Ethanol Assay Kit

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00:04  Introduction
01:00   Principle
02:37    Reagent Preparation
04:11     Procedure
09:14    Calculations

Ethanol Assay Kit K-ETOH Scheme
Reference code: K-ETOH
SKU: 700004279

60 assays (manual) / 600 assays (microplate) / 600 assays (auto-analyser)

Content: 60 assays (manual) / 600 assays (microplate) / 600 assays (auto-analyser)
Shipping Temperature: Ambient
Storage Temperature: Short term stability: 2-8oC,
Long term stability: See individual component labels
Stability: > 2 years under recommended storage conditions
Analyte: Ethanol
Assay Format: Spectrophotometer, Microplate, Auto-analyser
Detection Method: Absorbance
Wavelength (nm): 340
Signal Response: Increase
Linear Range: 0.25 to 12 µg of ethanol per assay
Limit of Detection: 0.093 mg/L
Reaction Time (min): ~ 5 min
Application examples: Wine, beer, cider, alcoholic fruit juices, spirits, liqueurs, low-alcoholic / non-alcoholic beverages, pickles, fruit and fruit juice, chocolate products, vinegar, jam, bread and bakery products, honey, soy sauce, dairy products, cosmetics, pharmaceuticals and other materials (e.g. biological cultures, samples, etc.).
Method recognition: Methods based on this principle have been accepted by AOAC (AOAC Method 2019.08, First Action), IFU, EBC Method 9.3.1, MEBAK and ASBC Method Beer 4-F

The Ethanol test kit is a simple, reliable and accurate method for the measurement and analysis of ethanol in beverages and foodstuffs.

Note for Content: The number of manual tests per kit can be doubled if all volumes are halved.  This can be readily accommodated using the MegaQuantTM  Wave Spectrophotometer (D-MQWAVE).

View our full range of alcohol assay kits.

Scheme-K-ETOH ETOH megayzme

  • Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.
  • Simple format – aldehyde dehydrogenase supplied as stable suspension 
  • Very competitive price (cost per test) 
  • All reagents stable for > 2 years after preparation 
  • Rapid reaction 
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing 
  • Standard included
  • Suitable for manual, microplate and auto-analyser formats
Validation of Methods
Certificate of Analysis
Safety Data Sheet
FAQs Assay Protocol Data Calculator Product Performance Validation Report
Megazyme publication

Determination of ethanol concentration in Kombucha beverages: Single-laboratory validation of an enzymatic method, First Action Method 2019.08.

Ivory, R., Delaney, E., Mangan & McCleary, B. V. (2020). Journal of AOAC International, qsaa122.

The Ethanol Assay Kit is an enzymatic test kit developed by Megazyme for the determination of ethanol in a variety of samples. The kit has been validated in a single laboratory for use with Kombucha fermented drinks, fruit juices and low-alcohol beer samples. The commercially available Ethanol Assay Kit (Megazyme catalogue no. K-ETOH) contains all components required for the analysis. Quantification is based on the oxidation of ethanol to acetaldehyde by alcohol dehydrogenase and further oxidation of acetaldehyde by acetaldehyde dehydrogenase with conversion of NAD+ to NADH. The single laboratory validation (SLV) outlined in this document was performed on a sample set of eight different commercial Kombucha products purchased in Ireland, a set of five Cerilliant aqueous ethanol solutions, two BCR low-alcohol beer reference materials, two alcohol-free beer samples and two fruit juice samples against SMPR 2016.001 (1). Parameters examined during the validation included Working range, Selectivity, Limit of Detection (LOD), Limit of Quantification (LOQ), Trueness (bias), Precision (reproducibility and repeatability), Robustness and Stability.

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Megazyme publication

Megazyme “advanced” wine test kits general characteristics and validation.

Charnock, S. J., McCleary, B. V., Daverede, C. & Gallant, P. (2006). Reveue des Oenologues, 120, 1-5.

