Lignocellulosic bioethanol production: a review on pretreatment strategies, biofuel separation, and artificial intelligence/machine learning − based sustainable optimization

Potential for reduced water consumption in biorefining of lignocellulosic biomass to bioethanol and biogas

With diminishing oil supplies and growing political instability in oil‐producing nations, the world is facing a major energy threat which needs to be solved by virtue of alternative energy sources. Bioethanol has received considerable attention in the transportation sector because of its utility as an octane booster, fuel additive, and even as neat fuel. Brazil and the USA have been producing ethanol on a large scale from sugarcane and corn, respectively. However, due to their primary utility as food and feed, these crops cannot meet the global demand for ethanol production as an alternative transportation fuel. Lignocellulosic biomass is projected as a virtually eternal raw material for fuel ethanol production. The main bottleneck so far has been the technology concerns, which do not support cost‐effective and competitive production of lignocellulosic bioethanol. This review sheds light on some of the practical approaches that can be adopted to make the production of lignocellulosic bioethanol economically attractive. These include the use of cheaper substrates, cost‐effective pre‐treatment techniques, overproducing and recombinant strains for maximized ethanol tolerance and yields, improved recovery processes, efficient bioprocess integration, economic exploitation of side products, and energy and waste minimization. An integrated and dedicated approach can help in realizing large‐scale commercial production of lignocellulosic bioethanol, and can contribute toward a cleaner and more energy efficient world. Copyright © 2009 Society of Chemical Industry and John Wiley & Sons, Ltd

Continuous recycling of enzymes during production of lignocellulosic bioethanol in demonstration scale

Bioethanol production from lignocellulosic biomass has gained significance as an alternative renewable fuel source to mitigate the environmental impact caused by fossil fuels. Lignocellulosic biomass, such as agricultural scums, forestry waste, and dedicated energy crops, offers several advantages for bioethanol production due to its abundance, low cost, and non- competitiveness with food crops. This abstract explores the current state of bioethanol production from lignocellulosic biomass, its challenges, and futuristic trends. The conversion of lignocellulosic biomass to bioethanol involves three main steps: pre-treatment, enzymatic hydrolysis, and fermentation. Pre-treatment is crucial to overcome the obstinacy of lignocellulosic biomass, making it more susceptible to enzymatic attack. Various pre-treatment techniques, including physical, chemical and biological methods have been established to enhance biomass accessibility and enzymatic digestibility. Enzymatic hydrolysis involves the breakdown of complex polysaccharides into fermentable sugars like D-glucose or D-xylose using cellulolytic and hemicellulolytic enzymes while fermentation employs yeast or specific bacteria to convert sugars into bioethanol. In recent years, new trends have emerged to revolutionize lignocellulosic bioethanol production. One such trend is the utilization of consolidated bioprocessing (CBP), which objects to combine all three steps of bioethanol production into a single microorganism or enzyme system. CBP offers the potential for simplified process design, reduced costs, and increased efficiency. Various microorganisms, including engineered bacteria and fungi, are being explored for CBP to achieve higher bioethanol yields from lignocellulosic biomass. Moreover, advancements in synthetic biology and genetic engineering have flagged the way for tailor-made enzymes and microorganisms with improved characteristics for lignocellulosic bioethanol production. Researchers are focusing on designing enzymes with enhanced stability, activity, and specificity to achieve higher sugar release. Similarly, genetically engineered microorganisms capable of efficiently fermenting a broad spectrum of sugars and tolerating inhibitory compounds are being developed to maximize bioethanol yields. Furthermore, the integration of lignocellulosic bioethanol production with other biorefinery processes is gaining attention. By exploiting the by- products of bioethanol production, such as lignin and hemicellulose, for the production of value-added biofuels, or materials, the overall process finances can be improved. Integrated biorefineries offer the potential for a more sustainable and economically viable approach to utilizing lignocellulosic biomass. Bioethanol production from lignocellulosic biomass holds immense potential as a renewable and sustainable fuel source. Despite the challenges faced, trending approaches such as consolidated bioprocessing, synthetic biology and biorefinery integration are flagging the way for more effectual and economically feasible bioethanol manufacture. Continued research and development efforts in these areas will be crucial in realizing the full potential of plant tissue biomass for bioethanol manufacture and reducing dependence on fossil fuels

