Formulation of an enzyme cocktail, HoloMix, using cellulolytic and xylanolytic enzyme core-sets for effective degradation of various pre-treated hardwoods

2018 ◽  
Author(s):  
◽  
Samkelo Malgas
Keyword(s):  
Enzymatic Hydrolysis ◽  
Enzymatic Saccharification ◽  
Protein G ◽  
Sodium Chlorite ◽  
Commercial Enzyme ◽  
Protein Loading ◽  
Core Set ◽  
Enzyme Cocktail ◽  
Pre Treatment ◽  
Hydrolysis Of

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.

An evaluation of synergistic interactions between feruloyl esterases and xylanases during the hydrolysis of various pre-treated agricultural residues

2021 ◽  
Author(s):  
◽  
Lithalethu Mkabayi
Keyword(s):  
Enzymatic Hydrolysis ◽  
Reducing Sugars ◽  
Value Added ◽  
Agricultural Residues ◽  
Corn Cobs ◽  
Synergistic Interactions ◽  
Enzyme Cocktail ◽  
Enzyme Combination ◽  
Pre Treatment ◽  
Hydrolysis Of

Agricultural residues are readily available and inexpensive renewable resources that can be used as raw materials for the production of value-added chemicals. The application of enzymes to facilitate the degradation of agricultural residues has long been considered the most environmentally friendly strategy for converting this material into good quality value-added chemicals. However, agricultural residues are typically lignocellulosic in composition and recalcitrant to enzymatic hydrolysis. Due to this recalcitrant nature, the complete degradation of biomass residues requires the synergistic action of a broad range of enzymes. The development and optimisation of synergistic enzyme cocktails is an effective approach for achieving high hydrolysis efficiency of lignocellulosic biomass. The aim of the current study was to evaluate the synergistic interactions between two termite metagenome-derived feruloyl esterases (FAE6 and FAE5) and endo-xylanases for the production of hydroxycinnamic acids and xylo-oligosaccharides (XOS) from model substrates, and untreated and pre-treated agricultural residues. Firstly, the two fae genes were heterologously expressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The biochemical properties of the purified recombinant FAEs and xylanases (XT6 and Xyn11) were then assessed to determine the factors which influenced their activities and to select suitable operating conditions for synergy studies. An optimal protein loading ratio of xylanases to FAEs required to maximise the release of both reducing sugar and ferulic acid (FA) was established using 0.5% (w/v) insoluble wheat arabinoxylan (a model substrate). The enzyme combination of 66% xylanase and 33% FAE (on a protein loading basis) produced the highest amounts of reducing sugars and FA. The enzyme combination of XT6 (GH10 xylanase) and FAE5 or FAE6 liberated the highest amount of FA while a combination of Xyn11 (GH11 xylanase) and FAE5 or FAE6 produced the highest reducing sugar content. The synergistic interactions which were established between the xylanases and FAEs were further investigated using agricultural residues (corn cobs, rice straw and sugarcane bagasse). The three substrates were subjected to hydrothermal and dilute acid pre-treatment prior to synergy studies. It is generally known that, during pre-treatment, many compounds can be produced which may influence enzymatic hydrolysis. The effects of these by-products were assessed and it was found that lignin and its degradation products were the most inhibitory to the FAEs. The optimised enzyme cocktail was then applied to 1% (w/v) of untreated and pre-treated substrates for the efficient production of XOS and hydroxycinnamic acids. A significant improvement in xylanase substrate degradation was observed, especially with the combination of 66% Xyn11 and 33% FAE6 which displayed an improvement in reducing sugars of approximately 1.9-fold and 3.4-fold for hydrothermal and acid pre-treated corn cobs (compared to when Xyn11 was used alone), respectively. The study demonstrated that pre-treatment substantially enhanced the enzymatic hydrolysis of corn cobs and rice straw. Analysis of the hydrolysate product profiles revealed that the optimised enzyme cocktail displayed great potential for releasing XOS with a low degree of polymerisation. In conclusion, this study provided significant insights into the mechanism of synergistic interactions between xylanases and metagenome-derived FAEs during the hydrolysis of various substrates. The study also demonstrated that optimised enzyme cocktails combined with low severity pre-treatment can facilitate the potential use of xylan-rich lignocellulosic biomass for the production of valuable products in the future.

scholarly journals Improvement of Enzymatic Saccharification of Cellulose-Containing Raw Materials Using Aspergillus niger

