Efficient polyhydroxybutyrate production using acetate by engineered Halomonas sp. JJY01 harboring acetyl-CoA acetyltransferase.

Polyhydroxybutyrate (PHB) is a well-known biodegradable bioplastic synthesized by microorganisms and can be produced from volatile fatty acids (VFAs). Among VFAs acetate can be utilized by Halomonas sp. YLGW01 for growth and PHB production. In this study, Halomonas sp. JJY01 was developed through introducing acetyl-CoA acetyltransferase (atoAD) with LacIq-Ptrc promoter into Halomonas sp. YLGW01. The effect of expression of atoAD on acetate was investigated by comparison with acetate consumption and PHB production. Shake-flask study showed that Halomonas sp. JJY01 increased acetate consumption rate, PHB yield and PHB production (0.27 g/L/h, 0.075 g/g, 0.72 g/L) compared to the wild type strain (0.17 g/L/h, 0.016 g/g, 0.11 g/L). In 10 L fermenter scale fed-batch fermentation, the growth of Halomonas sp. JJY01 resulted in higher acetate consumption rate, PHB yield and PHB titer (0.55 g/L/h, 0.091 g/g, 4.6 g/L) than wild type strain (0.35 g/L/h, 0.067 h/h, 2.9 g/L). These findings demonstrate enhanced acetate utilization and PHB production through the introduction of atoAD in Halomonas strains.

Application of liquid-based colorimetric method for high throughput screening of bioplastic-degrading strains using esterase assay.

To alleviate environmental problems caused by using conventional plastics, bioplastics have garnered significant interest as alternatives to petroleum-based plastics. Despite possessing better degradability traits compared to traditional plastics, the degradation of bioplastics still demands a longer duration than initially anticipated. This necessitates the utilization of degradation strains or enzymes to enhance degradation efficiency, ensuring timely degradation. In this study, a novel screening method to identify bioplastic degraders faster was suggested to circumvent the time-consuming and laborious characteristics of solid-based plate assays. This liquid-based colorimetric method confirmed the extracellular esterase activity with p-nitrophenyl esters. It eliminated the needs to prepare plastic emulsion plates at the initial screening system, shortening the time for the overall screening process and providing more quantitative data. p-nitrophenyl hexanoate (C6) was considered the best substrate among the various p-nitrophenyl esters as substrates. The screening was performed in liquid-based 96-well plates, resulting in the discovery of a novel strain, Bacillus sp. SH09, with a similarity of 97.4% with Bacillus licheniformis. Furthermore, clear zone assays, degradation investigations, scanning electron microscopy, and gel permeation chromatography were conducted to characterize the biodegradation capabilities of the new strain, the liquid-based approach offered a swift and less labor-intensive option during the initial stages.

Optimization of polyhydroxyalkanoate production in Halomonas sp. YLGW01 using mixed volatile fatty acids: a study on mixture analysis and fed-batch strategy.

Polyhydroxyalkanoate (PHA) is one of the most promising materials for replacing petroleum-based plastics, and it can be produced from various renewable biomass sources. In this study, PHA production was conducted using Halomonas sp. YLGW01 utilizing mixed volatile fatty acids (VFAs) as carbon sources. The ratio and concentration of carbon and nitrogen sources were optimized through mixture analysis and organic nitrogen source screening, respectively. It was found that the highest cell dry weight (CDW) of 3.15 g/L and PHA production of 1.63 g/L were achieved when the ratio of acetate to lactate in the mixed VFAs was 0.45:0.55. Furthermore, supplementation of organic nitrogen sources such as soytone resulted in a ninefold increase in CDW (reaching 2.32 g/L) and a 22-fold increase in PHA production (reaching 1.60 g/L) compared to using inorganic nitrogen sources. Subsequently, DO-stat, VFAs consumption rate stat, and pH-stat fed-batch methods were applied to investigate and evaluate PHA productivity. The results showed that when pH-stat-based VFAs feeding was employed, a CDW of 7 g/L and PHA production of 5.1 g/L were achieved within 68 h, with a PHA content of 73%. Overall, the pH-stat fed-batch strategy proved to be effective in enhancing PHA production by Halomonas sp. YLGW01 utilizing VFAs.

Polyhydroxybutyrate (PHB) production from sugar cane molasses and tap water without sterilization using novel strain, Priestia sp. YH4.

