Biofuels for a sustainable future.

Rapid increases of energy consumption and human dependency on fossil fuels have led to the accumulation of greenhouse gases and consequently, climate change. As such, major efforts have been taken to develop, test, and adopt clean renewable fuel alternatives. Production of bioethanol and biodiesel from crops is well developed, while other feedstock resources and processes have also shown high potential to provide efficient and cost-effective alternatives, such as landfill and plastic waste conversion, algal photosynthesis, as well as electrochemical carbon fixation. In addition, the downstream microbial fermentation can be further engineered to not only increase the product yield but also expand the chemical space of biofuels through the rational design and fine-tuning of biosynthetic pathways toward the realization of "designer fuels" and diverse future applications.

Cellulose degradation by polysaccharide monooxygenases.

Polysaccharide monooxygenases (PMOs), also known as lytic PMOs (LPMOs), enhance the depolymerization of recalcitrant polysaccharides by hydrolytic enzymes and are found in the majority of cellulolytic fungi and actinomycete bacteria. For more than a decade, PMOs were incorrectly annotated as family 61 glycoside hydrolases (GH61s) or family 33 carbohydrate-binding modules (CBM33s). PMOs have an unusual surface-exposed active site with a tightly bound Cu(II) ion that catalyzes the regioselective hydroxylation of crystalline cellulose, leading to glycosidic bond cleavage. The genomes of some cellulolytic fungi contain more than 20 genes encoding cellulose-active PMOs, suggesting a diversity of biological activities. PMOs show great promise in reducing the cost of conversion of lignocellulosic biomass to fermentable sugars; however, many questions remain about their reaction mechanism and biological function. This review addresses, in depth, the structural and mechanistic aspects of oxidative depolymerization of cellulose by PMOs and considers their biological function and phylogenetic diversity.

The role of AdhE on ethanol tolerance and production in Clostridium thermocellum.

Many anaerobic microorganisms use the bifunctional aldehyde and alcohol dehydrogenase enzyme, AdhE, to produce ethanol. One such organism is Clostridium thermocellum, which is of interest for cellulosic biofuel production. In the course of engineering this organism for improved ethanol tolerance and production, we observed that AdhE was a frequent target of mutations. Here, we characterized those mutations to understand their effects on enzymatic activity, as well ethanol tolerance and product formation in the organism. We found that there is a strong correlation between NADH-linked alcohol dehydrogenase (ADH) activity and ethanol tolerance. Mutations that decrease NADH-linked ADH activity increase ethanol tolerance; correspondingly, mutations that increase NADH-linked ADH activity decrease ethanol tolerance. We also found that the magnitude of ADH activity did not play a significant role in determining ethanol titer. Increasing ADH activity had no effect on ethanol titer. Reducing ADH activity had indeterminate effects on ethanol titer, sometimes increasing and sometimes decreasing it. Finally, this study shows that the cofactor specificity of ADH activity was found to be the primary factor affecting ethanol yield. We expect that these results will inform efforts to use AdhE enzymes in metabolic engineering approaches.

Methanol biotransformation toward high-level production of fatty acid derivatives by engineering the industrial yeast Pichia pastoris.

Methanol-based biorefinery is a promising strategy to achieve carbon neutrality goals by linking CO2 capture and solar energy storage. As a typical methylotroph, Pichia pastoris shows great potential in methanol biotransformation. However, challenges still remain in engineering methanol metabolism for chemical overproduction. Here, we present the global rewiring of the central metabolism for efficient production of free fatty acids (FFAs; 23.4 g/L) from methanol, with an enhanced supply of precursors and cofactors, as well as decreased accumulation of formaldehyde. Finally, metabolic transforming of the fatty acid cell factory enabled overproduction of fatty alcohols (2.0 g/L) from methanol. This study demonstrated that global metabolic rewiring released the great potential of P. pastoris for methanol biotransformation toward chemical overproduction.

Acoustic force spectroscopy reveals subtle differences in cellulose unbinding behavior of carbohydrate-binding modules.

