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Enzyme mediated multi-product process: A concept of bio-based refinery
Abstract
The bio-based refinery has evolved as a strong alternative to the fossil-based refinery. Fossil fuels have been critical to meet the global energy and chemical needs. The rapid utilization of fossil fuel reserves and environmental deterioration has led to the search for alternative sources. The naturally occurring biomass (forest residues, microalgae), and waste biomass generated through different anthropogenic activity (agricultural residues, industrial wastes) have emerged as a potential substitute for the fossil fuels. These biomasses consists of complex polymers such as cellulose, lignin, hemicelluloses, starch, pectin, lipids that can be used as starting material for fuels and building block chemicals. This biomass can be converted to suitable liquid or gaseous fuel and essential chemicals through a series of steps. Such an approach is called bio-based refinery as analogous to the petroleum refinery. The role of different hydrolytic enzymes is important to the biobased refinery. These enzymes act on different biomass components and result in the generation of specific oligomers and monomers. These oligomeric and monomeric compounds are subsequently converted to biofuels and biochemicals. The enzymes are industrially produced by various microorganisms and have characteristics of structural and mechanistic properties. There are several limitations associated with the application of enzymes in biorefineries. Therefore, to overcome the existing limitations of enzyme-based technologies in biorefinery there is a need to understand the current status of these hydrolyzing enzymes and enzyme-mediated technologies. The review will first give an insight into enzyme structure, mode of action, and its current role in the biobased biorefinery. This review also focuses on recent advances, techno-economic and environmental concerns associated with enzyme assisted biobased refinery. The review suggests that the application of the advanced biotechnological approach can help in the development of a consolidated bioprocessing approach. This approach will further lead to futuristic and self-sustainable “integrated biorefineries” that can fulfill the dream of “circular bioeconomy” with zero-waste.
... Catalyst mixed drinks characterized by a combination of a-amylase, b-amylase, and glucoamylase of different beginnings are more successful for food squander saccharification. Cellulases, xylanases, and pullulanases (a specific kind of glucanase, an amylolytic exoenzyme, that degrades pullulan) have likewise been added to the list of saccharifying compounds [166][167][168]. ...
To switch to renewable energy sources from fossil fuels, there is an urgent global need for an inventive technique for turning food waste into biofuels. The production of bioethanol from food waste might result in a long-lasting method that would satisfy both the growing population's energy needs and the problem of disposing of food waste. According to estimates, one-third of the food produced worldwide, or 1.3 billion tonnes annually, is wasted. Biofuels like bioethanol reduce dependency on fossil fuels and can operate with a fleet of internal combustion engines. Using biofuels can decrease carbon dioxide emissions from internal combustion engine fleets. Typically, bioethanol production involves the microbial fermentation of fermentable carbohydrates into ethanol. Fermentation processes used in bioethanol production generally employ yeast (Saccharomyces cerevisiae) to convert sugars from biomass into ethanol and CO 2. Batch, fed-batch, and continuous fermentation techniques are used, with advances such as immobilized cell reactors and genetic engineering improving output and efficiency. Furthermore, combining enzymatic hydrolysis with fermentation (simultaneous saccharification and fermentation) enhances the conversion of complex carbohydrates to ethanol. Traditional feedstocks (first-generation feedstock) consist of cereal grains, sugar cane, and sugar beets. However, lignocellulosic (second-generation), microbial biomass (third-generation), and genetically modified microalgae (fourth-generation) based feedstocks have been researched due to concerns about the sustainability of food. This paper discusses available methods, such as fermentation techniques, and compares bioethanol generation from various feedstocks. The objectives of this review are to compare different generations of biofuel production. The current review also provides industrial producers with knowledge of the technologies and resources that are currently accessible and the possibilities for future innovation.
... A method similar to the one studied by Placido et al. [47] is mentioned by the authors [41,48,50,51]. Placido et al. applied the pre-treatment method with ultrasound, liquid hot water and lignolytic enzymes in the presence of 15% NaOH [47]. ...
Today, the growth in textile consumption is influenced by the increasing population. This leads to the generation of a large volume of textile waste, which has a negative impact on the environment. Textile waste is mainly disposed of by landfill or incineration. To reduce the amount of textile waste disposed of, it can be recovered and transformed into valuable compounds, such as ethanol (bioethanol). This article describes how pre- and post-consumer textile waste is used as a feedstock for ethanol.
... The main benefits of SSF include low capital expenditure, reduced levels of catabolite repression and end-product inhibition, low wastewater output, improved productivity, higher enzyme yields, and better product recovery. SSF has been used predominantly as it triggers the production of various enzymes directly from raw materials rich in lignocellulose (Kumar and Verma 2020). SSF is highly favourable for fungal microflora as it is like their natural habitat. ...
The fungal system holds morphological plasticity and metabolic versatility which makes it unique. Fungal habitat ranges from the Arctic region to the fertile mainland, including tropical rainforests, and temperate deserts. They possess a wide range of lifestyles behaving as saprophytic, parasitic, opportunistic, and obligate symbionts. These eukaryotic microbes can survive any living condition and adapt to behave as extremophiles, mesophiles, thermophiles, or even psychrophile organisms. This behaviour has been exploited to yield microbial enzymes which can survive in extreme environments. The cost-effective production, stable catalytic behaviour and ease of genetic manipulation make them prominent sources of several industrially important enzymes. Pectinases are a class of pectin-degrading enzymes that show different mechanisms and substrate specificities to release end products. The pectinase family of enzymes is produced by microbial sources such as bacteria, fungi, actinomycetes, plants, and animals. Fungal pectinases having high specificity for natural sources and higher stabilities and catalytic activities make them promising green catalysts for industrial applications. Pectinases from different microbial sources have been investigated for their industrial applications. However, their relevance in the food and textile industries is remarkable and has been extensively studied. The focus of this review is to provide comprehensive information on the current findings on fungal pectinases targeting diverse sources of fungal strains, their production by fermentation techniques, and a summary of purification strategies. Studies on pectinases regarding innovations comprising bioreactor-based production, immobilization of pectinases, in silico and expression studies, directed evolution, and omics-driven approaches specifically by fungal microbiota have been summarized.
... The sugars produced (glucose and pentoses) should be separated and concentrated through evaporators to feed the fermentation stage. The efficient separation of the sugar-rich liquid from the unreacted solid is usually conducted by a cost-intensive centrifugation stage [226,227]. Several reports in the literature show different separation strategies to dewater the biomass and recover the sugar-rich liquid by a process that mimics the wet end of a paper machine [228][229][230]. Sugar yield and separation efficiency can be improved with an approach similar to the one used for paper making in two ways: (i) by using separation and concentration of solids in two-stage enzymatic hydrolysis processes and, (ii) by separating the final sugar-rich liquid. ...
50 days open access link -> https://authors.elsevier.com/c/1hG8MRCId3N9 Efficient utilization of forestry, agriculture, and marine resources in various manufacturing sectors requires optimizing fiber transformation, dewatering, and drying energy consumption. These processes play a crucial role in reducing the carbon footprint and boosting sustainability within the circular bioeconomy framework. Despite efforts made in the paper industry to enhance productivity while conserving resources and energy through lower grammage and higher machine speeds, reducing thermal energy consumption during papermaking remains a significant challenge. A key approach to address this challenge lies in increasing dewatering of the fiber web before entering the dryer section of the paper machine. Similarly, the production of high-value-added products derived from alternative lignocellulosic feedstocks, such as nanocellulose and microalgae, requires advanced dewatering techniques for techno-economic viability. This critical and systematic review aims to comprehensively explore the intricate interactions between water and lignocellulosic surfaces, as well as the leading technologies used to enhance dewatering and drying. Recent developments in technologies to reduce water content during papermaking, and advanced dewatering techniques for nanocellulosic and microalgal feedstocks are addressed. Existing research highlights several fundamental and technical challenges spanning from the nano- to macroscopic scales that must be addressed to make lignocellulosics a suitable feedstock option for industry. By identifying alternative strategies to improve water removal, this review intends to accelerate the widespread adoption of lignocellulosics as feasible manufacturing feedstocks. Moreover, this review aims to provide a fundamental understanding of the interactions, associations, and bonding mechanisms between water and cellulose fibers, nanocellulosic materials, and microalgal feedstocks. The findings of this review shed light on critical research directions necessary for advancing the efficient utilization of lignocellulosic resources and accelerating the transition towards sustainable manufacturing practices.
... Lignin [C 9 H 10 O 3 (OCH 3 ) 0.9-1.7 ] n is a complex organic aromatic polymer composed of cross-linked phenolic precursors which gives structural support to the plant tissue (Demirbas, 2008). The main building units of lignin are hydroxyl coumaryl alcohols, coniferyl alcohol, sinapyl alcohol, and p-coumaryl alcohol (Gall et al., 2017;Kumar & Verma 2020). Cellulose and hemicellulose are firmly associated with lignin by hydrogen bonds and glycosidic linkages . ...
... For designing an enzyme puri cation strategy, some information about the enzyme is required beforehand, such as the source of enzyme, some unique characteristics of the enzyme, and other information such as the biochemical properties of the enzyme and whether the enzyme is tagged for generation of recombinant enzyme. Whether the enzyme is expressed intracellularly or extracellularly, any contaminants present, and the nal application of the puri ed enzyme, are some other factors that need to be known prior to deciding the puri cation process (Banerjee 2006;Kumar & Verma, 2020;Kumar et al., 2018). Most essential is the knowledge of source, and assay techniques applicable to all samples since all enzyme puri cation processes require the sample to be prepared. ...
... Bioenergy from biomass can be made through enzyme-mediated biocatalysis, which is one of the most sustainable and renewable ways to make clean energy. A number of potential bioenergy options, such as biodiesel, biomethanol, bioethanol, biobutanol, biogas, and biohydrogen, are currently running via the enzyme-mediated biocatalysis process [24]. Figure 4 (a) depicts the main procedure for producing biofuels from waste biomass by enzymatic hydrolysis. ...
Enzyme based biocatalysis is emerging as advanced technique to develop green processes that help to maintain the sustainability of the environment. The bioremediations of toxic organic pollutants and waste to bioenergy production using enzymes as biocatalysts is rapidly growing due to its eco-friendly and sustainable nature. Additionally, a range of microbial species that typically grow on organic wastes can be used to produce these enzymes in an efficient manner. This is seen as a potential strategy for the development of cost-effective manufacturing for a number of biotechnological applications. The present study discusses biocatalysis as a promising and sustainable method towards the bioremediation of hazardous organic pollutants as well as for bioenergy production, based on the immense potential of enzymes as biocatalysts. Emphasis has been placed on evaluating the critical elements that can enhance the production of enzymes used as biocatalysts, as well as their functional effectiveness and stability.
... To reach the desired reduction of 2G bioethanol production costs, many researches evaluated developments of traditional methods in different aspects. The use of enzymatic cocktails for hydrolysis is one of the most critical steps in terms of process costs [188]. In addition, the traditionally used Saccharomyces yeasts only ferment C6 sugars [189]. ...
The world has been searching for clean and renewable alternatives to provide new materials and energy and, consequently reducing the dependence on petroleum fossil fuels. Sugarcane has become an important raw material as a solution for this demand since it is grown in more than 100 countries and can potentially reduce greenhouse gas (GHG) emissions. In this context, the production of biomaterials and biofuels and their effective use are promising ways to reach these goals. Brazilian government policies have created the optimal scenario to consolidate the first sugarcane 1G biorefinery with biofuels, sugar and bioelectricity production, trying to reach sustainable and low-cost solutions. After the establishment of the 1G technology, the 2G and 3G technologies are being developed, bringing new possibilities for value-added biochemicals production, in integrated systems with minimized emissions with a non-waste vison and circular bioeconomy. This review presents the current status and future perspectives of sugarcane as a potential biofactory. The present and future of Brazilian sugarcane biorefinery are discussed in terms of the exploitation of sugarcane, research and innovation with the integrated production of sugar, biofuels, bioelectricity, biopolymers, organic acids, enzymes and other biomolecules, as well as the use of process-generated liquid and solid by-products and/or wastes.
... The past 20 years has marked several major breakthroughs in our fundamental understanding of lignocellulose bioconversion [1][2][3]. Some of these breakthroughs are already incorporated in enzyme formulations for the efficient conversion of lignocellulosic biomass to fuels, chemicals and new bio-based materials [4][5][6]. Despite the tremendous achievements by excellent research groups world-wide, currently available enzyme formulations fall short of transformation efficiencies achieved in nature. ...