The production cost of biodegradable polymer like polyhydroxybutyrate (PHB) is still higher than that of petroleum-based plastics. A potential solution for reducing its production cost is using a cheap carbon source and avoiding a process of sterilization. In this study, a novel PHB-producing microbial strain, Priestia sp. YH4 was screened from the marine environment using sugarcane molasses as the carbon source without sterilization. Culture conditions, such as carbon, NaCl, temperature, pH, inoculum size, and cultivation time, were optimized for obtaining the highest PHB production by YH4 resulting in 5.94 g/L of dry cell weight (DCW) and 61.7 % of PHB content in the 5 mL culture. In addition, it showed similar PHB production between the cultures with or without sterilization in Marine Broth media. When cultured using only tap water, sugarcane molasses, and NaCl in a 5 L fermenter, 24.8 g/L DCW was produced at 41 h yielding 13.9 g/L PHB. Finally, DSC (Differential Scanning Calorimetry) and GPC (Gel Permeation Chromatography) were used to analyze thermal properties and molecular weights resulting in Tm = 167.2 °C, Tc = 67.3 °C, Mw = 2.85 × 105, Mn = 1.05 × 105, and PDI = 2.7, respectively. Therefore, we showed the feasibility of more economical process for PHB production by finding novel strain, utilizing molasses with minimal media components and avoiding sterilization.

Macroalgae as a source of sugar and detoxifier biochar for polyhydroxyalkanoates production by Halomonas sp. YLGW01 under the unsterile condition.

Macroalgae (seaweed) is considered a favorable feedstock for polyhydroxyalkanoates (PHAs) production owing to its high productivity, low land and freshwater requirement, and renewable nature. Among different microbes Halomonas sp. YLGW01 can utilize algal biomass-derived sugars (galactose and glucose) for growth and PHAs production. Biomass-derived byproducts furfural, hydroxymethylfurfural (HMF), and acetate affects Halomonas sp. YLGW01 growth and poly(3-hydroxybutyrate) (PHB) production i.e., furfural > HMF > acetate. Eucheuma spinosum biomass-derived biochar was able to remove 87.9 % of phenolic compounds from its hydrolysate without affecting sugar concentration. Halomonas sp. YLGW01 grows and accumulates a high amount of PHB at 4 % NaCl. The use of detoxified unsterilized media resulted in high biomass (6.32 ± 0.16 g cdm/L) and PHB production (3.88 ± 0.04 g/L) compared to undetoxified media (3.97 ± 0.24 g cdm/L, 2.58 ± 0.1 g/L). The finding suggests that Halomonas sp. YLGW01 has the potential to valorize macroalgal biomass into PHAs and open a new avenue for renewable bioplastic production.

Strategy for efficiently utilizing Escherichia coli cells producing isobutanol by combining isobutanol and indigo production systems.

Isobutanol is a potential biofuel, and its microbial production systems have demonstrated promising results. In a microbial system, the isobutanol produced is secreted into the media; however, the cells remaining after fermentation cannot be used efficiently during the isobutanol recovery process and are discarded as waste. To address this, we aimed to investigate the strategy of utilizing these remaining cells by combining the isobutanol production system with the indigo production system, wherein the product accumulates intracellularly. Accordingly, we constructed E. coli systems with genes, such as acetolactate synthase gene (alsS), ketol-acid reductoisomerase gene (ilvC), dihydroxyl-acid dehydratase (ilvD), and alpha-ketoisovalerate decarboxylase gene (kivD), for isobutanol production and genes, such as tryptophanase gene (tnaA) and flavin-containing monooxygenase gene (FMO), for indigo production. This system produced isobutanol and indigo simultaneously while accumulating indigo within cells. The production of isobutanol and indigo exhibited a strong linear correlation up to 72 h of production time; however, the pattern of isobutanol and indigo production varied. To our knowledge, this study is the first to simultaneously produce isobutanol and indigo and can potentially enhance the economy of biochemical production.

Polyhydroxybutyrate production from crude glycerol using a highly robust bacterial strain Halomonas sp. YLGW01.

