Second-generation Biorefineries Research Articles

Drivers and Barriers in the Production and Utilization of Second-Generation Bioethanol in India

Second-generation biorefinery refers to the production of different types of biofuels, biomaterials, and biochemicals by using agri-based and other lignocellulosic biomasses as substrates, which do not compete with arable lands, water for irrigation, and food supply. From the perspective of transportation fuels, second-generation bioethanol plays a crucial role in minimizing the dependency on fossil-based fuels, especially gasoline. Significant efforts have been invested in the research and development of second-generation bioethanol for commercialization in both developing and developed countries. However, in different developing countries like India, commercialization of second-generation bioethanol has been obstructed despite the abundance and variety of agricultural feedstocks. This commercial obstruction was majorly attributed to the recalcitrance of the feedstock, by-product management, and marginal subsidies compared to other nations. This article reviews the major roadblocks to the viability and commercialization of second-generation biofuels, especially bioethanol in India and a few other leading developed and developing nations. This article also reviews the biomass availability, technological advancements, investments, policies, and scale-up potential for biorefineries. A thorough discussion is made on the prospects and barriers to research, development, and demonstration as well as strengths, weaknesses, opportunities, and threats for the commercialization of second-generation bioethanol.

Calcium signaling positively regulates cellulase translation and secretion in a Clr-2-overexpressing, catabolically derepressed strain of Penicillium funiculosum

BackgroundLow-cost cellulase production is vital to sustainable second-generation biorefineries. The catabolically derepressed strain of Penicillium funiculosum NCIM1228 (PfMig188 or ∆Mig1) secretes a superior set of cellulolytic enzymes, that are most suitable for 2G biorefineries. At a 3% (w/w) load, the ∆Mig1 secretome can release > 80% of fermentable sugars from lignocellulose at a 15% (w/v) biomass load, irrespective of the type of biomass and pretreatment. The robustness of the secretome can be further increased by improving the cellulase production capacity of the fungal strain.ResultsWe began by identifying the transcription factor responsible for cellulase production in NCIM1228. An advanced RNA-seq screen identified three genes, clr-2, ctf1a and ctf1b; the genes were cloned under their native promoters and transformed into NCIM1228. Of the three, clr-2 overexpression led to twofold higher cellulase production than the parent strain and was thus identified as the transcriptional activator of cellulase in NCIM1228. Next, we overexpressed clr-2 in ∆Mig1 and expected an exponential increase in cellulolytic attributes accredited to the reinforced activation mechanisms, conjoint with diminished negative regulation. Although clr-2 overexpression increased the transcript levels of cellulase genes in ∆Mig1, there was no increase in cellulase yield. Even a further increase in the transcript levels of clr-2 via a stronger promoter was ineffective. However, when the CaCO3 concentration was increased to 5 g/l in the growth medium, we achieved a 1.5-fold higher activity of 6.4 FPU/ml in the ∆Mig1 strain with clr-2 overexpression. Enthused by the calcium effect, a transcriptomic screen for genes encoding Ca2+-activated kinase identified ssp1, whose overexpression could further increase cellulase yield to ~ 7.5 FPU/ml. Investigation of the mechanism revealed that calcium signaling exclusively enhances the translation and secretion of cellulase in Penicillium funiculosum.ConclusionsOur study identifies for the first time that cellulose activates two discrete signaling events to govern cellulase transcription and posttranscriptional processes (translation, processing and secretion) in P. funiculosum NCIM1228. Whereas Clr-2, the transcriptional activator of cellulase, governs transcription, calcium signaling specifically activates cellulase translation and secretion.

Filling in the gaps in second-generation biorefineries: evaluating rice straw and its bioethanol residue for the production of biogenic silica nanoparticles

Filling in the gaps in second-generation biorefineries: evaluating rice straw and its bioethanol residue for the production of biogenic silica nanoparticles

A comprehensive analysis of bioethanol and ethyl lactate joint production in second-generation biorefinery: Simulation, techno-economic, and profitability assessments

Second-generation biorefineries (2GBR) represent an innovative application of bioresources technologies to produce bioenergy and valuable products. This paper aims to introduce and analyze the joint production of bioethanol and ethyl lactate in a 2GBR. Techno-economic and profitability perspectives are considered in the analysis which is conducted via simulation considering corn stover as raw material. A key aspect in the analysis is a joint production parameter named α, whose values can dictate either the sole production of bioethanol (α = 0), joint production (0 < α < 1), or the unique production of ethyl lactate (α = 1). In other words, the proposed joint production scheme provides versatility in production. Simulations show that the lowest Total Capital Investment, Unit Production Cost, and Operating Cost values were associated with low values of α. Furthermore, when α ≥ 0.4, the 2GBR under study can achieve internal rates of return above 30%, which implies that the project offers a potentially high profitability.

