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.
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.
Lactose fermentation by Kombucha – a process to obtain new milk–based beverages.
Iličić, M., Kanurić, K., Milanović, S., Lončar, E., Djurić, M. & Malbaša, R. (2012). Romanian Biotechnological Letters, 17(1), 7013-7021.
This paper focuses on fermentation of lactose from a model system (black tea) and from two types of milk (0.9% w/w and 2.2% w/w of fat) by application of Kombucha. Quantities of the applied Kombucha starter were 10% v/v and 15% v/v. All fermentations were performed at 42°C. The process to achieve a desirable pH=4.5 was slower in the model system (16 h) than in milks (9 - 10 h). Regarding starter quantity, 10% v/v proved the optimal. Regarding types of milk, higher fat content guarantees shorter fermentation and higher yield of metabolites. Utilization of lactose was found at a level of ≈20% and ≈30% in milks with 0.9% w/w and 2.2% w/w of fat, respectively. This was correlated with an appearance of intermediates and/or products. Glucose underwent further transformations almost entirely, while galactose showed much lower reactivity. Seven to twelve times higher contents of lactic acid were found compared to acetic acid. Milk-based beverage from the reduced fat sample, inoculated with 10% v/v of Kombucha starter, has the best physical characteristics (syneresis and water holding capacity). It also developed a good texture (especially cohesiveness and index of viscosity). Milk lactose fermentation was a process that could have been used for obtaining new milk-based products.
Changes in the volatile compound production of fermentations made from musts with increasing grape content.
Keyzers, R. A. & Boss, P. K. (2010). Journal of Agricultural and Food Chemistry, 58(2), 1153-1164.
Wine is a complex consumer product produced predominately by the action of yeast upon grape juice. Model must systems have proven to be ideal for studies into the effects of fermentation conditions on the production of certain wine volatiles. To clarify the contribution of grape juice to the production of wine volatiles, we have employed a model must system spiked with increasing amounts of grape juice (Riesling or Cabernet Sauvignon). The resulting fermented wines were analyzed by SPME-GC-MS and the data obtained grouped using ANOVA and cluster analyses to reveal those compounds that varied in concentration with reproducible trends relative to juice concentration. Such grouping highlights those compounds that are grape-dependent or for which production is modulated by grape composition. In some cases, increasing the proportion of grape juice in the fermentations stimulated the production of certain esters to levels between 2- and 140-fold higher than those seen in fermentations made with model grape juice media alone. The identification of the grape components responsible for the increased production of these wine volatiles will have implications for the impact of grape production and enology on wine flavor and aroma.
Metabolic engineering of Saccharomyces cerevisiae to minimize the production of ethyl carbamate in wine.
Coulon, J., Husnik, J. I., Inglis, D. L., van der Merwe, G. K., Lonvaud, A., Erasmus, D. J. & van Vuuren, H. J. J. (2006). American Journal of Enology and Viticulture, 57(2), 113-124.
Saccharomyces cerevisiae metabolizes arginine, one of the major amino acids in grape musts, to ornithine and urea during wine fermentations. Wine yeast strains of S. cerevisiae do not fully metabolize urea during grape must fermentation. Urea is secreted by yeast cells and it reacts spontaneously with ethanol in wine to form ethyl carbamate, a potential carcinogenic agent for humans. The lack of urea catabolism by yeast in wine may be ascribed to the transcriptional repression of the DUR1,2 gene by good nitrogen sources present in the grape must. We expressed the DUR1,2 gene under control of the S. cerevisiae PGK1 promoter and terminator signals and integrated this DUR1,2 expression cassette, flanked by ura3 sequences, into the URA3-locus of the industrial wine yeast UC Davis 522. In vivo assays showed that the metabolically engineered industrial strain reduced ethyl carbamate in Chardonnay wine by 89.1%. Analyses of the genotype, phenotype, and transcriptome revealed that the engineered yeast 522EC− is substantially equivalent to the parental 522 strain.
Proso millet (Panicum miliaceum L.) fermentation for fuel ethanol production.
Rose, D. J. & Santra, D. (2013). Industrial Crops and Products, 43, 602-605.
