Optimising pretreatment methodologies to improve the use of nuisance macroalgal biomass from bloom Ulva spp. prior to enzyme hydrolysis for downstream processing and application

Currently, there is a growing interest in utilising hardwoods as feedstocks for bioethanol production due to the vast advantages they have over other feedstocks for fermentable sugar production. In this study, two selected hardwoods, Acacia and Populus spp., were subjected to two pre-treatment processes (Sodium chlorite delignification and Steam explosion) and compared with respect to how these pre-treatments affect their enzymatic saccharification. Hardwoods were selected for this study, because hardwoods are easier to delignify when compared to softwoods, and therefore their polysaccharides are more easily accessible by enzymes for the purpose of producing fermentable sugars. Currently available commercial enzyme mixtures have been developed for optimal hydrolysis of acid-pre-treated corn stover and are therefore not optimal for saccharification of pre-treated hardwoods. In this work, we attempted the empirical design of a hardwood specific enzyme cocktail, HoloMix. Firstly, a cellulolytic core-set, CelMix (in a ratio of Egl 68%: Cel7A 17%: Cel6A 6%: Bgl1 9%), for the optimal release of glucose, and a xylanolytic core-set, XynMix (in a ratio of Xyn2A 60%: XT6 20%: AguA 11%: SXA 9%), for the optimal release of xylose, were formulated using an empirical enzyme ratio approach after biochemically characterising these enzymes. As it is well ̶ known that biomass pre-treatment may result in the generation of compounds that hamper enzymatic hydrolysis and microbial fermentation, the effects of these compounds on CelMix and XynMix were evaluated. Using the optimised CelMix and XynMix cocktails, a HoloMix cocktail was established for optimal reducing sugar, glucose and xylose release from the various pre-treated hardwoods. For delignified biomass, the optimized HoloMix consisted of CelMix to XynMix at 75% to 25% protein loading, while for the untreated and steam exploded biomass the HoloMix consisted of CelMix to XynMix at 93.75% to 6.25% protein loading. Sugar release by the HoloMix at a loading of 27.5 mg protein/g of biomass (or 55 mg protein/g of glucan) after 24 h gave 70-100% sugar yield. Treatment of the hardwoods with a laccase from Agaricus bisporus, especially wood biomass with a higher proportion of lignin, significantly improved saccharification by the formulated HoloMix enzyme cocktails. This study provided insights into the enzymatic hydrolysis of various pre-treated hardwood substrates and assessed whether the same lignocellulolytic cocktail can be used to efficiently hydrolyse different hardwood species. The present study also demonstrated that the hydrolysis efficiency of the optimised HoloMix was comparable to (if not better) than commercial enzyme preparations during hardwood biomass saccharification.

Microwave assisted pretreatment of eucalyptus sawdust enhances enzymatic saccharification and maximizes fermentable sugar yield

Lignocellulosic biomass (LB) despite its huge potential as a renewable bioenergy resource faces bottlenecks due to its recalcitrance and lack of appropriate pretreatment approaches. The current study evaluates the combinatorial application of alkali and acid pretreatment of pine needle biomass (PNB), for achieving high sugar release upon enzymatic saccharification. Pine needle accumulation poses a big threat to the forest soil fertility and overall ecosystem and environment. However, pine needle waste can be valorized after appropriate pretreatment and enzymatic saccharification for production of renewable energy, i.e. biofuel–ethanol. In combinatorial pretreatment strategy, first PNB was subjected to ammonium carbonate pretreatment, and parameters like ammonium carbonate concentration, incubation time and pretreatment temperature were optimized using design of experiment (DoE) approach. The relative influence of parameters on efficacy of pretreatment was established individually and in interactive terms. Based on DoE, sugar yield of 7.56 mg/g of PNB was obtained. Furthermore, DoE-based pretreated PNB was subjected to sulphuric acid pretreatment, followed by enzymatic saccharification. The sugar released during various steps was pooled (8.19 g/100 g), concentrated and subjected to ethanol fermentation with dual yeast cultures using Saccharomyces cerevisiae and Pichia stipitis. An ethanol yield of 8.8%, v/v (6.94% w/v), was obtained. This represents the process efficiency of 19.34% for bioethanol production from PNB.