Petrochemical-based plastics are hardly biodegradable and a major cause of environmental pollution, and polyhydroxybutyrate (PHB) is attracting attention as an alternative due to its similar properties. However, the cost of PHB production is high and is considered the greatest challenge for its industrialization. Here, crude glycerol was used as a carbon source for more efficient PHB production. Among the 18 strains investigated, Halomonas taeanenisis YLGW01 was selected for PHB production due to its salt tolerance and high glycerol consumption rate. Furthermore, this strain can produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3 HV)) with 17 % 3 HV mol fraction when a precursor is added. PHB production was maximized through medium optimization and activated carbon treatment of crude glycerol, resulting in 10.5 g/L of PHB with 60 % PHB content in fed-batch fermentation. Physical properties of the produced PHB were analyzed, i.e., weight average molecular weight (6.8 × 105), number average molecular weight (4.4 × 105), and the polydispersity index (1.53). In the universal testing machine analysis, the extracted intracellular PHB showed a decrease in Young's modulus, an increase in Elongation at break, greater flexibility than authentic film, and decreased brittleness. This study confirmed that YLGW01 is a promising strain for industrial PHB production using crude glycerol.

Two-Stage Bio-Hydrogen and Polyhydroxyalkanoate Production: Upcycling of Spent Coffee Grounds.

Coffee waste is an abundant biomass that can be converted into high value chemical products, and is used in various renewable biological processes. In this study, oil was extracted from spent coffee grounds (SCGs) and used for polyhydroxyalkanoate (PHA) production through Pseudomonas resinovorans. The oil-extracted SCGs (OESCGs) were hydrolyzed and used for biohydrogen production through Clostridium butyricum DSM10702. The oil extraction yield through n-hexane was 14.4%, which accounted for 97% of the oil present in the SCGs. OESCG hydrolysate (OESCGH) had a sugar concentration of 32.26 g/L, which was 15.4% higher than that of the SCG hydrolysate (SCGH) (27.96 g/L). Hydrogen production using these substrates was 181.19 mL and 136.58 mL in OESCGH and SCGH media, respectively. The consumed sugar concentration was 6.77 g/L in OESCGH and 5.09 g/L in SCGH media. VFA production with OESCGH (3.58 g/L) increased by 40.9% compared with SCGH (2.54 g/L). In addition, in a fed-batch culture using the extracted oil, cell dry weight was 5.4 g/L, PHA was 1.6 g/L, and PHA contents were 29.5% at 24 h.

Unraveling Biohydrogen Production and Sugar Utilization Systems in the Electricigen Shewanella marisflavi BBL25.

Identification of novel, electricity-producing bacteria has garnered remarkable interest because of the various applications of electricigens in microbial fuel cell and bioelectrochemical systems. Shewanella marisflavi BBL25, an electricity-generating microorganism, uses various carbon sources and shows broader sugar utilization than the better-known S. oneidensis MR-1. To determine the sugar-utilizing genes and electricity production and transfer system in S. marisflavi BBL25, we performed an in-depth analysis using whole-genome sequencing. We identified various genes associated with carbon source utilization and the electron transfer system, similar to those of S. oneidensis MR-1. In addition, we identified genes related to hydrogen production systems in S. marisflavi BBL25, which were different from those in S. oneidensis MR-1. When we cultured S. marisflavi BBL25 under anaerobic conditions, the strain produced 427.58 ± 5.85 µl of biohydrogen from pyruvate and 877.43 ± 28.53 µl from xylose. As S. oneidensis MR-1 could not utilize glucose well, we introduced the glk gene from S. marisflavi BBL25 into S. oneidensis MR-1, resulting in a 117.35% increase in growth and a 17.64% increase in glucose consumption. The results of S. marisflavi BBL25 genome sequencing aided in the understanding of sugar utilization, electron transfer systems, and hydrogen production systems in other Shewanella species.

Finding of Novel Galactose Utilizing Halomonas sp. YK44 for Polyhydroxybutyrate (PHB) Production.

Polyhydroxybutyrate (PHB) is a biodegradable bioplastic with potential applications as an alternative to petroleum-based plastics. However, efficient PHB production remains difficult. The main cost of PHB production is attributed to carbon sources; hence, finding inexpensive sources is important. Galactose is a possible substrate for polyhydroxyalkanoate production as it is abundant in marine environments. Marine bacteria that produce PHB from galactose could be an effective resource that can be used for efficient PHB production. In this study, to identify a galactose utilizing PHB producer, we examined 16 Halomonas strains. We demonstrated that Halomonas cerina (Halomonas sp. YK44) has the highest growth and PHB production using a culture media containing 2% galactose, final 4% NaCl, and 0.1% yeast extract. These culture conditions yielded 8.98 g/L PHB (78.1% PHB content (w/w)). When galactose-containing red algae (Eucheuma spinosum) hydrolysates were used as a carbon source, 5.2 g/L PHB was produced with 1.425% galactose after treatment with activated carbon. Since high salt conditions can be used to avoid sterilization, we examined whether Halomonas sp. YK44 could produce PHB in non-sterilized conditions. Culture media in these conditions yielded 72.41% PHB content. Thus, Halomonas sp. YK44 is robust against contamination, allowing for long-term culture and economical PHB production.