Background
Substrate accessibility remains a key limitation to the efficient enzymatic deconstruction of lignocellulosic biomass. Limited substrate accessibility is often addressed by increasing enzyme loading, which increases process and product costs. Alternatively, considerable efforts are underway world-wide to identify amorphogenesis-inducing proteins and protein domains that increase the accessibility of carbohydrate-active enzymes to targeted lignocellulose components.
Results
We established a three-dimensional assay, PACER (plant cell wall model for the analysis of non-catalytic and enzymatic responses), that enables analysis of enzyme migration through defined lignocellulose composites. A cellulose/azo-xylan composite was made to demonstrate the PACER concept and then used to test the migration and activity of multiple xylanolytic enzymes. In addition to non-catalytic domains of xylanases, the potential of loosenin-like proteins to boost xylanase migration through cellulose/azo-xylan composites was observed.
Conclusions
The PACER assay is inexpensive and parallelizable, suitable for screening proteins for ability to increase enzyme accessibility to lignocellulose substrates. Using the PACER assay, we visualized the impact of xylan-binding modules and loosenin-like proteins on xylanase mobility and access to targeted substrates. Given the flexibility to use different composite materials, the PACER assay presents a versatile platform to study impacts of lignocellulose components on enzyme access to targeted substrates.
Broad industrial application makes cellulolytic enzymes always in industrial demand with economic and sustainable production method. Production of cellulase enzymes via solid state fermentation mode using solid waste can resolve enzyme production and solid waste management issue in eco-friendly way. In present investigation, co-fermentation of solid wastes and microbial co-cultivation using potential cellulase producers strains have been applied as promising strategy for enhancement of cellulase at economical scale. Under the optimized bioprocess, co-fermentative substrate ratio of 5:4 of coconut waste (Ccw) and jamun fruits (JFs) gave 16 IU/gds filter paper (FP) activity at 12 h of incubation via bacillus strains in solid state fermentation (SSF). Additionally, at optimum production temperature of 42 °C and pH 6.0, enzyme showed 19 and 21 IU/gds FP activity at 12 h of incubation. Further, using different organic and inorganic sources, enzyme produced 26 IU/gds FP activity at 12 h peptone as nitrogen source. Additionally, at 60% optimum, moisture content, enzyme gave highest 31 IU/gds FP activity, 239 IU/gds, β-glucosidase (BGL) activity and 176 IU/gds endoglucanase (EG) activity in 12 h of incubation of SSF confirms the efficient production of all cellulolytic components of enzyme. The study has prominent scope in economical industrial application of this enzyme with promising waste management for environment sustainability applications.
Food additives are substances intentionally added to products to achieve desired functional properties. Many enzymes are commercially utilized as food additives in food processing to maintain or improve food products’ physicochemical and sensory properties. Enzymes also produce food ingredients through enzymatic reactions such as synthesis, hydrolysis and biocatalysis. The research efforts are ongoing to improve the properties of enzymes, such as catalytic efficiencies, thermostabilities and specificities. The utilization of enzymes in the food industry has surged with the advent of advanced technologies such as recombinant DNA and protein engineering. The production of enzymes using advanced techniques offers a technological advantage over enzymes produced through conventional techniques without altering the sensory attributes of food products. This chapter of the book covers the production of enzymes, applications of enzymes for food product development, preparation of food ingredients through enzymatic reaction, and antimicrobial properties of enzymes.
Bioeconomy is currently perceived by scientists and policymakers as a solution to reduce human dependence on fossil resources by using biomass as an alternative energy and material input. Although bio-based economies are not new, the concept of the bioeconomy has considerably evolved over the last decades. It has become a technology-driven economic paradigm expected to bring substantial benefits to people and the environment thanks to the potential of technological innovations to eliminate or mitigate some of the negative impacts caused by fossil resources. However, despite the considerable potential that several bio-based innovations are bringing to the market, the level of sustainability of the current and future state of the bioeconomy is uncertain, and the side effects during and after its implementation are often underestimated. This is especially relevant for the early stages of the value chain when biomass is produced and pre-treated. In this context, the chapter examines the nexus between sustainability and bioeconomy and analyses how the sustainable development framework has reshaped the understanding and purpose of such an economic paradigm. It explores the bioeconomy evolution as a concept and investigates how the circular economy, chemistry, biotechnology, and Power-to-X processes could support the transition towards the ‘sustainable and circular bioeconomy’.
There has been a gradual and challenging paradigm shift towards a biomass-based economy from fossil- and chemical-based economy. In accordance with the United Nations’ (UN) criteria of sustainability, it is quintessential that while meeting our current needs, the capacity of future generations to address their own is not endangered. Biorefinery has thus evolved as a potent catalyst for new fossil fuel-free bioeconomy. Biorefinery refers to a sustainable platform that brings about the conversion of biomass waste into a variety of bioproducts such as bioenergy, biopolymers, biochemicals, etc. In parallel with the development of the concept of green chemistry, the idea of biorefinery also arose during the late 1990s. Biorefinery aims at elevating the economic potential of bioproduct production through the incorporation of conversion technologies that ensures the reuse of byproducts. Biorefinery system is categorized on the basis of four important features, viz., platform, processes, feedstocks, and products. Employment of life cycle perspective and circular bioeconomy framework in biorefineries can result in sustainable production of bioproducts through waste biorefineries and negative carbon emission in biorefinery supply chain.
This review explores the transformative potential of aquatic biorefineries in advancing sustainable resource utilisation. As global demands for renewable resources intensify, biorefineries have emerged as versatile solutions. Focusing on aquatic environments, this paper delves into diverse biomass resources, encompassing microorganisms, algae and aquatic plants. It navigates through key biorefinery processes, including hydrothermal liquefaction, algae cultivation and enzymatic conversion, illuminating their roles in sustainable biofuel and high-value chemical production. Thermochemical conversion processes, such as pyrolysis and gasification, offer additional pathways for bio-based product generation. The review critically assesses challenges in these processes, ranging from technical intricacies to regulatory considerations. Examining products derived from aquatic biorefineries (i.e. biofuels, chemicals and biomaterials) underscores their versatility. Looking ahead, the paper identifies technical challenges, regulatory landscapes and emerging technologies as focal points for future research. The review concludes by envisioning aquatic biorefineries as key players in sustainable resource management, advocating for research and technological innovation to propel this transformative field into the mainstream of the bio-based economy.
This comprehensive and critical review explores the synthesis and applications of carbohydrate‐based surfactants within the biorefinery concept, focusing on biobased sugar‐head molecules suitable for use across several manufacturing sectors, including cosmetics, pharmaceuticals, household products, detergents, and foods. The main focus relies on sustainable alternatives to conventional surfactants, which could reduce the final manufacturing carbon footprint of several industrial feedstocks and products. A thorough analysis of raw materials, highlighting the significance of feedstock sources, and the current biobased surfactants and rhamnolipid biosurfactants production trends, is presented. Key organic reactions for the production of sorbitan esters, sucrose esters, alkyl polyglycosides, and fatty acid glucamines, such as glycosidation, acylation, and etherification, as well as the production of rhamnolipids through fermentation are described. Given the scarce literature on the characterization of these surfactant types within the hydrophilic–lipophilic deviation (HLD) framework, the surfactant contribution parameter (SCP) in the HLD equation for sugar‐head surfactants is critically assessed. The economic landscape is also discussed, noting the significant growth in the biobased surfactants and biosurfactant market, driven by environmental awareness and regulatory changes, with projections indicating a substantial market increase in the forthcoming years. Finally, the promising potential of generative artificial intelligence (AI) in developing customized surfactant molecules, with optimized properties for targeted applications, is emphasized as a promising avenue for future research.
In recent times, resources based on fossil fuels have been considered the basis for the generation of energy. Currently, there has been a paradigm shift in this conventional practice, and the investigation of more sustainable, cost-effective, and eco-friendly feedstocks is being pursued for the generation of fuels and prebiotics. Lignocellulosic biomasses (LCBs) are a sustainable and alternate renewable resource that has been recognized as a substitute to curtail the dependency of this sector on fuels derived from fossil resources and to remove their imprints on the environment. Lignocellulosic biomasses are not only abundant but also renewable resources. The concept of biorefineries has set a stage for numerous biomasses, principally lignocellulosic biomasses, to be investigated for the production of fuels and prebiotics. The production of biofuels in a biorefinery can substantially curtail the emission of greenhouse gases (GHG) and are sustainable and eco-friendly. The industrial production of biofuels has taken novel dimensions, and approaches are reorganized for their production. Their production helps in uplifting the renewable economy under a sustainability regime. This chapter provides a deep insight into various aspects of oligosaccharides and biofuel, along with the strategies employed in the production of various oligosaccharides and biofuels.
Carbohydrate biopolymers have drawn much attention due to their eco-friendly approach and origin from agro-food-based industry waste which solves the problem of waste material disposition. Biopolymers exhibit similar properties to petroleum-based polymers that replace existing polymers in several applications. However, such biopolymers show poor essential physicochemical and mechanical qualities. Integration of nanotechnology with biopolymers strengthens the essential qualities of such biopolymers. The present review gives deep knowledge about the recent trends in extracting biopolymers from agro-food waste. The development of various carbohydrate nanopolymer films was discussed through the blending of biopolymers with other nanoparticles and the reapplications of agro-food waste-derived carbohydrate nanocomposite to the food and agriculture sectors.
Graphical abstract
Antecedentes:
Los rumiantes utilizan pastos y forrajes para su alimentación, pero en ocasiones no tienen la calidad requerida por lo que se afecta su digestibilidad. Una de las estrategias para mejorar la calidad es el tratamiento con enzimas exógenas que descomponen las estructuras de la pared celular y permiten un mejor aprovechamiento de los nutrientes.
Objetivo.
Profundizar en los mecanismos de acción de la aplicación de enzimas fibrolíticas exógenas en la alimentación de los rumiantes.
Desarrollo:
La pared celular vegetal está compuesta por: celulosa, hemicelulosa y lignina. Las celulasas, hemicelulasas y enzimas lignocelulolíticas participan en su degradación y se utilizan satisfactoriamente a las dietas para mejorar su digestibilidad con efectos positivos en la producción de estas especies.
Conclusiones:
Esta práctica favorece una mayor disponibilidad de los nutrientes para su digestión, absorción y contribuye a mejorar los procesos fisiológicos, y en muchas ocasiones, se evidencia a través de incrementos de la productividad ganadera.
Biorefineries are facilities in which lignocellulosic biomasses are converted in a wide range of bioproducts facilitating the transition from the use of petrochemical resources to renewable ones. Organic acids are considered very attractive for their utilization in different industrial areas as building blocks or as final bioproducts leading to a considerable market growth. They are metabolites which are naturally produced by microbials. The production of these molecules by filamentous fungi are attracting more attention due to their ability to hydrolyze lignocellulosic biomasses and to contextually produce different organic acids. Contrarily to a lot of other microorganisms, fungi have the ability to ferment pentoses, broadening the substrate utilization. The integrated use of lignocellulosic biomasses as material input and fungi as biocatalyst can contribute to make biorefineries more successful. This review gives an overview about the lignocellulosic biomass structure and hydrolysis, fungal morphology, and how they are connected. Further, it describes some relevant organic acids with regard to their processes, biocatalysts, industrial applications, and market considerations.
Thermostable enzymes are enzymes that can withstand elevated temperatures as high as 50 °C without altering their structure or distinctive features. The potential of thermostable enzymes to increase the conversion rate at high temperature has been identified as a key factor in enhancing the efficiency of industrial operations. Performing procedures at higher temperatures with thermostable enzymes minimises the risk of microbial contamination, which is one of the most significant benefits. In addition, it helps reduce substrate viscosity, improve transfer speeds, and increase solubility during reaction operations. Thermostable enzymes offer enormous industrial potential as biocatalysts, especially cellulase and xylanase, which have garnered considerable amount of interest for biodegradation and biofuel applications. As the usage of enzymes becomes more common, a range of performance-enhancing applications are being explored. This article offers a bibliometric evaluation of thermostable enzymes. Scopus databases were searched for scientific articles. The findings indicated that thermostable enzymes are widely employed in biodegradation as well as in biofuel and biomass production. Japan, the United States, China, and India, as along with the institutions affiliated with these nations, stand out as the academically most productive in the field of thermostable enzymes. This study's analysis exposed a vast number of published papers that demonstrate the industrial potential of thermostable enzymes. These results highlight the significance of thermostable enzyme research for a variety of applications.
