RESUMEN

BACKGROUND: The polysaccharides in lignocellulosic biomass hold potential for production of biofuels and biochemicals. However, achieving efficient conversion of this resource into fermentable sugars faces challenges, especially when operating at industrially relevant high solid loadings. While it is clear that combining classical hydrolytic enzymes and lytic polysaccharide monooxygenases (LPMOs) is necessary to achieve high saccharification yields, exactly how these enzymes synergize at high solid loadings remains unclear. RESULTS: An LPMO-poor cellulase cocktail, Celluclast 1.5 L, was spiked with one or both of two fungal LPMOs from Thermothielavioides terrestris and Thermoascus aurantiacus, TtAA9E and TaAA9A, respectively, to assess their impact on cellulose saccharification efficiency at high dry matter loading, using Avicel and steam-exploded wheat straw as substrates. The results demonstrate that LPMOs can mitigate the reduction in saccharification efficiency associated with high dry matter contents. The positive effect of LPMO inclusion depends on the type of feedstock and the type of LPMO and increases with the increasing dry matter content and reaction time. Furthermore, our results show that chelating free copper, which may leak out of the active site of inactivated LPMOs during saccharification, with EDTA prevents side reactions with in situ generated H2O2 and the reductant (ascorbic acid). CONCLUSIONS: This study shows that sustaining LPMO activity is vital for efficient cellulose solubilization at high substrate loadings. LPMO cleavage of cellulose at high dry matter loadings results in new chain ends and thus increased water accessibility leading to decrystallization of the substrate, all factors making the substrate more accessible to cellulase action. Additionally, this work highlights the importance of preventing LPMO inactivation and its potential detrimental impact on all enzymes in the reaction.

RESUMEN

Effectively pairing diverse lignocellulolytic enzyme cocktails with intricately structured lignocellulosic substrates is an enduring challenge for science and technology. To date, extensive trial-and-error remains the primary approach and no deep-learning methods were developed to address it due to limited experimental data and incomplete expert-level knowledge of enzyme-cocktail-substrate structure-dynamics-function relationships. Here, a novel model is developed to tackle this issue in efficient, cost-effective, and high-throughput manners. It needs no pre-labeled datasets, instead utilizing simple features, eliminating the reliance on expert-level prior knowledge of reaction mechanisms. Experimentally optimal combinations were found within predicted ranges of tailor-made combinations with precision of 91.98%, covering 80.00% of overall top-100. Practical tests demonstrated its effectiveness in narrowing down potential optimal combinations, speeding up targeted screening, and enabling efficient degradation of lignocellulosic biomass. The method has good applications in artificial proteins biosynthesis from low-value lignocellulosic straw, providing alternative solutions for biomass biorefining challenges in complex enzyme-cocktail-substrate interactions.

Asunto(s)

RESUMEN

Converting woody biomass to bioethanol might be more affordable, environmentally friendly, and efficient for making biofuel commercially feasible, but it would still need a significant optimization process and expand pilot-scale research. A combination of commercial low enzymes loading at 10 FPU/g glucan and compound additives utilizing Tween 80, PEG8000 and sophorolipid applied from lab-scale to pilot-scale have been studied in this work at economically viable dosages for enhancing bioethanol production. In lab-scale saccharification and fermentation, pretreated poplar at a high solid loading of 20% yielded the highest ethanol titers of 30.96 g/L and theoretical ethanol yield of 92.79%. Additionally, pilot-scale operation was used to investigate the bioethanol amplification, a final volume of 33 m3 which yielded the greatest ethanol amount of 599.6 kg from poplar wood while gaining on-site value-added production of hemicellulosic and cellobiose liquor 1122 kg and lignin residues 2292 kg.