Many of the enzymatic test kits are official methods of prestigious organisations such as the Association of Official Analytical Chemicals (AOAC) and the American Association of Cereal Chemists (AACC) in response to the interest from oenologists. Megazyme decided to use its long history of enzymatic bio-analysis to make a significant contribution to the wine industry, by the development of a range of advanced enzymatic test kits. This task has now been successfully completed through the strategic and comprehensive process of identifying limitations of existing enzymatic bio-analysis test kits where they occurred, and then using advanced techniques, such as molecular biology (photo 1), to rapidly overcome them. Novel test kits have also been developed for analytes of emerging interest to the oenologist, such as yeast available nitrogen (YAN; see pages 2-3 of issue 117 article), or where previously enzymes were simply either not available, or were too expensive to employ, such as for D-mannitol analysis.

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Megazyme publication

Grape and wine analysis: Oenologists to exploit advanced test kits.

Charnock, S. C. & McCleary, B. V. (2005). Revue des Enology, 117, 1-5.

It is without doubt that testing plays a pivotal role throughout the whole of the vinification process. To produce the best possible quality wine and to minimise process problems such as “stuck” fermentation or troublesome infections, it is now recognised that if possible testing should begin prior to harvesting of the grapes and continue through to bottling. Traditional methods of wine analysis are often expensive, time consuming, require either elaborate equipment or specialist expertise and frequently lack accuracy. However, enzymatic bio-analysis enables the accurate measurement of the vast majority of analytes of interest to the wine maker, using just one piece of apparatus, the spectrophotometer (see previous issue No. 116 for a detailed technical review). Grape juice and wine are amenable to enzymatic testing as being liquids they are homogenous, easy to manipulate, and can generally be analysed without any sample preparation.

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Growth-coupled anaerobic production of isobutanol from glucose in minimal medium with Escherichia coli.

Boecker, S., Schulze, P. & Klamt, S. (2023). Biotechnology for Biofuels and Bioproducts, 16(1), 148.

Background: The microbial production of isobutanol holds promise to become a sustainable alternative to fossil-based synthesis routes for this important chemical. Escherichia coli has been considered as one production host, however, due to redox imbalance, growth-coupled anaerobic production of isobutanol from glucose in E. coli is only possible if complex media additives or small amounts of oxygen are provided. These strategies have a negative impact on product yield, productivity, reproducibility, and production costs. Results: In this study, we propose a strategy based on acetate as co-substrate for resolving the redox imbalance. We constructed the E. coli background strain SB001 (ΔldhA ΔfrdA ΔpflB) with blocked pathways from glucose to alternative fermentation products but with an enabled pathway for acetate uptake and subsequent conversion to ethanol via acetyl-CoA. This strain, if equipped with the isobutanol production plasmid pIBA4, showed robust exponential growth (µ = 0.05 h−1) under anaerobic conditions in minimal glucose medium supplemented with small amounts of acetate. In small-scale batch cultivations, the strain reached a glucose uptake rate of 4.8 mmol gDW−1 h−1, a titer of 74 mM and 89% of the theoretical maximal isobutanol/glucose yield, while secreting only small amounts of ethanol synthesized from acetate. Furthermore, we show that the strain keeps a high metabolic activity also in a pulsed fed-batch bioreactor cultivation, even if cell growth is impaired by the accumulation of isobutanol in the medium. Conclusions: This study showcases the beneficial utilization of acetate as a co-substrate and redox sink to facilitate growth-coupled production of isobutanol under anaerobic conditions. This approach holds potential for other applications with different production hosts and/or substrate–product combinations.

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The marula and elephant intoxication myth: assessing the biodiversity of fermenting yeasts associated with marula fruits (Sclerocarya birrea).

Makopa, T. P., Modikwe, G., Vrhovsek, U., Lotti, C., Sampaio, J. P. & Zhou, N. (2023). FEMS microbes, 4, xtad018.