Enzymes and related products derived from microorganisms form the foundation for the working of bio-based technologies due to their enzymatic activities. The microorganisms offer wide applications such as delignification, oxidation, reduction, hydrolysis, etc., with the help of enzymes and related products derived from them. They can be used to produce various value-added products/chemicals and biofuels. Numerous bio-based alternatives have been explored worldwide to develop sustainable technologies via the utilization of microorganisms and enzymes. These bio-based products can be used to replace the conventional chemicals and fuels currently used, leading to a greener society. Applications of microbes and their enzymatic system have also been extended to various domains such as healthcare, bioremediation, food industry, bioelectricity, etc. Bioelectricity by using microorganisms in microbial fuel cells (MFCs) has been receiving attention globally as it can be an efficient source for a steady supply of energy and job opportunities. Also, the utilization of wastewater in MFCs for electricity generation adds up to its positive environmental impacts. The current chapter deals with the use of microorganisms and derived enzymes in various industrial sectors. The principle behind the utilization of microbes in MFCs and wastewater mitigation strategies are also discussed. Lastly, the limitations and prospects are highlighted to understand its potential future role and possible opportunities.
To protect the future of human beings is to protect the environment by utilizing the idea of bioenergy. Bioenergy is a renewable source of energy produced from biomass. Bioenergy like biodiesel, bioethanol, biobutanol, biogas, bioelectricity, etc., uses biomass sources like plants, edible vegetable oil, food crops with high content of sugar and starch, such as corn and sugarcane, and oilseeds to produce biofuel. Second generation bioethanol and biodiesel are produced using feedstock like nonedible vegetable oil, lignocellulosic biomass, cooking oil, animal fats, wood, and pulp, which provides numerous advantages over the first generation feedstock, it overcomes the food vs fuel issues. Biogas and biohydrogen are produced from the anaerobic digestion of wastes (sewage, manure, agricultural and industrial waste). The third generation bioenergy utilized the feedstock like oleaginous microorganisms such as algae. The major advantage of using algae lies in the fact that it can grow on saline water, municipal wastewater, and inappropriate land for agriculture and doubles the biomass in 2-5 days as compared to other feedstock. Bioelectricity is an advanced form of bioenergy that is produced by a microbial fuel cell that directly converts chemical energy into electricity by using biofilm as a catalyst. Therefore, there is a need to study bioenergy as it has the potential to replace fossil fuel-derived energy being a sustainable, renewable, and environmentally friendly alternative.
This chapter provides a nonexhaustive insight on the industrial applications of immobilized enzymes, including the food industry. The last two decades have seen a continuous advance in protein engineering and genetic engineering of enzymes (with many recombinant proteins available from genetically modified organisms), new supports and reactor configurations such as magnetic nanomaterials and 3D microreactors, new reaction media such as ionic liquids, and also many improvements in bioreactors and bioprocesses. However, there are still several problems to solve to go forward to a more widespread use of enzymatic technologies in classic applications and in fields like environmental remediation or materials.
Compared with single-enzyme catalysis, multienzyme cascade reactions play a significant role in the production of many compounds at an industrial level because they permit to perform very complex reactions. However, multienzymes in free forms are difficult to recover, causing high costs and low-production efficiency, which limits their use in industrial applications. Immobilization of multiple enzymes combines the potential of multiple enzymes to catalyze complex chemical reactions with the advantages of enzyme immobilization in reducing the cost and improving stability. Furthermore, a rational immobilization strategy can enhance the catalytic activity of immobilized multiple enzymes by achieving substrate channeling, reducing product inhibition, or increasing the concentration of local substrates. The current reviews on multienzyme immobilization focus on the selection of materials and methods. This chapter provides an overview of the immobilization techniques for multiple enzymes from a methodological perspective and analyzes their advantages as well as shortcomings.
The development of alternative fuels has been promoted by the extreme fossil fuel consumption brought on by urbanisation and deteriorating pollution. Due to its high energy and combustible qualities, biohydrogen has been perceived as a potential fuel substitute in dealing with issues related to the rising emission of greenhouse gases and global warming. As a source of carbon sequestration and sustainable renewable energy, biohydrogen synthesis by algae species has been prevalent in research scale. This review focuses on the novel and recent metabolic approaches for enhanced algal based biohydrogen production. Pretreatment methods available and scaling techniques used for enhancing the biohydrogen productivity using algal species have been elaborated in the review. Algal characteristics that make them suitable alternative for biohydrogen production are discussed briefly. Various pretreatment methods such as physical, chemical, biological and thermal are elaborated. In addition, the factors involved in influencing the biohydrogen productivity and the metabolic engineering approaches for modifying the pathway in algae are highlighted. Scaling up of process using different types of photobioreactors such as tubular, flat panel, airlift and stirred tank are reported that briefs about merits and demerits of each photobioreactor.
The generation of renewable energy resources as an alternative to fossil fuels is essential to sustain the growing human population. Lignocellulosic biomass is considered an important renewable resource for various value-added compounds and biofuels, as the world is currently poised toward a carbohydrate-based economy. Analogous to petroleum refineries, biorefineries deal with the carbohydrate polymers (cellulose, hemicellulose) and aromatic compounds (lignin), which can be processed into different bioproducts. However, the complex architecture of crystalline cellulose, hemicellulose, and lignin creates high recalcitrance, which requires significant pretreatment steps. Thus, developing cost-effective pretreatment is crucial for the effective separation of the biomass components. In this chapter, first, the basic components of the lignocellulosic biomass have been briefly described followed by the various conventional physical and chemical pretreatment methods. In addition, the efficiency of different biomass-specific pretreatment operations and their combinations has been discussed in detail. Moreover, challenges of the pretreatment processes, like chemical recovery, inhibitory byproducts formation, prolonged and costly methods, and feedstock utilization are also highlighted. Overcoming the challenges has demonstrated the potentiality of the available pretreatment methods in the advanced biological refinery process for the production of biofuels and various value-added compounds.
Biorefinery is a sustainable system used to produce bioproducts from non‐value‐added material as feedstock while reducing environmental impacts and creating new pathways for socio‐economic development. There are various concepts of biorefinery, and one of them is the use of lignocellulosic biomass (LB). It has attracted interest since LB is described as the most abundant and cheapest bio‐renewable resource on Earth. The main challenge in LB biorefinery is developing a technology that allows the full use of LB to produce bioenergy associated with bioproducts focused on environmental sustainability. In this way, studies have shown that the best vision of sustainable management for green product production is based on a vision of the circular economy and involves the implementation of Industry 4.0. The Industry 4.0 technologies have been used in downstream steps individually or combined, allowing detection problems and reducing costs in energy production. Moreover, the application of Industry 4.0 in LB biorefinery can link a set of physics, computer science, and biology technologies such as the internet of things and life‐cycle assessment. Thus, the improvement of biorefineries in downstream steps can significantly contribute to new economic, environmental, and social developments.
Biorefineries represent an integrated approach to facilitate biomass conversion to produce useful fuels, energy, and bulk chemicals from biomass. It employs microbes in the production process, employing methods that have a low carbon footprint. This chapter reviews the various approaches inside a modern biorefinery, from enhanced production of ethanol which can be used as a petrol alternative, to genetic engineering, allowing the production of solvents, bulk chemicals, plastics, and fibers. These advances make production in biorefineries greener as they counter various environmental implications by using lesser energy and being less toxic, producing lesser waste and cost-effective high-end products compared to their traditional manufacturing counterparts. Additionally, the extensive development in applications of biotechnological tools in biorefineries that convert biomass feedstocks to energy and other useful products is also reviewed. From pyrolysis-based processes to gasification, sugar-based biorefineries, and energy crops along with oilseed and lignocellulosic biorefineries. Further, the challenges to integrating higher value chemicals production systems with commodities, for energy and fuel, are also presented, with scope for optimization through resource utilization while minimizing wastes also discussed. Such advances catalyze diversification in feedstocks and products and contribute to sustainability from both economic and environmental perspectives.
Agricultural, industrial, and household practices generate a wide variety of waste biomass that is generally underutilized and often contributes significantly to environmental pollution. Such waste streams are rich in complex polysaccharides (cellulose, hemicellulose, and pectin), proteins, and lipids that can be hydrolyzed into fermentable sugars (hexoses and pentoses) or other added-value products (peptides, fatty acids, organic acids, carotenoids, etc.). However, the conversion of complex polymeric substrates into fermentable sugars is carried out by means of various physical and chemical methods. Physical methods of biomass treatment such as grinding, milling, microwave radiation, and ultrasonication are primarily aimed at reducing the size of the structural biopolymers and exposing the lignocelluloses to chemical reagents or enzymes for further hydrolysis. Conventionally, acid or alkali is used for hydrolysis of pretreated lignocellulosic biomass (such as agro and forestry residues). Other methods of physicochemical treatment such as liquid hot water treatment, autoclaving, or ammonia fiber expansion can be selected depending upon the biomass characteristics. Similarly, wastewater rich in proteins or lipids from industries such as dairy, oil refineries, and poultry is traditionally treated with hydrolytic enzymes (proteases and lipases) prior to anaerobic biodegradation. This chapter provides a comprehensive review of the various physical, chemical, and physicochemical methods for a breakdown of complex polymeric substrates in the waste streams into either simpler fermentable sugars or other bioproducts of commercial value.KeywordsHydrolysisLignocellulosic biomassWastewaterChemical methodsPhysical methodsHydrolytic enzymes
Now-a-days, the rapid depletion of fossil fuels has created a global crisis of natural resources. Researchers are constantly focusing on the utilization of environment-friendly technologies and fuels. Integrated biorefinery possesses the ability to provide long-lasting, self-dependent, strong, environment-friendly alternatives for the production of various chemicals and biofuels. An integrated biorefinery is a modern idea derived from oil refineries that uses biomass to produce a plethora of products. The biorefinery can be divided into three major categories on the basis of biomass composition, namely triglycerides-protein based biomass, sugar and starchy, and lignocellulose. Enzymes have a great influence on biochemical processes in the transformation of carbohydrates and starch, and lignocellulosic biomass to bioproducts like biofuels and bioethanol. The optimization of various enzymes can be applied to different processes such as enzymatic hydrolysis and fermentation to increase the efficacy and maintain the stability of these enzymes used in bioprocesses.KeywordsBiorefineryBiomassLignocellulosic biomassBiorefinery processesOptimization
Global population growth has increased the need for energy resources. The increased demand for energy has already begun to have negative consequences for the environment. This involves climate change and energy resource depletion, including fossil fuels. In order to avoid the exhaustion of such resources, fresh opportunities for the current scenario must be explored. Lignocellulosic Biomass (LB) is the easily available source form of renewable and sustainable energy. The LB consists majorly of cellulose, hemicelluloses, and lignin. Wheat straw, rice straw, sugarcane, and maize stover are the source of lignocellulosic biomass used for production of biofuels and high valued compounds such as ethanol, acids, and phenols. However, it is limited by recalcitrance phenomena which are subjected to various pretreatment methods leading to the production of biorefineries. Thus, this chapter will discuss methods in biomass pretreatment, factors contributing to recalcitrance, and the role of omics techniques in knowing the elements affecting recalcitrance.KeywordsBiomass conversionMicroorganismsBioenergyMicrofibrilsRenewable energyOmics
Agro residues could be utilized as a fuel in solid form or by converting to liquid or gaseous fuel such as bio-oil and biogas. Fuel conversion of agro residues either involves biochemical conversion or thermochemical conversion processes. The biochemical conversion process includes anaerobic digestion and fermentation, whereas thermochemical conversion includes direct combustion, gasification, and pyrolysis. These technologies have been believed to produce drop-in grade fuels which show promising potential for decarbonizing the transportation sector and supplying clean energy as an alternative to fossil fuels. Recent advancements and large-scale demonstrations of these technologies show its readiness to replace/substitute the centuries-old fossil fuel-based energy supply system. However, it is essential to investigate the economic, technological, and environmental trade-offs of these technologies. Life cycle assessment is a tool to access and quantify the possible environmental impacts and sustainability of these technologies. In this chapter, the authors elaborate on biochar, syngas, and bio-oil conversion of agro residues and discuss its sustainability and possible environmental impacts.
Microbes are important for various desired products via bioconversion routes to produce food, fuel, and feed, which not only include industrially important products but also energy options and bio-molecules. In this regard, microbial bioprospecting is the systematic identification, evaluation, and exploitation of microbial diversity for commercial purposes. A biorefinery is the facility that integrates biomass conversion processes and equipment to produce fuels, power, and valuable chemicals from these microbial communities. Microbial pretreatment of lignocellulosic biomass for biofuel applications, fermentation of organic materials for biofuels, enzymes, and valuable products are shaping new pathways for biorefinery applications. Thus, this chapter is aiming to summarize the microbial bioprospecting routes, products from biorefining applications with various challenges of economic stability and energy security in respect of greenhouse gas emission and promote sustainable options.