The objective of this research was to determine the conversion efficiency of proso millet to ethanol compared to corn in a bench-scale dry-grind procedure. Seven proso millet cultivars and six advanced breeding lines containing waxy starch were fermented with Saccharomyces cerevisiae and ethanol production was compared with normal corn and “highly fermentable” corn. The highly fermentable corn exhibited the highest fermentation efficiency (97.0 ± 1.4%). Among proso millet lines, those with the highest fermentation efficiencies were: Huntsman (85.9 ± 0.6%), 172-2-9 (90.8 ± 0.2%), 172-2-13 (85.1 ± 2.5%), and 182-4-24 (84.7 ± 2.1). Waxy proso millet lines resulted in higher fermentation efficiencies than the non-waxy proso millet varieties containing normal starch (82.4 ± 5.5% vs. 75.5 ± 7.4%, respectively, p = 0.01). Proso millet distiller's dried grains with solubles (DDGS) contained more protein (26.6–33.4%) than the DDGS from corn (17.2–23.4%). These data indicate that proso millet exhibits promise as a feedstock for ethanol production, especially if breeding programs focus on selecting “highly fermentable” lines for advancement.
Biosynthesis of ethanol and hydrogen by glycerol fermentation using Escherichia coli.
Chaudhary, N., Ngadi, M. O., Simpson, B. K. & Kassama, L. S. (2011). Advances in Chemical Engineering and Science, 1, 83-89.
Production of high value products from glycerol via anaerobic fermentation is of utmost importance for the biodiesel industry. The microorganism Escherichia coli (E. coli) K12 was used for fermentation of glycerol. The effects of glycerol concentration and headspace conditions on the cell growth, ethanol and hydrogen production were investigated. A full factorial experimental design with 3 replicates was conducted in order to test these factors. Under the three headspace conditions tested, the increase of glycerol concentration accelerated glycerol fermentation. The yields of hydrogen and ethanol were the lowest when glycerol concentration of 10 g/L was used. The maximum production of hydrogen was observed with an initial glycerol concentration of 25 g/L at a final concentration of hydrogen was 32.15 mmol/L. This study demonstrated that hydrogen production negatively affects cell growth. Maximum ethanol yield was obtained with a glycerol concentration of 10 g/L and was up to 0.40 g/g glycerol under membrane condition headspace. Statistical optimization showed that optimal conditions for hydrogen production are 20 g/L initial glycerol with initial sparging of the reactor headspace. The optimal conditions for ethanol production are 10 g/L initial glycerol with membrane.
Quantification of full range ethanol concentrations by using pH sensor.
Al-Mhanna, N. M. M. & Huebner, H. (2011). International Journal of Chemistry, 3(1), 47-56.
A differential pH measurement device was used to achieve operation conditions of alcohol dehydrogenase reaction. Optimum operating conditions were temperature of 30°C, 10 μl of alcohol dehydrogenase enzyme volume (with a final activity of 563.75 units ml-1) per 50 μl of sample, NAD+ concentration of 0.05 mM and 20 mM glycine-pyrophosphate buffer solution of pH 9.1. In this method a range of ethanol concentrations from 0-99,985%, which means 0.000001714 - 17.14 M, were used. The maximum obtained change in pH, delta pH, was (-33) mpH. A calibration curve of logarithmic values of ethanol concentrations against change in pH for standard ethanol samples was done. Since this calibration curve is a linear with a correlation coefficient (R) of 0.998, this calibration curve can be used in quantification of ethanol concentration. End point of equilibrium concentrations of reactants and products of ethanol oxidation reaction was measured within spectrophotometer. The results indicated 100 seconds of process time is required to reach the end point for all ethanol standard samples. This required time was satisfied with results of measuring change in pH within differential pH analyzer system.
Screening Thermo- and Ethanol Tolerant Bacteria for Ethanol Fermentation.
Dung, N. T. P. & Huynh, P. X. (2013). American Journal of Microbiological Research, 1(2), 25-31.
