scholarly journals Strategies towards Reduction of Cellulases Consumption: Debottlenecking the Economics of Lignocellulosics Valorization Processes

2021 ◽  
Vol 2 (2) ◽  
pp. 287-310
Author(s):  
Daniel Gomes ◽  
Joana Cunha ◽  
Elisa Zanuso ◽  
José Teixeira ◽  
Lucília Domingues
Keyword(s):  
Enzyme Immobilization ◽  
Consolidated Bioprocessing ◽  
Enzyme Stability ◽  
Cellulolytic Enzymes ◽  
Special Focus ◽  
Food Crops ◽  
Lignocellulosic Materials ◽  
Lignocellulosic Residues ◽  
Potential Reduction ◽  
Promising Source

Lignocellulosic residues have been receiving growing interest as a promising source of polysaccharides, which can be converted into a variety of compounds, ranging from biofuels to bioplastics. Most of these can replace equivalent products traditionally originated from petroleum, hence representing an important environmental advantage. Lignocellulosic materials are theoretically unlimited, cheaper and may not compete with food crops. However, the conversion of these materials to simpler sugars usually requires cellulolytic enzymes. Being still associated with a high cost of production, cellulases are commonly considered as one of the main obstacles in the economic valorization of lignocellulosics. This work provides a brief overview of some of the most studied strategies that can allow an important reduction of cellulases consumption, hence improving the economy of lignocellulosics conversion. Cellulases recycling is initially discussed regarding the main processes to recover active enzymes and the most important factors that may affect enzyme recyclability. Similarly, the potential of enzyme immobilization is analyzed with a special focus on the contributions that some elements of the process can offer for prolonged times of operation and improved enzyme stability and robustness. Finally, the emergent concept of consolidated bioprocessing (CBP) is also described in the particular context of a potential reduction of cellulases consumption.

scholarly journals Consolidated Bioprocessing: Synthetic Biology Routes to Fuels and Fine Chemicals

2021 ◽  
Vol 9 (5) ◽  
pp. 1079
Author(s):  
Alec Banner ◽  
Helen S. Toogood ◽  
Nigel S. Scrutton
Keyword(s):  
Enzymatic Degradation ◽  
Consolidated Bioprocessing ◽  
Carbon Sources ◽  
Cellulolytic Enzymes ◽  
Waste Biomass ◽  
Chemical Production ◽  
Iterative Design ◽  
Biomass Feedstocks ◽  
Advanced Fuels ◽  
Design Build

The long road from emerging biotechnologies to commercial “green” biosynthetic routes for chemical production relies in part on efficient microbial use of sustainable and renewable waste biomass feedstocks. One solution is to apply the consolidated bioprocessing approach, whereby microorganisms convert lignocellulose waste into advanced fuels and other chemicals. As lignocellulose is a highly complex network of polymers, enzymatic degradation or “saccharification” requires a range of cellulolytic enzymes acting synergistically to release the abundant sugars contained within. Complications arise from the need for extracellular localisation of cellulolytic enzymes, whether they be free or cell-associated. This review highlights the current progress in the consolidated bioprocessing approach, whereby microbial chassis are engineered to grow on lignocellulose as sole carbon sources whilst generating commercially useful chemicals. Future perspectives in the emerging biofoundry approach with bacterial hosts are discussed, where solutions to existing bottlenecks could potentially be overcome though the application of high throughput and iterative Design-Build-Test-Learn methodologies. These rapid automated pathway building infrastructures could be adapted for addressing the challenges of increasing cellulolytic capabilities of microorganisms to commercially viable levels.

scholarly journals Current Developments in Lignocellulosic Biomass Conversion into Biofuels Using Nanobiotechology Approach

Energies ◽  
2020 ◽  
Vol 13 (20) ◽  
pp. 5300
Author(s):  
Mamata Singhvi ◽  
Beom Soo Kim
Keyword(s):  
Lignocellulosic Biomass ◽  
Enzyme Immobilization ◽  
Enzyme Stability ◽  
Biomass Conversion ◽  
Cost Effective ◽  
Reaction Rates ◽  
Biofuel Production ◽  
Potential Strategy ◽  
Lignocellulosic Biomass Conversion ◽  
Review Current

