitation (HC) is a process technology with potential for application in different areas including environmental, food processing, and biofuels production. Although HC is an undesirable phenomenon for hydraulic equipment, the net energy released during this process is enough to accelerate certain chemical reactions. The application of cavitation energy to enhance the efficiency of lignocellulosic biomass pretreatment is an interesting strategy proposed for integration in biorefineries for the production of bio-based products. Moreover, the use of an HC-assisted process was demonstrated as an attractive alternative when compared to other conventional pretreatment technologies. This is not only due to high pretreatment efficiency resulting in high enzymatic digestibility of carbohydrate fraction, but also, by its high energy efficiency, simple configuration, and construction of systems, besides the possibility of using on the large scale. This paper gives an overview regarding HC technology and its potential for application on the pretreatment of lignocellulosic biomass. The parameters affecting this process and the perspectives for future developments in this area are also presented and discussed.     

Dilute acid pretreatment and enzymatic saccharification of sugarcane tops for bioethanol production

The aim of this work was to study the feasibility of using sugarcane tops as feedstock for the production of bioethanol. The process involved the pretreatment using acid followed by enzymatic saccharification using cellulases and the process was optimized for various parameters such as biomass loading, enzyme loading, surfactant concentration and incubation time using Box–Behnken design. Under optimum hydrolysis conditions, 0.685 g/g of reducing sugar was produced per gram of pretreated biomass. The fermentation of the hydrolyzate using Saccharomyces cerevisae produced 11.365 g/L of bioethanol with an efficiency of about 50%. This is the first report on utilization of sugarcane tops for bioethanol production.     

Enhanced enzymatic hydrolysis of rapeseed straw by popping pretreatment for bioethanol production

The objective of this study was to find a pretreatment process that enhances enzymatic conversion of biomass to sugars. Rapeseed straw was pretreated by two processes: a wet process involving wet milling plus a popping treatment, and a dry process involving popping plus dry milling. The effects of the pretreatments were studied both in terms of structural and compositional changes and change in susceptibility to enzymatic hydrolysis. After application of the wet and dry processes, the amounts of cellulose and xylose in the straw were 37-38% and 14-15%, respectively, compared to 31% and 12% in untreated counterparts. In enzymatic hydrolysis performance, the wet process presented the best glucose yield, with a 93.1% conversion, while the dry process yielded 69.6%, and the un-pretreated process yielded <20%. Electron microscopic studies of the straw also showed a relative increase in susceptibility to enzymatic hydrolysis with pretreatment.     

Lactic acid production from sugarcane bagasse hydrolysates by Lactobacillus pentosus: Integrating xylose and glucose fermentation

Lactic acid, traditionally obtained through fermentation process, presents numerous applications in different industrial segments, including production of biodegradable polylactic acid (PLA). Development of low cost substrate fermentations could improve economic viability of lactic acid production, through the use of agricultural residues as lignocellulosic biomass. Studies regarding the use of sugarcane bagasse hydrolysates for lactic acid production by Lactobacillus spp. are reported. First, five strains of Lactobacillus spp. were investigated for one that had the ability to consume xylose efficiently. Subsequently, biomass fractionation was performed by dilute acid and alkaline pretreatments, and the hemicellulose hydrolysate (HH) fermentability by the selected strain was carried out in bioreactor. Maximum lactic acid concentration and productivity achieved in HH batch were 42.5 g/L and 1.02 g/L h, respectively. Hydrolyses of partially delignified cellulignin (PDCL) by two different enzymatic cocktails were compared. Finally, fermentation of HH and PDCL hydrolysate together was carried out in bioreactor in a hybrid process: saccharification and co-fermentation with an initial enzymatic hydrolysis. The high fermentability of these process herein developed was demonstrated by the total consumption of xylose and glucose by Lactobacillus pentosus, reaching at 65.0 g/L of lactic acid, 0.93 g/g of yield, and 1.01 g/L h of productivity. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2718, 2019     

Microwave pretreatment of rape straw for bioethanol production: focus on energy efficiency

The energy efficiency of microwave-assisted dilute sulfuric acid pretreatment of rape straw for the production of ethanol was investigated. Different microwave energy inputs and solid loadings were tested to find economic pretreatment conditions. The lowest energy consumption was observed when solid loading and energy input were fixed at 50% (w/w) and 54 kJ (900 W for 1 min), respectively, and amounted to 5.5 and 10.9 kJ to produce 1 g of glucose after enzymatic hydrolysis and 1 g ethanol after fermentation, respectively. In general, 1 g ethanol can produce about 30 kJ of energy, and therefore, the energy input for the pretreatment was only 35% of the energy output. The approach developed in this study resulted in 92.9% higher energy savings for producing 1 g ethanol when compared with the results of microwave pretreatments previously reported.     