The inebriation of wild African elephants from eating the ripened and rotting fruit of the marula tree is a persistent myth in Southern Africa. However, the yeasts responsible for alcoholic fermentation to intoxicate the elephants remain poorly documented. In this study, we considered Botswana, a country with the world's largest population of wild elephants, and where the marula tree is indigenous, abundant and protected, to assess the occurrence and biodiversity of yeasts with a potential to ferment and subsequently inebriate the wild elephants. We collected marula fruits from over a stretch of 800 km in Botswana and isolated 106 yeast strains representing 24 yeast species. Over 93% of these isolates, typically known to ferment simple sugars and produce ethanol comprising of high ethanol producers belonging to Saccharomyces, Brettanomyces, and Pichia, and intermediate ethanol producers Wickerhamomyces, Zygotorulaspora, Candida, Hanseniaspora, and Kluyveromyces. Fermentation of marula juice revealed convincing fermentative and aromatic bouquet credentials to suggest the potential to influence foraging behaviour and inebriate elephants in nature. There is insufficient evidence to refute the aforementioned myth. This work serves as the first work towards understanding the biodiversity marula associated yeasts to debunk the myth or approve the facts.

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The phenotype and genotype of fermentative prokaryotes. 

Hackmann, T. J. & Zhang, B. (2023). Science Advances, 9(39), eadg8687.

Fermentation is a type of metabolism pervasive in oxygen-deprived environments. Despite its importance, we know little about the range and traits of organisms that carry out this metabolism. Our study addresses this gap with a comprehensive analysis of the phenotype and genotype of fermentative prokaryotes. We assembled a dataset with phenotypic records of 8350 organisms plus 4355 genomes and 13.6 million genes. Our analysis reveals fermentation is both widespread (in ~30% of prokaryotes) and complex (forming ~300 combinations of metabolites). Furthermore, it points to previously uncharacterized proteins involved in this metabolism. Previous studies suggest that metabolic pathways for fermentation are well understood, but metabolic models built in our study show gaps in our knowledge. This study demonstrates the complexity of fermentation while showing that there is still much to learn about this metabolism. All resources in our study can be explored by the scientific community with an online, interactive tool.

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Treatment of food processing wastes for the production of medium chain fatty acids via chain elongation.

Battista, F., Zeni, A., Andreolli, M., Salvetti, E., Rizzioli, F., Lampis, S. & Bolzonella, D. (2024). Environmental Technology & Innovation, 33, 103453.

The production of medium chain fatty acids (MCFAs) through reverse β-oxidation was investigated both on synthetic and real substrates. From preliminary batch tests emerged that caproic acid was maximized under an acetate/ethanol molar ratio of 5:1 at neutral pH. This ratio was then adopted in different semi-continuous tests operating with different amounts of the two reactants. It emerged that the MCFAs yield reached the maximum level of 6.7% when the total molar substrate amount was around 40–45 mmol/d, while the process was inhibited for values higher than 400 mmol/d. Semi-continuous tests using real waste as substrates, namely food waste condensate, cheese whey, and winery wastewater, confirmed the results obtained with the synthetic substrates. Better performances were obtained when an adequate molar ratio of the acetate and the electron-donor compound was naturally present. Therefore, a MCFAs yield of 25% and 10.5% was obtained for condensate of food waste and acidic cheese whey, respectively. Regarding MCFAs composition, caproic acid was the dominant form but small concentrations of octanoic acid were also found in the tests where ethanol was the electron donor (synthetic substrates and food waste condensate). Octanoic acid was not produced in test where lactic acid represented the electron donor molecules (cheese whey). Condensate and synthetic samples were dominated by Pseudoclavibacter caeni with an abundance of 38.19% and 33.38% respectively, while Thomasclavelia (24.13%) and Caproiciproducens (11.68%) was the most representative genus in acidic cheese whey sample.

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Extracellular DNA secreted in yeast cultures is metabolism-specific and inhibits cell proliferation.

de Alteriis, E., Incerti, G., Cartenì, F., Chiusano, M. L., Colantuono, C., Palomba, E., et al. (2023). Microbial Cell, 10(12), 292.