Enhanced tolerance of Cupriavidus necator NCIMB 11599 to lignocellulosic derived inhibitors by inserting NAD salvage pathway genes.

Polyhydroxybutyrate (PHB) is a bio-based, biodegradable and biocompatible plastic that has the potential to replace petroleum-based plastics. Lignocellulosic biomass is a promising feedstock for industrial fermentation to produce bioproducts such as polyhydroxybutyrate (PHB). However, the pretreatment processes of lignocellulosic biomass lead to the generation of toxic byproducts, such as furfural, 5-HMF, vanillin, and acetate, which affect microbial growth and productivity. In this study, to reduce furfural toxicity during PHB production from lignocellulosic hydrolysates, we genetically engineered Cupriavidus necator NCIMB 11599, by inserting the nicotine amide salvage pathway genes pncB and nadE to increase the NAD(P)H pool. We found that the expression of pncB was the most effective in improving tolerance to inhibitors, cell growth, PHB production and sugar consumption rate. In addition, the engineered strain harboring pncB showed higher PHB production using lignocellulosic hydrolysates than the wild-type strain. Therefore, the application of NAD salvage pathway genes improves the tolerance of Cupriavidus necator to lignocellulosic-derived inhibitors and should be used to optimize PHB production.

Finding a Benign Plasticizer to Enhance the Microbial Degradation of Polyhydroxybutyrate (PHB) Evaluated by PHB Degrader Microbulbifer sp. SOL66.

As a biodegradable plastic, polyhydroxybutyrate (PHB) has relatively poor mechanical properties, preventing its wider use. Various plasticizers have been studied to improve the mechanical properties of PHB; however, due to the slow degradation speed in the soil environment and lack of evaluation methods, studies on the degradation of PHB with plasticizers are rarely reported. In this study, by applying Microbulbifer sp. SOL66, which is able to degrade PHB very quickly, a benign plasticizer was evaluated with good properties and good degradability, not inhibiting microbial activities. Eight different plasticizers were applied with PHB and Microbulbifer sp. SOL66, PHB film containing 10% and 20% tributyl citrate showed significant biodegradability of PHB. It was confirmed that tributyl citrate could increase the speed of PHB degradation by Microbulbifer sp. SOL66 by 88% at 1 day, although the degree of degradation was similar after 3 days with and without tributyl citrate. By the analysis of microbial degradation, physical, chemical, and mechanical properties, tributyl citrate was shown not only to improve physical, chemical, and mechanical properties but also the speed of microbial degradation.

Synthesis of fatty acid-based ammonium ionic liquids and their application for extraction of Co(II) and Ni(II) metals ions from aqueous solution.

Leaching of cobalt and nickel into diverse water streams has become an environmental hazard and is continuously impacting human health through the food chain. Solvent extraction is the most widely accepted for separating these metals, but traditional extractants employed in conjunction with molecular diluents often lack selectivity and caused major environmental hurdles. Therefore, the development of cost-effective, environmentally friendly technologies for recovering these heavy metals has been strongly encouraged in recent years. Herein, two halogens free, low viscous, biocompatible fatty acid-based hydrophobic ionic liquids (ILs), i.e., methytrioctylammonium oleate, methytrioctylammonium linoleate were synthesized, analytically characterized and employed for recovery of cobalt, Co(II) and nickel, Ni(II) from their aqueous solutions. Extraction behaviour of Co(II) and Ni(II) was further evaluated by varying equilibrium time, ILs molar concentration, metal loading, and temperature. Thermodynamic parameters such as enthalpy change and Gibbs free energy change were also studied during extraction process. Slope analysis suggested that the extraction mechanism was an exothermic process that followed ion-transfer from the aqueous phase to the organic phase. Results showed that both fatty acid based-ILs were found to be capable of extracting >99% of Co(II) and Ni(II) from aqueous solutions at 298 K, in 15 min of shaking time using a 1:1 (org: aq.) ratio at low concentrations of 2.5-10 g L-1. Furthermore, for methyltrioctylammonium oleate IL, Co(II) extraction was selectively preferred over Ni(II) extraction when the metal concentration was increased to above to 10 g L-1. The stripping results showed that 2 M H2SO4, and 2 M HCl successfully stripped out >99% of Co(II) and Ni(II) from the organic phase, respectively compared to HNO3.