Genome engineering for breaking barriers in lignocellulosic bioethanol production

Biomass Conversion 190 7.7 Recent Innovative Approaches to Combat the Bottlenecks inLignocellulosic Bioconversion to Bioethanol 191 7.7.1 Enzyme Concoction Approach 1927.7.2 Direct Glucose Production Using Clostridium thermocellum Cultures Supplemented with a Thermostable β-glucosidase 1927.7.3 Single Step Bioethanol Production by Employing Novel Strains 1937.7.4 Use of Super Computer in Bioethanol Production 194 7.8 Summary 195 Keywords 196 References 196ABSTRACTThrust towards embarking on building a new economy based on nonfossil fuel energy has imparted impetus to research on lignocellulosic biofuel industry. Lignocellulosics form a large class of renewable feedstocks, which include agricultural residues, municipal solid waste and food processing and industrial wastes. Although lignocellulosics arevery energy rich substrates, unlocking its potential and extending it to practical large scales has a number of bottlenecks. The challenges in instituting and exploiting these resources for biofuel production compel us to rethink current paradigm of supply and usage. Hence there is an urgent need to combat these issues to scale it up and execute at practical scales. Technological constraints do not support cost-effective and competitive production of lignocellulosic bioethanol. In order to produce lignocellulosic bioethanol economically attractive, use of cheaper substrates, inexpensive pretreatment techniques, use of overproducing and recombinant strains for maximized ethanol tolerance and yields, better recovery processes, effective bioprocess integration, exploitation of side products, and reduction of energy and waste may be encouraged. A cohesive and dedicated approach can help in realizing large-scale commercial production of lignocellulosic bioethanol. This chapter is an exhaustive overview of this aspect of lignocellulosic biofuel production. Special case studies have been included to highlight the probable strategies that may be adopted in future.

Life cycle assessment of lignocellulosic bioethanol: Environmental impacts and energy balance

Intensification of lignocellulosic bioethanol production process using continuous double-staged immersed membrane bioreactors

The production of cellulase enzymes (CE) has been identified as one major contributor towards the life cycle environmental and economic impacts of second-generation lignocellulosic bioethanol (LCB) production. Despite this knowledge, the literature lacks consistent and transparent life cycle assessments (LCA) which compare CE production based on the three more commonly proposed carbon sources: cornstarch glucose, sugar cane molasses and pre-treated softwood. Furthermore, numerous LCAs of LCB omit CE production from their system boundaries, with several authors citing the lack of available production data. In this article, we perform a comparative attributional LCA for the on-site production of 1 kg CE in full broth via submerged aerobic fermentation (SmF) based on the three alternative carbon sources, cases A, B and C, respectively. We determine life cycle inventory (LCI) material consumption using stoichiometric equations and volume flow, supplemented with information from the literature. All LCIs are provided in a consistent and transparent manner, filling the existing data gaps towards performing representative LCAs of LCB production with on-site CE production. Life cycle impact assessment (LCIA) results are determined with SimaPro 8 software using CML 1A baseline and non-baseline methods along with cumulative energy demand and are compared to results of similar studies. Sensitivity analysis is performed both for all major assumptions and for market changes with the application of advanced attributional LCA (AALCA). We find that CE production from pre-treated softwood (case C) provides the lowest environmental impacts, followed by sugar cane molasses (case B) and then cornstarch glucose (case A), with global warming potentials of 7.9, 9.1 and 10.6 kg CO2 eq./kg enzyme, respectively. These findings compare well with those of similar studies, though great variation exists in the literature. Through sensitivity analysis, we determine that results are sensitive to assumptions made concerning carbon source origin, applied allocation, market changes, process efficiency and electricity supply. Furthermore, we find that the contribution of CE production towards the overall life cycle impacts of LCB is significant and that the omission of this sub-process in LCAs of LCB production can compromise their representativeness.