The thermophilic bacteria receive considerably interest nowadays because of a current challenge of increasing global temperature. Particularly for ethanol production, the thermo-ethanologenic bacteria possess advantages due to lower contamination risk, cost saving in industrial scale, and the wide range of sugars utilization. In this study, 13 bacterial isolates obtained from the previous isolation study were tested for their fermentative capacity and ethanol tolerance at high temperatures. Five bacterial isolates HM2, M2, MC3, MR1 and RD were found to be tolerant up to 12% ethanol. Of which HM2, M2 and MR1 could ferment glucose well at 30, 35 and 40°C, particularly isolates HM2 and MR1 could perform the fermentative capacity at 45°C and even 50°C. In the presence of 12, 16, and 20% w/v glucose, isolates HM2, M2, and MR1 showed the high fermentation rate by giving high gas production; however, the rate slightly decreased in the presence of 24% w/v glucose. The fermentative performance by these three isolates could happen at different pH levels of 4.0, 5.0 and 6.0. The favourable conditions of ethanol fermentation were found at 18.5% glucose, pH 5.0, and 33°C for isolate HM2 and at 14% glucose, pH 5.5, and 40°C for isolate MR1. The results of sequencing analysis of partial 16S rRNA gene showed that the gene sequences of the selected isolate HM2 shared 99% similarity with Bacillus subtilis.
Microbiological and chemical properties of kefir manufactured by entrapped microorganisms isolated from kefir grains.
Chen, T. H., Wang, S. Y., Chen, K. N., Liu, J. R. & Chen, M. J. (2009). Journal of Dairy Science, 92(7), 3002-3013.
In this study, various yeasts (Kluyveromyces marxianus, Saccharomyces turicensis, Pichia fermentans) and lactic acid bacteria (Lactobacillus kefiranofaciens, Lactobacillus kefiri, Leuconostoc mesenteroides) were entrapped in 2 different microspheres using an entrapment ratio for the strains that was based on the distribution ratio of these organisms in kefir grains. The purpose of this study was to develop a new technique to produce kefir using immobilized starter cultures isolated from kefir grains. An increase in cell counts with fermentation cycles was observed for both the lactic acid bacteria (LAB) and yeasts, whereas the cell counts of kefir grains were very stable during cultivation. Scanning electron microscopy showed that the short-chain lactobacilli and lactococci occupied the surface of the LAB microspheres, whereas the long-chain lactobacilli were inside the microspheres. When the yeasts were analyzed, cells at a high density were entrapped in cracks on the surface and within the microspheres, where they were surrounded by the short-chain lactobacilli. The distribution of the LAB and yeast species in kefir produced from grains and microspheres showed that there was no significant difference between the kefirs produced by the 2 methods; moreover, Leu. mesenteroides and K. marxianus were the predominating microflora in both types of kefir. There was no significant difference in the ethanol and exopolysaccharide contents between the 2 kefirs, although the acidity was different.
Taraxerone enhances alcohol oxidation via increases of alcohol dehyderogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities and gene expressions.
Sung, C. K., Kim, S. M., Oh, C. J., Yang, S. A., Han, B. H. & Mo, E. K. (2012). Food and Chemical Toxicology, 50(7), 2508-2514.
The present study, taraxerone (D-friedoolean-14-en-3-one) was isolated from Sedum sarmentosum with purity 96.383%, and its enhancing effects on alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities were determined: EC50 values were 512.42 ± 3.12 and 500.16 ± 3.23 μM for ADH and ALDH, respectively. In order to obtain more information on taraxerone related with the alcohol metabolism, 40% ethanol (5 mL/kg body weight) with 0.5–1 mM of taraxerone were administered to mice. The plasma alcohol and acetaldehyde concentrations of taraxerone-treated groups were significantly lowered than those of the control group (p < 0.01): approximately 20–67% and 7–57% lowered for plasma alcohol and acetaldehyde, respectively. Compare to the control group, the ADH and ALDH expressions in the liver tissues were abruptly increased in the taraxerone-treated groups after ethanol exposure. In addition, taraxerone prevented catalase, superoxide dismutase, and reduced glutathione concentrations from the decrease induced by ethanol administration with the concentration dependent manner.
Fermentation of high concentrations of maltose by Saccharomyces cerevisiae is limited by the COMPASS methylation complex.