Asunto(s)

RESUMEN

Agricultural development over the past decade has majorly contributed to the world's bioeconomy, but is the rise in agricultural activities just resulting in the best? Farming, food processing, livestock handling and other agro-based actions show an incremental rise in environmental deterioration by generating millions of tonnes of organic and inorganic solid waste across the globe. Incautious waste handling practices (incineration and landfilling) is resulting in greenhouse gas emissions, land pollution, groundwater contamination, soil erosion and chronic health hazards. Lately the concept of bioconversion has gained importance in valorising agro-waste (lignocellulosic biomasses) into value added products like biofuels, biogas, single cell proteins and biochar to effectively control waste and reduce the dependency on non-renewable feedstocks (fossil fuels). Biomass hydrolysis via enzymes is improved in terms of cost, efficiency, catalysis, stability and specificity by enrolling the use of enzyme cocktails to synergistically degrade lignocellulose into monomeric sugars and further into valued products. Enzyme blends like that of Xylanase + Pectinase + Cellulase shows 76.5% fermentation within 30 h by using banana peel as substrate for biofuel production. Other sectors like paper industries have also explored the use of enzyme blends of Xylanase + Pectinase + α-amylase + Protease+ lipase for bio-bleaching showing reduction in 50% chemical usage and 19.5% kappa number with adjacent increase in tensile strength by 23.55%. The scope of the present review is to highlight the technicalities of the concepts mentioned above, include qualitative data from different relatable studies and prove how the use of enzyme cocktails is an eco-friendly approach towards agro-waste management.

Asunto(s)

RESUMEN

With the transformation and revolution of the global plastics recycling system, recycling and upcycling of mixed plastics waste not only reduces the carbon emissions of plastics during its life cycle, but also addresses its potential ecological and environmental hazards. This article summarizes an international cooperation project, "MIXed plastics biodegradation and UPcycling using microbial communities" (MIX-UP) which was funded by the National Natural Science Foundation of China and the European Union (NSFC-EU) in 2019. The consortium of MIX-UP consists of 14 partners from European Union and China. Focusing on the global issue of "plastics pollution", this Sino-European MIX-UP project took the mixed waste of petroleum-based plastics (PP, PE, PUR, PET and PS) and bio-based plastics (PLA and PHA) as starting materials for biotechnological conversion into value-added, sustainable biomaterials. MIX-UP has three subprojects: 1) identification of plastics biodegradation pathway and design & engineering of key degrading elements, 2) construction and functional regulation of microbial consortia/enzyme cocktails with high-efficiency for degradation of plastics mixtures, 3) strategy of design and utilization of plastics degradation products for production of high value materials. Through NSFC-EU complementary and cross-disciplinary cooperation, MIX-UP proposes the engineering of a new-to-nature biological route for upcycling, a low carbon and sustainable bio-treatment that is different from the traditional physico-chemical treatment, which will empower the recycling industry to a new dimension. The implementation of the project will not only help to promote innovation and development in the field of biotechnology in China, but also contribute to the achievement of China's carbon neutral goal.

Asunto(s)

RESUMEN

The recalcitrance of lignocellulosic biomass is a major constraint to its high-value use at industrial scale. In nature, microbes play a crucial role in biomass degradation, nutrient recycling and ecosystem functioning. Therefore, the use of microbes is an attractive way to transform biomass to produce clean energy and high-value compounds. The microbial degradation of lignocelluloses is a complex process which is dependent upon multiple secreted enzymes and their synergistic activities. The availability of the cutting edge proteomics and highly sensitive mass spectrometry tools make possible for researchers to probe the secretome of microbes and microbial consortia grown on different lignocelluloses for the identification of hydrolytic enzymes of industrial interest and their substrate-dependent expression. This review summarizes the role of secretomics in identifying enzymes involved in lignocelluloses deconstruction, the development of enzyme cocktails and the construction of synthetic microbial consortia for biomass valorization, providing our perspectives to address the current challenges.