Renewable bio ionic liquids‐water mixtures‐mediated selective removal of lignin from rice straw: Visualization of changes in composition and cell wall structure

Pretreatment of rice straw by using renewable cholinium amino acids ionic liquids ([Ch][AA] ILs)‐water mixtures and the subsequent enzymatic hydrolysis of the residues were conducted in the present work. Of the eight mixtures composed of ILs and water, most were found to be effective for rice straw pretreatment. After pretreatment with 50% ILs‐water mixtures, the enzymatic digestion of the lignocellulosic biomass was enhanced significantly, thus leading to satisfactory sugar yields of >80% for glucose and approximately 50% for xylose. To better understand the ILs pretreatment mechanism, confocal laser scanning microscopy combined with immunolabeling and transmission electron microscopy were used to visualize changes in the contents and distribution of two major components—lignin and xylan. The results coupled with changes in chemical structures (infrared spectra) of the substrates indicated occurrence of extensive delignification, especially in cell corner and compound middle lumen of cell walls, which made polysaccharides more accessible to enzymes. This pretreatment process is promising for large‐scale application because of the high sugar yields, easy handling, being environmentally benign and highly tolerant to moisture, and significantly reduced cost and energy consumption. Biotechnol. Bioeng. 2013; 110: 1895–1902. © 2013 Wiley Periodicals, Inc.     

预处理对木质纤维素生物质细胞壁超微结构的影响

预处理是提高木质纤维素生物质向生物燃料转化的有效途径,但生物质的天然抗降解屏障严重阻碍了这一转化的进行,因此全面了解预处理过程中植物细胞壁的微观结构及区域化学变化是实现农林生物质高效转化的基础。本文总结了多种预处理方法对植物细胞壁超微结构影响的研究进展,对生物质科学研究可能有一定的促进和指导作用。     

Inhibition analysis of inhibitors derived from lignocellulose pretreatment on the metabolic activity of Zymomonas mobilis biofilm and planktonic cells and the proteomic responses

Lignocellulose pretreatment produces various toxic inhibitors that affect microbial growth, metabolism, and fermentation. Zymomonas mobilis is an ethanologenic microbe that has been demonstrated to have potential to be used in lignocellulose biorefineries for bioethanol production. Z. mobilis biofilm has previously exhibited high potential to enhance ethanol production by presenting a higher viable cell number and higher metabolic activity than planktonic cells or free cells when exposed to lignocellulosic hydrolysate containing toxic inhibitors. However, there has not yet been a systematic study on the tolerance level of Z. mobilis biofilm compared to planktonic cells against model toxic inhibitors derived from lignocellulosic material. We took the first insight into the concentration of toxic compound (formic acid, acetic acid, furfural, and 5‐HMF) required to reduce the metabolic activity of Z. mobilis biofilm and planktonic cells by 25% (IC₂₅), 50% (IC₅₀), 75% (IC₇₅), and 100% (IC₁₀₀). Z. mobilis strains ZM4 and TISTR 551 biofilm were two‐ to three fold more resistant to model toxic inhibitors than planktonic cells. Synergetic effects were found in the presence of formic acid, acetic acid, furfural, and 5‐HMF. The IC₂₅ of Z. mobilis ZM4 biofilm and TISTR 551 biofilm were 57 mm formic acid, 155 mm acetic acid, 37.5 mm furfural and 6.4 mm 5‐HMF, and 225 mm formic acid, 291 mm acetic acid, 51 mm furfural and 41 mm 5‐HMF, respectively. There was no significant difference found between proteomic analysis of the stress response to toxic inhibitors of Z. mobilis biofilm and planktonic cells on ZM4. However, TISTR 551 biofilms exhibited two proteins (molecular chaperone DnaK and 50S ribosomal protein L2) that were up‐regulated in the presence of toxic inhibitors. TISTR 551 planktonic cells possessed two types of protein in the group of 30S ribosomal proteins and motility proteins that were up‐regulated.     