Protein adsorption to solid carbohydrate interfaces is critical to many biological processes, particularly in biomass deconstruction. To engineer more-efficient enzymes for biomass deconstruction into sugars, it is necessary to characterize the complex protein-carbohydrate interfacial interactions. A carbohydrate-binding module (CBM) is often associated with microbial surface-tethered cellulosomes or secreted cellulase enzymes to enhance substrate accessibility. However, it is not well known how CBMs recognize, bind, and dissociate from polysaccharides to facilitate efficient cellulolytic activity, due to the lack of mechanistic understanding and a suitable toolkit to study CBM-substrate interactions. Our work outlines a general approach to study the unbinding behavior of CBMs from polysaccharide surfaces using a highly multiplexed single-molecule force spectroscopy assay. Here, we apply acoustic force spectroscopy (AFS) to probe a Clostridium thermocellum cellulosomal scaffoldin protein (CBM3a) and measure its dissociation from nanocellulose surfaces at physiologically relevant, low force loading rates. An automated microfluidic setup and method for uniform deposition of insoluble polysaccharides on the AFS chip surfaces are demonstrated. The rupture forces of wild-type CBM3a, and its Y67A mutant, unbinding from nanocellulose surfaces suggests distinct multimodal CBM binding conformations, with structural mechanisms further explored using molecular dynamics simulations. Applying classical dynamic force spectroscopy theory, the single-molecule unbinding rate at zero force is extrapolated and found to agree with bulk equilibrium unbinding rates estimated independently using quartz crystal microbalance with dissipation monitoring. However, our results also highlight critical limitations of applying classical theory to explain the highly multivalent binding interactions for cellulose-CBM bond rupture forces exceeding 15 pN.

Environmental outcomes of the US Renewable Fuel Standard.

The Renewable Fuel Standard (RFS) specifies the use of biofuels in the United States and thereby guides nearly half of all global biofuel production, yet outcomes of this keystone climate and environmental regulation remain unclear. Here we combine econometric analyses, land use observations, and biophysical models to estimate the realized effects of the RFS in aggregate and down to the scale of individual agricultural fields across the United States. We find that the RFS increased corn prices by 30% and the prices of other crops by 20%, which, in turn, expanded US corn cultivation by 2.8 Mha (8.7%) and total cropland by 2.1 Mha (2.4%) in the years following policy enactment (2008 to 2016). These changes increased annual nationwide fertilizer use by 3 to 8%, increased water quality degradants by 3 to 5%, and caused enough domestic land use change emissions such that the carbon intensity of corn ethanol produced under the RFS is no less than gasoline and likely at least 24% higher. These tradeoffs must be weighed alongside the benefits of biofuels as decision-makers consider the future of renewable energy policies and the potential for fuels like corn ethanol to meet climate mitigation goals.

Elongated galactan side chains mediate cellulose-pectin interactions in engineered Arabidopsis secondary cell walls.

The plant secondary cell wall is a thickened matrix of polysaccharides and lignin deposited at the cessation of growth in some cells. It forms the majority of carbon in lignocellulosic biomass, and it is an abundant and renewable source for forage, fiber, materials, fuels, and bioproducts. The complex structure and arrangement of the cell wall polymers mean that the carbon is difficult to access in an economical and sustainable way. One solution is to alter the cell wall polymer structure so that it is more suited to downstream processing. However, it remains difficult to predict what the effects of this engineering will be on the assembly, architecture, and properties of the cell wall. Here, we make use of Arabidopsis plants expressing a suite of genes to increase pectic galactan chain length in the secondary cell wall. Using multi-dimensional solid-state nuclear magnetic resonance, we show that increasing galactan chain length enhances pectin-cellulose spatial contacts and increases cellulose crystallinity. We also found that the increased galactan content leads to fewer spatial contacts of cellulose with xyloglucan and the backbone of pectin. Hence, we propose that the elongated galactan side chains compete with xyloglucan and the pectic backbone for cellulose interactions. Due to the galactan topology, this may result in comparatively weak interactions and disrupt the cell wall architecture. Therefore, introduction of this strategy into trees or other bioenergy crops would benefit from cell-specific expression strategies to avoid negative effects on plant growth.

Efficient utilization of xylose requires CO2 fixation in Synechococcus elongatus PCC 7942.

Cyanobacteria show great promise as autotrophic hosts for the renewable biosynthesis of useful chemicals from CO2 and light. While they can efficiently fix CO2, cyanobacteria are generally outperformed by heterotrophic production hosts in terms of productivity and titer. Photomixotrophy, or co-utilization of sugars and CO2 as carbon feedstocks, has been implemented in cyanobacteria to greatly improve productivity and titers of several chemical products. We introduced xylose photomixotrophy to a 2,3-butanediol producing strain of Synechococcus elongatus PCC 7942 and characterized the effect of gene knockouts, changing pathway expression levels, and changing growth conditions on chemical production. Interestingly, 2,3-butanediol production was almost completely inhibited in the absence of added CO2. Untargeted metabolomics implied that RuBisCO was a significant bottleneck, especially at ambient CO2 levels, restricting the supply of lower glycolysis metabolites needed for 2,3-butanediol production. The dependence of the strain on elevated CO2 levels suggests some practical limitations on how xylose photomixotrophy can be efficiently carried out in S. elongatus.

