Isopropanol production via the thermophilic bioconversion of sugars and syngas using metabolically engineered Moorella thermoacetica.

BACKGROUND: Isopropanol (IPA) is a commodity chemical used as a solvent or raw material for polymeric products, such as plastics. Currently, IPA production depends largely on high-CO2-emission petrochemical methods that are not sustainable. Therefore, alternative low-CO2 emission methods are required. IPA bioproduction using biomass or waste gas is a promising method. RESULTS: Moorella thermoacetica, a thermophilic acetogenic microorganism, was genetically engineered to produce IPA. A metabolic pathway related to acetone reduction was selected, and acetone conversion to IPA was achieved via the heterologous expression of secondary alcohol dehydrogenase (sadh) in the thermophilic bacterium. sadh-expressing strains were combined with acetone-producing strains, to obtain an IPA-producing strain. The strain produced IPA as a major product using hexose and pentose sugars as substrates (81% mol-IPA/mol-sugar). Furthermore, IPA was produced from CO, whereas acetate was an abundant byproduct. Fermentation using syngas containing both CO and H2 resulted in higher IPA production at the specific rate of 0.03 h-1. The supply of reducing power for acetone conversion from the gaseous substrates was examined by supplementing acetone to the culture, and the continuous and rapid conversion of acetone to IPA showed a sufficient supply of NADPH for Sadh. CONCLUSIONS: The successful engineering of M. thermoacetica resulted in high IPA production from sugars. M. thermoacetica metabolism showed a high capacity for acetone conversion to IPA in the gaseous substrates, indicating acetone production as the bottleneck in IPA production for further improving the strain. This study provides a platform for IPA production via the metabolic engineering of thermophilic acetogens.

Characterization of Biogenic PbS Quantum Dots.

Heavy metals in a polluted environment are toxic to life. However, some microorganisms can remove or immobilize heavy metals through biomineralization. These bacteria also form minerals with compositions similar to those of semiconductors. Here, this bioprocess was used to fabricate semiconductors with low energy consumption and cost. Bacteria that form lead sulfide (PbS) nanoparticles were screened, and the crystallinity and semiconductor properties of the resulting nanoparticles were characterized. Bacterial consortia that formed PbS nanoparticles were obtained. Extracellular particle size ranged from 3.9 to 5.5 nm, and lattice fringes were observed. The lattice fringes and electron diffraction spectra corresponded to crystalline PbS. The X-ray diffraction (XRD) patterns of bacterial PbS exhibited clear diffraction peaks. The experimental and theoretical data of the diffraction angles on each crystal plane of polycrystalline PbS were in good agreement. Synchrotron XRD measurements showed no crystalline impurity-derived peaks. Thus, bacterial biomineralization can form ultrafine crystalline PbS nanoparticles. Optical absorption and current-voltage measurements of PbS were obtained to characterize the semiconductor properties; the results showed semiconductor quantum dot behavior. Moreover, the current increased under light irradiation when PbS nanoparticles were used. These results suggest that biogenic PbS has band gaps and exhibits the general fundamental characteristics of a semiconductor.

A targeted liquid cultivation method for previously uncultured non-colony forming microbes.

A large number of microbes are not able to form colonies using agar-plating methods, which is one of the reasons that cultivation based on solid media leaves the majority of microbial diversity in the environment inaccessible. We developed a new Non-Colony-Forming Liquid Cultivation method (NCFLC) that can selectively isolate non-colony-forming microbes that exclusively grow in liquid culture. The NCFLC method involves physically separating cells using dilution-to-extinction (DTE) cultivation and then selecting those that could not grow on a solid medium. The NCFLC was applied to marine samples from a coastal intertidal zone and soil samples from a forest area, and the results were compared with those from the standard direct plating method (SDP). The NCFLC yielded fastidious bacteria from marine samples such as Acidobacteriota, Epsilonproteobacteria, Oligoflexia, and Verrucomicrobiota. Furthermore, 62% of the isolated strains were potential new species, whereas only 10% were novel species from SDP. From soil samples, isolates belonging to Acidobacteriota and Armatimonadota (which are known as rare species among identified isolates) were exclusively isolated by NCFLC. Colony formation capabilities of isolates cultivated by NCFLC were tested using solid agar plates, among which approximately one-third of the isolates were non-colony-forming, approximately half-formed micro-colonies, and only a minority could form ordinary size colonies. This indicates that the majority of the strains cultivated by NCFLC were previously uncultured microbial species unavailable using the SDP method. The NCFCL method described here can serve as a new approach to accessing the hidden microbial dark matter.