The conversion of lignocellulosic biomass (LB) to sugar is an intricate process which is the costliest part of the biomass conversion process. Even though acid/enzyme catalysts are usually being used for LB hydrolysis, enzyme immobilization has been recognized as a potential strategy nowadays. The use of nanobiocatalysts increases hydrolytic efficiency and enzyme stability. Furthermore, biocatalyst/enzyme immobilization on magnetic nanoparticles enables easy recovery and reuse of enzymes. Hence, the exploitation of nanobiocatalysts for LB to biofuel conversion will aid in developing a lucrative and sustainable approach. With this perspective, the effects of nanobiocatalysts on LB to biofuel production were reviewed here. Several traits, such as switching the chemical processes using nanomaterials, enzyme immobilization on nanoparticles for higher reaction rates, recycling ability and toxicity effects on microbial cells, were highlighted in this review. Current developments and viability of nanobiocatalysts as a promising option for enhanced LB conversion into the biofuel process were also emphasized. Mostly, this would help in emerging eco-friendly, proficient, and cost-effective biofuel technology.

scholarly journals Rapid optimisation of cellulolytic enzymes ratios in Saccharomyces cerevisiae using in vitro SCRaMbLE

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Elizabeth L. I. Wightman ◽  
Heinrich Kroukamp ◽  
Isak S. Pretorius ◽  
Ian T. Paulsen ◽  
Helena K. M. Nevalainen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzyme Activities ◽  
Consolidated Bioprocessing ◽  
Synergistic Action ◽  
Cellulosic Biomass ◽  
Gene Copy ◽  
Cellulolytic Enzymes ◽  
Plasmid Library ◽  
Substrate Hydrolysis

Abstract Background For the economic production of biofuels and other valuable products from lignocellulosic waste material, a consolidated bioprocessing (CBP) organism is required. With efficient fermentation capability and attractive industrial qualities, Saccharomyces cerevisiae is a preferred candidate and has been engineered to produce enzymes that hydrolyze cellulosic biomass. Efficient cellulose hydrolysis requires the synergistic action of several enzymes, with the optimum combined activity ratio dependent on the composition of the substrate. Results In vitro SCRaMbLE generated a library of plasmids containing different ratios of a β-glucosidase gene (CEL3A) from Saccharomycopsis fibuligera and an endoglucanase gene (CEL5A) from Trichoderma reesei. S. cerevisiae, transformed with the plasmid library, displayed a range of individual enzyme activities and synergistic capabilities. Furthermore, we show for the first time that 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-d-cellopentaoside (BPNPG5) is a suitable substrate to determine synergistic Cel3A and Cel5A action and an accurate predictive model for this synergistic action was devised. Strains with highest BPNPG5 activity had an average CEL3A and CEL5A gene cassette copy number of 1.3 ± 0.6 and 0.8 ± 0.2, respectively (ratio of 1.6:1). Conclusions Here, we describe a synthetic biology approach to rapidly optimise gene copy numbers to achieve efficient synergistic substrate hydrolysis. This study demonstrates how in vitro SCRaMbLE can be applied to rapidly combine gene constructs in various ratios to allow screening of synergistic enzyme activities for efficient substrate hydrolysis.

Metal–organic frameworks and inorganic nanoflowers: a type of emerging inorganic crystal nanocarrier for enzyme immobilization

2015 ◽  
Vol 5 (12) ◽  
pp. 5077-5085 ◽  
Author(s):  
Xiaoling Wu ◽  
Miao Hou ◽  
Jun Ge
Keyword(s):  
Enzyme Immobilization ◽  
Self Assembly ◽  
Enzyme Stability ◽  
Physical Adsorption ◽  
Metal Organic Frameworks ◽  
Great Promise ◽  
Inorganic Crystal ◽  
Metal Organic

By the methods of physical adsorption, covalent conjugation and self-assembly, enzymes can be immobilized on metal–organic frameworks (MOFs) and inorganic crystal nanoflowers with the great promise of enhancing enzyme stability, activity and even selectivity.

Immobilization of thermophilic enzymes in miniaturized flow reactors

2007 ◽  
Vol 35 (6) ◽  
pp. 1621-1623 ◽  
Author(s):  
A.M. Hickey ◽  
L. Marle ◽  
T. McCreedy ◽  
P. Watts ◽  
G.M. Greenway ◽  
...  
Keyword(s):  
Enzyme Immobilization ◽  
Flow Reactor ◽  
Capillary Flow ◽  
Enzyme Stability ◽  
Substrate Inhibition ◽  
Cost Effective ◽  
Microfluidic Channels ◽  
Flow Reactors ◽  
Thermophilic Archaeon ◽  
The Cost