Cultivar variation and selection potential relevant to the production of cellulosic ethanol from wheat straw

Bioethanol from the Portuguese forest residue Pterospartum tridentatum – An evaluation of pretreatment strategy for enzymatic saccharification and sugars fermentation

High-efficiency combination washing agents with eco-friendliness simultaneously removing Cd, Cu and Ni from soil of e-waste recycling site: A lab-scale experiment

Preprocessing with air classification, followed by a hybrid biochemical / thermochemical conversion scheme, was utilized to improve the quality of short rotation woody coppice for biofuels production. Air classification improved sugar release during enzymatic hydrolysis by 6-12 % for poplar and willow coppice respectively. Total theoretical sugar release for these hardwood coppices was ~70 %, which suggests that they could be utilized for biochemical conversion. Improved sugar yields after air classification were tied to compositional changes of reduced ash and extractives which can neutralize dilute acid pretreatment and inhibit fermentation. However, air classification was shown to have little to no effect on pyrolytic thermochemical conversion as it removed material without returning a significant improvement in liquid yield. It was also shown that pyrolysis of biochemical conversion lignin rich residue gives liquid yields comparable to whole tree (without any fractionation) pyrolysis, with a higher quality oil that has ~60 % reduced total acid number. Using this combined biochemical / thermochemical conversion strategy can improve yields of fermentable sugars and pyrolysis liquid above 80 %, instead of the 60 % yield of sugars or bio-oil when using a single conversion strategy. Overall, it has been shown that preprocessing and hybrid conversion pathways are a viable strategy for maximizing biorefinery viability.

Pretreatment of biomass is one of the most challenging steps in the process of second-generation (2G) ethanol and biochemical production. Dilute acid pretreatment is a widely adapted and convenient method to recover pentose (C5) as well as hexose (C6) sugars due to its featured solubilization of hemicellulose and cellulose before and after enzymatic saccharification, respectively. In the present study, dilute sulfuric acid (H2SO4) pretreatment of sugarcane bagasse (SCB) was statistically optimized using the face-centered composite design (FCCD) of response surface methodology (RSM) in terms of acid concentration (0.1–3% v/v), solid loading (5–20% w/v) and residence time (15–60 min) at constant temperature of 121 °C followed by enzymatic hydrolysis using commercial cellulase (Novozymes Cellic CTec2) for enhanced combined sugar yield (CSY) comprising of C5 and C6 sugars in pretreated as well as saccharified hydrolysates. Optimized process parameters found in the study were 2.18% (v/v) acid; 14.35% (w/v) solid loading; and 29.49 min residence time. CSY under optimized conditions was found to be 521.42 ± 7.2 g/kg raw SCB with 72.06 ± 1.0% sugars recovered out of the maximum theoretical sugars present in the raw biomass. Total reducing sugar yields in pretreated and saccharified hydrolysates were found to be 215.28 ± 2.4 and 306.14 ± 5.3 g/kg raw SCB, respectively. Morphological and structural changes in optimized pretreated and saccharified biomass further validated the efficiency of optimized pretreatment applied in the present study. The maximum ethanol concentration, volumetric productivity and yield from released sugars were calculated as 10.82 ± 2.2 g/L, 0.45 ± 0.9 g/L/h and 0.42 g/g-glucose consumed or 71.45 ± 2.5 g/kg raw SCB, respectively. Ethanol yield obtained from the fermentation of dilute H2SO4-pretreated SCB was corresponding to 82.4% of the theoretical ethanol yield.

Present study evaluated saccharification potential of marine microalgae N. oculata by employing conventional techniques of hydrolysis and subsequent saccharification often used for lignocellulosic biomass. N.oculata was first hydrolyzed using dilute acid such as, 5% (v/v) sulfuric acid, and 5% (v/v) and 2% (v/v) phosphoric acid at 160C before subjecting to enzymatic saccharification by two commercial cellulases, EI and EII. Neither dilute acid hydrolysis nor enzymatic saccharification alone released any sugars. However, hydrolysates after acid hydrolysis were readily saccharified on addition of enzymes EI or EII. The extent of saccharification ranged between 8 and 100% in all experiments. Sulfuric acid hydrolysis produced furfurals whereas no side products were detected after phosphoric acid hydrolysis. Maximum sugar yield using EI was 345 g sugars/kg ash free dry matter (afdm) within 4 hours whereas EII yielded 360 g sugars/kg afdm within 12 hours. Twice of the nominal enzyme loading facilitated 35% more sugar release and half the nominal enzyme loading yielded 64% less sugars. It was concluded that conventional dilute phosphoric acid hydrolysis followed by enzymatic saccharification using commercially available enzymes could be efficient for saccharification of marine microalgae.