Enhancing acetone production from H2 and CO2 using supplemental electron acceptors in an engineered Moorella thermoacetica.

Acetogens grow autotrophically and use hydrogen (H2) as the energy source to fix carbon dioxide (CO2). This feature can be applied to gas fermentation, contributing to a circular economy. A challenge is the gain of cellular energy from H2 oxidation, which is substantially low, especially when acetate formation coupled with ATP production is diverted to other chemicals in engineered strains. Indeed, an engineered strain of the thermophilic acetogen Moorella thermoacetica that produces acetone lost autotrophic growth on H2 and CO2. We aimed to recover autotrophic growth and enhance acetone production, in which ATP production was assumed to be a limiting factor, by supplementing with electron acceptors. Among the four selected electron acceptors, thiosulfate and dimethyl sulfoxide (DMSO) enhanced both bacterial growth and acetone titers. DMSO was the most effective and was further analyzed. We showed that DMSO supplementation enhanced intracellular ATP levels, leading to increased acetone production. Although DMSO is an organic compound, it functions as an electron acceptor, not a carbon source. Thus, supplying electron acceptors is a potential strategy to complement the low ATP production caused by metabolic engineering and to improve chemical production from H2 and CO2.

Reversible Hydrogenase Activity Confers Flexibility to Balance Intracellular Redox in Moorella thermoacetica.

Hydrogen (H2) converted to reducing equivalents is used by acetogens to fix and metabolize carbon dioxide (CO2) to acetate. The utilization of H2 enables not only autotrophic growth, but also mixotrophic metabolism in acetogens, enhancing carbon utilization. This feature seems useful, especially when the carbon utilization efficiency of organic carbon sources is lowered by metabolic engineering to produce reduced chemicals, such as ethanol. The potential advantage was tested using engineered strains of Moorella thermoacetica that produce ethanol. By adding H2 to the fructose-supplied culture, the engineered strains produced increased levels of acetate, and a slight increase in ethanol was observed. The utilization of a knockout strain of the major acetate production pathway, aimed at increasing the carbon flux to ethanol, was unexpectedly hindered by H2-mediated growth inhibition in a dose-dependent manner. Metabolomic analysis showed a significant increase in intracellular NADH levels due to H2 in the ethanol-producing strain. Higher NADH level was shown to be the cause of growth inhibition because the decrease in NADH level by dimethyl sulfoxide (DMSO) reduction recovered the growth. When H2 was not supplemented, the intracellular NADH level was balanced by the reversible electron transfer from NADH oxidation to H2 production in the ethanol-producing strain. Therefore, reversible hydrogenase activity confers the ability and flexibility to balance the intracellular redox state of M. thermoacetica. Tuning of the redox balance is required in order to benefit from H2-supplemented mixotrophy, which was confirmed by engineering to produce acetone.

Removal of Acetic Acid from Bacterial Culture Media by Adsorption onto a Two-Component Composite Polymer Gel.

Organic acids, including acetic acid, are the metabolic products of many microorganisms. Acetic acid is a target product useful in the fermentation process. However, acetic acid has an inhibitory effect on microorganisms and limits fermentation. Thus, it would be beneficial to recover the acid from the culture medium. However, conventional recovery processes are expensive and environmentally unfriendly. Here, we report the use of a two-component hydrogel to adsorb dissociated and undissociated acetic acid from the culture medium. The Langmuir model revealed the maximum adsorption amount to be 44.8 mg acetic acid/g of dry gel at neutral pH value. The adsorption capacity was similar to that of an ion-exchange resin. In addition, the hydrogel maintained its adsorption capability in a culture medium comprising complex components, whereas the ion-exchange did not adsorb in this medium. The adsorbed acetic acid was readily desorbed using a solution containing a high salt concentration. Thus, the recovered acetic acid can be utilized for subsequent processes, and the gel-treated fermentation broth can be reused for the next round of fermentation. Use of this hydrogel may prove to be a more sustainable downstream process to recover biosynthesized acetic acid.