Extracellular DNA (exDNA) can be actively released by living cells and different putative functions have been attributed to it. Further, homologous exDNA has been reported to exert species-specific inhibitory effects on several organisms. Here, we demonstrate by different experimental evidence, including 1H-NMR metabolomic fingerprint, that the growth rate decline in Saccharomyces cerevisiae fed-batch cultures is determined by the accumulation of exDNA in the medium. Sequencing of such secreted exDNA represents a portion of the entire genome, showing a great similarity with extrachromosomal circular DNA (eccDNA) already reported inside yeast cells. The recovered DNA molecules were mostly single strands and specifically associated to the yeast metabolism displayed during cell growth. Flow cytometric analysis showed that the observed growth inhibition by exDNA corresponded to an arrest in the S phase of the cell cycle. These unprecedented findings open a new scenario on the functional role of exDNA produced by living cells.

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The Effect of Dekkera bruxellensis Concentration and Inoculation Time on Biochemical Changes and Cellulose Biosynthesis by Komagataeibacter intermedius.

Devanthi, P. V. P., Pratama, F., Kho, K., Taherzadeh, M. J. & Aslanzadeh, S. (2022). Journal of Fungi, 8(11), 1206.

Bacterial Cellulose (BC) is a biopolymer with numerous applications. The growth of BC-producing bacteria, Komagataeibacter intermedius, could be stimulated by Dekkera bruxellensis, however, the effect on BC yield needs further investigation. This study investigates BC production and biochemical changes in the K. intermedius-D. bruxellensis co-culture system. D. bruxellensis was introduced at various concentrations (103 and 106 CFU/mL) and inoculation times (days 0 and 3). BC yield was ~24% lower when D. bruxellensis was added at 103 CFU/mL compared to K. intermedius alone (0.63 ± 0.11 g/L). The lowest BC yield was observed when 103 CFU/mL yeast was added on day 0, which could be compromised by higher gluconic acid production (10.08 g/L). In contrast, BC yields increased by ~88% when 106 CFU/mL D. bruxellensis was added, regardless of inoculation time. High BC yield might correlate with faster sugar consumption or increased ethanol production when 106 CFU/mL D. bruxellensis was added on day 0. These results suggest that cell concentration and inoculation time have crucial impacts on species interactions in the co-culture system and product yield.

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Turmeric extract (Curcuma longa L.) regulates hepatic toxicity in a single ethanol binge rat model.

Lee, H. Y., Lee, G. H., Hoang, T. H., Kim, S. W., Kang, C. G., Jo, J. H., Chung, M. J., Min, K. & Chae, H. J. (2022). Heliyon, 8(9), e10737.

Hepatic alcohol clearance is a key factor to overcome alcohol hangovers, and over the period, alcohol hangovers may lead to inflammation and oxidative stress. Natural food products with high antioxidant and anti-inflammatory effects might contribute to hepatic alcohol clearance, a hypothesis in this study. The present study aimed to evaluate the influence of turmeric (Curcuma longa L., Zingiberaceae) is an herbal product having antioxidant and anti-inflammatory activities, on alcohol metabolism using binge alcohol drinking rat model. In vivo investigations revealed that pretreatment with turmeric extract enhanced alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities upon binge ethanol (3 g/kg). Additionally, pretreatment with turmeric extract regulated CYP2E1 activity and levels of reactive oxygen species (ROS), Bax, Bcl-2, and inflammatory mediators like IL-1β, IL-6, and TNF-α. Moreover, turmeric extract upregulated superoxide dismutase, catalase, and glutathione peroxidase activities in liver tissues. Together, these observations shed light on the potential beneficial effects of turmeric extract against acute liver toxicity. The results offer an alternative natural functional food product, turmeric extract, to prevent the negative implications of binge drinking.

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Effect of herbal extracts and supplement mixture on alcohol metabolism in Sprague Dawley-rats.

Choe, H., Yun, I., Kim, Y., Lee, J. H., Shin, H. A., Lee, Y. K. & Kim, M. Y. (2022). Journal of Food Science and Technology, 1-9.