Optimization of cost-effective enzymatic saccharification using low-cost protic ionic liquid as pretreatment agent in Agave bagasse

Edible seaweeds are a promising substrate for microbial fermentations and the development of novel food products. However, the unique composition of their cell wall can limit microbial growth. To address this, the edible seaweed Ulva sp. was subjected to enzymatic saccharification using: (i) a crude extract of Aspergillus oryzae grown on Ulva sp.; (ii) a commercial enzymatic cocktail; and (iii) a combination of both. Saccharification with the latter substantially increased the release of fermentable sugars, with glucose (157.08mg/gsubstrate) and galacturonic acid (153.83mg/gsubstrate) as the dominant products, indicating disruption of key structural polysaccharides. Follow-up fermentations with Lactiplantibacillus plantarum and Cyberlindnera jadinii (GRAS microorganisms) exhibited a two-log growth increase compared to cultures in non-saccharified media. Saccharification also led to increased acidification and free amino nitrogen release by L. plantarum, and an increased nitrogen intake by C. jadinii, changes directly relevant to food fermentations. Despite these advances, the limited release of certain sugars suggests that some cell wall components remain resistant to hydrolysis. Overall, this work highlights enzymatic saccharification as a key enabling step for converting Ulva sp. into a viable and functional substrate for microbial fermentation and novel food development. KEY POINTS: • A. oryzae extract combined with cellulolytic enzymes led to highest sugar yield. • Saccharification improved growth, FAN release, and acidification by L. plantarum. • Evidence of cellulose hydrolysis but not of the remaining cell wall polysaccharides.

This paper examines the efficacy of ionic liquid (IL) pretreatment on seven different commercially harvested biomass types: corn stover, miscanthus, pine, sorghum, sugarcane bagasse, switchgrass, and wheat straw in an effort to improve the production of renewable fuels and chemicals from biomass derived sugars. Initial experiments screened the pretreatment of lodgepole pine, a particularly recalcitrant biomass feedstock, with nine different imidazolium based ionic liquids. After screening, one hydrophilic and one hydrophobic ionic liquid was selected for pretreatment tests on six commercially harvested biomasses. Ultimately, the hydrophilic ionic liquid functioned better for biomass pretreatment than the hydrophobic ionic liquid. These results were then compared to a traditional dilute acid pretreatment to examine the relative effectiveness of ionic liquid pretreatment across a variety of biomass and ionic liquid types. Total theoretical sugar yields after IL pretreatment varied widely by IL and biomass type and ranged from 4.9 to 90.2%. Dilute acid pretreatment showed consistent sugar yields for herbaceous material (from 71.4 to 80.8%) but low yield for lodgepole pine (22.8%). Overall, ILs showed the potential to reach slightly higher sugar yields than dilute acid and were particularly effective for woody feedstocks. More importantly, the sugar release kinetics for IL pretreatment were three times faster than dilute acid and gave maximum sugar yields after about 24 hours. Additional characterization of IL treated materials included scanning electron microscopy (SEM), x-ray diffraction (XRD), and compositional analysis. SEM and XRD showed qualitative and quantitative reductions in cellulose crystallinity (respectively) that correlated well to improved sugar release during enzymatic hydrolysis for hydrophilic ionic liquids. However, reductions in crystallinity associated with hydrophobic ionic liquids resulted in lower sugar release during enzymatic hydrolysis. Compositional analysis generally showed increased sugars content for hydrophilic ILs and increased lignin content for hydrophobic ILs.

In this study detoxification of 3 Chinese Cd-contaminated cultivars (Jinyou463, Yuchi, and Xiangzaoxian 32) of rice was explored. By separation with an alkaline method, Cd concentrations of the starch isolates were decreased from 0.2769, 0.4037, and 0.5156 mg/kg in starting milled rice to 0.1056, 0.1585, and 0.1923 mg/kg, respectively. However, the Cd concentrations reached up to 2.5905, 3.1628, and 4.8593 mg/kg in the protein isolates, respectively. Therefore, 10 common acids in food industry were investigated to remove Cd from protein isolate by washing process. The optimal washing conditions were 0.5 M citric acid, acid to rice protein isolate ratio of 6:1 v/w, shaking time of 1 h at room temperature. The rice protein isolate showed a significant decrease in Cd concentration and the removal efficiency was more than 95% after 2 washings at optimized conditions. Rice proteins were not degraded at all and had very little loss during citric acid washing process. The study presents a promising way of depurating Cd-contaminated rice, and meanwhile it reduces the risk of heavy metal causing food safety issues effectively.