Single-Cell Time-Lapse Observation Reveals Cell Shrinkage upon Cell Death in Batch Culture of Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a model organism for aging and longevity studies. In a clonal population of S. cerevisiae, the timing of cell death in the stationary phase is not synchronized, indicating that heterogeneity exists in survival at a single-cell level. Heterogeneity also exists in the cell size, and its correlation with the death rate has been discussed in past studies. However, the direct cause of the heterogeneity in survival remains unknown. In this report, we revisited this question and asked whether the death rate has any correlation with cell size. Past studies did not exclude a possibility that cells change their size upon or after death. If such a change exists, the size dependence of cell death could be misinterpreted. Therefore, we analyzed the correlation between the death rate and cell size before death by time-lapse imaging. It turned out that the size dependence of the death rate varied from one strain to another, suggesting that general principles between cell size and death do not exist. Instead, cells shrink upon cell death, resulting in the accumulation of small dead cells. The degree of cell shrinkage was proportional to the cell size, and the ratio was constant in two strains, which is between 25 and 28%, suggesting the presence of general principles and mechanisms behind the shrinkage event upon cell death. Further investigation of the cause and mechanism of the shrinkage will help us to understand the process of cell death and the origin of the heterogeneity in survival. IMPORTANCE Cells display various behaviors even though they originate from a clonal population. Such diversity is also observed in cell survival in the stationary phase of Saccharomyces cerevisiae. However, we know little about the causes of heterogeneity in the timing of cell death at a single-cell level. To deepen our understanding of the cause of heterogeneity, we observed the process of cell death in S. cerevisiae by time-lapse imaging. Our analysis showed that cells shrank upon cell death, resulting in the accumulation of small dead cells, while a general principle in the correlation between cell size and death was not seen. The degree of cell shrinkage was proportional to cell size before cell death, and it was constant under all conditions tested, indicating the presence of general principles behind the shrinkage event. Future studies to identify the cause of cell shrinkage must contribute to finding the origin of the heterogeneity in survival.

Optimization of Biomethane Production via Fermentation of Chicken Manure Using Marine Sediment: A Modeling Approach Using Response Surface Methodology.

In this study, marine sediment (MS) was successfully used as a source of methanogenic bacteria for the anaerobic digestion (AD) of chicken manure (CM). Using MS showed high production in liquid and semi-solid conditions. Even in solid conditions, 169.3 mL/g volatile solids of chicken manure (VS-CM) was produced, despite the accumulation of ammonia (4.2 g NH3-N/kg CM). To the best of our knowledge, this is the highest methane production from CM alone, without pretreatment, in solid conditions (20%). Comparing MS to Ozouh sludge (excess activated sewage sludge) (OS), using OS under semi-solid conditions resulted in higher methane production, while using MS resulted in more ammonia tolerance (301 mL/gVS-CM at 8.58 g NH3-N/kg). Production optimization was carried out via a response surface methodology (RDM) model involving four independent variables (inoculum ratio, total solid content, NaCl concentration, and incubation time). Optimized methane production (324.36 mL/gVS-CM) was at a CM:MS ratio of 1:2.5 with no NaCl supplementation, 10% total solid content, and an incubation time of 45 days.

Autotrophic growth and ethanol production enabled by diverting acetate flux in the metabolically engineered Moorella thermoacetica.

Gas fermentation is a promising biological process for the conversion of CO2 or syngas into valuable chemicals. Homoacetogens are microorganisms growing autotrophically using CO2 and H2 or CO and metabolizing them to form acetate coupled with energy conservation. The challenge in the metabolic engineering of the homoacetogens is divergence of the acetate formation, whose intermediate is acetyl-CoA, to a targeted chemical with sufficient production of adenosine triphosphate (ATP). In this study, we report that an engineered strain of the thermophilic homoacetogen Moorella thermoacetica, in which a pool of acetyl-CoA is diverted to ethanol without ATP production, can maintain autotrophic growth on syngas. We estimated the ATP production in the engineered strains under different gaseous compositions by considering redox-balanced metabolism for ethanol and acetate formation. The culture test showed that the combination of retaining a level of acetate production and supplying the energy-rich CO allowed maintenance of the autotrophic growth during ethanol production. In contrast, autotrophy was collapsed by complete elimination of the acetate pathway or supplementation of H2-CO2. We showed that the intracellular level of ATP was significantly lowered on H2-CO2 in consistent with the incompetence. In the meantime, the complete disruption of the acetate pathway resulted in the redox imbalance to produce ethanol from CO, albeit a small loss in the ATP production. Thus, preservation of a fraction of acetate formation is required to maintain sufficient ATP and balanced redox in CO-containing gases for ethanol production.