This study aimed to investigate the effect of mixture of herbal extracts and supplementary formula (FNP-C) on hangovers and antioxidant enzymes in alcohol-induced liver damage in rats. HepG2 cells were used as the experimental cells and divided into five groups: non-treated control (normal), alcohol-induced control (control), mixture of herbal extracts (FNP-B), FNP-C, and a commercial treatment of liver diseases (Livers®); inhibition of detoxification and alcohol-induced damage was confirmed in vivo. Blood alcohol and acetaldehyde concentration after alcohol consumption were measured in a timely manner; alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), superoxide dismutase (SOD), glutathione (GSH), glutathione transferase (GST), and lactate dehydrogenase (LDH) levels were measured in the liver. FNP-C exhibited the highest effect. When FNP-C was administered to alcohol-induced animals, blood alcohol and acetaldehyde concentration decreased compared to FNP-B and Livers®. FNP-C reduced ADH levels and improved LDH, GSH, GST, and SOD levels. The FNP-C group was effective in preventing alcohol-induced hangovers and liver damage. Thus, FNP-C improves hangovers and increases antioxidant activity in an alcohol-induced model. Adding amino acids and vitamins to natural ingredients can potentially enhance the effect of improving hangovers.

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Blocking mitophagy does not significantly improve fuel ethanol production in bioethanol yeast Saccharomyces cerevisiae.

Eliodório, K. P., de Gois e Cunha, G. C., White, B. A., Patel, D. H., Zhang, F., Hettema, E. H., Basso, T. O., Gombert, A. K. & Raghavendran, V. (2022). Applied and Environmental Microbiology, 88(5), e02068-21.

Ethanolic fermentation is frequently performed under conditions of low nitrogen. In Saccharomyces cerevisiae, nitrogen limitation induces macroautophagy, including the selective removal of mitochondria, also called mitophagy. Previous research showed that blocking mitophagy by deletion of the mitophagy-specific gene ATG32 increased the fermentation performance during the brewing of Ginjo sake. In this study, we tested if a similar strategy could enhance alcoholic fermentation in the context of fuel ethanol production from sugarcane in Brazilian biorefineries. Conditions that mimic the industrial fermentation process indeed induce Atg32-dependent mitophagy in cells of S. cerevisiae PE-2, a strain frequently used in the industry. However, after blocking mitophagy, no significant differences in CO2 production, final ethanol titers, or cell viability were observed after five rounds of ethanol fermentation, cell recycling, and acid treatment, which is commonly performed in sugarcane biorefineries. To test if S. cerevisiae’s strain background influenced this outcome, cultivations were carried out in a synthetic medium with strains PE-2, Ethanol Red (industrial), and BY (laboratory) with and without a functional ATG32 gene and under oxic and oxygen restricted conditions. Despite the clear differences in sugar consumption, cell viability, and ethanol titers, among the three strains, we did not observe any significant improvement in fermentation performance related to the blocking of mitophagy. We concluded, with caution, that the results obtained with Ginjo sake yeast were an exception and cannot be extrapolated to other yeast strains and that more research is needed to ascertain the role of autophagic processes during fermentation.

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Production of Bioactive Substances to Alleviates Hangover and Ethanol-Induced Liver Damage through Fermentation of Oenanthe javanica Using Lactiplantibacillus plantarum.

Gam, D. H., Park, J. H., Kim, S. H., Kang, M. H., Kim, S. B. & Kim, J. W. (2022). Molecules, 27(4), 1175.

The purpose of this study is to evaluate the effect of the bioconversion products of Oenanthe javanica extract fermented by Lactiplantibacillus plantarum (OEFL) on relieving hangovers and improving liver function. In addition, the bioactive substance of the OEFL, which alleviates hangover and ethanol-induced liver damage, was identified and its bioactive property was verified through in vivo experiments. In major substances analysis using high-performance liquid chromatography, OEFL produced 9.5-fold higher p-coumaric acid than the O. Javanica extract (OE). In addition, considering that quinic acid, which is not present in the OE, was produced in the OEFL it was confirmed that chlorogenic acid was decomposed into quinic acid by bioconversion. In the in vivo experiment using Sprague-Dawley rats, the OEFL and p-coumaric acid diets reduced blood ethanol, acetaldehyde, GPT, and ALP concentrations, increasing blood albumin concentrations compared to ethanol-administered groups, demonstrating that OEFL and p-coumaric acid, the main substance in the OEFL, improved ethanol-induced liver damage. Furthermore, the OEFL and its main bioactive substance, p-coumaric acid, alleviated liver fibrosis by downregulating TGF-β, SMAD-2, SMAD-4, α-SMA, and upregulating MMP-1. Therefore, OEFL is expected to be used as a functional food or pharmaceutical material as it has been confirmed to effectively relieve hangovers, prevent liver damage, and delay liver fibrosis in ethanol-induced liver damages.