The threat of climate change has intensified efforts toward the development of safer alternatives to depleting fossil fuels (Cox et al., 2000). Lignocellulosic bioethanol is considered to be a viable and environmentally friendly alternative to fossil fuels. Though lignocellulosic biomass is available in massive quantities and is renewable (Dillon and Dillon, 2003; Lynd et al., 2008; Pauly and Keegstra, 2008; Kricka et al., 2015), the presence of certain barriers makes lignocellulosic bioethanol expensive. Discovery of proteins with novel specificities is necessary to break these barriers and make lignocellulosic bioethanol economically viable (Horn et al., 2012; Ulaganathan et al., 2015). Cellulolytic bacteria isolated from various environments have been explored for proteins of potential use in lignocellulosic bioethanol production (Badger, 2002; Wang et al., 2012; Pinheiro et al., 2015). Bacteria belonging to the genera Bacillus, Bacteroides, Butyrivibrio, Cellulosimicrobium, Citrobacter, Clostridium, Devosia, Dyadobacter, Ensifer, Kaistia, Labrys, Methanobrevibacter, Microbacterium, Ochrobactrum, Paracoccus, Pseudomonas, Rhizobium, Ruminococcus, Shinella, Siphonobacter, Stenotrophomonas, Trichonympha, and Variovorax, were found to be cellulolytic (Saxena et al., 1993; Schwarz, 2001; Gupta et al., 2012; Huang et al., 2012; Yanga et al., 2014). Bacillus pumilus strains are known to produce cellulase enzyme up to a maximum of 11.4 mg/g of cell dry mass (Suzuki and Kaneko, 1976; Kotchoni and Shonukan, 2002; Ariffin et al., 2006). The cellulase enzyme produced by B. pumilus strain EB3 has been found to be superior to fungal cellulases due to its higher optimum pH and temperature (Ariffin et al., 2006). Further it has been shown that the B. pumilus cellulase enzyme could be mutated to remove the catabolite repression (Kotchoni et al., 2003). We have recently isolated bacterial strains from the gut contents of the wood boring Mesomorphus sp. These isolates were screened for cellulolytic and xylose isomerase activities and the isolate ku-bf1 which exhibited maximum cellulolytic and xylose isomerase activities was identified as B. pumilus by 16S rRNA sequencing. The whole genome of this strain has been sequenced. The dataset has been submitted to NCBI and is reported here.

Life cycle environmental benefits of recycling waste liquor and chemicals in production of lignocellulosic bioethanol

Measurement of the protein content in samples from production of lignocellulosic bioethanol is an important tool when studying the adsorption of cellulases. Several methods have been used for this, and after reviewing the literature, we concluded that one of the most promising assays for simple and fast protein measurement on this type of samples was the ninhydrin assay. This method has also been used widely for this purpose, but with two different methods for protein hydrolysis prior to the assay-alkaline or acidic hydrolysis. In samples containing glucose or ethanol, there was significant interference from these compounds when using acid hydrolysis, which was not the case when using the alkaline hydrolysis. We evaluated the interference from glucose, cellulose, xylose, xylan, lignin and ethanol on protein determination of BSA, Accellerase(®) 1500 and Cellic(®) CTec2. The experiments demonstrated that the presence of cellulose, lignin and glucose (above 50 g/kg) could significantly affect the results of the assay. Comparison of analyses performed with the ninhydrin assay and with a CN analyser revealed that there was good agreement between these two analytical methods, but care has to be taken when applying the ninhydrin assay. If used correctly, the ninhydrin assay can be used as a fast method to evaluate the adsorption of cellulases to lignin.