Microalgal hydrogen production: prospects of an essential technology for a clean and sustainable energy economy

Modern energy production is required to undergo a dramatic transformation. It will have to replace fossil fuel use by a sustainable and clean energy economy while meeting the growing world energy needs. This review analyzes the current energy sector, available energy sources, and energy conversion technologies. Solar energy is the only energy source with the potential to fully replace fossil fuels, and hydrogen is a crucial energy carrier for ensuring energy availability across the globe. The importance of photosynthetic hydrogen production for a solar-powered hydrogen economy is highlighted and the development and potential of this technology are discussed. Much successful research for improved photosynthetic hydrogen production under laboratory conditions has been reported, and attempts are underway to develop upscale systems. We suggest that a process of integrating these achievements into one system to strive for efficient sustainable energy conversion is already justified. Pursuing this goal may lead to a mature technology for industrial deployment.     

Combined Cell Surface Display of β‐d‐Glucosidase (BGL), Maltose Transporter (MAL11), and Overexpression of Cytosolic Xylose Reductase (XR) in Saccharomyces cerevisiae Enhance Cellobiose/Xylose Coutilization for Xylitol Bioproduction from Lignocellulosic Biomass

Xylitol is a highly valuable commodity chemical used extensively in the food and pharmaceutical industries. The production of xylitol from d ‐xylose involves a costly and polluting catalytic hydrogenation process. Biotechnological production from lignocellulosic biomass by micro‐organisms like yeasts is a promising option. In this study, xylitol is produced from lignocellulosic biomass by a recombinant strain of Saccharomyces cerevisiae (S. cerevisiae) (YPH499‐SsXR‐AaBGL) expressing cytosolic xylose reductase (Scheffersomyces stipitis xylose reductase [SsXR]), along with a β‐d ‐glucosidase (Aspergillus aculeatus β‐glucosidase 1 [AaBGL]) displayed on the cell surface. The simultaneous cofermentation of cellobiose/xylose by this strain leads to an ≈2.5‐fold increase in Yxylitol/xylose (=0.54) compared to the use of a glucose/xylose mixture as a substrate. Further improvement in the xylose uptake by the cell is achieved by a broad evaluation of several homologous and heterologous transporters. Homologous maltose transporter (ScMAL11) shows the best performance in xylose transport and is used to generate the strain YPH499‐XR‐ScMAL11‐BGL with a significantly improved xylitol production capacity from cellobiose/xylose coutilization. This report constitutes a promising proof of concept to further scale up the biorefinery industrial production of xylitol from lignocellulose by combining cell surface and metabolic engineering in S. cerevisiae.     

Sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) for robust enzymatic saccharification of hardwoods

This study demonstrates sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) for robust bioconversion of hardwoods. With only about 4% sodium bisulfite charge on aspen and 30‐min pretreatment at temperature 180°C, SPORL can achieve near‐complete cellulose conversion to glucose in a wide range of pretreatment liquor of pH 2.0–4.5 in only about 10 h enzymatic hydrolysis. The enzyme loading was about 20 FPU cellulase plus 30 CBU β‐glucosidase per gram of cellulose. The production of fermentation inhibitor furfural was less than 20 mg/g of aspen wood at pH 4.5. With pH 4.5, SPORL avoided reactor corrosion problem and eliminated the need for substrate neutralization prior to enzymatic hydrolysis. Similar results were obtained from maple and eucalyptus. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Supercritical carbon dioxide pretreatment of corn stover and switchgrass for lignocellulosic ethanol production

Supercritical CO₂ (SC-CO₂), a green solvent suitable for a mobile lignocellulosic biomass processor, was used to pretreat corn stover and switchgrass at various temperatures and pressures. The CO₂ pressure was released as quickly as possible by opening a quick release valve during the pretreatment. The biomass was hydrolyzed after pretreatment using cellulase combined with β-glucosidase. The hydrolysate was analyzed for the amount of glucose released. Glucose yields from corn stover samples pretreated with SC-CO₂ were higher than the untreated sample’s 12% glucose yield (12 g/100 g dry biomass) and the highest glucose yield of 30% was achieved with SC-CO₂ pretreatment at 3500 psi and 150 °C for 60 min. The pretreatment method showed very limited improvement (14% vs. 12%) in glucose yield for switchgrass. X-ray diffraction results indicated no change in crystallinity of the SC-CO₂ treated corn stover when compared to the untreated, while SEM images showed an increase in surface area.     