Valorization of lignocellulosic biomass (LCB) as a promising alternative to bioenergy and value-added products and for biorefinery application has fetched a lot of attention in the past few decades. LCB comprised of cellulose, hemicelluloses, and lignin, which converse a recalcitrant structure, that makes it inefficient for valorization through a biological approach. This review aims to emphasize the fate of recalcitrant LCB and understand their fate during biomass utilization. In specific, LCB recalcitrance for valorization is compared by various physio-chemical pretreatment techniques, and their combinations with enzyme treatment are discussed. It was found that the combined treatments are more effective, while their effects on recalcitrant removal were totally dependent on structural and functional factors of LCB. Therefore, a detailed investigation of efficient LCB conversion protocol into viable bioproducts needs more research.
Rising energy demands and depletion of fossil fuels have led the research community to investigate alternative fuel sources. Green and renewable biofuels have evolved to substitute for a non-renewable energy source. Biomass can be utilized as a raw material for producing low-carbon fuels. Although biomass-based fuels can replace fossil fuels, direct use is limited due to the low quality of the fuels and expensive process costs. An unrivaled solution to this problem is an integrated biorefinery concept involving generating hydrocarbon-grade fuels and valuable chemicals from pyrolysis-derived bio-oil. The chapter examines recent breakthroughs in bio-oil up-gradation processes and moisture removal techniques and bio-oil recovery of valuable compounds. One of the widely used and well-developed techniques for producing bio-oil is the fast pyrolysis of biomass. The catalytic cracking process has been identified as a viable technology for converting bio-crude to liquid fuel in bio-oil upgrading. The chapter examines recent trends and advances in the fast pyrolysis technique to improve overall profitability of the process. Critical analysis of the potential and existing techniques and necessary future steps are essential for adopting these methods industrially and in a feasible manner.KeywordsPyrolysisUpgraded fuelGreen fuelBiomass
Pretreatment of lignocellulosic waste is one of the costliest phases in transforming cellulosic material into fermentable sugars. It represents one-third of the overall process cost, and about 90% of the dry weight constitutes cellulose, hemicellulose, lignin, and pectin. Hydrogen bonds and some covalent bonds bind the carbohydrate polymers to lignin firmly. The existence of lignin in lignocelluloses barricades the plant cell against the breakdown action by fungi and bacteria. The purpose of the pretreatment procedure is to disrupt the crystalline phase of cellulose and disintegrate the lignin structure, improving the porosity of the lignocellulosic material.It further provides acids and enzymes access to hydrolyze cellulose by expanding the porosity of the lignocellulosic material so that it readily attacks to break down the cellulose. Pretreatment is therefore done: (i) to facilitate hydrolysis for the formation of sugars, (ii) to keep away the decaying or waste of carbohydrates, (iii) to prevent the creation of by-products that hinder the hydrolytic activities and fermentation that follow, and (iv) make it economical. Numerous pretreatment protocols are employed for treating biomass to overcome the problem faced during pretreatment. In this study, we have dealt with various pretreatment adopted against lignocellulosic biomass, which is a measure of their potential as feedstock for biofuels.KeywordsLignocellulosic wastePretreatmentCelluloseLignin
In this chapter, a brief introduction to various pyrolysis processes and a detailed analysis of fast pyrolysis has been explored. A wide variety of feedstocks suitable for the fast pyrolysis process and physicochemical properties of bio-oil has been incorporated. Various types of pyrolyzers that are available in the papers are also mentioned here. Furthermore, enrichment of bio-oil using various upgradation techniques and its physicochemical properties are also discussed. This includes the processes such as steam reforming, catalytic cracking, and supercritical extraction. The application of bio-oil as a fuel requires enhancement in its properties which is achieved using blending with other fuels. Thus, this chapter also strives to explain the recent advances made in bio-oil properties enhancement. Also, a brief analysis of the techno-economic feasibility of bio-oil production and its environmental sustainability is specified. This chapter attempts to give an overview of the whole concept of bio-oil production to its application.KeywordsPyrolysisBio-oilFast pyrolysis reactor (pyrolyzer)Upgradation and enhancement of bio-oilTechno-economicsEnvironmental sustainability
Finite petro-based reserves and a surge in environmental pollution demands the valorization of waste into revenue streams like biofuels and other industrial commodities. Enzymatic technology provides an eco-friendly platform for the same with higher product yields. Enzymes act as a catalyst in the reaction, and the matter of value addition in this technology is its requirement in low quantity and reusability. They have been included in the valorization of lignocellulosic (woody, agro, and food) waste, treatment of wastewater, and degradation of non-biodegradable hazardous waste. Microbial flora has enormously experimented as well as explored in the conversion of this waste into valuable products. In addition to that, protein engineering and metabolic engineering have been seen as new biotechnological trends in the same field. In this chapter, we will focus on different classes of hydrolytic enzymes based on the structural composition of different types of biomass with special attention to their catalytic activity. The mechanistic action of these enzymes will also be discussed in lieu of their use at various stages in the transformation of waste to value-added substances. We will also shed light on the future advancement through the biotechnological revolution in the field of enzyme technology.KeywordsHydrolytic enzymesValorization of wasteLignocellulosic wasteWastewaterBiorefinery
The potential of renewable energy and chemical sources is more important than ever before due to the combination of diminishing crude oil supplies and population increase. The bio-refinery concept is evolving from a fascinating notion to a viable replacement for a variety of fossil-fuel-based goods. Pre-treatment processes designed for a comprehensive bio-refinery shall show selective dissociation of each constituent of a biomass feedstock, ease of access to and detachment of the constituents after separation, high yield revival of every component, process components readily available for conversion into chemicals with negligible purification, as well as economic feasibility. These criteria are typically met by organosolv pre-treatments. To be broadly accepted by markets and the public, the generation of renewable chemicals, as well as biofuels, should be price and performance competitive employing crude oil-derived counterparts. The focus of this study is on developing a biomass conversion technique that maximizes the transformation of lignocellulosic biomass into commercially viable high-value products, allowing for effective translation to an economically feasible commercial process.KeywordsBio-refineryBiomass feedstockEconomyOrganosolv technologyHigh-value products
Plant biomass is an excellent lignocellulosic source that can produce renewable and environment-friendly biofuels. However, the natural physiochemical structure of plant lignocellulose has strong recalcitrance and heterogeneity, which results in low yields of biofuels, limiting its effective valorization in biorefineries. This rigidity of lignocellulose presents economic and technical challenges in biomass conversion processes. Various pretreatment methods are used separately and in combination to resolve this. Pretreatment methods change the structure and chemical composition of the plant biomass, which makes it more accessible to the conversion systems for biofuel production. This chapter will discuss the physical and chemical basis of lignocellulose recalcitrance and the biomass components contributing to it. This chapter will also explain the role of pretreatment strategies in biorefineries and their influence on the structure and composition of lignocellulosic biomass. The fundamental understanding of biomass recalcitrance and the role of pretreatment methods can aid in the efficient utilization of lignocellulosic biomass in biorefineries and the development of future pretreatment methodologies.KeywordsLignocelluloseRecalcitrancePretreatmentBiorefinery
Biomass-derived biorefineries seem a promising approach for the complete valorization of biomass into bioenergy and a range of bioproducts. However, the product quality and bioprocessing strategy highly depend on the nature, composition, and quality of the biomass feedstock. The source and cultivation conditions do not only affect the quality and composition of the biomass but also affect the cost of biomass. Therefore, the choice of cultivation conditions and selection of biomass suitable for its subsequent use is critically important. Accordingly, residual biomass from agricultural or industrial activities or biomass production on marginal lands using wastewater offers an opportunity to produce low-cost biomass without creating any competition with food or land for food. However, it is important to consider that how these conditions affect the nature and composition of biomass with reference to its downstream processing. This book chapter covers the desired characteristics of the biomass to consider it as a potential feedstock for biorefinery while achieving environmental and economic sustainability.KeywordsFeedstock characteristicsEnvironmental sustainabilityBioenergyBiorefinery
La biomasa lignocelulósica es reconocida como materia prima renovable y abundante en el planeta y útil en plataformas de procesamiento para la producción de biocombustibles y/o biomoléculas de alto valor agregado. Este tipo de proceso de producción integrado es llamado “biorrefinería” y es intensamente estudiado debido a que su implementación todavía es obstaculizada por factores como el consumo energético en las etapas de pretratamiento, la carencia de una comprensión profunda de la sinergia de las enzimas celulasas, y la dificultad de estandarización de los procesos de conversión dada la variabilidad de materias primas y escalas de aplicación. Así, este trabajo propone una revisión global de los tópicos anteriormente mencionados asociados a los fundamentos de la composición y características de la lignocelulosa, así como ejemplos de moléculas derivadas significativas por su valor comercial. Desde esta perspectiva se propone hacer una colección de conocimientos necesarios para el entendimiento de las plataformas de procesamiento de la biomasa y la valorización de biomoléculas derivadas mediante herramientas de la Ingeniería de Procesos y Sistemas que permitan la identificación de rutas tecnológicas de base biológica sostenibles, rentables y flexibles.
Lignocellulosic biomass (LCB) is an energy source that has a huge impact in today's world. The depletion of fossil fuels, increased pollution, climatic changes, etc. have led the public and private sectors to move towards sustainability i.e. using LCB for the production of biofuels and value-added compounds. A major bottleneck of the process is the recalcitrant nature of LCB. This can be overcome by using various pretreatment strategies like physical, chemical, biological, physicochemical, etc. Further, the pretreated biomass is made to undergo various steps like hydrolysis, saccharification, etc. for the conversion of value-added products and the remaining waste residues can be further utilized for the synthesis of secondary products thus favouring the zero-waste biorefinery concept. Currently, microorganisms are being explored for their use in biorefinery but the unavailability of commercial strains is a major limitation. Thus, the use of metagenomics can be used to overcome the limitation which is both cost-effective and environmentally friendly. The review deliberates the composition of LCBs, and their recalcitrance nature, followed by the structural changes caused by various pretreatment methods. The further steps in biorefineries, strategies for the development of zero-waste refineries, bottlenecks, and suggestions are also discussed. Special emphasis is given to the use of metagenomics for the discovery of microorganisms efficient for zero-waste biorefineries.