A framework for the design of sustainable multi-input second-generation biorefineries through process simulation: A case study for the valorization of lignocellulosic and starchy waste from the plantain agro-industry

The plantain agro-industry generates different residues in the harvest and post-harvest stages. Therefore, new processes for its valorization are required. The aim of this research was to propose a general methodological framework for the design and analysis of multi-input biorefineries for the valorization of residues with a high content of lignocellulosic and starchy materials. This approach was based on the independent processing of the starchy and lignocellulosic materials in the first steps of the biorefinery and relies on process simulation and hierarchic process design procedures. A case study for the design of a multi-input biorefinery for the valorization of residues from the plantain agro-industry was performed by applying the framework proposed. The biorefinery was simulated using SuperPro Designer software. The results obtained suggest that the best alternative for the valorization of plantain residues under Colombian conditions corresponded to the following distribution of key residues: 100% leaf sheaths processed into the natural fiber production section, and 25% peels and 25% rachis processed into the starch extraction and liquefaction section; with this distribution, an NPV of $21,348,000 can be achieved. This work shows that waste from the plantain agro-industry exhibits high potential as a feedstock for the production of value-added products.

Design of a High-Rate Wastewater Treatment Process for Energy and Water Recovery at Biorefineries

Industrial wastewaters rich in organic carbon have potential for value generation, but conventional, low-rate, anaerobic–aerobic wastewater treatment (WWT) processes often incur significant capital expenses and energy consumption. In this study, we leveraged experimental data for biorefinery-derived wastewaters to characterize the implications of transitioning from a conventional, low-rate process to a high-rate, multistage anaerobic process. We designed and simulated these WWT processes across seven first- and second-generation (1G/2G) biorefineries and evaluated the implications for biorefinery sustainability through techno-economic analysis (TEA) and life cycle assessment (LCA). Compared to the conventional design, the new process can substantially reduce capital costs and electricity usage. These improvements were particularly evident for 2G biorefineries, translating to 5%–13% lower minimum product selling prices (MPSPs) and 7%–135% lower 100-year global warming potentials (GWPs; the 135% reduction is due to the transition of one biorefinery from net emission [0.87 kg of CO2e·gal–1] to net sequestration [−0.31 kg of CO2e·gal–1]). Biorefineries could further reduce the MPSP through the renewable identification number (RIN) credits by upgrading and selling the biogas as renewable natural gas, but at the expense of increasing GWP. When normalized, the COD management cost ranged from $–56 to $465 per tonne of COD, indicating that wastewater could be a net source of revenue for some biorefineries.

Bioethanol Production from Lignocellulosic Biomass-Challenges and Solutions.

Regarding the limited resources for fossil fuels and increasing global energy demands, greenhouse gas emissions, and climate change, there is a need to find alternative energy sources that are sustainable, environmentally friendly, renewable, and economically viable. In the last several decades, interest in second-generation bioethanol production from non-food lignocellulosic biomass in the form of organic residues rapidly increased because of its abundance, renewability, and low cost. Bioethanol production fits into the strategy of a circular economy and zero waste plans, and using ethanol as an alternative fuel gives the world economy a chance to become independent of the petrochemical industry, providing energy security and environmental safety. However, the conversion of biomass into ethanol is a challenging and multi-stage process because of the variation in the biochemical composition of biomass and the recalcitrance of lignin, the aromatic component of lignocellulose. Therefore, the commercial production of cellulosic ethanol has not yet become well-received commercially, being hampered by high research and production costs, and substantial effort is needed to make it more widespread and profitable. This review summarises the state of the art in bioethanol production from lignocellulosic biomass, highlights the most challenging steps of the process, including pretreatment stages required to fragment biomass components and further enzymatic hydrolysis and fermentation, presents the most recent technological advances to overcome the challenges and high costs, and discusses future perspectives of second-generation biorefineries.