Bioconversion of Mixed Alkanes to Polyhydroxyalkanoate by Pseudomonas resinovornas: Upcycling of Pyrolysis Oil from Waste-Plastic.

Polyhydroxyalkanoate (PHA) is a biodegradable plastic that can be used to replace petroleum-based plastic. In addition, as a medium-chain-length PHA (mcl-PHA), it can be used to provide elastomeric properties in specific applications. Because of these characteristics, recently, there has been much research on mcl-PHA production using inexpensive biomass materials as substrates. In this study, mcl-PHA producers were screened using alkanes (n-octane, n-decane, and n-dodecane) as sources of carbon. The amount of PHA produced by Pseudomonas resinovorans using sole n-octane, n-decane, or n-dodecane was 0.48 g/L, 0.27 g/L, or 0.07 g/L, respectively, while that produced using mixed alkane was 0.74 g/L. As a larger amount of PHA was produced using mixed alkane compared with sole alkane, a statistical mixture analysis was used to determine the optimal ratio of alkanes in the mixture. The optimal ratio predicted by the analysis was a medium with 9.15% n-octane, 6.44% n-decane, and 4.29% n-dodecane. In addition, through several concentration-specific experiments, the optimum concentrations of nitrogen and phosphorus for cell growth and maximum PHA production were determined as 0.05% and 1.0%, respectively. Finally, under the determined optimal conditions, 2.1 g/L of mcl-PHA and 60% PHA content were obtained using P. resinovorans in a 7 L fermenter.

Acceleration of lactate-utilizing pathway for enhancing biohydrogen production by magnetite supplementation in Clostridium butyricum.

A conductive metal compound can be used as a catalyst for enhancing hydrogen production by dark fermentation. This study aimed to identify mechanisms of enhanced hydrogen production by magnetite supplementation. Experiments were performed with lactate and/or magnetite supplementation to confirm that the lactate-utilizing pathway is the key cause of enhanced hydrogen production. Also, ribonucleic acid sample was collected for monitoring gene regulation under each condition. Hydrogen production was significantly enhanced by approximately 25.6% and 58.9%, respectively, via magnetite alone and with lactate. Moreover, the expression of genes involved in hydrogen production, including pyruvate ferredoxin oxidoreductase, hydrogenase, and ferredoxin, via magnetite alone and with lactate was upregulated by 0.26, 0.71, and 3.50 and 1.06, 2.14, and 1.94 times, respectively.

Lignocellulosic hydrolysate based biorefinery for marine exopolysaccharide production and application of the produced biopolymer in environmental clean-up.

The present study deals with the utilization of lignocellulosic hydrolysate-based carbon source for exopolysaccharide (EPS) production using newly reported marine Echinicola sediminis BBL-M-12. This bacterium produced 7.56 g L-1 and 5.32 g L-1 of EPS on supplementing 30 g L-1 glucose and 10 g L-1 xylose as the sole carbon source, respectively. Whereas on feeding Miscanthus hydrolysate (MCH) with glucose content adjusting to 20 g L-1, E. sediminis BBL-M-12 produced 6.18 g L-1 of EPS. The inhibitors study showed bacterium could tolerate higher concentrations of fermentation inhibitors include furfural (0.05%), 5-hydroxymethylfurfural (0.1%), vanillin (0.1%) and acetate (0.5%). Moreover, the EPS composition was greatly altered with the type and concentration of carbon source supplied, although ß-D-Glucopyranose, ß-D-Galactopyranose, and ß-D-Xylopyranose were the dominant monomers detected. Interestingly, E. sediminis BBL-M-12 EPS revealed excellent environmental applications like clay flocculation, oil emulsification, and removal of humic acid, textile dye, and heavy metal from the aqueous phase.

Finding of novel polyhydroxybutyrate producer Loktanella sp. SM43 capable of balanced utilization of glucose and xylose from lignocellulosic biomass.