Pectinases are a group of enzymes that lyse pectin. Pectins are polysaccharides, and they are found abundantly in plant cell walls. The feature of pectinolytic activity is exploited by the industry. With the advances, the industry has developed a focus on various microbial sources for the efficient production of pectinolytic enzymes. The bioprocess principles are applied extensively for the production of pectinase enzymes for efficient commercial production and harvest. Many factors affect the yield of pectinases, which are overlooked, and the shortcomings are improved; this is done with the help of understanding and the research made on studying the biochemical properties of pectinases. The purification and characterization of pectinase enzymes have a key role in controlling the purity and standards. With the help of studies on the mechanism of action of pectinases, it is found to have wide applications, which range in the field of the brewery, juice making, jam making, retting, plant fiber making, paper making, and so on.KeywordsPectinasesPectinolyticBreweryRettingPectin
Lignin is an abundant polyphenol found in the plant cell wall. In an enzyme-catalysed reaction, the monomeric monolignols p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol as phenylpropanoids form lignin. The composition of lignin differs across the plants with respect to the presence of monomers. It is difficult to degrade and act as a recalcitrant in the carbon cycle. Structural heterogeneity of lignin is a major hindrance in the bioconversion of specific by-products.Basidiomycetes, white-rot fungi, brown-rot fungi, and certain aerobic bacteria can partially and totally degrade lignin with or without the use of mediators. Major enzymes involved in lignin degradation include manganese peroxidase, lignin peroxidase, versatile peroxidase, and laccase. The microbial-assisted systems with their enzymes can modify and biotransform lignin into a wide range of small molecular weight products. Lignin is an economical relatively non-toxic and renewable substrate for biotransformation processes. Lignin and its degradation products can be utilised for a variety of industrial applications including flavouring agents, polymers, biodegradable plastics, adhesives, fillers foam, insulators, etc. They have proven to have therapeutic benefits such as anticancer, anti-inflammatory, antioxidants, antibiotics, and antimicrobials agent.KeywordsLigninRecalcitrantEnzymesLignicolous fungiBiotransformationIndustrial applications
Strain improvement is an advanced biotechnological strategy where various cellular pathways are modified by recombinant DNA technology to improve the yield of metabolic products that are beneficial to humanity. Strain improvements are directed toward improving product quality and yield by enhancing substrate utilization, regulating enzyme activity, resistance to phage infection, etc. The primary genetic routes to strain improvement include (1) mutagenesis for the creation of genetic variants, (2) screening to select improved strains, (3) identification of improved strains, and (4) mass culture optimization of operational and cellular responses and downstream processing. This chapter details the various strain improvement strategies and the respective computational and biotechnological methods that are used.KeywordsStrain improvementMutationGene expressionMicroarraySequencingPrincipal component analysis (PCA)
The highly acclaimed prospect of renewable lignocellulosic biocommodities as obvious replacement of their fossil-based counterparts is burgeoning within the last few years. However, the use of the abundant lignocellulosic biomass provided by nature to produce value-added products, especially bioethanol, still faces significant challenges. One of the crucial challenging factors is in association with the expression levels, stability, and cost-effectiveness of the cellulose-degrading enzymes (cellulases). Interestingly, several recommendable endeavors in the bid to curb these
challenges are in pursuance. However, the existing body of literature has not well provided the updated roadmap of the advancement and key players spearheading the current success. Moreover, the description of enzyme systems
and emerging paradigms with high prospects, for example, the cell-surface display system has been ill-captured in
the literature. This review focuses on the lignocellulosic biocommodity pathway, with emphasis on cellulase and hemicellulase systems. The paradigm shift towards cell-surface display system and its emerging recommendable developments have also been discussed. The attempts in supplementing cellulase with other enzymes, accessory
proteins, and chemical additives have also been discussed. Moreover, some of the prominent and influential discoveries in the cellulase fraternity have been discussed
Ligninolytic enzymes play a key role in degradation and detoxification of lignocellulosic waste in environment. The major ligninolytic enzymes are laccase, lignin peroxidase, manganese peroxidase, and versatile peroxidase. The activities of these enzymes are enhanced by various mediators as well as some other enzymes (feruloyl esterase, aryl-alcohol oxidase, quinone reductases, lipases, catechol 2, 3-dioxygenase) to facilitate the process for degradation and detoxification of lignocellulosic waste in environment. The structurally laccase is isoenzymes with monomeric or dimeric and glycosylation levels (10-45%). This contains four copper ions of three different types. The enzyme catalyzes the overall reaction: 4 benzenediol + O2 to 4 benzosemiquinone + 2H2O. While, lignin peroxidase is a glycoprotein molecular mass of 38-46 kDa containing one mole of iron protoporphyrin IX per one mol of protein, catalyzes the H2O2 dependent oxidative depolymerization of lignin. The manganese peroxidase is a glycosylated heme protein with molecular mass of 40-50kDa. It depolymerizes the lignin molecule in the presence of manganese ion. The versatile peroxidase has broad range substrate sharing typical features of the manganese and lignin peroxidase families. Although ligninolytic enzymes have broad range of industrial application specially the degradation and detoxification of lignocellulosic waste discharged from various industrial activities, its large scale application is still limited due to lack of limited production. Further, the extremophilic properties of ligninolytic enzymes indicated their broad prospects in varied environmental conditions. Therefore it needs more extensive research for understanding its structure and mechanisms for broad range commercial applications.
From the last two decades, white biotechnology, with particular reference to deploying enzyme bio-catalysis, has gained special research interest to valorize the bio-sources lignocellulosic biomass. In this context, ligninolytic enzymes from a white biotechnology background have tremendous potentialities to transform biomass following the green agenda. The enzyme-based white biotechnology is now considered a key endeavor of twenty-first century, as it offers socio-economic and environmental merits over traditional biotechnology, such as eco-friendlier processing conditions, no/limited use of harsh chemicals/reagents, high catalytic turnover, high yield, cost-effective ratio, low energy costs, green alternative of complex synthesis, renewability, reusability, and recyclability. Research efforts are underway, around the globe, to exploit naturally occurring biomass, as a green feedstock and low-cost substrates, to generate value-added bio-products, bio-fuels, and bio-energy. One core problem in developing an eco-friendlier and economical bioprocess is the pre-treatment of lignocellulosic biomass to entirely or partially remove the lignin barrier from cellulose fibers, thereby allowing the enzymes to access the cellulose fibers and generate the products of industrial interests. The entire process requires lignocelluloses deconstruction where ligninolytic enzymes in synergies with redox mediators systems have not explored much. The limited exploitation of ligninolytic enzymes with tremendous catalytic efficiencies has created a massive research gap that we have tried to cover herein. This review further insights the white biotechnology, also termed industrial biotechnology, which uses microorganisms and their unique enzyme system to facilitate the clean and sustainable deconstruction process.
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The scaled-up production of biofuels and bioproducts in the US is likely to cause land use expansion and intensification domestically and internationally, possibly leading to undesirable environmental and socioeconomic consequences. Although these concerns have been widely recognized, sustainability governance systems are yet to be developed. Here, we review (1) the US bioenergy policies, (2) biofuel production and market trends, (3) major sustainability concerns, and (4) existing regulations and programs for sustainability governance, including potential interactions with markets and technology. US bioenergy policy dates back to the 1970s and has evolved over time with various tax incentives plus production mandates in recent key legislation. Commercial production of cellulosic biofuels is impeded largely by technology and cost barriers. Uncertainties exist in the estimates of environmental and socioeconomic impacts due to the lack of empirical data and knowledge of complex relationships among biofuel and bioeconomic development, natural ecosystems, and socioeconomic dimensions. There are various existing sustainability governance mechanisms on which a biofuel sustainability governance system can be built on. Considering all these, we propose an adaptive system that incorporates regulations, certification, social norms, market, and technology for sustainability monitoring and governance, and is able to contribute to addressing the overall environmental concerns associated with collective land use for food, fiber, and fuel production. Building on existing programs and mechanisms and with proper monitoring of biofuel and bioproduct development, such a governing system can be developed and implemented in response to sustainability concerns that may arise as biofuel and bioproduct production increases.
The use of immobilized enzymes is now a routine process for the manufacture of many industrial products in the pharmaceutical, chemical and food industry. Some enzymes, such as lipases, are naturally robust and efficient, can be used for the production of many different molecules and have a wide range of industrial applications thanks to their broad selectivity. As an example, lipase from Candida antarctica (CalB) has been used by BASF to produce chiral compounds, such as the herbicide Dimethenamide-P, which was previously made chemically. The use of the immobilized enzyme has provided significant advantages over a chemical process, such as the possibility to use equimolar concentration of substrates, obtain an enantiomeric excess > 99%, use relatively low temperatures (< 60 °C) in organic solvent, obtain a single enantiomer instead of the racemate as in the chemical process and use a column configuration that allows dramatic increases in productivity. This process would not have been possible without the use of an immobilized enzyme, since it runs in organic solvent [1].
Some more specific enzymes, like transaminases, have required protein engineering to become suitable for applications in production of APIs (Active Pharmaceutical Ingredients) in conditions which are extreme for a wild type enzyme. The process developed by Merck for sitagliptin manufacture is a good example of challenging enzyme engineering applied to API manufacture. The previous process of sitagliptin involved hydrogenation of enamine at high pressure using a rhodium-based chiral catalyst. By developing an engineered transaminase, the enzymatic process was able to convert 200 g/l of prositagliptin in the final product, with e.e. >99.5% and using an immobilized enzyme in the presence of DMSO as a cosolvent [2].
For all enzymes, the possibility to be immobilized and used in a heterogeneous form brings important industrial and environmental advantages, such as simplified downstream processing or continuous process operations. Here, we present a series of large-scale applications of immobilized enzymes with benefits for the food, chemical, pharmaceutical, cosmetics and medical device industries, some of which have been scarcely reported on previously.
In general, all enzymatic reactions can benefit from the immobilization, however, the final choice to use them in immobilized form depends on the economic evaluation of costs associated with their use versus benefits obtained in the process. It can be concluded that the benefits are rather significant, since the use of immobilized enzymes in industry is increasing.
Energy demand is constantly growing, and, nowadays, fossil fuels still play a dominant role in global energy production, despite their negative effects on air pollution and the emission of greenhouse gases, which are the main contributors to global warming. An alternative clean source of energy is represented by the lignocellulose fraction of plant cell walls, the most abundant carbon source on Earth. To obtain biofuels, lignocellulose must be efficiently converted into fermentable sugars. In this regard, the exploitation of cell wall lytic enzymes (CWLEs) produced by lignocellulolytic fungi and bacteria may be considered as an eco-friendly alternative. These organisms evolved to produce a variety of highly specific CWLEs, even if in low amounts. For an industrial use, both the identification of novel CWLEs and the optimization of sustainable CWLE-expressing biofactories are crucial. In this review, we focus on recently reported advances in the heterologous expression of CWLEs from microbial and plant expression systems as well as some of their industrial applications, including the production of biofuels from agricultural feedstock and of value-added compounds from waste materials. Moreover, since heterologous expression of CWLEs may be toxic to plant hosts, genetic strategies aimed in converting such a deleterious effect into a beneficial trait are discussed.
The global rise in urbanization and industrial activity has led to the production and incorporation of foreign contaminant molecules into ecosystems, distorting them and impacting human and animal health. Physical, chemical, and biological strategies have been adopted to eliminate these contaminants from water bodies under anthropogenic stress. Biotechnological processes involving microorganisms and enzymes have been used for this purpose; specifically, laccases, which are broad spectrum biocatalysts, have been used to degrade several compounds, such as those that can be found in the effluents from industries and hospitals. Laccases have shown high potential in the biotransformation of diverse pollutants using crude enzyme extracts or free enzymes. However, their application in bioremediation and water treatment at a large scale is limited by the complex composition and high salt concentration and pH values of contaminated media that affect protein stability, recovery and recycling. These issues are also associated with operational problems and the necessity of large-scale production of laccase. Hence, more knowledge on the molecular characteristics of water bodies is required to identify and develop new laccases that can be used under complex conditions and to develop novel strategies and processes to achieve their efficient application in treating contaminated water. Recently, stability, efficiency, separation and reuse issues have been overcome by the immobilization of enzymes and development of novel biocatalytic materials. This review provides recent information on laccases from different sources, their structures and biochemical properties, mechanisms of action, and application in the bioremediation and biotransformation of contaminant molecules in water. Moreover, we discuss a series of improvements that have been attempted for better organic solvent tolerance, thermo-tolerance, and operational stability of laccases, as per process requirements.
Abstract Xylan is the second most abundant naturally occurring renewable polysaccharide available on earth. It is a complex heteropolysaccharide consisting of different monosaccharides such as l-arabinose, d-galactose, d-mannoses and organic acids such as acetic acid, ferulic acid, glucuronic acid interwoven together with help of glycosidic and ester bonds. The breakdown of xylan is restricted due to its heterogeneous nature and it can be overcome by xylanases which are capable of cleaving the heterogeneous β-1,4-glycoside linkage. Xylanases are abundantly present in nature (e.g., molluscs, insects and microorganisms) and several microorganisms such as bacteria, fungi, yeast, and algae are used extensively for its production. Microbial xylanases show varying substrate specificities and biochemical properties which makes it suitable for various applications in industrial and biotechnological sectors. The suitability of xylanases for its application in food and feed, paper and pulp, textile, pharmaceuticals, and lignocellulosic biorefinery has led to an increase in demand of xylanases globally. The present review gives an insight of using microbial xylanases as an “Emerging Green Tool” along with its current status and future prospective.
Butanol is an important bulk chemical, as well as a promising renewable gasoline substitute, that is commonly produced by solventogenic Clostridia. The main cost of cellulosic butanol fermentation is caused by cellulases that are required to saccharify lignocellulose, since solventogenic Clostridia cannot efficiently secrete cellulases. However, cellulolytic Clostridia can natively degrade lignocellulose and produce ethanol, acetate, butyrate and even butanol. Therefore, cellulolytic Clostridia offer an alternative to develop consolidated bioprocessing (CBP), which combines cellulase production, lignocellulose hydrolysis and co‐fermentation of hexose/pentose into butanol in one step. This review focuses on CBP advances for butanol production of cellulolytic Clostridia and various synthetic biotechnologies that drive these advances. Moreover, the efforts to optimize the CBP‐enabling cellulolytic Clostridia chassis are also discussed. These include the development of genetic tools, pentose metabolic engineering and the improvement of butanol tolerance. Designer cellulolytic Clostridia or consortium provide a promising approach and resource to accelerate future CBP for butanol production.