Houghton-Larsen, J. & Brandt, A. Applied and Environmental Microbiology, 72(11), 7176-7182.
In Saccharomyces cerevisiae, genes encoding maltose permeases and maltases are located in the telomeric regions of different chromosomes. The COMPASS methylation complex, which methylates lysine 4 on histone H3, controls the silencing of telomeric regions. Yeast strains deleted for SWD1, SWD3, SDC1, SET1, BRE2, or SPP1, encoding components of the COMPASS complex, fermented a medium containing 22% maltose with noticeably higher attenuation than did the wild type, resulting in production of up to 29% more ethanol. The least effective strain was spp1. Absence of COMPASS components had no effect on the fermentation of media with 20% glucose, 20% sucrose, or 16% maltose. Deletion of SWD3 resulted in larger amounts of MAL12 transcript, encoding maltase, at the late stages of fermentation of 22% maltose. A similar effect on maltase activity and maltose uptake capability was seen. The lysine 4 residue of histone H3 was trimethylated in wild-type cells at the late stages, while only small amounts of the dimethylated form were detected. Trimethylation and dimethylation of this residue were not detected in strains deleted for SWD1, SWD3, SET1, BRE2, or SDC1. Trimethylated lysine 4 was apparent only at the early stages (48 and 96 h) of fermentation in an spp1 strain. This work indicates that the COMPASS complex represses the expression of maltose utilization genes during the late stages of fermentation of a high concentration of maltose.
Single-step, single-organism bioethanol production and bioconversion of lignocellulose waste materials by phlebioid fungal species.
Mattila, H., Kuuskeri, J. & Lundell, T. (2017). Bioresource Technology, 225, 254-261.
Ethanol production from non-pretreated lignocellulose was carried out in a consolidated bioprocess with wood-decay fungi of phlebioid Polyporales. Ethanol production was attempted on glucose, spruce wood sawdust and waste core board. Substantial quantities of ethanol were achieved, and isolate Phlebia radiata 0043 produced 5.9 g/L of ethanol reaching the yield of 10.4% ethanol from core board lignocellulose substrate. Acidic initial culture conditions (pH 3) induced ethanol fermentation compared to the more neutral environment. Together with bioethanol, the fungi were able to produce organic acids such as oxalate and fumarate, thus broadening their capacity and applicability as efficient organisms to be utilized for bioconversion of various lignocelluloses. In conclusion, fungi of Phlebia grow on, convert and saccharify solid lignocellulose waste materials without pre-treatments resulting in accumulation of ethanol and organic acids. These findings will aid in applying fungal biotechnology for production of biofuels and biocompounds.
Living composites of electrospun yeast cells for bioremediation and ethanol production.
Letnik, I., Avrahami, R., Rokem, J. S., Greiner, A., Zussman, E. & Greenblatt, C. (2015). Biomacromolecules, 16(10), 3322-3328.
The preparation of composites of living functional cells and polymers is a major challenge. We have fabricated such “living composites” by preparation of polymeric microtubes that entrap yeast cells. Our approach was the process of coaxial electrospinning in which a core containing the yeast was “spun” within a shell of nonbiodegradable polymer. We utilized the yeast Candida tropicalis, which was isolated from olive water waste. It is particularly useful since it degrades phenol and other natural polyphenols, and it is capable of accumulating ethanol. The electrospun yeast cells showed significant activity of bioremediation of phenol and produced ethanol, and, in addition, the metabolic processes remained active for a prolonged period. Comparison of electrospun cells to planktonic cells showed decreased cell activity; however, the olive water waste after treatment by the yeast was no longer toxic for Escherichia coli, suggesting that detoxification and prolonged viability and activity may outweigh the reduction of efficiency.
The role of lecithin and solvent addition in ethylcellulose-stabilized heat resistant chocolate.
Stortz, T. A., Laredo, T. & Marangoni, A. G. (2015). Food Biophysics, 10(3), 253-263.