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Synergy of Cellulase Systems between Acetivibrio thermocellus and Thermoclostridium stercorarium in Consolidated-Bioprocessing for Cellulosic Ethanol.

Wang, N., Yan, Z., Liu, N., Zhang, X. & Xu, C. (2022). Microorganisms, 10(3), 502.

Anaerobes harbor some of the most efficient biological machinery for cellulose degradation, especially thermophilic bacteria, such as Acetivibrio thermocellus and Thermoclostridium stercorarium, which play a fundamental role in transferring lignocellulose into ethanol through consolidated bioprocessing (CBP). In this study, we compared activities of two cellulase systems under varying kinds of hemicellulose and cellulose. A. thermocellus was identified to contribute specifically to cellulose hydrolysis, whereas T. stercorarium contributes to hemicellulose hydrolysis. The two systems were assayed in various combinations to assess their synergistic effects using cellulose and corn stover as the substrates. Their maximum synergy degrees on cellulose and corn stover were, respectively, 1.26 and 1.87 at the ratio of 3:2. Furthermore, co-culture of these anaerobes on the mixture of cellulose and xylan increased ethanol concentration from 21.0 to 40.4 mM with a high cellulose/xylan-to-ethanol conversion rate of up to 20.7%, while the conversion rates of T. stercorarium and A. thermocellus monocultures were 19.3% and 15.2%. The reason is that A. thermocellus had the ability to rapidly degrade cellulose while T. stercorarium co-utilized both pentose and hexose, the metabolites of cellulose degradation, to produce ethanol. The synergistic effect of cellulase systems and metabolic pathways in A. thermocellus and T. stercorarium provides a novel strategy for the design, selection, and optimization of ethanol production from cellulosic biomass through CBP.

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Ethanol Production from Oil Palm Trunk: A Combined Strategy Using an Effective Pretreatment and Simultaneous Saccharification and Cofermentation.

Wardani, A. K., Sutrisno, A., Faida, T. N., Yustina, R. D. & Murdiyatmo, U. (2021). International Journal of Microbiology, 2021, In Press.

Background: Oil palm trunk (OPT) with highly cellulose content is a valuable bioresource for bioethanol production. To produce ethanol from biomass, pretreatment is an essential step in the conversion of lignocellulosic biomass to fermentable sugars such as glucose and xylose. Several pretreatment methods have been developed to overcome biomass recalcitrance. In this study, the effects of different pretreatment methods such as alkali pretreatment, microwave-alkali, and alkaline peroxide combined with autoclave on the lignocellulosic biomass structure were investigated. Moreover, ethanol production from the treated biomass was performed by simultaneous saccharification and cofermentation (SSCF) under different temperatures, fermentation times, and cell ratios of Saccharomyces cerevisiae NCYC 479 and pentose-utilizing yeast, Pichia stipitis NCYC 1541. Results: Pretreatment resulted in a significant lignin removal up to 83.26% and cellulose released up to 80.74% in treated OPT by alkaline peroxide combined with autoclave method. Enzymatic hydrolysis of treated OPT resulted in an increase in fermentable sugar up to 93.22%. Optimization of SSCF by response surface method showed that the coculture could work together to produce maximum ethanol (1.89%) and fermentation efficiency (66.14%) under the optimized condition. Conclusion: Pretreatment by alkaline peroxide combined with autoclave method and SSCF process could be expected as a promising system for ethanol production from oil palm trunk and various lignocellulosic biomass.

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Safety Information
Symbol : GHS07, GHS08
Signal Word : Danger
Hazard Statements : H319, H334
Precautionary Statements : P261, P264, P280, P284, P304+P340, P305+P351+P338, P337+P313, P342+P311, P501
Safety Data Sheet
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