Dairy manufacturing sector generates large quantity of nutrient-rich wastewater which requires treatment before it can be released into the environment. This study aimed to use a low-cost food grade sodium lignosulphonate (Na-lignosulphonate) to recover proteins and lipids from dairy wastewater and reduce the BOD. The colloidal particles of wastewater sample were precipitated with varying the concentration of Na-lignosulphonate (0.002–0.032%, w/v) and pH of medium (1.0–3.5). Experiments were conducted at ambient (22 °C) and mildly elevated (40 °C) temperatures. At the optimum concentration (0.016%, w/v) of Na-lignosulphonate, the highest turbidity removal (98%) was achieved at pH 3.5 and at both temperatures. This method recovered 96% lipids and 46% proteins (mostly caseins) and reduced the BOD by 73% at 22 °C. Na-lignosulphonate is found to be an effective coagulant owing to its ability to readily complex with positively charged colloidal particles at acidic pH. Keywords: Dairy wastewater, Sodium lignosulphonate, Proteins, Lipid, Complexation, Coagulation, Recovery
The term biorefinery is related to the sustainable production of value-added bioproducts and bioenergy from biomass. Esters from fatty acids are important compounds synthesized from by-products of the oleochemical industry. In agreement with the biorefinery concept, it is important to search for catalysts that reduce the consumption of energy and water, using moderate operation conditions and low reaction times. In this work, response surface methodology (RSM) was used to optimize the enzymatic synthesis of pentyl oleate using Candida antarctica lipase B (CALB) immobilized on a polyethylene-aluminum structured support. A factorial design was employed to evaluate the effects of several parameters on the ester yield. To obtain a model with a good fit, an approach to reaction mechanism and enzyme kinetics was taken into consideration. Experimental findings were correlated and explained using equations of a ping-pong bi-bi kinetic model and considering the inhibitory effects of both substrates. The developed model was consistent with the experimental data predicting an increase in pentyl oleate production with increasing temperature and a decrease with higher oleic acid amounts and alcohol to acid molar ratios. This model could be useful in a future industrial application of CALB/LLDPE/Al to minimize the costs in oleochemical biorefineries.
Abstract Background Lipases are serine hydrolases that degrade triglycerides, an attribute that treasures wide applications in biodiesel production, detergent, chemical industries, etc. The most sought after the application is in the high quality and economical production of biodiesel under mild reaction conditions and simplified product separation. For the said application, fungal lipases are ideal catalysts that could effectively catalyze esterification and transesterification reactions with their specific ability to release fatty acids from 1, 3 positions of acylglycerols. Results In the present work, to facilitate bulk synthesis, lipase production using Aspergillus niger MTCC 872 was studied by solid-state fermentation (SSF). The chosen fungal strain was evaluated for lipase production using a mixture of agro-industrial substrates viz. rice husk, cottonseed cake, and red gram husk in various combinations at flask level. Tri-substrate mixture (rice husk, cottonseed cake, and red gram husk) combined in the ratio of 2:1:1 has shown the maximum lipase activity 28.19 U/gds at optimum cultivation conditions of temperature 40 °C, moisture content 75% (v/w), pH 6.0 and initial spore concentration of 5.4 million spores per mL. Further studies were performed for scale-up of lipase from flask level to lab scale using tray fermenter. Lipase activity was found to be 24.38 U/gds and 21.62 U/gds for 100 g and 1000 g substrate respectively. Conclusion This is the first report on the production of lipase from Aspergillus niger MTCC 872 using tri-substrate mixture of rice husk (RH), cottonseed cake (CSC), and red gram husk (RGH). Moreover, comparison between individual, binary, and tri-substrate mixture was carried out for which the highest lipase activity was observed for tri-substrate mixture. In addition, comparable results were found when scale-up was performed using tray fermenter. Thus, the current work signifies usage of agro-industrial residues as substrates for enzyme production by solid-state fermentation process as an effective alternative to submerged fermentation for industrial applications.
The current paper reviews the content and variation of fiber fractions in feed ingredients commonly used in swine diets. Carbohydrates serve as the main source of energy in diets fed to pigs. Carbohydrates may be classified according to their degree of polymerization: monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Digestible carbohydrates include sugars, digestible starch, and glycogen that may be digested by enzymes secreted in the gastrointestinal tract of the pig. Non-digestible carbohydrates, also known as fiber, may be fermented by microbial populations along the gastrointestinal tract to synthesize short-chain fatty acids that may be absorbed and metabolized by the pig. These non-digestible carbohydrates include two disaccharides, oligosaccharides, resistant starch, and non-starch polysaccharides. The concentration and structure of non-digestible carbohydrates in diets fed to pigs depend on the type of feed ingredients that are included in the mixed diet. Cellulose, arabinoxylans, and mixed linked β-(1,3) (1,4)-d-glucans are the main cell wall polysaccharides in cereal grains, but vary in proportion and structure depending on the grain and tissue within the grain. Cell walls of oilseeds, oilseed meals, and pulse crops contain cellulose, pectic polysaccharides, lignin, and xyloglucans. Pulse crops and legumes also contain significant quantities of galacto-oligosaccharides including raffinose, stachyose, and verbascose. Overall, understanding the structure, characteristics and measurable chemical properties of fiber in feed ingredients may result in more accurate diet formulations, resulting in an improvement in the utilization of energy from less expensive high-fiber ingredients and a reduction in reliance on energy from more costly cereal grains.
Abstract Lytic polysaccharide monooxygenases (LPMOs) are abundant in nature and best known for their role in the enzymatic conversion of recalcitrant polysaccharides such as chitin and cellulose. LPMO activity requires an oxygen co-substrate, which was originally thought to be O2, but which may also be H2O2. Functional characterization of LPMOs is not straightforward because typical reaction mixtures will promote side reactions, including auto-catalytic inactivation of the enzyme. For example, despite some recent progress, there is still limited insight into the kinetics of the LPMO reaction. Recent discoveries concerning the role of H2O2 in LPMO catalysis further complicate the picture. Here, we review commonly used methods for characterizing LPMOs, with focus on benefits and potential pitfalls, rather than on technical details. We conclude by pointing at a few key problems and potential misconceptions that should be taken into account when interpreting existing data and planning future experiments.
People have exploited the biocatalytic potential of microorganisms for centuries to produce wine, vinegar, bread, and so forth without understanding their biochemical basis. Microbial enzymes are also used as biocatalysts in various industrial processes in an economical and environmentally-friendly way, as compared with chemical catalysis. Over the past few decades, the use of microbial enzymes in bioprocesses has increased rapidly, because of their catalytic activity, as well as stability. Worldwide, enzymes produced from microorganisms have been extensively investigated for isolation, purification, characterization, and applications. Microbial enzymes have diverse applications in the food, pharmaceutical, and biotechnological industries, and so forth. Commercially, many recombinant enzymes from bacteria and fungi are used in various bioprocesses. Modern techniques such as metagenomics and genomics can be used to discover new microbial enzymes, whose catalytic properties can be improved further using molecular techniques. Moreover, the screening of novel enzymes that are capable of catalyzing new reactions is constantly required. The discovery of new enzymes will provide clues for the design of new enzymatic processes. This chapter provides an overview of industrially important microbial enzymes; particularly their sources and applications.
Plant biomass is a promising carbon source for producing value-added chemicals, including transportation biofuels, polymer precursors, and various additives. Most engineered microbial hosts and a select group of wild-type species can metabolize mixed sugars including oligosaccharides, hexoses, and pentoses that are hydrolyzed from plant biomass. However, most of these microorganisms consume glucose preferentially to non-glucose sugars through mechanisms generally defined as carbon catabolite repression. The current lack of simultaneous mixed-sugar utilization limits achievable titers, yields, and productivities. Therefore, the development of microbial platforms capable of fermenting mixed sugars simultaneously from biomass hydrolysates is essential for economical industry-scale production, particularly for compounds with marginal profits. This review aims to summarize recent discoveries and breakthroughs in the engineering of yeast cell factories for improved mixed-sugar co-utilization based on various metabolic engineering approaches. Emphasis is placed on enhanced non-glucose utilization, discovery of novel sugar transporters free from glucose repression, native xylose-utilizing microbes, consolidated bioprocessing (CBP), improved cellulase secretion, and creation of microbial consortia for improving mixed-sugar utilization. Perspectives on the future development of biorenewables industry are provided in the end.
Metagenomes from uncultured microorganisms are rich resources for novel enzyme genes. The methods used to screen the metagenomic libraries fall into two categories, which are based on sequence or function of the enzymes. The sequence-based approaches rely on the known sequences of the target gene families. In contrast, the function-based approaches do not involve the incorporation of metagenomic sequencing data and, therefore, may lead to the discovery of novel gene sequences with desired functions. In this review, we discuss the function-based screening strategies that have been used in the identification of enzymes from metagenomes. Because of its simplicity, agar plate screening is most commonly used in the identification of novel enzymes with diverse functions. Other screening methods with higher sensitivity are also employed, such as microtiter plate screening. Furthermore, several ultra-high-throughput methods were developed to deal with large metagenomic libraries. Among these are the FACS-based screening, droplet-based screening, and the in vivo reporter-based screening methods. The application of these novel screening strategies has increased the chance for the discovery of novel enzyme genes.
Lignocellulosic biomass (LCB) is the most abundantly available bioresource amounting to about a global yield of up to 1.3 billion tons per year. The hydrolysis of LCB results in the release of various reducing sugars which are highly valued in the production of biofuels such as bioethanol and biogas, various organic acids, phenols, and aldehydes. The majority of LCB is composed of biological polymers such as cellulose, hemicellulose and lignin, which are strongly associated with each other by covalent and hydrogen bonds thus forming a highly recalcitrant structure. The presence of lignin renders the bio-polymeric structure highly resistant to solubilization thereby inhibiting the hydrolysis of cellulose and hemicellulose which presents a significant challenge for the isolation of the respective bio-polymeric components. This has led to extensive research in the development of various pretreatment techniques utilizing various physical, chemical, physicochemical and biological approaches which are specifically tailored towards the source biomaterial and its application. The objective of this review is to discuss the various pretreatment strategies currently in use and provide an overview of their utilization for the isolation of high-value bio-polymeric components. The article further discusses the advantages and disadvantages of the various pretreatment methodologies as well as addresses the role of various key factors that are likely to have a significant impact on the pretreatment and digestibility of LCB.
Redox mediators could be used to improve the efficiency of microbial fuel cells (MFCs) by enhancing electron transfer rates and decreasing charge transfer resistance at electrodes. However, many artificial redox mediators are expensive and/or toxic. In this study, laccase enzyme was employed as a biocathode of MFCs in the presence of two natural redox mediators (syringaldehyde (Syr) and acetosyringone (As)), and for comparison, a commonly-used artificial mediator 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was used to investigate their influence on azo dye decolorization and power production. The redox properties of the mediator-laccase systems were studied by cyclic voltammetry. The presence of ABTS and As increased power density from 54.7 ± 3.5 mW m−2 (control) to 77.2 ± 4.2 mW m−2 and 62.5 ± 3.7 mW m−2 respectively. The power decreased to 23.2 ± 2.1 mW m−2 for laccase with Syr. The cathodic decolorization of Acid orange 7 (AO7) by laccase indicated a 12–16% increase in decolorization efficiency with addition of mediators; and the Laccase-Acetosyringone system was the fastest, with 94% of original dye (100 mgL−1) decolorized within 24 h. Electrochemical analysis to determine the redox properties of the mediators revealed that syringaldehyde did not produce any redox peaks, inferring that it was oxidized by laccase to other products, making it unavailable as a mediator, while acetosyringone and ABTS revealed two redox couples demonstrating the redox mediator properties of these compounds. Thus, acetosyringone served as an efficient natural redox mediator for laccase, aiding in increasing the rate of dye decolorization and power production in MFCs. Taken together, the results suggest that natural laccase redox mediators could have the potential to improve dye decolorization and power density in microbial fuel cells.
Lignocellulosic biomass is an abundant and renewable resource that potentially contains large amounts of energy. It is an interesting alternative for fossil fuels, allowing the production of biofuels and other organic compounds. In this paper, a review devoted to the processing of lignocellulosic materials as substrates for fermentation processes is presented. The review focuses on physical, chemical, physicochemical, enzymatic, and microbiologic methods of biomass pretreatment. In addition to the evaluation of the mentioned methods, the aim of the paper is to understand the possibilities of the biomass pretreatment and their influence on the efficiency of biofuels and organic compounds production. The effects of different pretreatment methods on the lignocellulosic biomass structure are described along with a discussion of the benefits and drawbacks of each method, including the potential generation of inhibitory compounds for enzymatic hydrolysis, the effect on cellulose digestibility, the generation of compounds that are toxic for the environment, and energy and economic demand. The results of the investigations imply that only the stepwise pretreatment procedure may ensure effective fermentation of the lignocellulosic biomass. Pretreatment step is still a challenge for obtaining cost-effective and competitive technology for large-scale conversion of lignocellulosic biomass into fermentable sugars with low inhibitory concentration.