RESUMEN

With the transformation and revolution of the global plastics recycling system, recycling and upcycling of mixed plastics waste not only reduces the carbon emissions of plastics during its life cycle, but also addresses its potential ecological and environmental hazards. This article summarizes an international cooperation project, "MIXed plastics biodegradation and UPcycling using microbial communities" (MIX-UP) which was funded by the National Natural Science Foundation of China and the European Union (NSFC-EU) in 2019. The consortium of MIX-UP consists of 14 partners from European Union and China. Focusing on the global issue of "plastics pollution", this Sino-European MIX-UP project took the mixed waste of petroleum-based plastics (PP, PE, PUR, PET and PS) and bio-based plastics (PLA and PHA) as starting materials for biotechnological conversion into value-added, sustainable biomaterials. MIX-UP has three subprojects: 1) identification of plastics biodegradation pathway and design & engineering of key degrading elements, 2) construction and functional regulation of microbial consortia/enzyme cocktails with high-efficiency for degradation of plastics mixtures, 3) strategy of design and utilization of plastics degradation products for production of high value materials. Through NSFC-EU complementary and cross-disciplinary cooperation, MIX-UP proposes the engineering of a new-to-nature biological route for upcycling, a low carbon and sustainable bio-treatment that is different from the traditional physico-chemical treatment, which will empower the recycling industry to a new dimension. The implementation of the project will not only help to promote innovation and development in the field of biotechnology in China, but also contribute to the achievement of China's carbon neutral goal.

Asunto(s)

RESUMEN

The production of bioethanol from rice straw can contribute to the rural economy and provide clean fuel in a sustainable manner. However, phenolic compounds, which are mostly produced during acid pretreatment of biomass, act as inhibitors of fermenting microorganisms. Laccase is well known for its ability to oxidize lignin and phenolic compounds derived from lignocellulosic biomass. In the present study, an immobilized enzyme cocktail containing laccase was isevaluated in regard to its ability to enhance the saccharification and fermentation processes by reducing the amount of phenolic compounds produced. Saccharification of rice straw with the laccase-supplemented immobilized enzyme cocktail reduced phenolic compounds by 73.8%, resulting in a saccharification yield of 84.6%. In addition, improved yeast performance was is noted during the fermentation process, resulting in a 78.3% conversion of sugar into ethanol with an ethanol productivity of 0.478 g/L/h. To the best of our knowledge, this is the first description of the use of an immobilized enzyme cocktail comprised of Celluclast 1.5L, ß-glucosidase, and laccase for the production of bioethanol from rice straw. This study details a potential approach to producing biofuels from agricultural biomass, the applicability of which can be improved through up-scaling.

Asunto(s)

RESUMEN

BACKGROUND: Conceptually, multi-functional enzymes are attractive because in the case of complex polymer hydrolysis having two or more activities defined by a single enzyme offers the possibility of synergy and reduced enzyme cocktail complexity. Nevertheless, multi-functional enzymes are quite rare and are generally multi-domain assemblies with each activity being defined by a separate protein module. However, a recent report described a GH51 arabinofuranosidase from Alicyclobacillus sp. A4 that displays both α-l-arabinofuranosidase and ß-d-xylanase activities, which are defined by a single active site. Following on from this, we describe in detail another multi-functional GH51 arabinofuranosidase and discuss the molecular basis of multifunctionality. RESULTS: THSAbf is a GH51 α-l-arabinofuranosidase. Characterization revealed that THSAbf is active up to 75 °C, stable at 60 °C and active over a broad pH range (4-7). THSAbf preferentially releases para-nitrophenyl from the l-arabinofuranoside (k cat/K M = 1050 s(-1) mM(-1)) and to some extent from d-galactofuranoside and d-xyloside. THSAbf is active on 4-O-methylglucuronoxylans from birch and beechwood (10.8 and 14.4 U mg(-1), respectively) and on sugar beet branched and linear arabinans (1.1 ± 0.24 and 1.8 ± 0.1 U mg(-1)). Further investigation revealed that like the Alicyclobacillus sp. A4 α-l-arabinofuranosidase, THSAbf also displays endo-xylanase activity, cleaving ß-1,4 bonds in heteroxylans. The optimum pH for THASAbf activity is substrate dependent, but ablation of the catalytic nucleophile caused a general loss of activity, indicating the involvement of a single active center. Combining the α-l-arabinofuranosidase with a GH11 endoxylanase did not procure synergy. The molecular modeling of THSAbf revealed a wide active site cleft and clues to explain multi-functionality. CONCLUSION: The discovery of single active site, multifunctional enzymes such as THSAbf opens up exciting avenues for enzyme engineering and the development of new biomass-degrading cocktails that could considerably reduce enzyme production costs.