BackgroundThe development of affordable woody biomass feedstocks represents a significant opportunity in the development of cellulosic biofuels. Primary woodchips produced by forest mills are considered an ideal feedstock, but the prices they command on the market are currently too expensive for biorefineries. In comparison, forestry residues represent a potential low-cost input but are considered a more challenging feedstock for sugar production due to complexities in composition and potential contamination arising from soil that may be present. We compare the sugar yields, changes in composition in Douglas-fir woodchips and forestry residues after pretreatment using ionic liquids and enzymatic saccharification in order to determine if this approach can efficiently liberate fermentable sugars.ResultsThese samples were either mechanically milled through a 2 mm mesh or pretreated as received with the ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate [C2mim][OAc] at 120°C and 160°C. IL pretreatment of Douglas-fir woodchips and forestry residues resulted in approximately 71-92% glucose yields after enzymatic saccharification. X-ray diffraction (XRD) showed that the pretreated cellulose was less crystalline after IL pretreatment as compared to untreated control samples. Two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) revealed changes in lignin and hemicellulose structure and composition as a function of pretreatment. Mass balances of sugar and lignin streams for both the Douglas-fir woodchips and forestry residues throughout the pretreatment and enzymatic saccharification processes are presented.ConclusionsWhile the highest sugar yields were observed with the Douglas-fir woodchips, reasonably high sugar yields were obtained from forestry residues after ionic liquid pretreatment. Structural changes to lignin, cellulose and hemicellulose in the woodchips and forestry residues of Douglas-fir after [C2mim][OAc] pretreatment are analyzed by XRD and 2D-NMR, and indicate that significant changes occurred. Irrespective of the particle sizes used in this study, ionic liquid pretreatment successfully allowed high glucose yields after enzymatic saccharification. These results indicate that forestry residues may be a more viable feedstock than previously thought for the production of biofuels.

Recalcitrance of lignocellulosic cell wall is a major hurdle that needs to be disrupted for biochemical refinery to be successful. Pretreatment of lignocellulosic materials play a key role for the conversion of lignocellulosic feed stocks to fermentable sugars. At present, various pretreatment technologies mainly achieved by empirical methods are available for the production of sugars from lignocellulosic polysaccharides. These methods depend largely upon the "demolition" of the cell wall by strong chemicals or using high energy input. As a result, these processes release "smaller compounds" as by-products or end products which inhibits downstream fermentation of monomeric sugars.;This project is a preliminary study to understand the combined effect of mechanical action and enzyme hydrolysis on the yield of sugars from two lignocellulosic biomass viz, hard maple (Acer saccharum Marsh.) and corn stover (Zea mays L.). The mechanical treatment involves wet attrition milling and the application electron beams. The enzyme treatments are done by using six cell wall depolymerizing enzymes. The amounts of total sugars release after each treatment are measured by Dinitrosalicylic acid (DNS) assay and types of sugars release were determined by High Performance Liquid Chromatography (HPLC) analysis. Particle size were measured by Microtract particle size analyzer and also by image analysis. Treatments that required minimum number of enzymes to produce significantly higher amount of sugars were selected for the studying the effect of increased enzyme concentration on the amount of sugars released. Further, the residues from enzyme hydrolysis are analyzed by ATR-FTIR and NMR to understand the degree of depolymerization of cell wall biopolymers. Depolymerizing properties of electron beams on corn stover particulates were also studied. The effects of irradiation on these specimens were evaluated by estimating the amount of total sugars released after the treatment with AccelleraseRTM 1000.;This study has shown that the enzymatic digestibility of a pretreated hard maple and corn stover are enhanced by reducing the particle size. A significant increase in the yield of sugars were also observed in the treatments that were pretreated with CDTA (1,2-Cyclohexylenedi nitrilo-tetraacetic Acid) and BSA (Bovine Serum Albumin). Presence of glucose and xylose were confirmed by HPLC (High Performance Liquid Chromatography) analysis. Lower sugar yields were measured after the treatment corn stover with electron beams. Future studies are required to understand the effect of the sequence of the enzymes for a higher sugar yield and also to combine other pretreatment methods with electron beams for a better deconstruction of