The exploitation of enzymes for biotransformation reactions for the production of new and safer drug intermediates has been the focus of much research. While a number of enzymes are commercially available, their use in an industrial setting is often limited to reactions that are cost-effective and they are rarely investigated further. However, the development of miniaturized flow reactor technology has meant that the cost of such research, once considered cost- and time-inefficient, would be much less prohibitive. The use of miniaturized flow reactors for enzyme screening offers a number of advantages over batch enzyme assay systems. Since the assay is performed on a miniaturized scale, enzyme, substrate and cofactor quantities are significantly reduced, thus reducing the cost of laboratory-scale investigations. Since flow reactors use microfluidic systems, where the substrate and products flow out of the system, the problems of substrate inhibition and product inhibition encountered by some enzymes are avoided. Quite often, enzymes fulfil a single-use function in biotransformation processes; however, enzyme immobilization allows enzyme reuse and often helps to increase enzyme stability. We have used an aminoacylase enzyme with potential use for industrial biotransformation reactions and have successfully immobilized it in miniaturized flow reactors. This L-aminoacylase is from the thermophilic archaeon Thermococcus litoralis. Two approaches to enzyme immobilization have been examined, both involving enzyme cross-linking. The first reactor type has used monoliths, to which the enzyme was attached, and the second contained previously cross-linked enzyme trapped using frits, in the microfluidic channels. Two different microreactor designs were used in the investigation: microreactor chips for the monoliths and capillary flow reactors for the cross-linked enzyme. These systems allowed passage of the substrate and product through the system while retaining the aminoacylase enzyme performing the catalytic conversion. The enzyme has been successfully immobilized and used to produce stable biocatalytic microreactors that can be used repeatedly over a period of several months.

scholarly journals Microbial Cellulose Utilization: Fundamentals and Biotechnology

2002 ◽  
Vol 66 (3) ◽  
pp. 506-577 ◽  
Author(s):  
Lee R. Lynd ◽  
Paul J. Weimer ◽  
Willem H. van Zyl ◽  
Isak S. Pretorius
Keyword(s):  
Consolidated Bioprocessing ◽  
Quantitative Description ◽  
Product Yield ◽  
Cellulose Hydrolysis ◽  
Taxonomic Diversity ◽  
Cellulosic Biomass ◽  
Limiting Factors ◽  
Cellulolytic Enzymes ◽  
Cellulase Enzymes ◽  
Microbial Cellulose

SUMMARY Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for “consolidated bioprocessing” (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.

scholarly journals Clostridium thermocellum as a Promising Source of Genetic Material for Designer Cellulosomes: An Overview

Catalysts ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 996
Author(s):  
Dung Minh Ha-Tran ◽  
Trinh Thi My Nguyen ◽  
Chieh-Chen Huang
Keyword(s):  
Clostridium Thermocellum ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Consolidated Bioprocessing ◽  
Genetic Material ◽  
Economic Feasibility ◽  
Biofuel Production ◽  
Cellulolytic Bacterium ◽  
Microbial Conversion ◽  
Promising Source

Plant biomass-based biofuels have gradually substituted for conventional energy sources thanks to their obvious advantages, such as renewability, huge quantity, wide availability, economic feasibility, and sustainability. However, to make use of the large amount of carbon sources stored in the plant cell wall, robust cellulolytic microorganisms are highly demanded to efficiently disintegrate the recalcitrant intertwined cellulose fibers to release fermentable sugars for microbial conversion. The Gram-positive, thermophilic, cellulolytic bacterium Clostridium thermocellum possesses a cellulolytic multienzyme complex termed the cellulosome, which has been widely considered to be nature’s finest cellulolytic machinery, fascinating scientists as an auspicious source of saccharolytic enzymes for biomass-based biofuel production. Owing to the supra-modular characteristics of the C. thermocellum cellulosome architecture, the cellulosomal components, including cohesin, dockerin, scaffoldin protein, and the plentiful cellulolytic and hemicellulolytic enzymes have been widely used for constructing artificial cellulosomes for basic studies and industrial applications. In addition, as the well-known microbial workhorses are naïve to biomass deconstruction, several research groups have sought to transform them from non-cellulolytic microbes into consolidated bioprocessing-enabling microbes. This review aims to update and discuss the current progress in these mentioned issues, point out their limitations, and suggest some future directions.

scholarly journals The use of lytic polysaccharide monooxygenases in anaerobic digestion of lignocellulosic materials

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Thales H. F. Costa ◽  
Vincent G. H. Eijsink ◽  
Svein Jarle Horn
Keyword(s):  
Anaerobic Digestion ◽  
Methane Production ◽  
Biogas Production ◽  
Process Efficiency ◽  
Cellulolytic Enzymes ◽  
Cellulolytic Enzyme ◽  
Lignocellulosic Materials ◽  
Active Components ◽  
Enzyme Preparations ◽  
The Impact