Large deformation mechanical testing and Fourier-transform infrared spectroscopy were used to gain further insights into the mechanism of heat resistance in ethylcellulose- (EC) stabilized chocolate prepared using the solvent substitution method. Here we show that the presence of lecithin at the surface of sucrose reduced heat resistance by impeding interactions between EC and sucrose. These techniques along with fluorescence microscopy also showed that the EtOH used in solvent substitution chocolate was able to remove the EtOH soluble lecithin phospholipids from the surface of the sucrose. Removal of the lecithin and the slight solubility of sucrose in EtOH both have positive impacts on heat resistance. It was also found that EtOH may reduce heat resistance by destabilizing the casein micelle in samples made with skim milk powder. Finally, results have indicated that EC is likely able to interact with the lactose in skim milk powder and the starch in cocoa powder leading to greater heat resistance. These findings will be useful in developing the ideal heat resistant chocolate formula.
The composition of readily available carbon sources produced by fermentation of fish faeces is affected by dietary protein: energy ratios.
Letelier-Gordo, C. O., Larsen, B. K., Dalsgaard, J. & Pedersen, P. B. (2017). Aquacultural Engineering, 77, 27-32.
Fish solid waste (faeces) produced in recirculated aquaculture systems (RAS) might be used for on-farm, single-sludge denitrification if transformed into soluble organic carbon substances. The current study investigated the effect of feeding diets with increasing protein to energy ratios (P:E_15, 17, 19, 21 and 23 g/MJ) to rainbow trout (Oncorhynchus mykiss) on the production of volatile fatty acids (VFAs) and ethanol during 7 days fermentation of the produced fish faeces. The total yields of VFAs and ethanol obtained (expressed as chemical oxygen demand (COD)) ranged between 0.21–0.24 gCOD/gTCOD, showing no differences between treatments. However, the type and quantities of individual VFAs and ethanol changed according to the dietary treatment. Lower P:E ratio diets resulted in higher production of butyric acid and ethanol, whereas higher P:E ratio diets resulted in an increased production of acetic and valeric acid. Changing the diet composition thus affects the composition of readily available carbon that can be derived from the faeces. This can be applied to enhance on-farm single sludge denitrification and reduce the need for adding external carbon sources such as e.g. methanol.
Effects of Cudrania Tricuspidata Root Extract (CTE) on Ethanol-Induced Hangover via Modulating Alcohol Metabolizing Enzyme Activities and Blood Gas Levels in Rats.
Choi, N. E., Ro, J. Y., Lee, J. Y., Ryu, J. H. & Cho, H. J. (2017). Journal of the Korea Academia-Industrial cooperation Society, 18(2), 218-225.
To investigate the anti-hangover effects of Cudrania tricuspidata root extract (CTE), the blood alcohol metabolism and blood gas imbalance of CTE in rats treated with 10 ml/kg alcohol were examined. CTE (500 mg/kg and 750 mg/kg) was administrated after 30 minutes of alcohol consumption (10 ml/kg). Blood collection was implemented from the tails of the animals after 1, 3, and 5 hours post alcohol consumption. The Condition drink (a commercial anti-hangover beverage) was used as a positive control. Single administration by the oral route was performed. The consumption of CTE (500 mg/kg and 750 mg/kg) decreased the serum alcohol concentration by increasing and maintaining both the alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) enzyme activity levels in the blood and liver. In addition, CTE led to recovery from the imbalances in the blood gas levels, including carbon dioxide (CO2-) and changes in pH, bicarbonate (HCO3-) and lactic acid levels due to alcohol ingestion. In conclusion, CTE exerted a more pronounced anti-hangover effect than a commercial anti-hangover drink. Therefore, CTE can be a novel and safe anti-hangover natural product agent for the prevention or treatment of symptoms caused by excessive alcohol consumption.
Production of Bioethanol from Agricultural Wastes Using Residual Thermal Energy of a Cogeneration Plant in the Distillation Phase.
Cutzu, R. & Bardi, L. (2017), 3(2), 24.