RESUMEN

The selection of microbes by enrichment on plant biomass has been proposed as an efficient way to develop new strategies for lignocellulose saccharification. Here, we report an in-depth analysis of soil-derived microbial consortia that were trained to degrade once-used wheat straw (WS1-M), switchgrass (SG-M) and corn stover (CS-M) under aerobic and mesophilic conditions. Molecular fingerprintings, bacterial 16S ribosomal RNA (rRNA) gene amplicon sequencing and metagenomic analyses showed that the three microbial consortia were taxonomically distinct. Based on the taxonomic affiliation of protein-encoding sequences, members of the Bacteroidetes (e.g. Chryseobacterium, Weeksella, Flavobacterium and Sphingobacterium) were preferentially selected on WS1-M, whereas SG-M and CS-M favoured members of the Proteobacteria (e.g. Caulobacter, Brevundimonas, Stenotrophomonas and Xanthomonas). The highest degradation rates of lignin (~59 %) were observed with SG-M, whereas CS-M showed a high consumption of cellulose and hemicellulose. Analyses of the carbohydrate-active enzymes in the three microbial consortia showed the dominance of glycosyl hydrolases (e.g. of families GH3, GH43, GH13, GH10, GH29, GH28, GH16, GH4 and GH92). In addition, proteins of families AA6, AA10 and AA2 were detected. Analysis of secreted protein fractions (metasecretome) for each selected microbial consortium mainly showed the presence of enzymes able to degrade arabinan, arabinoxylan, xylan, ß-glucan, galactomannan and rhamnogalacturonan. Notably, these metasecretomes contain enzymes that enable us to produce oligosaccharides directly from wheat straw, sugarcane bagasse and willow. Thus, the underlying microbial consortia constitute valuable resources for the production of enzyme cocktails for the efficient saccharification of plant biomass.

Asunto(s)

RESUMEN

BACKGROUND: Glycoside hydrolases (GHs) and accessory proteins are key components for efficient and cost-effective enzymatic hydrolysis of polysaccharides in modern, biochemically based biorefineries. Currently, commercialized GHs and accessory proteins are produced by ascomycetes. However, the role of wood decay basidiomycetes proteins in biomass saccharification has not been extensively pursued. Wood decay fungi degrade polysaccharides in highly lignified tissues in natural environments, and are a promising enzyme source for improving enzymatic cocktails that are designed for in vitro lignocellulose conversion. RESULTS: GHs and accessory proteins were produced by representative brown- and white-rot fungi, Laetiporus sulphureus and Pleurotus ostreatus, respectively. Concentrated protein extracts were then used to amend commercial enzymatic cocktails for saccharification of alkaline-sulfite pretreated sugarcane bagasse. The main enzymatic activities found in the wood decay fungal protein extracts were attributed to endoglucanases, xylanases and ß-glucosidases. Cellobiohydrolase (CBH) activities in the L. sulphureus and P. ostreatus extracts were low and nonexistent, respectively. The initial glucan conversion rates were boosted when the wood decay fungal proteins were used to replace half of the enzymes from the commercial cocktails. L. sulphureus proteins increased the glucan conversion levels, with values above those observed for the full load of commercial enzymes. Wood decay fungal proteins also enhanced the xylan conversion efficiency due to their high xylanase activities. Proteomic studies revealed 104 and 45 different proteins in the P. ostreatus and L. sulphureus extracts, respectively. The enhancement of the saccharification of alkaline-pretreated substrates by the modified enzymatic cocktails was attributed to the following protein families: GH5- and GH45-endoglucanases, GH3-ß-glucosidases, and GH10-xylanases. CONCLUSIONS: The extracellular proteins produced by wood decay fungi provide useful tools to improve commercial enzyme cocktails that are currently used for the saccharification of alkaline-pretreated lignocellulosic substrates. The relevant proteins encompass multiple glycoside hydrolase families, including the GH5- and GH45-endoglucanases, GH3-ß-glucosidases, and GH10-xylanases.