Processes ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 1360
Author(s):  
Ekaterina Budenkova ◽  
Stanislav Sukhikh ◽  
Svetlana Ivanova ◽  
Olga Babich ◽  
Vyacheslav Dolganyuk ◽  
...  
Keyword(s):  
Enzymatic Hydrolysis ◽  
Aspergillus Niger ◽  
Filter Paper ◽  
Raw Materials ◽  
Enzymatic Saccharification ◽  
Wood Chip ◽  
Cellulase Activity ◽  
Wood Chips ◽  
Acid Pretreatment ◽  
Hydrolysis Of

Enzymatic hydrolysis of cellulose-containing raw materials, using Aspergillus niger, were studied. Filter paper, secondary cellulose-containing or starch-containing raw materials, miscanthus cellulose after alkaline or acid pretreatment, and wood chip cellulose, were used as substrates. The study focused on a wild A. niger strain, treated, or not (control), by ultraviolet (UV) irradiations for 45, 60, or 120 min (UV45, UV60, or UV120), or by UV irradiation for 120 min followed by a chemical treatment with NaN3 + ItBr for 30 min or 80 min (UV120 + CH30 or UV120 + CH80). A mixture of all the A. niger strains (MIX) was also tested. A citrate buffer, at 50 mM, wasthe most suitable for enzymatic hydrolysis. As the UV exposure time increased to 2 h, the cellulase activity of the surviving culturewas increased (r = 0.706; p < 0.05). The enzymatic activities of the obtained strains, towards miscanthus cellulose, wood chips, and filter paper, were inferior to those obtained with commercial enzymes (8.6 versus 9.1 IU), in some cases. Under stationary hydrolysis at 37 °C, pH = 4.7, the enzymatic activity of A. niger UV120 + CH30 was 24.9 IU. The enzymatic hydrolysis of secondary raw materials, using treated A. niger strains, was themost effective at 37 °C. Similarly, the most effective treatment of miscanthus cellulose and wood chips occurred at 50 °C. The maximum conversion of cellulose to glucose was observed using miscanthus cellulose (with alkaline pretreatment), and the minimum conversion was observed when using wood chips. The greatest value of cellulase activity was evidenced in the starch-containing raw materials, indicating that A. niger can ferment not only through cellulase activity, but also via an amylolytic one.

Pre-treatment and enzymatic hydrolysis of lettuce residues as feedstock for bio-butanol production

2017 ◽  
Vol 96 ◽  
pp. 172-179 ◽  
Author(s):  
Alessandra Procentese ◽  
Francesca Raganati ◽  
Giuseppe Olivieri ◽  
Maria Elena Russo ◽  
Antonio Marzocchella
Keyword(s):  
Enzymatic Hydrolysis ◽  
Butanol Production ◽  
Pre Treatment ◽  
Hydrolysis Of

scholarly journals Effects of Biosurfactants on Enzymatic Saccharification and Fermentation of Pretreated Softwood

Molecules ◽  
2020 ◽  
Vol 25 (16) ◽  
pp. 3559 ◽  
Author(s):  
Alfredo Oliva-Taravilla ◽  
Cristhian Carrasco ◽  
Leif J. Jönsson ◽  
Carlos Martín
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pseudomonas Aeruginosa ◽  
Enzymatic Hydrolysis ◽  
Enzymatic Saccharification ◽  
Horse Chestnut ◽  
Yeast Growth ◽  
Cellulose Conversion ◽  
Enzymatic Hydrolysis Of Cellulose ◽  
Hydrolysis Of Cellulose ◽  
Hydrolysis Of

The enzymatic hydrolysis of cellulose is inhibited by non-productive adsorption of cellulases to lignin, and that is particularly problematic with lignin-rich materials such as softwood. Although conventional surfactants alleviate non-productive adsorption, using biosurfactants in softwood hydrolysis has not been reported. In this study, the effects of four biosurfactants, namely horse-chestnut escin, Pseudomonas aeruginosa rhamnolipid, and saponins from red and white quinoa varieties, on the enzymatic saccharification of steam-pretreated spruce were investigated. The used biosurfactants improved hydrolysis, and the best-performing one was escin, which led to cellulose conversions above 90%, decreased by around two-thirds lignin inhibition of Avicel hydrolysis, and improved hydrolysis of pretreated spruce by 24%. Red quinoa saponins (RQS) addition resulted in cellulose conversions above 80%, which was around 16% higher than without biosurfactants, and it was more effective than adding rhamnolipid or white quinoa saponins. Cellulose conversion improved with the increase in RQS addition up to 6 g/100 g biomass, but no significant changes were observed above that dosage. Although saponins are known to inhibit yeast growth, no inhibition of Saccharomyces cerevisiae fermentation of hydrolysates produced with RQS addition was detected. This study shows the potential of biosurfactants for enhancing the enzymatic hydrolysis of steam-pretreated softwood.