Mass spectrometry-based methods for the advanced characterization and structural analysis of lignin: A review.

Lignin is currently one of the most promising biologically derived resources, due to its abundance and application in biofuels, materials and conversion to value aromatic chemicals. The need to better characterize and understand this complex biopolymer has led to the development of many different analytical approaches, several of which involve mass spectrometry and subsequent data analysis. This review surveys the most important analytical methods for lignin involving mass spectrometry, first looking at methods involving gas chromatography, liquid chromatography and then continuing with more contemporary methods such as matrix assisted laser desorption ionization and time-of-flight-secondary ion mass spectrometry. Following that will be techniques that directly ionize lignin mixtures-without chromatographic separation-using softer atmospheric ionization techniques that leave the lignin oligomers intact. Finally, ultra-high resolution mass analyzers such as FT-ICR have enabled lignin analysis without major sample preparation and chromatography steps. Concurrent with an increase in the resolution of mass spectrometers, there have been a wealth of complementary data analyses and visualization methods that have allowed researchers to probe deeper into the "lignome" than ever before. These approaches extract trends such as compound series and even important analytical information about lignin substructures without performing lignin degradation either chemically or during MS analysis. These innovative methods are paving the way for a more comprehensive understanding of this important biopolymer, as we seek more sustainable solutions for our human species' energy and materials needs.

Fast-growing cyanobacterial chassis for synthetic biology application.

Carbon neutrality by 2050 has become one of the most urgent challenges the world faces today. To address the issue, it is necessary to develop and promote new technologies related with CO2 recycling. Cyanobacteria are the only prokaryotes performing oxygenic photosynthesis, capable of fixing CO2 into biomass under sunlight and serving as one of the most important primary producers on earth. Notably, recent progress on synthetic biology has led to utilizing model cyanobacteria such as Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 as chassis for "light-driven autotrophic cell factories" to produce several dozens of biofuels and various fine chemicals directly from CO2. However, due to the slow growth rate and low biomass accumulation in the current chassis, the productivity for most products is still lower than the threshold necessary for large-scale commercial application, raising the importance of developing high-efficiency cyanobacterial chassis with fast growth and/or higher biomass accumulation capabilities. In this article, we critically reviewed recent progresses on identification, systems biology analysis, and engineering of fast-growing cyanobacterial chassis. Specifically, fast-growing cyanobacteria identified in recent years, such as S. elongatus UTEX 2973, S. elongatus PCC 11801, S. elongatus PCC 11802 and Synechococcus sp. PCC 11901 was comparatively analyzed. In addition, the progresses on their recent application in converting CO2 into chemicals, and genetic toolboxes developed for these new cyanobacterial chassis were discussed. Finally, the article provides insights into future challenges and perspectives on the synthetic biology application of cyanobacterial chassis.

Valorization of Agriculture Residues into Value-Added Products: A Comprehensive Review of Recent Studies.

Global agricultural by-products usually go to waste, especially in developing countries where agricultural products are usually exported as raw products. Such waste streams, once converted to "value-added" products could be an additional source of revenue while simultaneously having positive impacts on the socio-economic well-being of local people. We highlight the utilization of thermochemical techniques to activate and convert agricultural waste streams such as rice and straw husk, coconut fiber, coffee wastes, and okara power wastes commonly found in the world into porous activated carbons and biofuels. Such activated carbons are suitable for various applications in environmental remediation, climate mitigation, energy storage, and conversions such as batteries and supercapacitors, in improving crop productivity and producing useful biofuels.

Comparative genomics reveals probable adaptations for xylose use in Thermoanaerobacterium saccharolyticum.

Second-generation ethanol, a promising biofuel for reducing greenhouse gas emissions, faces challenges due to the inefficient metabolism of xylose, a pentose sugar. Overcoming this hurdle requires exploration of genes, pathways, and organisms capable of fermenting xylose. Thermoanaerobacterium saccharolyticum is an organism capable of naturally fermenting compounds of industrial interest, such as xylose, and understanding evolutionary adaptations may help to bring novel genes and information that can be used for industrial yeast, increasing production of current bio-platforms. This study presents a deep evolutionary study of members of the firmicutes clade, focusing on adaptations in Thermoanaerobacterium saccharolyticum that may be related to overall fermentation metabolism, especially for xylose fermentation. One highlight is the finding of positive selection on a xylose-binding protein of the xylFGH operon, close to the annotated sugar binding site, with this protein already being found to be expressed in xylose fermenting conditions in a previous study. Results from this study can serve as basis for searching for candidate genes to use in industrial strains or to improve Thermoanaerobacterium saccharolyticum as a new microbial cell factory, which may help to solve current problems found in the biofuels' industry.