Reaction Rate of Hydrothermal Ammonia Production from Chicken Manure.

Ammonia is an important fertilizer feedstock and an expected next-generation hydrogen carrier. Thus, it is necessary to ensure effective production of ammonia from the waste biomass. In this regard, chicken manure was treated in an autoclave under hydrothermal reaction conditions, and the ammonia release rate was determined in the temperature range of 250-400 °C for holding times ranging from 2 to 120 min. A reaction network for ammonia production was proposed, and the reaction rate constants were determined. A nitrogen yield as high as 0.8 was obtained, corresponding to a hydrogen potential of 88.1 billion m3/year from chicken manure. Consequently, chicken manure was identified as a potentially favorable feedstock for ammonium production.

Visualization of Gene Reciprocity among Lactic Acid Bacteria in Yogurt by RNase H-Assisted Rolling Circle Amplification-Fluorescence In Situ Hybridization.

Recently, we developed an in situ mRNA detection method termed RNase H-assisted rolling circle amplification-fluorescence in situ hybridization (RHa-RCA-FISH), which can detect even short mRNA in a bacterial cell. However, because this FISH method is sensitive to the sample condition, it is necessary to find a suitable cell permeabilization and collection protocol. Here, we demonstrate its further applicability for detecting intrinsic mRNA expression using lactic acid bacteria (LAB) as a model consortium. Our results show that this method can visualize functional gene expression in LAB cells and can be used for monitoring the temporal transition of gene expression. In addition, we also confirmed that data obtained from bulk analyses such as RNA-seq or microarray do not always correspond to gene expression in individual cells. RHa-RCA-FISH will be a powerful tool to compensate for insufficient data from metatranscriptome analyses while clarifying the carriers of function in microbial consortia. By extending this technique to capture spatiotemporal microbial gene expression at the single-cell level, it will be able to characterize microbial interactions in phytoplankton-bacteria interactions.

Metabolic engineering of Moorella thermoacetica for thermophilic bioconversion of gaseous substrates to a volatile chemical.

Gas fermentation is one of the promising bioprocesses to convert CO2 or syngas to important chemicals. Thermophilic gas fermentation of volatile chemicals has the potential for the development of consolidated bioprocesses that can simultaneously separate products during fermentation. This study reports the production of acetone from CO2 and H2, CO, or syngas by introducing the acetone production pathway using acetyl-coenzyme A (Ac-CoA) and acetate produced via the Wood-Ljungdahl pathway in Moorella thermoacetica. Reducing the carbon flux from Ac-CoA to acetate through genetic engineering successfully enhanced acetone productivity, which varied on the basis of the gas composition. The highest acetone productivity was obtained with CO-H2, while autotrophic growth collapsed with CO2-H2. By adding H2 to CO, the acetone productivity from the same amount of carbon source increased compared to CO gas only, and the maximum specific acetone production rate also increased from 0.04 to 0.09 g-acetone/g-dry cell/h. Our development of the engineered thermophilic acetogen M. thermoacetica, which grows at a temperature higher than the boiling point of acetone (58 °C), would pave the way for developing a consolidated process with simplified and cost-effective recovery via condensation following gas fermentation.

Improvement of fatty acid productivity of thraustochytrid, Aurantiochytrium sp. by genome editing.

Thraustochytrid strains belonging to the genus Aurantiochytrium accumulate significant amounts of lipids including polyunsaturated fatty acids and carotenoids and, therefore, are expected to be used for industrial production of various valuable materials. Although various efforts such as chemical mutagenesis and homologous gene recombination have been made to improve lipid productivity of Aurantiochytrium species, low specificity and efficiency in the conventional methods hinder the research progress. Here, we attempted to apply a genome editing technology, the CRISPR-Cas9 system as an alternative molecular breeding technique for Aurantiochytrium species to accelerate the metabolic engineering. The efficiency of specific gene knock-in by the homologous recombination increased more than 10-folds by combining the CRISPR-Cas9 system. As a result of disrupting the genes associated with ß-oxidation of fatty acids by the improved method, the genome edited strains with higher fatty acid productivity were isolated, demonstrating for the first time that the CRISPR-Cas9 system was effective for molecular breeding of the strains in the genus Aurantiochytrium to improve lipid productivity.