A locally isolated strain of Aspergillus niger van Tieghem was found to produce thermostable β-xylosidase activity. The enzyme was purified by cation and anion exchange and hydrophobic interaction chromatography. Maximum activity was observed at 70-75°C and pH 4.5. The enzyme was found to be thermostable retaining 91 and 87% of its original activity after incubation for 72h at 60 and 65°C, respectively, with 52% residual activity detected after 18h at 70°C. Available data indicates that the purified β-xylosidase is more thermostable over industrially relevant prolonged periods at high temperature than those reported from other A. niger strains. Maximum activity was observed on p-nitrophenyl-β-D-xylopyranoside and the enzyme also hydrolysed p-nitrophenyl β-D-glucopyranoside and p-nitrophenyl α-L-arabinofuranoside. The purified enzyme acted synergistically with A. niger endo-1,4-β-xylanase in the hydrolysis of beechwood xylan at 65°C. During hydrolysis of pretreated straw lignocellulose at 70°C using a commercial lignocellulosic enzyme cocktail, inclusion of the purified enzyme resulted in a 19-fold increase in the amount of xylose produced after 6h. The results observed indicate potential suitability for industrial application in the production of lignocellulosic bioethanol where thermostable β-xylosidase activity is of growing interest to maximise the enzymatic hydrolysis of lignocellulose.

The production of lignocellulosic ethanol calls for a robust fermentative yeast able to tolerate a wide range of toxic molecules that occur in the pre-treated lignocellulose. The concentration of inhibitors varies according to the composition of the lignocellulosic material and the harshness of the pre-treatment used. It follows that the versatility of the yeast should be considered when selecting a robust strain. This work aimed at the validation of seven natural Saccharomyces cerevisiae strains, previously selected for their industrial fitness, for their application in the production of lignocellulosic bioethanol. Their inhibitor resistance and fermentative performances were compared to those of the benchmark industrial yeast S. cerevisiae Ethanol Red, currently utilized in the second-generation ethanol plants. The yeast strains were characterized for their tolerance using a synthetic inhibitor mixture formulated with increasing concentrations of weak acids and furans, as well as steam-exploded lignocellulosic pre-hydrolysates, generally containing the same inhibitors. The eight non-diluted liquors have been adopted to assess yeast ability to withstand bioethanol industrial conditions. The most tolerant S. cerevisiae Fm17 strain, together with the reference Ethanol Red, was evaluated for fermentative performances in two pre-hydrolysates obtained from cardoon and common reed, chosen for their large inhibitor concentrations. S. cerevisiae Fm17 outperformed the industrial strain Ethanol Red, producing up to 18 and 39 g/L ethanol from cardoon and common reed, respectively, with ethanol yields always higher than those of the benchmark strain. This natural strain exhibits great potential to be used as superior yeast in the lignocellulosic ethanol plants.

Considering the possible threats to the oil supply due to the rapid depletion of oil reservoirs and the negative environmental impacts of petroleum use, developing an environmentally friendly biofuel such as bioethanol is needed. Pretreatment is a critical step in the production of lignocellulosic bioethanol. In this study, the effect of sulfuric acid on the pretreatment of the YSS-10R variety of sorghum was evaluated. Response Surface Methodology (RSM) was employed to develop an experimental design matrix and evaluate the effect of pretreatment parameters on the release of fermentable sugars. Sorghum straw was treated with sulfuric acid concentrations of 0.5, 1.75, and 3% (V/V) at temperatures of 70, 100, and 130 °C for reaction times of 10, 20, and 30 min. The maximum glucose yield was 7.66 g/L (0.064 g/g) and was obtained via pretreatment with 0.5% H2SO4 at 100 °C for 10 min. That of xylose was 7.62 g/L (0.064 g/g), obtained via pretreatment with 0.50% H2SO4 at 130 °C for 20 min. The pretreatment conditions for maximum xylose yield were determined to be 2% H2SO4, 130 °C, and 20 min. Results indicate that sulfuric acid is an efficient catalyst for pretreatment at high temperatures and relatively long reaction times.