A Holistic Approach to Circular Bioeconomy Through the Sustainable Utilization of Microalgal Biomass for Biofuel and Other Value-Added Products.

Emissions from transportation and industry primarily cause global warming, leading to floods, glacier melt, and rising seas. Widespread greenhouse gas emissions and resulting global warming pose significant risks to the environment, economy, and society. The need for alternative fuels drives the development of third-generation feedstocks: microalgae, seaweed, and cyanobacteria. These microalgae offer traits like rapid growth, high lipid content, non-competition with human food, and growth on non-arable land using brackish or waste water, making them promising for biofuel. These unique phototrophic organisms use sunlight, water, and carbon dioxide (CO2) to produce biofuels, biochemicals, and more. This review delves into the realm of microalgal biofuels, exploring contemporary methodologies employed for lipid extraction, significant value-added products, and the challenges inherent in their commercial-scale production. While the cost of microalgae bioproducts remains high, utilizing wastewater nutrients for cultivation could substantially cut production costs. Furthermore, this review summarizes the significance of biocircular economy approaches, which encompass the utilization of microalgal biomass as a feed supplement and biofertilizer, and biosorption of heavy metals and dyes. Besides, the discussion extends to the in-depth analysis and future prospects on the commercial potential of biofuel within the context of sustainable development. An economically efficient microalgae biorefinery should prioritize affordable nutrient inputs, efficient harvesting techniques, and the generation of valuable by-products.

Cytotoxicity and Epithelial Barrier Toxicity of Fine Particles from Residential Biomass Pellet Burning.

Rising environmental concerns associated with the domestic use of solid biofuels have driven the search for clean energy alternatives. This study investigated the in vitro toxicological characteristics of PM2.5 emissions from residential biomass pellet burning using the A549 epithelial cell line. The potential of modern pellet applications to reduce PM2.5 emissions was evaluated by considering both mass reduction and toxicity modification. PM2.5 emissions from raw and pelletized biomass combustion reduced cell viability, indicative of acute toxicity, and also protein expression associated with epithelial barrier integrity, implying further systemic toxicity, potentially via an oxidative stress mechanism. Toxicity varied between PM2.5 emissions from raw biomass and pellets, with pelletized straw and wood inducing cytotoxicity by factors of 0.54 and 1.30, and causing epithelial barrier damage by factors of 1.76 and 2.08, respectively, compared to their raw counterparts. Factoring in both mass reduction and toxicity modifications, PM2.5 emissions from pelletized straw and wood dropped to 1.83 and 5.07 g/kg, respectively, from 30.1 to 9.32 g/kg for raw biomass combustion. This study underscores the effectiveness of pelletized biomass, particularly straw pellets, as a sustainable alternative to traditional biofuels and highlights the necessity of considering changes in toxicity when assessing the potential of clean fuels to mitigate emissions of the PM2.5 complex.

Effect of NH4Cl supplementation on growth, photosynthesis, and triacylglycerol content in Chlamydomonas reinhardtii under mixotrophic cultivation.

AIM: Ammonium chloride (NH4Cl) is one of the nitrogen sources for microalgal cultivation. An excessive amounts of NH4Cl are toxic for microalgae. However, combining mixotrophic conditions and excessive quantities of NH4Cl positively affects microalgal biomass and lipid production. In this study, we investigated the impact of NH4Cl on the growth, biomass, and triglyceride (TAG) content of the green microalga Chlamydomonas reinhardtii especially under mixotrophic conditions. METHODS AND RESULTS: Under photoautotrophic conditions (without organic carbon supplementation), adding 25 mM NH4Cl had no significant effect on microalgal growth or TAG content. However, under mixotrophic condition (with acetate supplementation), NH4Cl interfered with microalgal growth while inducing TAG content. To explore these effects further, we conducted a two-step cultivation process and found that NH4Cl reduced microalgal growth, but induced total lipid and TAG content, especially after 4-day cultivation. The photosynthesis performances showed that NH4Cl completely inhibited oxygen evolution on day 4. However, NH4Cl slightly reduced the Fv/Fm ratio indicating that the NH4Cl supplementation directly affects microalgal photosynthesis. To investigate the TAG induction effect by NH4Cl, we compared the protein expression profiles of microalgae grown mixotrophically with and without 25 mM NH4Cl using a proteomics approach. This analysis identified 1782 proteins, with putative acetate uptake transporter GFY5 and acyl-coenzyme A oxidase being overexpressed in the NH4Cl-treated group. CONCLUSION: These findings suggested that NH4Cl supplementation may stimulate acetate utilization and fatty acid synthesis pathways in microalgae cells. Our study indicated that NH4Cl supplementation can induce microalgal biomass and lipid production, particularly when combined with mixotrophic conditions.