Protection of environment is of immediate concern and this can only be achieved by avoiding the use of chemicals for fuel production. Lignocellulosic waste is becoming popular as a feedstock for biofuel production. The can be converted into usable form for biofuel production by using a suitable pretreatment method. Different pretreatment methods have been used by researchers which are physical, chemical, physico-chemical, biological and combined pretreatments. Evidently chemical pretreatment is found to be more expensive as a large amount of chemicals are used for pretreating the lignocellulosic substrate. It has been shown that combined pretreatments are more effective as compared to single pretreatment and there is an extensive scope of combinations which can also be applied in future. Recent review critically discusses and compares different pretreatment methods, biomass resources, chemical composition of different agricultural biomass and the use of this biomass for bioenergy generation. Various pretreatment processes used for bio-hydrogen, bio-methane, bio-ethanol, bio-methanol bio-butanol and bio-diesel production are also discussed.
Agave bagasse is a lignocellulosic agroindustrial waste suitable for biofuel production. This study aimed to compare biological, enzymatic, chemical, and hydrothermal pretreatments applied to Agave bagasse in terms of solubilization of carbohydrates (CHO) and chemical oxygen demand (COD), as well as the biochemical methane potential (BMP) from hydrolyzates. Most pretreatments behave similarly, with an average yield of 0.16 ± 0.02gCOD/g, only the chemical pretreatment overcame this yield by a factor of 2.6 with a CHO/COD ratio of 0.95. The concentrations of inhibitors – furfural, hydroxymethylfurfural, and phenols – were higher in the chemical hydrolyzates than in the biological and enzymatic hydrolyzates. BMP from most hydrolyzates was the same, on average 219 ± 15 mL/gCODin, hydrolyzates differentiate only in terms of lag phase and the methane production rates.
Conversion of lignocellulosic biomass to biofuels using microbial fermentation is an attractive option to substitute petroleum-based production economically and sustainably. The substantial efforts to design yeast strains for biomass hydrolysis have led to industrially applicable biological routes. Saccharomyces cerevisiae is a robust microbial platform widely used in biofuel production, based on its amenability to systems and synthetic biology tools. The critical challenges for the efficient microbial conversion of lignocellulosic biomass by engineered S. cerevisiae include heterologous expression of cellulolytic enzymes, co-fermentation of hexose and pentose sugars, and robustness against various stresses. Scientists developed many engineering strategies for cellulolytic S. cerevisiae strains, bringing the application of consolidated bioprocess at an industrial scale. Recent advances in the development and implementation of engineered yeast strains capable of assimilating lignocellulose will be reviewed.
In the present investigation, cloning and overexpression of xylanase (XynF1), the main xylanase of A. oryzae LC1, was performed in prokaryotic system E. coli BL21(DE3) to produce recombinant xylanase with high titer of specific activity (1037.3 U/mg), which was 9.3-fold higher than the native strain. Further, the recombinant XynF1 of size 37 kDa was purified using Ni2+-NTA resins followed by cation exchange chromatography, which showed an 1.8-fold increase in purity with 71.4% yield. The r-XynF1 exhibited a wide range of activity at different pH (3.0-10.0) range and temperature (30-70 °C) with an optimum pH at 5.0 and temperature at 30 °C. The results from the current study, clearly demonstrate that this is an effective method to generate a recombinant enzyme with improved activity, making it useful for possible industrial applications.
The present work aimed at investigating the isolation and application of cellulose from sugarcane bagasse in two different processes according to the biorefinery concept. The cellulose isolation step involved, in the following order, an H2SO4 (10% v/v) treatment, an NaOH (5% w/v) treatment, and a chemical bleaching with H2O2 (5% v/v). The isolated cellulose was characterized to determine its chemical composition and physical structure. The bleached cellulose was used for the synthesis of carboxymethylcellulose (CMC), and the materials obtained after acid and alkaline treatments were applied in the process of enzymatic hydrolysis. CMC degree of substitution was 0.59, evidencing that it is soluble in water. The enzymatic conversion of cellulose achieved 73.35% and the content of lignin was demonstrated to interfere directly in the efficiency of the enzymes. The results showed that cellulose from sugarcane bagasse can be successfully applied in the two investigated technological processes.
A comprehensive study on lignin contents and composition of 30 Indonesian sorghum accessions has been performed. Lignocellulose compositional analyses, i.e., thioglycolic acid assay, neutral sugar analysis, ash content measurement, and cell-wall-bound phenolic quantification, and lignin structural analyses, i.e., thioacidolysis and HSQC NMR analysis, of sorghum stem biomass were performed. In addition, their enzymatic saccharification efficiencies (ESEs) were also evaluated. The results showed that the total biomass was varied among accessions, ranging from 615.97 kg/plant (Pahat) and up to 1425.17 kg/plant (JP-1). Total lignin contents ranged from 14.35% to 22.89% of cell wall residues (CWRs), which were obtained from Pahat and KS, respectively. Analytical thioacidolysis and NMR analysis of selected 9 accessions with the lowest (Pahat, JP-1, and Buleleng Empok), moderate (Kawali, Super 1, and Super 2–300 Gy) and the highest (Sorgum Malai Mekar, 4183A, and KS) total lignin contents showed that lignin syringyl (S)/guaiacyl (G) unit ratio varied from around 0.8 to 1.5, where KS and 4183A had a relatively low and high S/G ratio, respectively. Correlation analysis of lignocellulose composition/structures with ESE showed significant associations of ESE with total lignin content, amorphous glucan content, and relative frequency of cinnamyl alcohol end-units in lignin polymers (P < 0.05, P < 0.01, P < 0.05, respectively). Taken together, this study provides a foundation toward further optimization of sorghum biomass composition for various biorefinery applications.
This work exploited the concept of mechanical fractionation of sugarcane stalks prior to processing in an integrated biorefinery. The use of four sugarcane hybrids assured the broad evaluation of sugarcane genotypes. In all cases, the outermost region of the stalk contained denser tissue, which was suitable for burning in cogeneration systems. In contrast, the core cane was processed for sucrose extraction, which generated a less recalcitrant lignocellulose fraction more amenable to pretreatment and enzymatic hydrolysis. The mass balance of the lignocellulose components along with biorefining indicated that the core cane provided up to 34% enhanced utilization of polysaccharides compared to the sugarcane bagasse, whereas the highest heating value from the sugarcane bagasse and the outermost fractions were similar.
Agro- and industrial processes that utilises medicinal and aromatic plants (MAPs) generates various kinds of residues like residual biomasses from distillation of aromatic plant and non-utilized parts of medicinal plant. These residual biomasses cannot be considered as waste as these can actually be recycled and converted into value added products. So, value addition to these residual biomasses through processing, extraction, hydrolysis, pyrolysis and fermentation, etc. could be an exciting avenue especially for the underutilized part of medicinal plant and residual biomass from the distillation of aromatic plant. These biomasses are suitable for isolation of phytochemicals like phenolics-antioxidants which can be used in pharmaceutical, cosmetic and perfumery industry. After extraction of phytochemicals, the residual biomass can be used directly as animal feed/or organic mulch. Besides, preparation of value added product, like bio sorbent for waste water purification, composts and biochar for an effective soil amendment. These value added products are found to be more promising. In this article, the potential uses of these residual biomasses as valued products have been discussed including the technology developed at laboratory scale and their application in industry. Effective recycling of residual biomass from MAPs is not only for an economic gain, but also a practical solution for its disposal. Thus, dual utilization of the residual biomasses is of great interest and will open windows of opportunity in MAPs sector.
In the present work attempts have been made to mimic the natural system of biomass delignification using laccase from Myrothecium verrucaria ITCC-8447 and synergistic effect of cellulase and xylanase from Schizophyllum commune NAIMCC-F-03379 and Aspergillus oryzae ITCC-8571 respectively for enhanced saccharification and subsequent bioethanol production. The laccase mediated pretreatment resulted in 6.7% delignification of rice straw (RS). The structural analysis of enzymatic pretreated RS using XRD and FTIR suggested high crystallinity index (CrI) and improved accessibility of cellulose and hemicelluloses as compared to untreated RS. The single step simultaneous delignification, saccharification and fermentation (SDSF) of RS using partially purified in-house enzyme cocktail with Saccharomyces cerevisiae MTCC-173 resulted in sugar yield of 26.7 ± 0.2 g/L with an experimental ethanol yield of 6.47 ± 0.16 g/L which is comparable to maximum sugar and ethanol yield of 31.2 ± 0.7 g/L and 7.34 ± 0.39 g/L respectively using commercial enzyme cocktail with S. cerevisiae MTCC-173. The SDSF was obtained as a suitable single step process for the ethanol production from RS.
A critical factor which controls the socioeconomic development of any nation is "energy". With depleting fossil fuels, there is a shift in focus to lignocellulosic biorefinery. The review aims to present an insight on currently available pre-treatment technologies for deconstruction and fractionation of lignocellulosic biomass for development of lignocellulosic feedstock based biorefinery. These bio-refineries facilitate generation of biofuels and value-added products e.g. sugar, bioethanol. Thus, in order to improve the sugar and subsequent biofuel yield, several pretreatment techniques have been investigated and have been categorized into physical, chemical, physicochemical and biological methods. The current status of each pretreatment technology on sugar and biofuel yield along with their limitations has been discussed. The present study will enable better understanding of already available processes and help overcome the limitations and develop an improvised technology to ease the pretreatment process to make the concept of biorefinery a reality.
Fast, non-destructive methods for determining the seed composition of Camelina sativa (L.) Crantz would be beneficial in evaluating germplasm for important agronomic traits. In this study, near infrared spectroscopy (NIRS) methods were developed and evaluated for conducting non-destructive, high throughput phenotyping of seed quality traits. Crude protein and total oil content for 85 accessions (63 summer- and 22 winter-biotypes) were first determined by established wet chemistry methodology; whereas, for fatty acid profiles 173 accessions (149 summer- and 24 winter-biotypes) were determined using Gas Chromatography (GC). The wet chemistry and GC data were used to develop NIRS calibration equations for each trait. Based on the wet chemistry data obtained from 85 accessions, mean crude protein content was significantly less in summer (300 g kg⁻¹) than in winter (315 g kg⁻¹) biotypes (P ≤ 0.05) and total oil was greater in seeds of summer (351 g kg⁻¹) than that of winter (326 g kg⁻¹) biotypes. Coefficient of determination (r² = 0.979 and 0.894, respectively) and ratio of performance to deviation (RPD = 9.15 and 4.33, respectively) for crude protein and oil content indicated a high level of confidence for predicting these traits using NIRS. Evaluation of all 173 accessions by NIRS did not appreciably change the predicted mean crude protein content of summer- and winter-biotypes; however, it did change the predicted mean total oil content of summer biotypes (260 g kg⁻¹), which was significantly less than predicted for winter biotypes (323 g kg⁻¹). Fatty acids contents were not significantly different between summer- and winter-biotypes. The most abundant fatty acid was linolenic acid (18:3) ranging from 22.8 to 38.4%, followed by linoleic acid (18:2) at 15.2–27.1%, eicosenoic acid (20:1) at 11.6–18.2%, and oleic acid (18:1) at 9.1–22.1%. Calibration models for the main fatty acids oleic, linoleic, linolenic, and eicosenoic acids had r² values of 0.718, 0.790, 0.828, and 0.586, respectively. Results of this study indicate that NIRS has potential as a non-destructive, high throughput method for determining quality traits of camelina seed.
Development of advanced biofuels such as bioethanol and biodiesel from renewable resources is critical for the earth's sustainable management and to slow down the global climate change by partial replacement of gasoline and diesel in the transport sector. Being a diverse group of aquatic micro-organisms, algae are the most prominent resources on the planet, distributed in an aquatic system, a potential source of bioenergy, biomass and secondary metabolites. Microalgae-based biofuel production is widely accepted as non-food fuel sources and better choice for achieving goals of incorporation of a clean fuel source into the transportation sector. The present review article provides a comprehensive literature survey as well as a novel approach on the application of microalgae for their simultaneous cultivation and bioremediation of high nutrient containing wastewater. In addition to that, merits and demerits of different existing conventional techniques for microalgae culture reactors, harvesting of algal biomass, oil recovery, use of different catalysts for transesterification reactions and other by-products recovery have been discussed and compared with the membrane-based system to find out the best optimal conditions for higher biomass as well as lipid yield. This article also deals with the use of a tailor-made membrane in an appropriate module that can be used in upstream and downstream processes during algal-based biofuels production. Such membrane-integrated system has the potential of low-cost and eco-friendly separation, purification and concentration enrichment of biodiesel as well as other valuable algal by-products which can bring the high degree of process intensification for scale-up at the industrial stage.