An integrated assessment of the potential of agricultural and forestry residues for energy production in China

Biomass has been widely recognized as an important energy source with high potential to reduce greenhouse gas emissions while minimizing environmental pollution. In this study, we employ the Global Change Assessment Model to estimate the potential of agricultural and forestry residue biomass for energy production in China. Potential availability of residue biomass as an energy source was analyzed for the 21st century under different climate policy scenarios. Currently, the amount of total annual residue biomass, averaged over 2003–2007, is around 15 519 PJ in China, consisting of 10 818 PJ from agriculture residues (70%) and 4701 PJ forestry residues (30%). We estimate that 12 693 PJ of the total biomass is available for energy production, with 66% derived from agricultural residue and 34% from forestry residue. Most of the available residue is from south central China (3347 PJ), east China (2862 PJ) and south‐west China (2229 PJ), which combined exceeds 66% of the total national biomass. Under the reference scenario without carbon tax, the potential availability of residue biomass for energy production is projected to be 3380 PJ by 2050 and 4108 PJ by 2095, respectively. When carbon tax is imposed, biomass availability increases substantially. For the CCS 450 ppm scenario, availability of biomass increases to 9002 PJ (2050) and 11 524 PJ (2095), respectively. For the 450 ppm scenario without CCS, 9183 (2050) and 11 150 PJ (2095) residue biomass, respectively, is projected to be available. Moreover, the implementation of CCS will have a little impact on the supply of residue biomass after 2035. Our results suggest that residue biomass has the potential to be an important component in China's sustainable energy production portfolio. As a low carbon emission energy source, climate change policies that involve carbon tariff and CCS technology promote the use of residue biomass for energy production in a low carbon‐constrained world.     

Utilization of sugarcane bagasse for bioethanol production: sono-assisted acid hydrolysis approach

In this study, the production of sugar monomers from sugarcane bagasse (SCB) by sono-assisted acid hydrolysis was performed. The SCB was subjected to sono-assisted alkaline pretreatment. The cellulose and hemicellulose recovery observed in the solid content was 99% and 78.95%, respectively and lignin removal observed during the pretreatment was about 75.44%. The solid content obtained was subjected to sono-assisted acid hydrolysis. Under optimized conditions, the maximum hexose and pentose yield observed was 69.06% and 81.35% of theoretical yield, respectively. The hydrolysate obtained was found to contain very less inhibitors, which improved the bioethanol production and the ethanol yield observed was 0.17 g/g of pretreated SCB.     

Selectivity and delignification kinetics for oxidative short‐term lime pretreatment of poplar wood,part I: Constant‐pressure

Kinetic models applied to oxygen bleaching of paper pulp focus on the degradation of polymers, either lignin or carbohydrates. Traditionally, they separately model different moieties that degrade at three different rates: rapid, medium, and slow. These models were successfully applied to lignin and carbohydrate degradation of poplar wood submitted to oxidative pretreatment with lime at the following conditions: temperature 110–180°C, total pressure 7.9–21.7 bar, and excess lime loading of 0.5 g Ca(OH)₂ per gram dry biomass. These conditions were held constant for 1–6 h. The models properly fit experimental data and were used to determine pretreatment selectivity in two fashions: differential and integral. By assessing selectivity, the detrimental effect of pretreatment on carbohydrates at high temperatures and at low lignin content was determined. The models can be used to identify pretreatment conditions that selectively remove lignin while preserving carbohydrates. Lignin removal ≥50% with glucan preservation ≥90% was observed for differential glucan selectivities between ～10 and ～30 g lignin degraded per gram glucan degraded. Pretreatment conditions complying with these reference values were preferably observed at 140°C, total pressure ≥14.7 bars, and for pretreatment times between 2 and 6 h depending on the total pressure (the higher the pressure, the less time). They were also observed at 160°C, total pressure of 14.7 and 21.7 bars, and pretreatment time of 2 h. Generally, at 110°C lignin removal is insufficient and at 180°C carbohydrates do not preserve well. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011