Abstract Background The recent discovery that LPMOs can work under anaerobic conditions when supplied with low amounts H2O2 opens the possibility of using LPMOs as enzyme aids in biogas reactors to increase methane yields from lignocellulosic materials. We have explored this possibility by studying anaerobic digestion of various lignocellulosic materials: Avicel, milled spruce and birch wood, and a lignin-rich hydrolysis residue from steam-exploded birch. The digestions were added LPMOs and various cellulolytic enzyme cocktails and were carried out with or without addition of H2O2. Results In several cases, enzyme addition had a beneficial effect on methane production, which was partly due to components present in the enzyme preparations. It was possible to detect LPMO activity during the initial phases of the anaerobic digestions of Avicel, and in some cases LPMO activity could be correlated with improved methane production from lignocellulosic materials. However, a positive effect on methane production was only seen when LPMOs were added together with cellulases, and never upon addition of LPMOs only. Generally, the experimental outcomes showed substrate-dependent variations in process efficiency and the importance of LPMOs and added H2O2. These differences could relate to variations in the type and content of lignin, which again will affect the activity of the LPMO, the fate of the added H2O2 and the generation of potentially damaging reactive-oxygen species. The observed effects showed that the interplay between cellulases and LPMOs is important for the overall efficiency of the process. Conclusion This study shows that it may be possible to harness the power of LPMOs in anaerobic digestion processes and improve biogas production, but also highlight the complexity of the reaction systems at hand. One complicating factor was that the enzymes themselves and other organic components in the enzyme preparations acted as substrates for biogas production, meaning that good control reactions were essential to detect effects caused by enzyme activity. As also observed during regular aerobic enzymatic digestion of lignocellulosic biomass, the type and contents of lignin in the substrates likely plays a major role in determining the impact of LPMOs and of cellulolytic enzymes in general. More work is needed to unravel the interplay between LPMOs, O2, H2O2, and the multitude of redox-active components found in anaerobic bioreactors degrading lignocellulosic substrates.

scholarly journals Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolytica

2021 ◽  
Vol 12 ◽  
Author(s):  
Hui Wei ◽  
Wei Wang ◽  
Eric P. Knoshaug ◽  
Xiaowen Chen ◽  
Stefanie Van Wychen ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Cell Growth ◽  
Yarrowia Lipolytica ◽  
Lipid Accumulation ◽  
Consolidated Bioprocessing ◽  
Cellulosic Biomass ◽  
Host Cells ◽  
Selection Marker ◽  
Cellulolytic Enzymes ◽  
Cellulolytic Activity

Yarrowia lipolytica is known to be capable of metabolizing glucose and accumulating lipids intracellularly; however, it lacks the cellulolytic enzymes needed to break down cellulosic biomass directly. To develop Y. lipolytica as a consolidated bioprocessing (CBP) microorganism, we previously expressed the heterologous CBH I, CBH II, and EG II cellulase enzymes both individually and collectively in this microorganism. We concluded that the coexpression of these cellulases resulted in a metabolic drain on the host cells leading to reduced cell growth and lipid accumulation. The current study aims to build a new cellulase coexpressing platform to overcome these hinderances by (1) knocking out the sucrose non-fermenting 1 (Snf1) gene that represses the energetically expensive lipid and protein biosynthesis processes, and (2) knocking in the cellulase cassette fused with the recyclable selection marker URA3 gene in the background of a lipid-accumulating Y. lipolytica strain overexpressing ATP citrate lyase (ACL) and diacylglycerol acyltransferase 1 (DGA1) genes. We have achieved a homologous recombination insertion rate of 58% for integrating the cellulases-URA3 construct at the disrupted Snf1 site in the genome of host cells. Importantly, we observed that the disruption of the Snf1 gene promoted cell growth and lipid accumulation and lowered the cellular saturated fatty acid level and the saturated to unsaturated fatty acid ratio significantly in the transformant YL163t that coexpresses cellulases. The result suggests a lower endoplasmic reticulum stress in YL163t, in comparison with its parent strain Po1g ACL-DGA1. Furthermore, transformant YL163t increased in vitro cellulolytic activity by 30%, whereas the “total in vivo newly formed FAME (fatty acid methyl esters)” increased by 16% in comparison with a random integrative cellulase-expressing Y. lipolytica mutant in the same YNB-Avicel medium. The Snf1 disruption platform demonstrated in this study provides a potent tool for the further development of Y. lipolytica as a robust host for the expression of cellulases and other commercially important proteins.

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