Polyhydroxybutyrate (PHB) is a potential substitute for plastics derived from fossil fuels, owing to its biodegradable and biocompatible properties. Lignocellulosic biomass could be used to reduce PHB production costs; however, the co-utilization of sugars, such as glucose and xylose, without catabolite repression is a difficult problem to be solved. Here, we selected a novel Loktanella sp. SM43 from a marine environment and optimized the conditions for PHB production. Loktanella sp. SM43 showed high PHB production (66.5% content) from glucose. When glucose and xylose were used together, this strain showed high utilization of both substrates compared to other high PHB-producers such as Halomonas sp. and Cupriavidus necator, which showed glucose preference. Loktanella sp. SM43 showed high growth and PHB production with lignocellulosic hydrolysates. When pine tree hydrolysates were used, PHB production was the highest at 3.66 ± 0.01 g/L, followed by Miscanthus (3.46 ± 0.09 g/L) and barley straw hydrolysate (3.36 ± 0.36 g/L). Overall, these results reveal the potential of Loktanella sp. SM43 to produce PHB using various lignocellulosic hydrolysates as feedstock and the first systematic study for PHB production with Loktanella sp. The approach of screening novel strains is a strategy to overcome co-utilization of sugars without genetic engineering.

Gamma aminobutyric acid (GABA) production in Escherichia coli with pyridoxal kinase (pdxY) based regeneration system.

Gamma-aminobutyric acid (GABA) is a non-proteinogenic amino acid act as a major neurotransmitter inhibitor in the nervous system of mammals. It also used as a precursor of bioplastics synthesis such as N-methylpyrolidone and polyamide 4. Chemical-based synthesis methods have many environmental-related issues, so efforts have been made to develop biosynthetic methods to produce GABA. Glutamate decarboxylase (GAD) transforms L-glutamate to GABA using pyridoxal 5'-phosphate (PLP) as a cofactor. Bioconversion of GABA with whole cells overexpressing the glutamate decarboxylase has advantages of fewer byproducts and rapid reaction. However, there is a bottleneck in the whole-cell bioconversion system i.e., higher GABA production require a large amount of cofactor PLP which make the process costly. Therefore, pyridoxal kinase (PdxY) able to regenerate PLP was introduced in the whole-cell system to construct a new GABA producing system. Culture and reaction conditions were optimized, and 100% conversion of 0.6 M MSG was obtained. This study reports that a competitive level of GABA production could be achieved without supplying additional PLPs.

Finding of novel lactate utilizing Bacillus sp. YHY22 and its evaluation for polyhydroxybutyrate (PHB) production.

Polyhydroxyalkanoates (PHAs) and their derivatives are biopolymers that have the potential of replacing petroleum-based plastics and can be produced and degraded via bacterial metabolism. However, there are only a few studies on polyhydroxybutyrate (PHB) production using lactate, one of the major waste organic acids that could be implemented in the production of polylactic acid (PLA). Herein, we screened and characterized the PHA-producing microbial strains isolated from saltern soil from Docho Island (South Korea). Among the 24 identified microorganisms that can use lactate as a carbon source, Bacillus sp. YHY22, a newly reported strain, produced the highest amount of PHB: 4.05 g/L with 6.25 g/L dry cell weight, which is 64.7% PHB content under optimal production conditions. Bacillus sp. YHY22 could form the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer with propionate addition. Moreover, Bacillus sp. YHY22 produced PHB in non-sterilized 2% lactate and 8% NaCl marine broth culture medium, suggesting that its production can occur in high salinity media without additional sterilization steps, rendering fermentation cost- and time-efficient.

Recent advances in commercial biorefineries for lignocellulosic ethanol production: Current status, challenges and future perspectives.

Cellulosic ethanol production has received global attention to use as transportation fuels with gasoline blending virtue of carbon benefits and decarbonization. However, due to changing feedstock composition, natural resistance, and a lack of cost-effective pretreatment and downstream processing, contemporary cellulosic ethanol biorefineries are facing major sustainability issues. As a result, we've outlined the global status of present cellulosic ethanol facilities, as well as main roadblocks and technical challenges for sustainable and commercial cellulosic ethanol production. Additionally, the article highlights the technical and non-technical barriers, various R&D advancements in biomass pretreatment, enzymatic hydrolysis, fermentation strategies that have been deliberated for low-cost sustainable fuel ethanol. Moreover, selection of a low-cost efficient pretreatment method, process simulation, unit integration, state-of-the-art in one pot saccharification and fermentation, system microbiology/ genetic engineering for robust strain development, and comprehensive techno-economic analysis are all major bottlenecks that must be considered for long-term ethanol production in the transportation sector.