Direct detection of mRNA expression in microbial cells by fluorescence in situ hybridization using RNase H-assisted rolling circle amplification.

Meta-analyses using next generation sequencing is a powerful strategy for studying microbiota; however, it cannot clarify the role of individual microbes within microbiota. To know which cell expresses what gene is important for elucidation of the individual cell's function in microbiota. In this report, we developed novel fluorescence in situ hybridization (FISH) procedure using RNase-H-assisted rolling circle amplification to visualize mRNA of interest in microbial cells without reverse transcription. Our results show that this method is applicable to both Gram-negative and Gram-positive microbes without any noise from DNA, and it is possible to visualize the target mRNA expression directly at the single-cell level. Therefore, our procedure, when combined with data of meta-analyses, can help to understand the role of individual microbes in the microbiota.

Combined simultaneous enzymatic saccharification and comminution (SESC) and anaerobic digestion for sustainable biomethane generation from wood lignocellulose and the biochemical characterization of residual sludge solid.

Simultaneous enzymatic saccharification and comminution (SESC) was used for large-scale anaerobic digestion of wood lignocellulose to generate methane and unmodified lignin. During SESC, 10% aqueous mixture of powdered debarked wood from various species was subjected to bead milling with hydrolytic enzymes to generate particles below 1 µm. This slurry was directly used as a cosubstrate for anaerobic digestion in a 500 L stirred-tank reactor. Temperature and hydraulic retention time (HRT) were maintained at 50 °C and 30 days, respectively. At stable operation periods, an average yield of 224 L of methane per kg of cedar was attained. Comparable yields were achieved with red pine, elm, oak, and cedar bark. High-throughput microbial analysis established the presence of a relevant community to support the elevated level of methane production. The stability of the unmodified lignin in anaerobic digestion was also confirmed, allowing for its recovery as an important by-product.

Ethanol yield and sugar usability in thermophilic ethanol production from lignocellulose hydrolysate by genetically engineered Moorella thermoacetica.

Bioconversion from inexpensive renewable resource, such as biomass, to liquid fuel is one of the promising technologies to reduce the use of petroleum. We previously reported the genetically engineered Moorella thermoacetica could produce ethanol from the lignocellulosic feedstock. However, it was still unclear which carbon source in the substrate was preferentially consumed to produce ethanol. To identify the hierarchy of the sugar utilization during ethanol fermentation of this strain, we analyzed the sugar composition of lignocellulosic feedstock, and consumption rate of sugars during the fermentation process. The hydrolysates after acid pretreatment and enzymatic saccharification contained glucose, xylose, galactose, arabinose, and mannose. Time course data suggested that xylose was the most preferred carbon source among those sugars during ethanol fermentation. Ethanol yield was 0.40 ± 0.06 and 0.40 ± 0.12 g/g-total sugar, from lignocellulosic hydrolysates of Japanese cedar (Cryptomeria japonica) and rice straw (Oryza sativa), respectively. The results demonstrated that the genetically engineered M. thermoacetica is a promising candidate for thermophilic ethanol fermentation of lignocellulosic feedstocks, especially hemicellulosic sugars.

Complete genome sequence of Nitratireductor sp. strain OM-1: A lipid-producing bacterium with potential use in wastewater treatment.

Reducing CO2 emissions is necessary to alleviate rising global temperature. Renewable sources of energy are becoming an increasingly important substitute for fossil fuels. An important step in this direction is the isolation of novel, technologically relevant microorganisms. Nitratireductor sp. strain OM-1 can convert volatile short-chain fatty acids in wastewater into 2-butenoic acid and its ester and can accumulate intracellularly esterified compounds up to 50% of its dried cell weight under nitrogen-depleted conditions. It is believed that a novel fatty acid biosynthesis pathway including an esterifying enzyme is encoded in its genome. In this study, we report the whole-genome sequence (4.8 Mb) of OM-1, which comprises a chromosome (3,977,827 bp) and a megaplasmid (857,937 bp). This sequence information provides insight into the genome organization and biochemical pathways of OM-1. In addition, we identified lipid biosynthesis pathways in OM-1, paving the way to a better understanding of its biochemical characterization.

The mechanosensitive channel YbdG from Escherichia coli has a role in adaptation to osmotic up-shock.