On the Oxidative Valorization of Lignin to High-Value Chemicals: A Critical Review of Opportunities and Challenges.

The efficient valorization of lignin is crucial if we are to replace current petroleum-based feedstock and establish more sustainable and competitive lignocellulosic biorefineries. Pulp and paper mills and second-generation biorefineries produce large quantities of low-value technical lignin as a by-product, which is often combusted on-site for energy recovery. This Review focuses on the conversion of technical lignins by oxidative depolymerization employing heterogeneous catalysts. It scrutinizes the current literature describing the use of various heterogeneous catalysts in the oxidative depolymerization of lignin and includes a comparison of the methods, catalyst loadings, reaction media, and types of catalyst applied, as well as the reaction products and yields. Furthermore, current techniques for the determination of product yields and product recovery are discussed. Finally, challenges and suggestions for future approaches are outlined.

Enzymes as an environmental bottleneck in cellulosic ethanol production: Does on-site production solve it?

With the progressive implementation of Low-Carbon Policies based on Life Cycle Assessment (LCA) methodologies to assess climate change mitigation, biofuels’ potential environmental advantages could finally be translated into economic revenues, especially for residue-based production chains. This implies, however, tackling environmental hotspots along the Life Cycle, such as the use of enzymes, an important input in cellulosic ethanol production. While relevant, however, aspects of enzyme manufacturing, crucial to the LCA results, are often oversimplified in databases or even undisclosed, which might render the accounting for this flow unreliable. A potential solution for this is to model enzyme production, disclosing aspects such as carbon source uptakes, energy consumption and purification steps, all of which may carry significant environmental burdens, especially for fossil-reliant procedures. Integrating these steps into the biorefinery process design as an on-site operation, however, would allow access to renewable energy and carbon sources for enzyme production, potentially reducing their burden in the final biofuel LCA profile, when compared with off-site supply. This work, then, aims to evaluate the effect of inserting on-site enzyme production within a stand-alone second-generation (2G) biorefinery, using sugarcane straw as feedstock, to produce ethanol and electricity, as opposed to off-site enzyme supply from manufacturers. The results show that adopting on-site production can reduce the overall environmental impact profile for cellulosic ethanol, in comparison to off-site supply, even though off-site data present significant variability among databases and literature. This trend persisted for varied enzyme dosages in the saccharification process and for different inventories to model off-site supply. The reference inventories for off-site supply lack transparency and details, while also displaying large discrepancies between impact indicators, indicating the need to pursue more consistent, reliable, and complete inventories for its modelling in LCA. Compared to first-generation (1G) sugarcane ethanol, 2G-ethanol (with on-site enzyme production) demonstrated a reduction in the Global Warming Potential category of around 80%. However, relevant trade-offs for 2G ethanol (with off-site enzyme supply) were observed – regarding ecotoxicity, mineral scarcity, eutrophication and even the potential GHG reduction – when different enzyme inventories are used.

Availability and Environmental Performance of Wood for a Second-Generation Biorefinery

The current global climate change, the 2030 Agenda, and the planetary boundaries have driven new development strategies, such as the circular economy, bioeconomy, and biorefineries. In this framework, this study analyzes the potential availability and sustainability of the wood supply chain for a small-scale biorefinery aiming at producing 280–300 L of bioethanol per ton of dry biomass, consuming 30,000 t of dry biomass per year harvested in a 50 km radius. This wood production goal was assessed from Eucalyptus grandis stands planted for solid wood in northeastern Uruguay. Moreover, to understand the environmental performance of this biomass supply chain, the energy return on investment (EROI), carbon footprint (CF), and potential soil erosion were also assessed. The results showed that the potential wood production would supply an average of 81,800 t of dry mass per year, maintaining the soil erosion below the upper threshold recommended, an EROI of 2.3, and annual CF of 1.22 kg CO2−eq m−3 (2.6 g CO2−eq MJ−1). Combined with the environmental performance of the bioethanol biorefinery facility, these results would show acceptable values of sustainability according to EU Directive 2009/28/ec because the bioethanol CF becomes 1.7% of this petrol’s CF.