Evaluation of steam explosion pre-treatment for enzymatic hydrolysis of sunflower stalks

2008 ◽  
Vol 42 (2) ◽  
pp. 160-166 ◽  
Author(s):  
Encarnación Ruiz ◽  
Cristóbal Cara ◽  
Paloma Manzanares ◽  
Mercedes Ballesteros ◽  
Eulogio Castro
Keyword(s):  
Enzymatic Hydrolysis ◽  
Steam Explosion ◽  
Sunflower Stalks ◽  
Pre Treatment ◽  
Hydrolysis Of

scholarly journals Biological pretreatment of wheat straw: Effect of fungal culturing on enzymatic hydrolysis of carbohydrate polymers

2021 ◽  
Author(s):  
Aleksandar Knežević ◽  
Ivana Đokić ◽  
Tomislav Tosti ◽  
Slađana Popović ◽  
Dušanka Milojković-Opsenica ◽  
...  
Keyword(s):  
Enzymatic Hydrolysis ◽  
Wheat Straw ◽  
Lignin Degradation ◽  
Enzymatic Saccharification ◽  
White Rot Fungi ◽  
Fungal Species ◽  
White Rot ◽  
Irpex Lacteus ◽  
Fungal Pretreatment ◽  
Hydrolysis Of

Abstract The aim of the study was comparative analysis of degradation of wheat straw lignin by white-rot fungi and its implications on the efficiency of enzymatic hydrolysis of holocellulose. Cyclocybe cylindracea, Ganoderma resinaceum, Irpex lacteus, Pleurotus ostreatus and Trametes versicolor were the species studied. Peroxidases were predominantly responsible for lignin degradation even though high laccase activities were detected, except in the case of Irpex lacteus where laccase activity was not detected. Studied fungal species showed various ability to degrade lignin in wheat straw which further affected release of reducing sugars during enzymatic saccharification. The highest rate of lignin degradation was noticed in sample pretreated with Irpex lacteus (50.9 ± 4.1%). Among all tested species only Ganoderma resinaceum was suitable lignin degrader with the 2-fold higher hydrolysis yield (51.1 ± 4.7%) than in the control, and could have significant biotechnological application due to lower cellulose loss. A key mechanism of carbohydrate component convertibility enhancement was lignin removal in the biomass. Long time consumption, the low sugar yields and unpredictable fungal response still remain the challenge of the fungal pretreatment process.

scholarly journals PRE-TREATMENT AND ENZYMATIC HYDROLYSIS OF BANANA (Musa acuminata x balbisiana) PSEUDOSTEM FOR ETHANOL PRODUCTION

2020 ◽  
Vol 3 (2) ◽  
pp. 98-107
Author(s):  
Galileo E. Araguirang ◽  
Arianne Joyce R. Arizala ◽  
Eden Beth B. Asilo ◽  
Jamie Louise S. Batalon ◽  
Erin B. Bello ◽  
...  
Keyword(s):  
Enzymatic Hydrolysis ◽  
Ethanol Production ◽  
Incubation Time ◽  
Enzyme Concentration ◽  
The Philippines ◽  
Solid Loading ◽  
Hydrolysis Process ◽  
Pre Treatment ◽  
Hydrolysis Of ◽  
Second Generation Ethanol

Banana (M. acuminata x balbisiana) is an abundant lignocellulosic waste material in large plantations all over the Philippines, especially in Mindanao, which can be utilized as substrate in producing high-value products like ethanol. To compensate for the low yield based on total weight of substrate due to the high moisture content of banana pseudostem, there is the primary challenge to make the conversion of this lignocellulosic biomass into monomeric sugar and then into ethanol more efficiently in order to achieve yields that would make it cost-competitive. Hence, this study evaluated the effects of solid loading, incubation time and amount of enzyme on yield of reducing sugars in the enzymatic hydrolysis process and attempted to optimize the significant factors by Response Surface Methodology (RSM), specifically using Box-Behnken design. There was significant improvement on the reducing sugar yield of the pretreated banana pseudostem at 20 h incubation time, 15 g solid loading and 0.55 % enzyme concentration. Ethanol production was observed to be higher in the detoxified substrate although biomass was higher for the non-detoxified substrate. As to our knowledge, the present study is the first attempt to produce second generation ethanol using banana pseudostem waste as feedstock in the Philippines.

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