Alcoholic fermentations were performed adapting the technology to exploit the residual thermal energy (hot water at 83-85°C) of a cogeneration plant and to valorize agricultural wastes. Substrates were apple, kiwifruit and peaches wastes and Corn Threshing Residue (CTR). Saccharomyces bayanus was chosen as biocatalyst. The fruits, fresh or blanched, were mashed; CTR was gelatinized and liquefied by adding Liquozyme® SC DS (Novozyme); saccharification simultaneous to fermentation was carried out using the enzyme Spirizyme® Ultra (Novozyme). Lab-scale static fermentations were carried out at 28°C and 35°C, using raw fruits, blanched fruits and CTR, monitoring the ethanol production. The highest ethanol production was reached with CTR (10,22%9 and among fruits with apple (8,71%). Distillations at low temperatures and under vacuum, to exploit warm water from cogeneration plant, were tested; distillation at 80°C and 200 mbar or 400 mbar allowed to recover 93,35 and 89,59 % of ethanol respectively. These results support a fermentation process coupled to a cogeneration plant, fed with apple wastes and with CTR when apple wastes are not available, where hot water from cogeneration plant is used in blanching and distillation phases. The scale up in a pilot plant was also carried out.
During yeast chronological aging resveratrol supplementation results in a short-lived phenotype Sir2-dependent.
Orlandi, I., Stamerra, G., Strippoli, M. & Vai, M. (2017). Redox Biology, 12, 745-754.
Resveratrol (RSV) is a naturally occurring polyphenolic compound endowed with interesting biological properties/functions amongst which are its activity as an antioxidant and as Sirtuin activating compound towards SIRT1 in mammals. Sirtuins comprise a family of NAD+-dependent protein deacetylases that are involved in many physiological and pathological processes including aging and age-related diseases. These enzymes are conserved across species and SIRT1 is the closest mammalian orthologue of Sir2 of Saccharomyces cerevisiae. In the field of aging researches, it is well known that Sir2 is a positive regulator of replicative lifespan and, in this context, the RSV effects have been already examined. Here, we analyzed RSV effects during chronological aging, in which Sir2 acts as a negative regulator of chronological lifespan (CLS). Chronological aging refers to quiescent cells in stationary phase; these cells display a survival-based metabolism characterized by an increase in oxidative stress. We found that RSV supplementation at the onset of chronological aging, namely at the diauxic shift, increases oxidative stress and significantly reduces CLS. CLS reduction is dependent on Sir2 presence both in expired medium and in extreme Calorie Restriction. In addition, all data point to an enhancement of Sir2 activity, in particular Sir2-mediated deacetylation of the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (Pck1). This leads to a reduction in the amount of the acetylated active form of Pck1, whose enzymatic activity is essential for gluconeogenesis and CLS extension.
The Biorefinery Concept Applied to Bioethanol and Biomethane Production from Manure.
Bona, D., Vecchiet, A., Pin, M., Fornasier, F., Mondini, C., Guzzon, R. & Silvestri, S. Waste and Biomass Valorization, 1-11.
Production of biofuels from farm animal waste represents a promising approach to diversifying green energy production and reducing competition for cultivable lands between fuel and food-oriented crops. This work was aimed to define the technical feasibility and the specific suitability of cattle, swine and poultry manure to integrating bioethanol and biomethane production, using the biorefinery concept. Saccharification obtained by dilute acid pretreatment (3.5% H2SO4 , 121°C, 30 min) followed by enzymatic hydrolysis resulted in total sugar recovery of 230.16, 160.40, and 98.40 mg g-1 (of dry matter) for cattle, pig, and poultry manure respectively. The sugar was then fermented using free yeast co-cultures. The ethanol obtained was 56.32 mg g-1 of dry matter for cattle (about 52.59% of the theoretical ethanol yield); 27.98 mg g-1 for swine (about 88.66% of the theoretical ethanol yield); 12.69 mg g-1 for poultry (about 31.32% of the theoretical ethanol yield). Methane production from distillation waste was 72.95 mg g−1 from dry raw faeces for cattle, 126.48 mg g-1 for swine and 119.03 mg g-1 for poultry. Cattle manure showed the best energy balance in terms of ethanol production with about 824.16 kJ kg-1 of dry faeces, but the two integrated processes generated a net energy balance of 1.28 MJ kg-1 for cattle, 4.57 MJ kg-1 for swine and 4.79 MJ kg-1 for poultry.