Multi-criteria analysis of detoxification alternatives: Techno-economic and socio-environmental assessment

The transformation of fermentable sugars provided from lignocellulosic wastes into biofuels or bioproducts is a key point at second-generation biorefineries. Spent sulfite liquor is a xylose-rich hydrolysate constituting the main residue of sulfite mills producing dissolving cellulose. Due to the presence of the inhibitors in the spent liquor, the most promising valorization options require detoxification before sugars bioconversion.In this work, a multi criteria analysis was implemented to select techno-economic and socio-environmental feasible detoxification alternatives that can be adapted to a wide variety of fermenting scenarios. Total inhibitors removal, phenolics removal, acetic acid removal, lignosulfonates removal, total sugar losses, fixed capital invested, manufacturing costs, waste toxicity, social acceptance, and employment were chosen as the most relevant criteria. The maximum allowable concentration of undesirable inhibitors cannot be established with a general character, and thereby decision-making tools result in feasible and efficient solutions. From a technical viewpoint best solution was anionic resins with a score of 0.68; the most economical alternative was the overliming with a score of 0.76; finally, from a socio-environmental perspective, overliming reached the highest score of 0.78. In addition, three spent liquor biorefinery models were proposed. Based on the multi-criteria analysis and based on the inhibitor's concentration affecting fermentation yields and productivity, the best detoxification alternatives were (1) anionic resins for polyhydroxyalkanoate production; (2) activated carbon for ethanol biorefinery; (3) overliming for xylitol biorefinery.

Counter-Current Suspension Extraction Process of Lignocellulose in Biorefineries to Reach Low Water Consumption, High Extraction Yields, and Extract Concentrations

The processing of large quantities of water in biorefining processes can lead to immense costs for heating, evaporation, and wastewater disposal. These costs may prohibit the exploitation of alternative products, e.g., xylooligosaccharides from straw, which are regarded as too costly. A new counter-current extractions method is proposed that aims at low solvent (water) consumption, as well as high yields and extract concentrations. This process was evaluated with suspension extraction experiments with steam pretreated wheat straw and the process window analysis based on a mass balance for a washing and a leaching scenario. The latter was conducted with two other suspension extraction processes as a comparison. The equilibration time was found to be well below 10 min. While the suspension extraction with and without recycling need to be designed to achieve a high yield or a high concentration and low solvent consumption, the proposed extraction method can reach all three simultaneously. Thus, this new process is evaluated as a potential method to spare water and downstream costs and allow new processing pathways in second-generation biorefineries.

Optimal design and operation of a second-generation biofuels supply chain

This article investigates how climate influences the value of adding preprocessing depots to a second-generation biorefinery’s supply chain. This is vital because humidity determines the amount of dry matter loss—exponential decay of energy content—suffered by biomass stored without preprocessing. The large volume of biomass required to fuel a biorefinery poses challenges in storage and in transportation. Further complications arise because the biomass is produced seasonally by hundreds of growers. Thus, recent failures of biorefineries may have been avoided, and future success may be achieved, by adding an intermediate layer to a biorefinery’s supply chain. A rigorous climate-based analysis of cost functions for each potential grower/depot pair leads to a stochastic program that optimizes the locations of depots, the assignment of growers to depots, and the volume of biomass stored fieldside vs. the volume stored as pelleted feedstock at depots. A computational study reveals that the humidity of the climate has much greater influence on the value of adding pre-processing depots to a second-generation biofuel supply chain than does transportation consolidation or any other parameter, endogenous or exogenous. Under favorable circumstances, our process reduces a biorefinery’s costs by over 30%, on average.