A biorefinery scheme was proposed for manufacturing platform chemicals (furfural, denoted F, and 5-hydroxymethylfurfural, denoted HMF) from Miscanthus polysaccharides (including hemicelluloses and cellulose). The feedstock was first subjected to hydrothermal processing, which caused an extensive hemicellulose solubilization, yielding solids enriched in cellulose and aqueous solutions rich in saccharides. The acidic processing of the aqueous solutions in the presence of methyl isobutyl ketone (MIBK), which acted as an extracting agent, led to the formation and the in situ separation of F from pentoses at high molar yields (up to 78%). Moreover, HMF and levulinic acid were obtained from the hexoses released from the feedstock.
The cellulose-enriched solids from hydrothermal processing were used as a substrate for HMF manufacture, employing a combination of enzymatic and chemical treatments. The enzymatic hydrolysis yielded glucose (in concentration up to 59 g /L), which was enzymatically isomerized into fructose in the presence of sodium tetraborate at yields up to 80%. Acidic treatments of the resulting reaction media at low temperature (134 °C) in the presence of H2SO4 enabled the formation of HMF at yields about 49%. Under the assayed conditions, glucose remained practically unaltered, facilitating its reutilization in the isomerization stage.
In this study, Box–Behnken design Response Surface Methodology (BBD-RSM) was used to develop microwave-assisted pretreatment (MAP) for Rice Straw (RS) using FeCl3-H3PO4 system. The concentration of FeCl3 and H3PO4, pretreatment temperature and time was used as independent variables and complex severity considering the effect of all variables were also calculated. Based on BBD-RSM, 57.5% of sugar yield per biomass was obtained for pretreated (MAP) RS using FeCl3 (0.35 M), H3PO4 (3%), pretreatment temperature and time of 155 °C and 10 min respectively with intermediate complex severity of 3.0. Based on the structural analysis using FTIR and XRD, it was suggested that the pretreatment results in reduction of crystallinity of RS that result in high crystallinity index (CrI) of 49.9% for efficient hydrolysis. Simultaneous saccharification and fermentation (SSF) of pretreated (MAP) RS resulted in maximum ethanol production of 7.0% (v/v) using in-house cellulase and Saccharomyces cerevisiae MTCC-173. The hydrolysis of pretreated (MAP) RS pulp using in-house produced endoxylanase resulted in generation of oligosaccharides.
Xylitol is a highly valuable commodity chemical intensively used in food and pharmaceutical industries. Production of xylitol from D‐xylose involves costly and polluting catalytic hydrogenation process. Biotechnological production from lignocellulosic biomass by microorganisms like yeasts is a promising option. In this study, xylitol was produced from lignocellulosic biomass by a recombinant strain of Saccharomyces cerevisiae (YPH499‐SsXR‐AaBGL) expressing cytosolic xylose reductase (SsXR), along with a β‐D‐glucosidase (AaBGL) displayed on the cell surface. The simultaneous co‐fermentation of cellobiose/xylose by this strain led to a ≈2.5‐fold increase of Yxylitol/xylose (=0.54) compared to using glucose/xylose mixture as substrate. Further improvement of the xylose uptake by the cell has been obtained by a broad evaluation of several homologous and heterologous transporters. Homologous maltose transporter (ScMAL11) showed the best performance in xylose transport and was used to generate the strain YPH499‐XR‐ScMAL11‐BGL with a significantly improved xylitol production capacity from cellobiose/xylose co‐utilization. This report constitutes a promising proof of concept to further scale up bio‐refinery industrial production of xylitol from lignocellulose by combining cell surface and metabolic engineering in S. cerevisiae. This article is protected by copyright. All rights reserved.
Soluble carbohydrates in sweet sorghum juices and syrups are the main sugars converted to ethanol during fermentation. Recently, it was found that sweet sorghum contains a substantial amount of insoluble starch in sweet sorghum by-products: juice sediment and clarification mud, which is an untapped source of fermentable sugars. In this study, a response surface method was used to optimize hydrolysis, liquefaction, and saccharification conditions and enzymes to customize a two-step process to convert starch in grain sorghum flour, as well as juice sediment, and clarification mud. Optimal starch liquefaction with the industrial α-amylase (Termamyl SC™) was best achieved at 80 °C in 90 min when <18% w/w flour was used, since the solid concentration significantly (P < 0.05) affected starch hydrolysis efficiency. Subsequent studies revealed that an industrial enzyme cocktail comprised of 63% SAN Extra™ (α-glucoamylase), 16% Promozyme D2™ (pullulanase), and 21% Viscozyme L™ (β-carbohydrase mixture) was most effective in improving the saccharification of starch, with particular emphasis on insoluble starch granules, to fermentable sugars at 60 °C in 90 min. Application of the optimal conditions tripled fermentable glucose and doubled total sugars in juice sediment; its application to clarification mud did not show much improvement (P < 0.05). Practical applications of this enzyme cocktail will also depend on cost effectiveness.
The discovery of lytic polysaccharide monooxygenases (LPMOs) has revolutionized enzymatic processing of polysaccharides, in particular recalcitrant insoluble polysaccharides such as cellulose. These monocopper enzymes display intriguing and unprecedented catalytic chemistry, which make them highly valuable in industrial bioprocessing, but also generate considerable challenges in terms of scientific understanding and optimal implementation. One issue of particular interest is the fact that both molecular oxygen and hydrogen peroxide can drive LPMO reactions. Here we review recent insights into the catalytic mechanism of LPMOs derived from structural, spectroscopic and functional studies. We then turn to the question of how one can optimally harness the potential of LPMOs in biomass processing, given the current knowledge of their catalytic mechanism. Finally, we review recent, more applied studies that have addressed the importance of LPMOs in enzymatic conversion of lignocellulosic biomass, and discuss how the impact of these powerful enzymes could be improved.
Oyster mushrooms use different lignocellulosic substrates with different biological efficiency, whereas deep understanding of the molecular mechanism is lacking. The extracellular/intracellular proteomes, lignocellulosic composition were analyzed after 21-day cultivation of P. ostreatus in sawdust, cottonseed hull and corncob. Lignin and hemicellulose content of three substrates significantly decreased, and cottonseed hull showed the highest saccharification rate. 297, 333, and 312 soluble proteins were identified in hardwood sawdust, cottonseed hull and corncob, respectively. P. ostreatus mobilized the corresponding antioxidant and carbon metabolism pathways and produced more abundant ligninolytic enzymes, especially class II peroxidases, to accommodate lignin-rich substrate hardwood. Ligninolytic enzymes and carbohydrate oxidases showed higher expression levels in sawdust, while carbohydrate active enzymes were highly expressed in polysaccharide-rich cottonseed hull and corncob. These results suggested P. ostreatus adapts to different substrates through regulating extra/intracellular proteins expression, and cottonseed hull is a potential source for biorefinery and oyster mushroom cultivation.
The profitability of biorefinery scenarios utilizing sugarcane lignocellulose (bagasse and harvesting residues), annexed to an existing sugar mill for the co-production of electricity and bioproducts was investigated. Biorefinery scenarios for xylitol, citric acid or glutamic acid production, each in combination with electricity, were simulated in Aspen Plus ® to generate mass and energy balances, and perform economic analyses. The profitability was compared to a combined heat and power (CHP) plant baseline scenario that solely combusts biomass for electricity co-production. Scenario 2, citric acid and electricity co-production was not profitable at a selling price of 680 /t and 3625 $/t, respectively. These IRR values exceeded the baseline CHP plant IRR of 10.3% and hurdle rate of 9.7%.
Xylitol is a major commodity chemical widely used in both the food and pharmaceutical industries. Although the worldwide demand of xylitol is constantly growing, its industrial production from purified D-xylose involves a costly and polluting catalytic hydrogenation process. Biotechnological production of xylitol from biomass is a promising strategy to establish an environmentally-friendly sustainable conversion process. In this study, xylitol was produced from woody Kraft pulp (KP) by using an engineered strain of Saccharomyces cerevisiae (YPH499-XR-BGL-XYL-XYN) expressing cytosolic xylose reductase (XR), along with β-D-glucosidase (BGL), xylosidase (XYL) and xylanase (XYN) enzymes co-displayed on the cell surface. All these enzymes contributed to the consolidated bioprocessing of KP to xylitol with a yield of 2.3 g/L (28% conversion) after 96 hours, along with a significantly reduced amount of commercial enzymes required for pre-treatment (commercial hemicellulases cocktail (CHC), [CHC] = 0.02 g-DW/g). Further improvement of the cell surface display of XYL and XYN was obtained by using a SED1 “SSS” cassette, containing the coding sequences of the SED1 promoter, the SED1 secretion signal, and the SED1 anchoring domain, to generate the improved strain YPH499-XR-BGL-XYLsss-XYNsss. This improved strain showed a significantly enhanced xylitol production capacity reaching a yield of 3.7 g/L (44% conversion) after 96 hours. The cellulosic content of KP residues was also significantly increased, from 78% to 87% after 96 hours of fermentation, and nanofibrillation of KP residues was observed by scanning electron microscopy. Pre-treatment and fermentation were successfully performed as a proof of concept to further scale up bio-refinery industrial production of xylitol from lignocellulose.
This chapter discusses opportunities and limitations of bioenergy production integrated into organic farming systems when considering the global challenge of a food-energy-nexus. An overview is presented on the specific organic principles and constraints regarding agricultural biomass utilization for energetic purposes. A history of the developments of bioenergy in organic agriculture is briefly outlined. Several cases of bioenergy systems implemented on organic farms are highlighted. From these cases, general synergies can be deducted and potentials for integrated organic bioenergy systems are discussed. Research and development needs are identified.
The white laccase was produced from Myrothecium verrucaria ITCC-8447 under submerged fermentation. The media components were optimized by response surface methodology (CCD-RSM). The nutritional components (glucose and peptone) and physical parameters (pH and temperature) were optimized by response surface methodology for enhanced laccase production by Myrothecium verrucaria ITCC-8447. The enzyme activity under optimum condition exhibited 1.45 fold increases in laccase activity. The white laccase was subjected to ion exchange chromatography with 6 fold purification. The molecular weight of white laccase was ~63–75 kDa as estimated by SDS-PAGE followed by the activity staining with ABTS where green bands confirmed the presence of laccase. The enzyme was stable over an alkaline pH range of 7–9 and the temperature range of 30–40 °C. The characterization of white laccase was done by CD spectra, UV–visible absorption, FTIR and XRD. The Km and Vmax values of the purified laccase were 2.5 mM and1818.2 μmol/min/L. The delignification capability of the white laccase was determined by reduction in Kappa number (58.8%) and Klason lignin (64.7%) of wheat straw after 12 h of incubation. Further the delignification was confirmed FTIR and XRD.
India is one among the world's largest economies and its energy demand accounts for 3.5% of world's commercial energy consumption. According to the International Energy Agency oil demand in India is expected to grow by a factor 2.2 by 2030, increasing the oil import dependency from 69% now to 91%. Rising energy prices and climate change are increasing the demand for biofuel production. The Planning Commission of India recommends replacing 20% of India's diesel consumption mainly by non-edible Jatropha oil and Pongamia. Biorefinery could be one of the best solutions to overcome the problem. A review on the progress of biorefinery in India is attempted.
Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes discovered within the last 10 years. By degrading recalcitrant substrates oxidatively, these enzymes are major contributors to the recycling of carbon in nature and are being used in the biorefin-ery industry. Recently, two new families of LPMOs have been defined and structurally characterized, AA14 and AA15, sharing many of previously found structural features. However, unlike most LPMOs to date, AA14 degrades xylan in the context of complex substrates, while AA15 is particularly interesting because they expand the presence of LPMOs from the predominantly microbial to the animal kingdom. The first two neutron crystallography structures have been determined, which, together with high-resolution room temperature X-ray structures, have putatively identified oxygen species at or near the active site of LPMOs. Many recent computational and experimental studies have also investigated the mechanism of action and substrate-binding mode of LPMOs. Perhaps, the most significant recent advance is the increasing structural and biochemical evidence, suggesting that LPMOs follow different mechanistic pathways with different substrates, co-substrates and reductants, by behaving as monooxygenases or peroxygenases with molecular oxygen or hydrogen peroxide as a co-substrate, respectively.