Mechanosensitive channels play an important role in the adaptation of cells to hypo-osmotic shock. Among members of this channel family in Escherichia coli, the exact function and physiological role of the mechanosensitive channel homolog YbdG remain unclear. Characterization of YbdG's physiological role has been hampered by its lack of measurable transport activity. Using a nitrosoguanidine mutagenesis-aided screen in combination with next-generation sequencing, here we isolated a mutant with a point mutation in ybdG This mutation (resulting in a I167T change) conferred sensitivity to high osmotic stress, and the mutant cells differed from WT cells in morphology during hyperosmotic stress at alkaline pH. Interestingly, unlike the cells containing the I167T variant, a null-ybdG mutant did not exhibit this sensitivity and phenotype. Although I167T was located near the putative ion-conducting pore in a transmembrane region of YbdG, no change in ion channel activities of YbdG-I167T was detected. Of note, introduction of the WT C-terminal cytosolic region of YbdG into the I167T variant complemented the osmo-sensitive phenotype. Co-precipitation of proteins interacting with the C-terminal YbdG region led to the isolation of HldD and FbaA, whose overexpression in cells containing the YbdG-I167T variant partially rescued the osmo-sensitive phenotype. This study indicates that YbdG functions as a component of a mechanosensing system that transmits signals triggered by external osmotic changes to intracellular factors. The cellular role of YbdG uncovered here goes beyond its predicted function as an ion or solute transport protein.

Metabolite Profile Analysis of Aurantiochytrium limacinum SR21 Grown on Acetate-based Medium for Lipid Fermentation.

Thraustochytrids, a group of marine protists, are continuously gaining attention due to their capability in producing lipids for various biotechnological applications towards foods, medicines, chemicals, and biofuels. Although various substrates, predominantly glucose, have been used as carbon source for this microalga, it is desirable to adopt cheaper and more diversified substrate to expand their application range. In this study, we aimed to examine the ability of acetate, which can be easily generated from various resources by acetogenic microorganisms, as a substrate of Aurantiochytrium limacinum SR21. As a result of flask-scale analysis, specific growth rates (µ) of the strain SR21 grown in 3% acetate- or glucose-based medium were 0.55 and 0.98 h-1, respectively. The maximum yield of total fatty acid in acetate medium was 4.8 g/L at 48 h while that in glucose medium was 6.8 g/L at 30 h, indicating that acetate has potential as substrate. Metabolome analysis was performed to comprehensively elucidate characteristic metabolic fluctuations caused by acetate assimilation and identify targets to improve the fatty acid productivity from acetate. It was found that the use of glyoxylate cycle, which bypasses release of energy molecules such as NADH and GTP, and the inhibition of utilization of compounds from TCA cycle for anabolic reactions, may cause the slow growth in acetate which has an effect also in lipid productivity. The activity of the pentose phosphate pathway was found to be weak in acetate cultivation, thus NADPH was mainly produced in malate-pyruvate cycle. Lastly, mevalonate pathway was found to be activated in acetate cultivation which additionally competes with acetyl-CoA as starting material of fatty acid synthesis.

Simultaneous enzymatic saccharification and comminution for the valorization of lignocellulosic biomass toward natural products.

BACKGROUND: Large-scale processing of lignocellulosics for glucose production generally relies on high temperature and acidic or alkaline conditions. However, extreme conditions produce chemical contaminants that complicate downstream processing. A method that mainly rely on mechanical and enzymatic reaction completely averts such problem and generates unmodified lignin. Products from this process could find novel applications in the chemicals, feed and food industry. But a large-scale system suitable for this purpose is yet to be developed. In this study we applied simultaneous enzymatic saccharification and communition (SESC) for the pre-treatment of a representative lignocellulosic biomass, cedar softwood, under both laboratory and large-scale conditions. RESULTS: Laboratory-scale comminution achieved a maximum saccharification efficiency of 80% at the optimum pH of 6. It was possible to recycle the supernatant to concentrate the glucose without affecting the efficiency. During the direct alcohol fermentation of SESC slurry, a high yield of ethanol was attained. The mild reaction conditions prevented the generation of undesired chemical inhibitors. Large-scale SESC treatment using a commercial beads mill system achieved a saccharification efficiency of 60% at an energy consumption of 50 MJ/kg biomass. CONCLUSION: SESC is very promising for the mild and clean processing of lignocellulose to generate glucose and unmodified lignin in a large scale. Economic feasibility is highly dependent on its potential to generate high value natural products for energy, specialty chemicals, feed and food application.