Hot Compressed Water Pretreatment and Surfactant Effect on Enzymatic Hydrolysis Using Agave Bagasse

Agave bagasse is a residual biomass in the production of the alcoholic beverage tequila, and therefore, it is a promising raw material in the development of biorefineries using hot compressed water pretreatment (hydrothermal processing). Surfactants application has been frequently reported as an alternative to enhance monomeric sugars production efficiency and as a possibility to reduce the enzyme loading required. Nevertheless, the surfactant’s action mechanisms in the enzymatic hydrolysis is still not elucidated. In this work, hot compressed water pretreatment was applied on agave bagasse for biomass fractionation at 194 °C in isothermal regime for 30 min, and the effect of non-ionic surfactants (Tween 20, Tween 80, Span 80, and Polyethylene glycol (PEG 400)) was studied as a potential enhancer of enzymatic saccharification of hydrothermally pretreated solids of agave bagasse (AGB). It was found that non-ionic surfactants show an improvement in the conversion yield of cellulose to glucose (100%) and production of glucose (79.76 g/L) at 15 FPU/g glucan, the highest enhancement obtained being 7% regarding the control (no surfactant addition), using PEG 400 as an additive. The use of surfactants allows improving the production of fermentable sugars for the development of second-generation biorefineries.

Microwave subcritical water pre-treatment and enzymatic hydrolysis of geographical identification (GI) tag Indian black rice (Chakhao Poireiton) straw for fermentable sugar production

Rice straw is an important feedstock for second-generation biorefineries, the majority being conventional white rice. This research investigated the potential of the straw of black rice Chakhao Poireiton, a rice variety of increasing importance, for biorefining. Pre-treatment of black rice straw was carried out in a pressurised microwave reactor and free sugar release was measured following digestion with Cellic® CTec3. This was compared to white rice straw. Pre-treatment (100 − 200° C for 5 min) did not drastically impact the sugar composition but brought about an enhanced release of glucose after enzymatic hydrolysis from 25 to 50% (black rice) and from 26 to 55% (white rice). For xylose digestibility, the increase was around seven-fold for black rice (8 to 57%) and five-fold for white rice (13 to 64%). This improvement in digestibility of the straw samples could have come from modification in the lignocellulose structural features making the polysaccharides more susceptible to hydrolysis. Black rice could be a suitable feedstock for a combined biorefinery – food and nutraceuticals from grain (much higher than white rice) and the straw for fermentable sugars comparable to white rice straw for biofuels.

Subcritical water pretreatment for agave bagasse fractionation from tequila production and enzymatic susceptibility

This work focuses on the effect of subcritical water pretreatment conditions on agave bagasse chemical composition, biomass fractionation, and enzymatic hydrolysis obtained from the different tequila production processes. The pretreatment was carried out in a batch pressurized reactor within an isothermal regime. The operational conditions for subcritical water pretreatment were (150–190 °C) and (10–50 min). The best operational conditions were selected, based on the increased cellulose content (>50%) in the pretreated solid phase. The conditions for 190 °C for 50 and 30 min of pretreated agave bagasse solids were chosen for enzymatic hydrolysis susceptibility (15 FPU/g of the substrate). The maximum conversion yield (cellulose to glucose) during enzymatic hydrolysis achieved was up to 61.62% (5.86 g/L) in industrial bagasse at 72 h and initial saccharification rate was 0.34 g/(L*h) at 12 h. This study indicates that the agave bagasse is a promising raw material in the development of second-generation biorefineries.

Insights into Glucose-6-phosphate Allosteric Activation of β-Glucosidase A.

Second-generation ethanol production involves the use of agricultural and forestry waste as feedstock, being an alternative to the first-generation technology as it relies on low-cost abundant residues and does not affect food agriculture. However, the success of second-generation biorefineries relies on energetically efficient processes and effective enzyme cocktails to convert cellulose into fermentable sugars. β-glucosidases catalyze the last step on the enzymatic hydrolysis of cellulose; however, they are often inhibited by glucose. Previous studies demonstrated that glucose-6-phosphate (G6P) is a positive allosteric modulator of Bacillus polymyxa β-glucosidase A, improving enzymatic efficiency, providing thermoresistance, and imparting glucose tolerance. However, the precise molecular details of G6P-β-glucosidase A interactions have not yet been described so far. We investigated the molecular details of G6P binding into B. polymyxa β-glucosidase A through in silico docking using the site identification by ligand competitive saturation technology followed by site-directed mutagenesis studies, from which an allosteric binding site for G6P was identified. In addition, a mechanistic shift toward the transglycosylation reaction as opposed to hydrolysis was observed in the presence of G6P, suggesting a new role of G6P allosteric modulation of the catalytic activity of β-glucosidase A.