Nanomaterials - A promising solution for textile and fossil fuel generated pollutants.

This review approach is divided into two scopes to focus the pollution threats. We cover the applications of nanomaterials to curtail the pollution induced by fossil fuel combustion, and textile dye effluents. Toxic emissions released from automobile exhaust that comprise of NOX. SOX and PAHs compile to harsh breathing and respiratory troubles. The effluents generated from the mammoth textile and leather industry is potential threat to beget massive health issues to human life, and environmental problem. Part I projects the broad envisage on role of nano materials in production of alternative biofuels. In addition, green sources for synthesizing nanomaterials are given special importance. Nano catalyst's utilization in bio-derived fuels such as biogas, bio-oil, bioethanol, and biodiesel are catered to this article. Part II cover the current statistics of textile effluent pollution level in India and its steps in confronting the risks of pollution are discussed. A clear picture of the nano techniques in pre-treatment, and the recent nano related trends pursued in industries to eliminate the dyes and chemicals from the discharges is discussed. The substantial aspect of nano catalysis in achieving emission-free fuel and toxic-free effluents and the augmentation in this field is conferred. This review portrays the dependency on nano materials & technology for sustainable future.

Progress and challenges in valorisation of biomass waste from ornamental trees pruning through pyrolysis processes. Prospects in the bioenergy sector.

Nowadays, the scarcity of energy resources is promoting the search for alternative energy sources, boosting interest in the use of forest lignocellulosic residue in the energy sector. In this study, the focus is on the energy recovery from two lignocellulosic residues originated during the pruning of ornamental trees (Horse Chestnut, CI, and False Acacia, FA). Both conventional and flash pyrolysis techniques were applied. The experimental pyrolysis variables were obtained from the study of the thermal behaviour of the pruning residues in thermogravimetric analysis. It was carried out under 5 heating rates and kinetic parameters were estimated using Flynn-Wall-Ozawa method. Results denoted higher maximum mass loss rate values for the same release temperature regions under FA experiments. Also, FA samples had lower final residues for the processes. However, activation energy values were so close for both species. FA was also linked to the faster reactions according frequency factor outcomes. Conventional pyrolysis of pruning residues was carried out in a horizontal oven of original design at a heating rate of 25 °C/min, at 750 °C and 60 min of permanence at that temperature; flash pyrolysis was tested in that oven at 750 and 850 °C. In these pyrolysis processes, three fractions were obtained: bio-char, bio-oil and gas. The physicochemical attributes of the bio-chars suggested their potential utility as biofuels (28.4-29.8 MJ/kg), adsorbent precursors or soil additives. Conventional pyrolysis bio-oils had a dominant monoaromatic hydrocarbons nature, with phenols being the most abundant (≥60%), while flash bio-oils contain mainly polycyclic aromatic hydrocarbons. Conventional pyrolysis gases contained up to 60 vol% of CO2; flash pyrolysis gases had high combustible gas content (CO, CH4, H2) and a low CO2 content (<25 vol%). As a result, their calorific value (18.06 MJ/kg) exhibited a threefold increase compared to the gas produced through conventional pyrolysis (6.04 MJ/kg).

Nannochloropsis as an Emerging Algal Chassis for Light-Driven Synthesis of Lipids and High-Value Products.

In light of the escalating global energy crisis, microalgae have emerged as highly promising producers of biofuel and high-value products. Among these microalgae, Nannochloropsis has received significant attention due to its capacity to generate not only triacylglycerol (TAG) but also eicosapentaenoic acid (EPA) and valuable carotenoids. Recent advancements in genetic tools and the field of synthetic biology have revolutionized Nannochloropsis into a powerful biofactory. This comprehensive review provides an initial overview of the current state of cultivation and utilization of the Nannochloropsis genus. Subsequently, our review examines the metabolic pathways governing lipids and carotenoids, emphasizing strategies to enhance oil production and optimize carbon flux redirection toward target products. Additionally, we summarize the utilization of advanced genetic manipulation techniques in Nannochloropsis. Together, the insights presented in this review highlight the immense potential of Nannochloropsis as a valuable model for biofuels and synthetic biology. By effectively integrating genetic tools and metabolic engineering, the realization of this potential becomes increasingly feasible.