Inventory review and environmental evaluation of first- and second-generation sugars through life cycle assessment.

In recent years, there has been a great movement towards the generation of knowledge related to the biorefinery concept. First-generation biorefineries bear the stigma of using arable land and edible crops for fuel instead of as sources of food and feed. However, second-generation biorefineries have not reached the level of full technical feasibility. Bearing in mind the objective of sugar production from sugar, starch, or lignocellulosic raw materials, the purpose of this study is to assess the environmental impact of first- and second-generation biorefineries, considering as an example for the comparative evaluation, the production of sugar fractions from crops (starch and sugar crops), and lignocellulosic biomass (hardwood and softwood). The characterization results were obtained using the ReCiPe 1.1 model, implemented through the SimaPro 9.0 software. Both production systems are inherently different and have strengths and weaknesses that must be carefully analyzed. The resulting environmental profile shows that the silviculture of wood contributes less to the environmental impact than cropping activities in most impact categories. In general, this study suggests that first-generation systems are burdened environmentally by the use of fertilizers, which havea significant impact on categories such as marine and freshwater eutrophication and terrestrial acidification, while second-generation systems are limited by the intensive processing steps needed for delignification, typically involving the use of chemicals and/or energy. LCA in early stages of the production of bio-based building blocks, rather than on the manufacture of biofuels or bioplastics, allows the precise identification of the environmental burdens that may be influencing the overall environmental profile of a biorefinery.

Engineered LPMO Significantly Boosting Cellulase-Catalyzed Depolymerization of Cellulose.

Lytic polysaccharide monooxygenases (LPMOs) play a crucial role in the enzymatic depolymerization of cellulose through oxidative cleavage of the glycosidic bond in the highly recalcitrant crystalline cellulose region. Improving the activity of LPMOs is of considerable importance for second-generation biorefinery. In this study, we identified a beneficial amino acid substitution (N526S) located in the cellulose binding module (CBM) of HcLPMO10 (LPMO of Hahella chejuensis) using directed evolution. The improved variant HcLPMO10 M1 (N526S) exhibits 2.1-fold higher activity for the H2O2 production, 2.7-fold higher oxidation activity, and 1.9-fold higher binding capacity toward cellulose compared with those of the wild type (WT). Furthermore, M1 shows 2.1-fold higher activity for degradation of crystalline cellulose in synergy with cellulase, compared to the WT. Structural analysis through molecular modeling and molecular dynamics (MD) simulation revealed that the substitution N526S located in the CBM likely stabilizes the cellulose binding surface and enhances the binding capacity of HcLPMO10 to cellulose, thereby enhancing enzyme activity. These findings demonstrate the important role of the CBM in the catalytic function of LPMO.

Syngas Derived from Lignocellulosic Biomass Gasification as an Alternative Resource for Innovative Bioprocesses

A hybrid system based on lignocellulosic biomass gasification and syngas fermentation represents a second-generation biorefinery approach that is currently in the development phase. Lignocellulosic biomass can be gasified to produce syngas, which is a gas mixture consisting mainly of H2, CO, and CO2. The major challenge of biomass gasification is the syngas’s final quality. Consequently, the development of effective syngas clean-up technologies has gained increased interest in recent years. Furthermore, the bioconversion of syngas components has been intensively studied using acetogenic bacteria and their Wood–Ljungdahl pathway to produce, among others, acetate, ethanol, butyrate, butanol, caproate, hexanol, 2,3-butanediol, and lactate. Nowadays, syngas fermentation appears to be a promising alternative for producing commodity chemicals in comparison to fossil-based processes. Research studies on syngas fermentation have been focused on process design and optimization, investigating the medium composition, operating parameters, and bioreactor design. Moreover, metabolic engineering efforts have been made to develop genetically modified strains with improved production. In 2018, for the first time, a syngas fermentation pilot plant from biomass gasification was built by LanzaTech Inc. in cooperation with Aemetis, Inc. Future research will focus on coupling syngas fermentation with additional bioprocesses and/or on identifying new non-acetogenic microorganisms to produce high-value chemicals beyond acetate and ethanol.